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Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity., (© 2022. The Author(s).)

Biomedical imaging using cell labeling is an important technique to visualize cell dynamics in the body. To label cells, thiol-organosilica nanoparticles (thiol-OS) containing fluorescein (thiol-OS/Flu) and rhodamine B (thiol-OS/Rho) were surface-functionalized with polyethyleneimine (PEI) (OS/Flu-PEI and OS/Rho-PEI) with 4 molecular weights (MWs). We hypothesized PEI structures such as brush, bent brush, bent lie-down, and coiled types on the surface depending on MWs based on dynamic light scattering and thermal gravimetric analyses. The labeling efficacy of OS/Flu-PEIs was dependent on the PEI MW and the cell type. A dual-particle administration study using thiol-OS and OS-PEIs revealed differential endosomal sorting of the particles depending on the surface of the NPs. The endosomes in the labeled cells using OS/Flu-PEI and thiol-OS/Rho revealed various patterns of fluorescence termed barcoded endosomes. The cells labeled with OS-PEI in vitro were administrated to mice intraperitoneally after in situ labeling of peritoneal cells using thiol-OS/Rho. The in vitro labeled cells were detected and identified in cell aggregates in vivo seamlessly. The labeled cells with barcoded endosomes were also identified in cell aggregates. Biomedical imaging of in vitro OS-PEI-labeled cells combined with in situ labeled cells showed high potential for observation of cell dynamics., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Polarized optical microscopy (POM) and transmission electron microscopy (TEM) are widely used for imaging polymer spherulite structures. TEM provides nanometer resolution to image small spherulites of sub-micrometer in diameter, while POM is more suitable for observing large spherulites. However, high-resolution images with a large field of view are challenging to achieve due to the deformations of ultrathin sectioned samples used for TEM observations. In this study, we demonstrated that correlative light and electron microscopy (CLEM) combining POM and TEM could effectively characterize the spherulite structures in a wide range from nanometer to several hundred micrometers that neither TEM nor POM alone could cover. Furthermore, the deformations of the TEM ultrathin sections were corrected by referencing to the POM images at the same position of the sample, and large-area TEM images without deformations were successfully produced. The spherulite structures of poly(ʟ-lactic acid) were successfully analyzed using CLEM and TEM in a wide range of spatial scales at the same field of view. The large-area TEM image (250 µm × 250 µm), consisting of 702 TEM images stitched together, was subjected to machine learning to extract the essential structural information of spherulites. Analysis using the convolutional neural network, a well-known algorithm You Only Look Once (YOLO), demonstrated that it was practical to accurately obtain the diameter distribution and the space-filling factor (relative crystallinity) of the spherulites. This study presents a new approach for acquiring high-resolution images with a large field of view and processing the images at a fast speed., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Japanese Society of Microscopy.)

Several types of macrophages have been reported in the intestinal mucosa, but their histological localization remains ambiguous. Here, we obtained detailed information about ultrastructural and phenotypical diversity of macrophage-like cells (MLCs) in the rat ileal mucosa using immunofluorescent analysis and serial block-face scanning electron microscopy (SBF-SEM). The results revealed that the cells immunopositive for CD68, the pan-macrophage marker, included CD163 - CD4 + , CD163 + CD4 + , and CD163 - CD4 - cells in the lamina propria (LP) of the intestinal villus and around the crypt. CD68 + CD4 + CD163 - cells seemed to be preferentially localized in the intestinal villus, whereas CD68 + CD163 + CD4 + cells were frequently localized around the crypt. SBF-SEM analysis identified three types of MLCs in the ileal mucosa, which were tentatively named types I-III MLC based on aspects of the 3D-ultrastructure, such as the localization, quantity of lysosomes, endoplasmic reticulum, and exoplasm. Type I and II MLCs were localized in the villous LP, while type III MLCs were localized around the crypt, although type II MLCs were a minor population. All three MLC types extended their cellular processes into the epithelium, with type I MLCs showing the greatest abundance of extended processes. Type I MLCs in the upper portion of the intestinal villus showed a higher level of attachment to intraepithelial lymphocytes (IELs) compared to type III MLCs around the crypt. These findings suggest that macrophages of the rat ileal mucosa differed by region along the longitudinal axis of the villous tip-crypt from the perspective of ultrastructure, cellular composition, localization, and interactions with IELs., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

During autophagy, autophagosomes are formed to engulf cytoplasmic contents. p62/SQSTM-1 is an autophagic adaptor protein that forms p62 bodies. A unique feature of p62 bodies is that they seem to directly associate with membranous structures. We first investigated the co-localization of mKate2-p62 bodies with phospholipids using click chemistry with propargyl-choline. Propargyl-choline-labeled phospholipids were detected inside the mKate2-p62 bodies, suggesting that phospholipids were present inside the bodies. To clarify whether or not p62 bodies come in contact with membranous structures directly, we investigated the ultrastructures of p62 bodies using in-resin correlative light and electron microscopy of the Epon-embedded cells expressing mKate2-p62. Fluorescent-positive p62 bodies were detected as uniformly lightly osmificated structures by electron microscopy. Membranous structures were detected on and inside the p62 bodies. In addition, multimembranous structures with rough endoplasmic reticulum-like structures that resembled autophagosomes directly came in contact with amorphous-shaped p62 bodies. These results suggested that p62 bodies are unique structures that can come in contact with membranous structures directly.

Eosinophils are abundantly present in intestinal mucosa. However, the morphological characteristics of their cellular population are still largely unknown. In this study, we examine their characteristics in the rat ileal mucosa using histological and ultrastructural methods. The results indicated that ileal eosinophils could be distinguished into two main groups based on their nuclear shapes and distribution: eosinophils with spheric or reniform nuclei mainly localized in the villous region and eosinophils with annular or bacilliform nuclei as the major population around crypts. Immunohistochemical analysis revealed that all eosinophils in the lamina propria (LP) were immunopositive for CD11b, whereas eosinophils in LP of the intestinal villus but not those in LP around the crypt, were immunopositive for CD11c. Three-dimensional ultrastructural analysis using serial block-face scanning electron microscopy showed that the eosinophils with spheric or reniform nuclei were abundant in the upper portions of the intestinal villus, whereas those with annular nuclei were abundant in the lower portions of the intestinal villus and around crypts. The eosinophils with spheric or reniform nuclei possessed broader cellular bodies with greater abundance of surface projections compared with those with annular nuclei. Eosinophils in the upper portions of intestinal villus frequently extended their cellular bodies into the intraepithelial space. The number of total and eosinophil-specific granules was positively correlated with the minor axis of the nuclear holes in the annular nuclei. These data suggest that ileal eosinophils exhibit not homogenous but rather diverse characteristics, possible due to the mixture of eosinophils at different maturation and/or activation stages.

