Critical role of TSLP-responsive mucosal dendritic cells in the induction of nasal antigen-specific IgA response.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine involved in T helper 2 type immune responses. The primary target of TSLP is myeloid dendritic cells (DCs), however, little is known about the mechanism by which TSLP elicits respiratory IgA immune responses upon mucosal immunization. Here, we found that the levels of TSLP and TSLPR were upregulated in the mucosal DCs of mice nasally immunized with pneumococcal surface protein A (PspA) plus cholera toxin (CT) compared with those immunized with PspA alone. PspA-specific IgA responses, but not IgG Ab responses were significantly reduced in both serum and mucosal secretions of TSLPR knockout mice compared with wild-type mice after nasal immunization with PspA plus CT. Furthermore, CD11c+ mucosal DCs isolated from TSLPR knockout mice nasally immunized with PspA plus CT were less activated and exhibited markedly reduced expression of IgA-enhancing cytokines (e.g., APRIL, BAFF, and IL-6) compared with those from equivalently immunized wild-type mice. Finally, exogenous TSLP promoted production of IgAs in an in vitro DC-B cell co-culture system as exhibited by enhanced IL-6 production. These results suggest that TSLP-TSLPR signaling is pivotal in the induction of nasal respiratory immunity against pathogenic pneumococcal infection.

Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques.

We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [(18)F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [(18)F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia in humans.

Induction of IL-10-producing CD4+ T-cells in chronic periodontitis.

Precise immunological aspects of inflamed gingival mucosa remain to be elucidated in the murine experimental periodontitis model; therefore, we have characterized the mucosal immune cells in the inflamed gingiva of mice with alveolar bone reduction. Mice were orally infected with Porphyromonas gingivalis 15 times over 2 weeks. Gingival mononuclear cells (GMCs) were isolated from P. gingivalis- and sham-infected mice 1, 7, 15, and 30 days after the last infection. Although the greatest degree of periodontitis was seen in P. gingivalis-infected mice at 30 days after infection, the highest levels of IL-6 and TNF-α production were noted in the GMCs isolated 7 days after infection. Further, the frequency of RANKL(+)CD4(+) T-cells in GMCs of inflamed gingiva peaked 15 days after infection. Importantly, the number of Foxp3(+)CD4(+) CD25(+) regulatory T (Treg)-cells was increased only in the experimental group 30 days after infection. Thus, intracellular cytokine analysis revealed an increased number of IL-10-producing CD4(+) T-cells in inflamed gingiva when compared with the control group. These results suggest that there are potential roles for Treg cells during the chronic stage of periodontitis in the regulation of gingival inflammation and alveolar bone loss.

Hydrogen peroxide induces expression and activation of AMP-activated protein kinase in a dental pulp cell line.

AIM: To investigate the effects of hydrogen peroxide on cell viability and expression and activation of AMP-activated protein kinase (AMPK) in rat dental pulp cell line RPC-C2A. METHODOLOGY: RPC-C2A cells derived from rat dental pulp were maintained in MEM supplemented with 10% FBS at 37 degrees C, in a humidified atmosphere at 5% CO(2). Cells were cultured in the presence or absence of H(2)O(2) for up to 60 min at concentrations of from 0.1 to 3.0 mmol L(-1). Cell viability was analysed by WST-1 reduction assay. Expression of AMPK subunit isoforms was analysed by Western blotting using antibodies to the catalytic alpha1 and regulatory beta1 and gamma1 subunit isoforms. The effect of silencing AMPKalpha1 on cell viability was determined using siRNA. RESULTS: Exposure to H(2)O(2) decreased cell viability in a time- and dose-dependent manner. The catalytic AMPKalpha1 subunit and its activated form, phospho-AMPKalpha, increased with exposure to H(2)O(2) in a time- and dose-dependent manner, whereas the regulatory beta1 and gamma1 subunits showed no change. Downregulation of AMPKalpha1 resulted in a reduction in cell viability in H(2)O(2)-treated cells at a concentration of 0.1 mmol L(-1) for 30 min incubation, indicating an increased sensitivity to H(2)O(2). CONCLUSIONS: Reactive oxygen induced energy fuel gauge enzyme AMPKalpha expression and its activation by phosphorylation in RPC-C2A cells, suggesting that AMPK is essential for protection against H(2)O(2)-induced nonapoptotic cell death. Therefore, AMPK may be a therapeutic modulation target for treatment of the dentine-pulp complex injured by reactive oxygen.

Hypoxia induces expression and activation of AMPK in rat dental pulp cells.

AMP-activated protein kinase (AMPK) is a stress-responsive enzyme involved in cell adaptation to an energy crisis. We hypothesized that hypoxia suppresses oxidative phosphorylation and ATP production, resulting in AMPK activation to protect cells. We investigated the effects of hypoxia on cell proliferation, the expression of AMPK and hypoxia-inducible factor 1alpha (HIF-1alpha), the activation of AMPK, and the relationship between AMPK and HIF-1alpha expression in rat dental pulp RPC-C2A cells. AMPK in the cells was composed of catalytic alpha1, and regulatory beta1 and gamma1 subunit isoforms. Cell proliferation was initially suppressed under hypoxia, but it increased thereafter, together with an increase in the expression of AMPK and HIF-1alpha, and the activation of AMPK. Down-regulation of AMPKalpha1 by siRNA inhibited cell proliferation under both normoxia and hypoxia, revealing that AMPK induction and activation were required for cell proliferation, although HIF-1alpha expression under hypoxia was not affected.

Measurement of a long electronic spin relaxation time of cesium atoms in superfluid helium.