The comprehensive targets of innervation in the intestinal mucosa are unknown, partly because of the diversity of cell types and the complexity of the neural circuits. Herein, we investigated the comprehensive targets of neural connectivity and analyzed the precise characteristics of their contact structures in the mucosa of rat ileum. We examined target cells of neural connections and the characteristics of their contact structures by serial block-face scanning electron microscopy at four portions of the rat ileal mucosa: the apical and basal portions in the villi, and the lateral and basal portions around/in the crypts. Nerve fibers were in contact with several types of fibroblast-like cells (FBLCs), macrophage-like cells, eosinophils, lymphocyte-like cells, and other types of cells. The nerve fibers almost always ran more inside of lamina propria than subepithelial FBLC, and thus contacts with epithelial cells were very scarce. The contact structures of the nerve fibers were usually contained synaptic vesicle-like structures, and we classified them into patterns based on the number of nerve fiber contacting the target cells at one site, the maximum diameter of the contact structures, and the relationship between nerve fibers and nerve bundles. The contact structures for each type of cells occasionally dug into the cellular bodies of the target cells. We revealed the comprehensive targets of neural connectivity based on the characteristics of contact structures, and identified FBLCs, immunocompetent cells, and eosinophils as the candidate targets for innervation in the rat ileal mucosa.

In this study, we investigated use of local surface plasmon resonance (LSPR) of metal nanoparticles (NPs) as a correlative light and electron microscopy (CLEM) tag for biological samples. Gold NPs in ultra-thin sections for TEM revealed that LSPR could be observed by optical microscopy at sizes of 20 nm or larger. Gold NPs at sizes less than 20 nm could be observed using the gold enhancement method. Therefore, this CLEM tag could be applied to immunoelectron microscopy using this gold enhancement method., (© The Author(s) 2019. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In this study, we investigated the optical properties of a silicon nitride (SiN) film. The thin SiN film (30 nm thick) exhibited good light transmittance and little autofluorescence and could be used as a microscope slide for optical microscopy (OM). In addition, we developed a novel correlative light and electron microscopy (CLEM) that combines OM with transmission electron microscopy (TEM) using an SiN thin film. In this system, CLEM was performed by replacing a detachable retainer with a holder for TEM and an adaptor for OM. The advantage of this method is that the same specimens can be sequentially observed using suitable OM and TEM., (© The Author(s) 2019. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Serial block-face scanning electron microscopy (SBF-SEM) is useful for three-dimensional observation of tissues or cells at high-resolution. In this study, SBF-SEM was used to three-dimensionally analyze the characteristics of fibroblast-like cells (FBLCs) in the rat ileal lamina propria (LP). The results revealed that FBLCs in LP could be divided into four types, tentatively named FBLC type I-IV, based on the external cellular appearance, abundance or shape of each organelle, detailed distribution in the LP and relationship with surrounding cells. FBLC-I and -II localized around the intestinal crypt (InC), FBLC-III localized from the lateral portion of InC to the apical portion of the intestinal villus (InV), and FBLC-IV localized in the InV. FBLC-I, -II and -III, but not FBLC-IV, localized beneath the epithelium. FBLC-II possessed thin lamellar-shaped endoplasmic reticula, whereas FBLC-I possessed expanded endoplasmic reticula that occasionally showed a spherical shape. FBLC-III and -IV possessed a cytoplasmic region with high-electron density and no organelles immediately beneath the cellular membrane; this region was found at the epithelial sides in FBLC-III and scattered in FBLC-IV. FBLC-IV were in constant close proximity to villous myocytes throughout the InV and also in contact with FBLC-III especially in the apical portion of the InV. FBLC-I, -II and -IV, but not -III, were constantly found to be in contact with various immunocompetent cells in the LP. Three-dimensional analysis using SBF-SEM indicates that four types of FBLC localized in the rat ileal LP.

In transmission electron microscopy (TEM), silicon nitride (SiN) films are widely used as sample-supporting films owing to their robustness. We fabricated large-scale SiN films deposited by low-pressure chemical vapor deposition (LPCVD). This preparation method is advantageous for large window areas, since it yields films with control over properties such as tension and thickness. We fabricated large SiN windows for mounting large ultrathin sections and for acquiring large-area TEM images. Thus, sample sections sliced by conventional sample preparation techniques were successfully mounted on these sample-supporting films. We successfully obtained a 680 × 250 μm2 TEM montage image of a whole Drosophila embryo.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses., (© 2017 Wiley Periodicals, Inc.)

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

Paramyosin is a myosin-binding protein characteristic of invertebrate animals, while troponin is a Ca 2+ -dependent regulator of muscle contraction. Both proteins are widely distributed in protostomes, while in deuterostomes, their distribution is limited; namely, presence of paramyosin and absence of troponin are common features in echinoderm muscles, while muscles of chordates contain troponin but lack paramyosin. In this study, we examined the muscle of a hemichordate, acorn worm, to clarify whether this animal is like echinoderms or like the other deuterostome animals. We found a 100-kDa protein in the smooth muscle of acorn worm. This protein was identified with paramyosin, since the purified protein formed paracrystals with a constant axial periodicity in the presence of divalent cations as paramyosin of other animals, showed ability to interact with myosin and shared common antigenicity with echinoderm paramyosin. On the other hand, troponin band was not detected in isolated thin filaments, and the filaments increased myosin-ATPase activity in a Ca 2+ -independent manner. The results indicate that troponin is lacking in thin filaments of acorn worm muscle just as in those of echinoderms. The muscle of hemichordate acorn worm is quite similar to echinoderm muscles, but different from chordate muscles., (© The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Plant cytokinesis is achieved by formation of cell plates in the phragmoplast, a plant-specific cytokinetic apparatus, which consists of microtubules (MTs) and microfilaments. During cytokinesis, the cell plate is expanded centrifugally outward from the inside of cells in a process that is supported by dynamic turnover of MTs. M-phase-specific kinesin NACK1, which comprises the motor domain at the amino-terminal half to move on MT bundles and the stalk region in the carboxyl-terminal half, is a key player in the process of MT turnover. That is, the specific region in the stalk binds the MAP kinase kinase kinase to activate the whole MAP kinase cascade, which stimulates depolymerization of MTs for the MT turnover. The stalk is also responsible for recruiting the activated kinase cascade to the mid-zone of the phragmoplast, which corresponds to the cell-plate formation site. It should be crucial to uncover roles of the NACK1 kinesin stalk as well as the motor domain in the formation of cell plates in order to understand the mechanisms of cell plate formation. Using dissected Arabidopsis NACK1 (AtNACK1/HINKEL) molecules and AtNACK1-fused GFP, we showed that the C-terminal tail of the stalk in addition to the motor domain is critical for its proper localization to the site of cell plate formation in the phragmoplast, probably by affecting its motility activity.

Recurrent fever often occurs during the treatment of bacterial meningitis in children. Few studies have focused on the difference between the patients with and without recurrent fever. The pathogenesis and clinical significance of recurrent fever remain to be elucidated. The objectives of this study were to clarify the clinical features of the patients with recurrent fever, and to identify the predictive factors for recurrent fever in childhood bacterial meningitis. We conducted a hospital-based retrospective chart review and confirmed 52 cases of bacterial meningitis. Recurrent fever was observed in 27 of 52 (52%) patients overall, and 27 of 47 (57%) patients with dexamethasone (DEX) therapy. All patients with recurrent fever received DEX therapy. The mean date of recurrent fever occurrence was 6.2 +/- 2.0 days after admission. The median duration of recurrent fever was 5 days (range 1-19 days). The proportion of patients < 2 years of age was higher in patients with recurrent fever than those without recurrent fever (p = 0.041). Initial peripheral white blood cell count was lower in patients with recurrent fever (p = 0.008). Of the 52 children, 10 (19%) showed neurological sequelae. Neurological sequelae were more frequent in patients with recurrent fever than those without recurrent fever, although this was not statistically significant (p = 0.078). Univariate and multivariate logistic regression analysis showed that the most powerful predictive factor for recurrent fever in patients with DEX therapy was young age (< 2 years) (adjusted odds ratio, 5.1; 95% confidence interval, 1.3-20.7). These results suggest that recurrent fever is related to the two known factors for sequelae, low peripheral white blood cell count and young age. Recurrent fever should be recognized as a possible risk factor for neurological sequelae.

This study examined 19 patients with portosystemic shunt encephalopathy caused by a splenorenal shunt (SRS), which was treated with balloon-occluded retrograde transvenous obliteration (B-RTO). Long-term treatment outcomes were evaluated based on hepatic functional reserve and vital prognosis. Encephalopathy improved in all patients after shunt embolization and closure. Albumin, serum ammonia, and the Child-Pugh score, a measure of liver function, were significantly improved 3 years after B-RTO, and exacerbation of damage to liver function was avoided (p<0.01). During the follow-up period, three patients died from liver failure and two patients from hepatocellular carcinoma. Patients had a poor prognosis if their albumin levels were less than 2.8 mg / dL before B-RTO (p<0.05). Encephalopathy patients had complete response to B-RTO, but long-term prognosis was affected by hepatic functional reserve before B-RTO and by concurrent hepatocellular carcinoma. The results of this study suggest that in patients with SRS, it is important to perform B-RTO at an early stage when the hepatic functional reserve is still satisfactory.

Isolated gastric varices (IGV) have a lower risk of bleeding than esophageal varices, however IGV bleeding is associated with a higher mortality than bleeding of esophageal varices. In recent years, two widely used treatments for IGV have been balloon-occluded retrograde transvenous obliteration (B-RTO) and endoscopic injection sclerotherapy (EIS) using cyanoacrylate or ethanolamine oleate (EO). This study compared these two treatment methods for IGV. The subjects were 112 patients who were treated at our hospital for IGV bleeding between October 1990 and December 2003. Forty-nine (49) patients were treated with B-RTO and 63 patients with EIS. These two patient groups were compared as regards content of treatment, post-treatment incidence of variceal bleeding, incidence of IGV rebleeding, survival rate, cause of death, and complications. Multivariate analysis was performed on post-treatment variceal bleeding and survival. Although EO was used in higher amounts in the B-RTO group than in the EIS group, the B-RTO group had a significantly lower number of treatment sessions and a significantly shorter treatment period (p<0.05). The EIS group had significantly more patients with IGV rebleeding after treatment than the B-RTO group. Treatment method was the only independent prognostic factor of IGV bleeding after treatment (p=0.024). The two groups did not differ significantly in the percentage of patients with aggravated esophageal varices after treatment. Bleeding from ectopic varices was not observed in any patient. There was no significant difference in survival by treatment method. The presence of hepatocellular carcinoma was the only independent prognostic factor for survival (p=0.003). It is concluded that B-RTO was more effective than EIS in the eradication of IGV and prevention of IGV recurrence and rebleeding.

Ipratropium bromide (IPR) is an anticholinergic used to treat chronic obstructive pulmonary disease (COPD), and is a substrate of organic cation transporters. The present study aimed to assess the contribution of organic cation transporters to tracheobronchial absorption of IPR in vivo by directly injecting [(3) H]IPR into the tracheal lumen of mice and measuring its accumulation in tracheal tissue. RT-PCR and immunohistochemical analysis showed that Octn1, Octn2, and Oct2 were localized at epithelial cells in the respiratory tract. Electron-microscopic immunohistochemistry indicated that Octn1 and Octn2 were localized at the apical portions of ciliated epithelial cells of trachea. In vitro uptake studies in HEK293 cells expressing these transporters demonstrated that IPR is a preferred substrate of Octn2. Inhibition of mouse tracheal accumulation of [(3) H]IPR by carnitine was concentration-dependent, reaching a maximum of 42% at 1 mM, whereas inhibition by 0.1 mM MPP(+) amounted to 62%. Tracheal accumulation of [(3) H]IPR was unchanged when mice were simultaneously injected with Octn1 substrate ergothioneine and organic anion transporter substrate estrone sulfate. These results suggest that Octn2 is involved in membrane permeation of IPR in the respiratory tract in vivo. Targeting organic cation transporters may be an effective strategy for delivery of cationic anti-COPD drugs to patients., (Copyright © 2013 Wiley Periodicals, Inc.)

Background and Aims: Bleeding from esophageal and gastric varices is a fatal event in patients with liver cirrhosis and portal hypertension. However, the effects of Helicobacter pylori (H. pylori) infection on esophagogastric variceal bleeding are not known. The present study was aimed to elucidate the role of H. pylori infection in esophagogastric variceal bleeding., Methods: The subjects were 196 cirrhotic patients who were admitted to the Kurume University Hospital to treat their esophagogastric varices consisted of 95 with acute bleeding and 101 with nonbleeding but high risk of bleeding. For the diagnosis of H. pylori infection, a (13) C-urea breath test was used, and serum pepsinogen (PG) I and II levels and the PG I/II ratio were also measured., Results: Esophagogastric variceal bleeding was seen in 34.9% (n = 30) of the H. pylori-infected patients (n = 86) and in 59.1% (n = 65) of the noninfected patients (n = 110) (P < 0.0007). There was no significant difference in the infection rate between the bleeding sites of the esophagus and the stomach. The serum PG I and II levels and the PG I/II ratio were 65.6 ng/dL, 14.7 ng/dL, and 4.4, respectively, for the bleeding patients (n = 95), and 43.7 ng/dL, 17.7 ng/dL, and 3.1 for the nonbleeding patients (n = 101). Thus, the nonbleeding patients had significantly higher rate of H. pylori infection and lower acid secretion than bleeding patients (0.001). In addition, multivariate logistic regression analysis showed a significant negative association between H. pylori infection and esophagogastric variceal bleeding., Conclusions: These results suggest that H. pylori infection has a protective effect against esophagogastric variceal bleeding through the induction of gastric mucosal atrophy and concomitant hypoacidity., (© 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

To evaluate pathogens in pediatric inpatients with community-acquired pneumonia (CAP), an Acute Respiratory Diseases Study Group organized by ten Japanese medical institutions devised a rapid, reliable process based on real-time PCR results in nasopharyngeal swab samples plus admission blood test results. From April 2008 to April 2009, we enrolled 903 children with CAP based on chest radiographs and clinical findings who were hospitalized within 5 days of onset. Comprehensive real-time PCR was used to detect 6 bacteria and 11 respiratory viruses. The swab specimens also were used for bacterial cultures. After initial determination of presence or absence of viral and mycoplasmal infections, significant bacterial contributions were defined by bacterial identification, clinical efficacy of antimicrobial agent, and reference to blood test results. Children were stratified by age: below 1 year, 1 year, 2-5 years, or at least 6 years old. Among patients studied, 34.4 % were diagnosed with viral infection; 21.8 %, bacterial infection; 17.5 %, viral/bacterial co-infection; 5.9 %, mycoplasmal infection; 0.3 %, mycoplasmal/bacterial co-infection; and 1.7 %, viral/mycoplasmal co-infection. The remaining 18.4 % had unknown pathogens. Purely viral infection was suggested mainly in infants younger than 1 year; mycoplasmal infection typically occurred in children at least 6 years old. Our results suggest usefulness of real-time PCR for nasopharyngeal samples together with blood tests in estimating etiologic agents in clinical settings.

Members of the Japanese Society of Pediatric Pulmonology and the Japanese Society for Pediatric Infectious Diseases developed the Guidelines for the Management of Respiratory Infectious Diseases in Children with the objective of facilitating the appropriate diagnosis and treatment of childhood respiratory infections. To date, a first edition (2004) and a revised edition (2007) have been issued. Many problems complicate the diagnosis of the pathogens responsible for bronchopulmonary infections in children. The Guidelines were the first pediatric guidelines in the world to recommend treatment with antimicrobials suited to causative pathogens as identified from cultures of sputum and other clinical specimens collected from infection sites and satisfying assessment criteria. The major causative microorganisms for pneumonia in infants and children were revealed to be Streptococcus pneumoniae, Haemophilus influenzae and Mycoplasma pneumoniae. This manuscript describes the Guidelines for the Management of Respiratory Infectious Diseases in Children in Japan 2007, with a focus on pneumonia.

An evaluation committee studied the relationship between initial treatment drug and prognosis in 339 of 466 subjects with bacterial meningitis treated at 108 institutions between April 2004 and January 2007, after excluding those with uncertain diagnosis or non-assessable records. Prognosis was considered unfavorable if meningitis sequelae such as quadriplegia, deafness, or epilepsy were present in 3- month follow-up; Based on this definition, 43 (12.7%) had a poor prognosis. No significant relationship was seen between unfavorable prognosis and age or causative pathogen. More had an unfavorable prognosis if treatment was initiated 4 days or later after onset. The percentage with an unfavorable prognosis was 6.4% (4/64) in the group administered combined panipenem/betamipron (PAPM/BP) plus ceftriaxone (CTRX), 10.5% (6/57) administered MEPM plus cefotaxime (CTX), 14.0% (7/50) administered meropenem (MEPM) plus CTRX, and none of the 23 administered CTRX alone. The percentage with an unfavorable prognosis was 26.2% (11/42) in those administered MEPM, significantly higher than that in those administered PAPM/BP plus CTRX, MEPM plus CTX, or CTRX alone (p < 0.05). We concluded that in initial treatment, it would be more desirable to use MEPM combined with another drug than alone.

A 13-year-old boy was admitted to our hospital with altered states of consciousness coupled with a headache and nausea. Upon admission, the patient was afebrile and comatose with a decorticated posture and was subsequently intubated. All routine laboratory tests and cerebrospinal fluid analyses were normal. Brain T2-weighted MRI (figure 1A) revealed multiple hyperintense signals in the brainstem and cerebellum. A single gadolinium-enhanced lesion was observed in the left occipital lobe. These observations were indicative of acute disseminated encephalomyelitis (ADEM) and we subsequently started methylprednisolone pulse therapy. In the follow-up MRI study, the lesions were necrotic, suggesting changes after a stroke rather than ADEM. The MR angiography (figure 1B) and the conventional cerebral angiography (figure 1C,D) performed on days 25 and 28, respectively, revealed segmental stenoses ("beading") of the basilar artery and the left middle cerebral artery and the near occlusions of both posterior cerebral arteries with thrombus adjacent to the basilar artery bifurcation. No angiographic abnormalities were observed in the extracranial carotid and renal arteries. We diagnosed the lesions as angiitic infarctions and started plasma exchange and antiplatelet therapy.

Ipratropium bromide (ipratropium) and tiotropium bromide (tiotropium), anticholinergic agents with bronchodilating properties, are used to treat patients with chronic obstructive pulmonary disease. Because they are actively secreted into urine, the interaction of these agents with organic cation transporters (OCTs/Octs) was examined in rat kidney slices and in cultured cells expressing rat Oct (rOct) or human OCT (hOCT). Uptake of radiolabeled ipratropium in rat kidney slices was significantly inhibited by OCT/Oct substrates including cimetidine, imipramine, and quinidine, but not by organic anion transporter substrates (e.g., p-aminohippuric acid and estrone-3-sulfate). [(3)H]Tiotropium uptake showed similar characteristics. Reverse transcription-polymerase chain reaction showed that, in rat kidney, mRNA expression of rOct2 was the highest, followed by rOct1, but little rOct3 was detected. In vitro, rOct1 and rOct2 transported both anticholinergics, but rOct3 accepted only ipratropium. Ipratropium uptake by rat kidney slices consisted of two components with K(m) values of 0.114 ± 0.06 and 24.5 ± 2.21 μM. The K(m) value of rOct2-mediated ipratropium uptake (0.143 ± 0.03 μM) was consistent with that of the high-affinity component. The OCT/Oct inhibitor corticosterone, at a concentration of 1 μM (IC(50), 1.11 ± 0.20 μM for rOct2-mediated ipratropium transport), inhibited ipratropium by 18.4%, suggesting that rOct2 is involved in renal secretion of ipratropium. In a similar manner, ipratropium and tiotropium were taken up by cultured cells expressing hOCT1 and hOCT2 but not hOCT3. We conclude that OCT2/Oct2 plays a role in renal secretion of both anticholinergics in these species. Coadministration of these anticholinergics with cationic drugs recognized by OCT2/Oct2 may decrease renal clearance, resulting in increased systemic exposure.

We discuss the efficacy 3 pandemic influenza, measures planned against an anticipated outbreak. First was an exclusive influenza outpatient clinic. Second was a medical call center for febrile illness subjects needing with fever clinic recommendation. The last was isolation. Before the outbreak, we had thought that all confirmed or suspected new influenza case should be quarantined. May 2009 brought the first A1/H1 pandemic influenza outbreak to Kobe, Japan. After the first infection announcement, call center and fever clinic consultations skyrocketed, filling all 55 designated Kobe hospital bed within 48 hours. Inquiries at call centers increased more rapidly than numbers of subjects rushing to fever clinics. Just after designated hospital beds were filled, medical service restrictions were rapidly relaxed. Our experiences suggest that compulsory hospitalization broke down quickest in the fever case overflow, so medical call centers may be crucial in preventing fever clinic overflows by subjects with fever of unknown origin not recommended to consult fever clinics. Those with severe influenza symptoms should be given priority in hospitalization and flexible policies are recommended.

An evaluation committee was organized to evaluate 464 cases of bacterial meningitis treated at 108 nationwide medical facilities participating in this survey between April 2004 and January 2007. There were 413 evaluable cases of bacterial meningitis, including 342 children (82.8%) and 71 adults (17.2%). Haemophilus influenzae (217 cases, 63.5%) and Streptococcus pneumoniae (35 cases, 49.3%) were the most frequent pathogens for meningitis in children and adults, respectively. The most used initial therapy for children was carbapenem + cephalosporin therapy (212 cases, 61.9%). Of the 333 children included in efficacy evaluation, 320 (96.1%) were rated as remission, 10 (3.0%) as partial remission, and three (0.9%) as poor response. The combination therapy with two drugs was also most often used in adults (41 cases, 57.7%). In efficacy analysis in 60 adults, remission was observed in 50 (83.3%), partial remission in five (8.3%) and poor response in five (8.3%). In prognosis analysis, 273 (80.3%) among 340 children were alive at the end of treatment without sequelae, but one (0.3%) died by the end of treatment, and 64 (18.8%) had sequelae. Of all adults, six (8.5%) died of bacterial meningitis and 23 (32.4%) had sequelae at the end of treatment. Among the patients followed up for 1 year, 26 (12.3%) of 211 children and three (7.7%) of 39 adults had sequelae. The selection of drugs and its dose level of many cases were appropriate, but the dose level of several cases was inappropriate. It is necessary to spread the method of proper antibiotic therapy.

VIM-1 metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa was isolated from 35 Kobe City Medical Center General Hospital patients from September 2007 to July 2008. All but one were highly resistant to all beta-lactams, aminoglycoside, and fluoroquinolone, and one susceptible to amikacin. Strains negative to a disk diffusion screening test using sodium mercaptoacetate for detecting MBL numbered 35. PCR for MBL indicated all strains were positive for bla(VIWM-1). These strains were indistinguishable by pulsed-field gel electrophoresis, indicating an outbreak of infections caused by VIM-1 MBL producing Pseudomonas aeruginosa. After intervention to control contact, the outbreak was controlled.

The formation of the ventral furrow during Drosophila gastrulation is driven by coordinated apical constriction. Cell-cell adhesion is thought to regulate apical constriction, but the mechanisms are poorly understood. DE-cadherin, an epithelial classic cadherin, has in its membrane-proximal extracellular region a suite of domains absent from vertebrate/urochordate classic cadherins. We constructed DEΔP, a DE-cadherin derivative that lacks the membrane-proximal half of the extracellular region but retains the entire cytoplasmic domain and still exhibits strong cell-cell binding ability. The extracellular region of DEΔP consists of only cadherin repeats, mimicking vertebrate/urochordate classic cadherins. In animals lacking DE-cadherin, DEΔP organized adherens junction assembly and functioned fully in many cadherin-dependent processes, including oogenesis. Embryos in which DE-cadherin was entirely replaced by DEΔP established the blastoderm epithelium but failed to form a ventral furrow. Apical constrictions were initiated relatively normally but subsequently decelerated. These were then followed by catastrophic disruption of the junctional network. Our results suggest that although the membrane-proximal half of the DE-cadherin extracellular region is dispensable for many developmental events, it is essential for efficient and robust apical constriction during ventral furrow formation., (© 2010 The Authors. Journal compilation © 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.)

Ipratropium bromide, an anticholinergic drug used for the treatment of asthma and chronic obstructive pulmonary disease, has low oral bioavailability, but systemic exposure, superior to oral administration, can be achieved by inhalation. Therefore, we investigated the pulmonary absorption mechanism of ipratropium using human bronchial epithelial BEAS-2B cells. [3H]Ipratropium uptake by BEAS-2B cells was temperature-dependent and saturable, with a K(m) value of 78.0 microM, suggesting involvement of carrier-mediated uptake. An RT-PCR study showed that organic cation/carnitine transporters OCTN1 and OCTN2 are expressed in BEAS-2B cells, but organic cation transporters (OCTs) are not. Uptake of [3H]ipratropium by HEK293 cells expressing OCTN1 (HEK293/OCTN1) and OCTN2 (HEK293/OCTN2) was significantly increased, compared with mock-transfected cells, and the estimated K(m) values were 444 microM and 53.0 microM, respectively. Finally, the contributions of OCTN1 and OCTN2 to ipratropium uptake were evaluated by measuring [3H]ipratropium uptake by BEAS-2B cells in which OCTN1 or OCTN2 gene expression had been silenced. Knock-down of OCTN1 or OCTN2 suppressed the uptake of [3H]ipratropium to 78.2% and 14.8% of that by control BEAS-2B cells, respectively. In addition, another anticholinergic, tiotropium, was also taken up by both HEK293/OCTN1 and HEK293/OCTN2 cells. Therefore, ipratropium and tiotropium are taken up primarily by OCTN2, and to a lesser extent by OCTN1, in bronchial epithelial cells. These findings are consistent with the pharmacological activity of the drugs after administration via inhalation.

Aim: In a prospective, longitudinal study, we investigated the influence of the severity of motor impairment on changes in body characteristics in children with moderate-to-severe cerebral palsy (CP)., Method: Twenty-six single children and adolescents (15 females, 11 males; mean age 10y 6mo, SD 3y 3mo) with spastic (quadriplegia, hemiplegia, or diplegia), athetotic, or hypotonic CP at Gross Motor Function Classification System (GMFCS) levels III to V participated. They were categorized into moderate (GMFCS levels III or IV, n=11) and severe (GMFCS level V, n=15) groups. Muscle thickness of the quadriceps femoris (MTQ) and fat thickness of the anterior thigh (FTA) measured using ultrasound images, together with weight and height, were obtained at annual measurements over 3 years., Results: Significant increases in all variables were found in both groups. The areas under the curve (AUCs: an index of the gain) for weight, MTQ, and FTA were significantly higher in the moderate group. The AUC for weight, adjusted for height, was significantly correlated with the AUC for MTQ in the moderate group, and with the AUC for FTA in the severe group., Interpretation: Weight gain in children with severe CP does not necessarily reflect muscle growth.

Psittacosis outbreak due to Chlamydophila psittaci occurred among staff members at an avian exhibition of nearly 1,000 birds in Kobe, Japan, in December 2005. Staff members not trained about zoonosis or psittacosis used little protective attire such as masks and gloves when caring for their discharges. Two of 67 staff members contracted psittacosis pneumonia. Additional two suffered from pneumonia and 19 reported symptoms such as fever and cough, although none were diagnosed with psittacosis. The roughly 970 birds were kept without quarantine and identified by leg bands. Doxycycline administrated in drinking water and food failed to eradicate chlamydia, so all birds were captured, identified by leg band, and tested for chlamydia by PCR. Six were found to carry large amounts of chlamydia. Major outer membrane protein (MOMP) DNA sequence of chlamydia in a patient's bronchoalveolar lavage fluid (BALF) was identical to that derived from a channel-billed toucan kept in a closed aviary, and staff members may have been infected by inhaling excrement while working in the aviary. The MOMP DNA sequence was useful in comparing strains. We review the difficulty of diagnosing psittacosis and the knowledge and infection control measures required against it.

A reversible splenial lesion of the corpus callosum has been recognized in patients with mild encephalitis/encephalopathy associated with various infectious diseases. All reported cases of encephalitis/encephalopathy, by definition, present with neurologic signs, many of which involve seizures or mildly altered states of consciousness, such as drowsiness. We report an 8-year-old boy with influenza A infection not associated with neurologic signs, whose brain magnetic resonance images incidentally revealed a reversible splenial lesion in the corpus callosum. This case indicates that a transient splenial lesion in the corpus callosum may occur during a febrile illness, without neurologic manifestations. Therefore, physicians should not diagnose or treat a patient as encephalopathic merely because of the presence of a splenial lesion on magnetic resonance imaging.

Aim: Initial presentation with only cervical lymphadenopathy and fever is one of the pitfalls in the diagnosis of Kawasaki disease (KD). As the number of such patients is small, their clinical features have remained uncertain. The purpose of the present study is to characterise the features of such KD patients, especially in comparison with those of patients with common onset., Methods: We conducted a retrospective review of the medical records of 136 consecutive KD patients admitted to Kobe City General Hospital from April of 2000 to March of 2006. Twenty-nine of the 136 patients initially presented with only cervical lymphadenopathy and fever and were classified into the lymphadenopathy-KD (LKD); they were compared with the remaining 107 KD patients with other presentations (other-KD)., Results: Age, days of fever to diagnosis, and duration of fever were significantly higher or longer in LKD patients, who also showed higher C-reactive protein levels and neutrophil alkaline phosphatase activity. There were no significant differences between two groups in gender, duration of hospitalization, frequency of high-dose intravenous immunoglobulin (IVIG) administration, coronary artery lesions (CALs), white blood cell or platelet counts, and levels of hemoglobin or albumin on admission., Conclusions: Although a delay in diagnosis and stronger inflammation were found in LKD patients, such differences did not have any significant effect on patients' outcomes as assessed by the frequency of IVIG administration and the presence of CALs.

The aim of this study was to investigate the relation between muscle thickness of the quadriceps femoris muscle, knee joint function (spasticity and range of motion), and activity and participation measures in children and adolescents with cerebral palsy (CP). Thirty-eight children and adolescents with mild to severe CP (20 males, 18 females; mean age 12y 8mo [SD 3y 7mo], range 6-18y) participated. The severity and type of CP of participants covered all five levels of the Gross Motor Function Classification System and three types: spastic (quadriplegia, hemiplegia, and diplegia), athetotic, and hypotonic. The thickness of the quadriceps femoris muscle (MTQ) was measured from B-mode ultrasound images. Activity limitations were evaluated by the Gross Motor Function Measurement-66 (GMFM-66) and the Pediatric Evaluation of Disability Inventory (PEDI). Spasticity was assessed with the modified Ashworth scale (MAS). After adjustment for age and body mass index, the MTQ showed significant positive correlations with GMFM-66 and PEDI scores; however, there was no significant correlation with MAS ratings. The degree of knee flexion contracture correlated positively with the MAS rating of the knee flexor muscles and negatively with the MTQ. These results established the clinical relevance of assessment of muscle thickness across a broad spectrum of individuals with CP.

Serine phosphorylation of insulin receptor substrate (IRS)-1 and the induction of suppressor of cytokine signaling 3 (SOCS3) is recently well documented as the mechanisms for the insulin resistance. However, the relationship between these two mechanisms is not fully understood. In this study, we investigated the involvement of SOCS3 and IRS-1 serine phosphorylation in TNFalpha-induced insulin resistance in 3T3-L1 adipocytes. TNFalpha transiently stimulated serine phosphorylation of IRS-1 from 10 min to 1 h, whereas insulin-stimulated IRS-1 tyrosine phosphorylation was inhibited only after TNFalpha treatment longer than 4 h. These results suggest that serine phosphorylation of IRS-1 alone is not the major mechanism for the inhibited insulin signaling by TNFalpha. TNFalpha stimulation longer than 4 h enhanced the expression of SOCS3 and signal transducer and activator of transcription-3 phosphorylation, concomitantly with the production of IL-6. Anti-IL-6 neutralizing antibody ameliorated suppressed insulin signaling by 24 h TNFalpha treatment, when it partially decreased SOCS3 induction and signal transducer and activator of transcription-3 phosphorylation. These results suggest that SOCS3 induction is involved in inhibited insulin signaling by TNFalpha. However, low-level expression of SOCS3 by IL-6 or adenovirus vector did not affect insulin-stimulated IRS-1 tyrosine phosphorylation. Interestingly, when IRS-1 serine phosphorylation was enhanced by TNFalpha or anisomycin in the presence of low-level SOCS3, IRS-1 degradation was remarkably enhanced. Taken together, both IRS-1 serine phosphorylation and SOCS3 induction are necessary, but one of the pair is not sufficient for the inhibited insulin signaling. Chronic TNFalpha may inhibit insulin signaling effectively because it causes both IRS-1 serine phosphorylation and the following SOCS3 induction in 3T3-L1 adipocytes.

Neutrophil alkaline phosphatase (NAP) is used as a diagnostic marker in several hematological disorders. In regard to the role of NAP in infectious diseases, previous investigators have presented the hypothesis that NAP activity is useful to distinguish viral infections from bacterial infections. Because the numbers of patients enrolled in previous studies of viral infections were limited, we intended to evaluate the hypothesis by measuring NAP activity in a large number of pediatric patients with respiratory viral infections. A cytochemical analysis of NAP was performed in 160 patients with various types of respiratory infections. In patients with adenovirus or respiratory syncytial (RS) virus infection, NAP activity was significantly higher than the control value newly established at our department, while in patients with Epstein-Barr virus, measles, or influenza infection, it was comparable to the control value. On an individual basis, NAP scores (determined from NAP cytochemical activity) in 22 of 26 patients (84.6%) with adenovirus infection, and 31 of 42 patients (73.8%) with RS virus infection were found to exceed the 95% confidence upper limit of the control group. In conclusion, NAP activity is quite varied among different respiratory viral infections. When NAP activity is high in respiratory infections, adenovirus or RS virus infection, as well as bacterial infections, should be taken into consideration.

KL-6 is a useful marker for interstitial pneumonia of various origins. However, the role of KL-6 in common pediatric respiratory infections is largely unknown. In order to determine whether the KL-6 level is elevated during respiratory infection, and whether KL-6 is a useful biomarker for the disease activity, we evaluated serum KL-6 levels in 132 children with various respiratory infections. KL-6 levels were significantly higher in patients with measles, influenza, or respiratory syncytial virus infection than in the control subjects. On the other hand, KL-6 levels in patients with bacterial infections such as mycoplasma, chlamydia, or pertussis were comparable to the control values. In patients with viral infections, high KL-6 levels, as defined by the mean plus 2 standard deviations of the control group, significantly correlated with low SpO(2) or days of O(2) administration, but did not correlate with C-reactive protein or white blood cell counts. These results indicate that measurement of serum KL-6 levels is helpful for the management of common pediatric respiratory infections.

Proinflammatory cytokines are recently reported to inhibit insulin signaling causing insulin resistance. IL-1alpha is also one of the proinflammatory cytokines; however, it has not been clarified whether IL-1alpha may also cause insulin resistance. Here, we investigated the effects of IL-1alpha treatment on insulin signaling in 3T3-L1 adipocytes. IL-1alpha treatment up to 4 h did not alter insulin-stimulated insulin receptor tyrosine phosphorylation, whereas tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the association with phosphatidylinositol 3-kinase were partially inhibited with the maximal inhibition in around 15 min. IRS-1 was transiently phosphorylated on some serine residues around 15 min after IL-1alpha stimulation, when several serine kinases, IkappaB kinase, c-Jun-N-terminal kinase, ERK, and p70S6K were activated. Chemical inhibitors for these kinases inhibited IL-1alpha-induced serine phosphorylation of IRS-1. Tyrosine phosphorylation of IRS-1 was recovered only by the IKK inhibitor or JNK inhibitor, suggesting specific involvement of these two kinases. Insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake were not inhibited only by IL-1alpha. Interestingly, Akt phosphorylation was synergistically inhibited by IL-1alpha in the presence of IL-6. Taken together, short-term IL-1alpha treatment transiently causes insulin resistance at IRS-1 level with its serine phosphorylation. IL-1alpha may suppress insulin signaling downstream of IRS-1 in the presence of other cytokines, such as IL-6.

In 3T3-L1 adipocytes, insulin or anisomycin stimulated phosphorylation of IRS-1 at Ser(307) and Ser(636/639), both of which were partially reduced by the mTOR inhibitor, rapamycin, or the JNK inhibitor, SP600125, and were further inhibited by a combination of them. Interestingly, anisomycin-induced p70(S6K) phosphorylation was reduced by SP600125, while insulin-induced p70(S6K) phosphorylation was not. Furthermore, unlike insulin, anisomycin failed to elicit translocation or degradation of IRS-1. These results indicate that mTOR and JNK play roles in phosphorylating IRS-1 serine residues, and that insulin and anisomycin are different in terms of the relationship of activation between mTOR and JNK, and the effects on IRS-1 localization and stability.

We experimentally demonstrate the direct measurement of net group and reshaping delays for arbitrary optical pulses in dispersive media, verifying the earlier prediction of Peatross et al. [Phys. Rev. Lett. 84, 2370 (2000)]. Incoherent pulse propagation in an absorptionless system is well described by net group delay; even the medium causes a great deal of deformation in the transmitted pulse. However, in the case of phase modulated chirping pulses in a resonant absorber, the so-called superluminal or subluminal propagation velocity is strongly influenced by the reshaping delay.

Background: Adenoviruses are associated with a variety of diseases including upper respiratory tract infections, acute conjunctivitis, cystitis and gastroenteritis. Adenoviruses can also cause fatal disseminated infections in patients undergoing stem cell transplantation. Measurement of adenovirus load in clinical samples from localized adenovirus infections or disseminated adenovirus infections may provide important information for analyzing the pathogenesis of various adenovirus infections. The purpose of the present study was to develop and optimize a highly sensitive real-time polymerase chain reaction (PCR) assay to detect a wide range of adenoviruses and to detect adenovirus DNA in clinical samples from immunocompetent children., Methods: Clinical samples of throat swabs and blood were collected from 111 patients suspected of having adenovirus infection. The copy number of adenovirus DNA was measured by real-time PCR assay., Results: SYBR Green real-time PCR assay is able to detect 10-10(6) copies of standard adenovirus DNA per run. Adenovirus DNA was detected in all culture-positive samples serotyped as 1, 2, 3, 4, 5, 6, 8 and 11. Viral loads on throat swabs from immunocompetent children with adenovirus infection ranged from 10(5) to 10(11) copies/mL. Adenovirus DNA was detected in 60% of blood samples and copy number ranged from 10(3) to 10(5) copies/mL., Conclusion: SYBR Green real-time PCR is a useful quantitative tool for analysis of adenovirus DNA. The present results for immunocompetent children with adenovirus infections provided basic data for comparison with data obtained from immunocompromised patients.

Background: The aim of this study was to investigate the clinical spectrum of echovirus type 13 (E13) infection in hospitalized children., Methods: From April to August 2002, prospective viral surveillance was performed for hospitalized patients (aged 10 days to 14 years) irrespective of their presenting symptoms and severity. Medical records of laboratory-confirmed echovirus 13 infection were reviewed., Results: Of the 41 patients analyzed, the median age was 3.4 years and 30% of them were less than 1 year of age. The male:female ratio was 1.6:1. The main clinical features were non-specific febrile illness (nine patients), gastroenteritis (seven), bronchitis (seven), aseptic meningitis (16) and idiopathic thrombocytopenic purpura (two). Each age group had their representative symptoms: less than 1 year of age, non-specific febrile illness; from 1 to 6 years of age, enterocolitis and bronchitis; more than 6 years of age, aseptic meningitis., Conclusion: The representative symptoms of E13 infection in hospitalized patients were variable but strongly associated with age distribution. It was of interest to note that two patients developed idiopathic thrombocytopenic purpura along with the infection.

We report a male infant with congenital tuberculosis who developed cerebral hemorrhage associated with vitamin K deficiency during treatment with isoniazid and rifampin. Despite an absence of risk factors for vitamin K deficiency, the severe hemorrhagic disorder occurred at 4 months of age. We speculate that vitamin K deficiency in the present case may have resulted from a synergic effect of antituberculosis agents and immaturity of vitamin K metabolism and/or its absorption.

Animal studies have provided substantial evidence for a key role of reactive oxygen species, nitric oxide and its related compounds in the complex pathophysiology of bacterial meningitis. However, there is little information on changes in the redox status in human meningitis. In the present study, we evaluated the redox status and oxidative stress in the central nervous system of children with meningitis. Oxidant and antioxidant activities were assessed from cerebrospinal fluid levels of acrolein-lysine adducts (a marker of lipid peroxidation), nitrite (a marker of nitric oxide production) and bilirubin derivatives (a marker of antioxidant activity of bilirubin). All these markers were several times higher in children during the early phase of bacterial meningitis compared with those of children without meningitis and patients with aseptic meningitis. In the bacterial meningitis group, the levels of bilirubin derivatives correlated significantly with those of acrolein-lysine adducts and nitrite. Acrolein-lysine adducts and nitrite decreased significantly as the patients started to respond to treatment but bilirubin derivatives remained elevated. In conclusion, our data indicate the enhancement of both oxidant and antioxidant activities in the central nervous system of children with early bacterial meningitis, but not in those with aseptic meningitis. Clinical and laboratory improvement may be associated with a decrease in oxidant activities in the central nervous system.