The longitudinal electronic spin relaxation time of Cs atoms optically polarized in superfluid helium (He II, 1.5 K) has been measured with special care to cope with a serious decrease in the number of Cs atoms in the observation region. This decrease, mainly caused by helium convection in introducing the atoms into He II by laser sputtering, was significantly reduced using a new atom implantation method. Combined with a careful correction for the number of atoms, we have determined the relaxation time to be 2.24(19) s or longer, roughly twice as long as that in solid He.

The relationship between aberrant methylation and survival in non-small-cell lung cancers.

The present study examined the relationship between methylation of five genes (p16(INK4a), RASSF1A, APC, RARbeta and CDH13) and patient survival in 351 cases of surgically resected lung cancers. While there was no relationship between the other genes and survival, p16(INK4a) methylation was significantly related to unfavourable prognosis in lung adenocarcinomas.

Evaluation of pyridoindoles, pyridylindoles and pyridylpyridoindoles as matrices for ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In an effort to gain an understanding of the processes governing ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI), direct comparison was made of the mass spectra of proteins, carbohydrates and synthetic polymers (polyethylene glycol, polyester and polyamide) by using pyridylindoles, pyridoindoles and pyridylpyridoindoles as UV (337 nm)-MALDI-TOFMS matrices in positive and negative ion mode. In order to study the combined effect of the indole N-H and the pyridine nitrogen of the MALDI matrix on the desorption/ionization process in MALDI, compounds were selected that include either or both of these functions in their structure. Within the compounds studied only those that possess simultaneously both functions in a 1,4-relation behave as very good matrices for proteins. These compounds also work as matrices for some carbohydrates and synthetic polymers used as analytes in the present study. Some of the compounds were also found to be useful for the post-source decay (PSD) analysis of cyclodextrins in positive and negative ion mode. In several cases we also examined the matrix behavior of the corresponding N-methylindole derivatives.

Clinical and pedigree study on familial cases of West syndrome in Japan.

Nationwide survey on familial cases of West syndrome (WS) in first- and second-degree relatives was conducted by mailing a questionnaire to 64 major university hospitals, children's hospitals, and epilepsy centers in Japan, and by review of the Japanese cases in the literatures. Thirty-four familial cases, 20 males and 14 females, were obtained in 15 families including one with five affected members in two generations and another with three affected male siblings including a half brother by a different father (X-linked WS). A mother and the child or children were involved in three families. Nine families had 21 cryptogenic cases and six families had 13 symptomatic cases, and the etiologies were same among the affected members in each family. Familial cases of WS have characteristic clinical features and genetic mechanisms. Age of onset, seizure types, electroencephalographic abnormalities, early seizure outcome, effective treatment, long-term seizure prognosis, and long-term developmental prognosis were concordant among the affected members in each family. Long-term seizure and developmental prognoses were far better than those in WS in general, with seizure-free rate of 82% and normal mental development rate of 44%. Poor prognosis was limited to specific symptomatic cases. Adrenocorticotropic hormone (ACTH) was a treatment of choice, and even in relapse of WS after ACTH therapy, the patients well responded to antiepileptic drugs. Specific inheritance pattern was difficult to imagine in the majority of the present cases, except for one family with X-linked WS and another family with five patients of maternal inheritance. These results are helpful for the treatment choice and prognostication of clinical course for familial cases of WS.

Severe myoclonic epilepsy in infants--a review based on the Tokyo Women's Medical University series of 84 cases.

Severe myoclonic epilepsy in infants (SME) is one of the most malignant epileptic syndromes recognized in the latest classification of epileptic syndromes. The clinical details and electroencephalographic (EEG) characteristics have been elucidated by Dravet et al. The diagnosis of SME depends largely on the combination of clinical and EEG manifestations at different ages, of which the presence of myoclonic seizures appears to be the most important. However, because of the inclusion of different types of myoclonic attack and the lack of strict criteria for diagnosing SME, there has been some confusion as to whether patients without myoclonic seizures or myoclonus should be classified as SME, despite other identical clinical symptoms (SME borderlands (SMEB) group). Among the various clinical manifestations characterizing SME, special attention has been paid to seizures easily precipitated by fever and hot baths in Japan. We have demonstrated that the onset of myoclonic attack in these patients is very sensitive to the elevation of body temperature itself rather than its etiology. Using simultaneous EEG and rectal temperature monitoring during hot water immersion, we showed that epileptic discharges increased in frequency, and eventually developed into seizures at temperatures over 38 degrees C. We believe that the unique fever sensitivity observed in SME is similar to, but more intense than that of febrile convulsions. We have also identified a group of cases who have had innumerous myoclonic and atypical absence seizures daily which were sensitive to the constant bright light illumination. In these cases, spike discharges increased or decreased depending on the intensity of constant light illumination. Although these cases form the most resistant SME group, they lost the constant light sensitivity with increasing age, leaving only relatively common types of fever-sensitive grand mal seizures (FSGM) at the age of around 5 years. In the long run, only convulsive seizures continue, while myoclonic or absence seizures and photosensitivity disappear with advancing age, thus it is conceivable that SMEB constitutes a basic epileptic condition underlying SME. There is a clinical continuum that extends from the mildest end of SMEB to the severest end of SME with constant light sensitivity, with intermediates of frequent or infrequent myoclonic and absence seizures in-between. This spectrum concept appropriately explains the clinical variabilities between SME and SMEB during early childhood.

Myoclonic-astatic epilepsy of early childhood--clinical and EEG analysis of myoclonic-astatic seizures, and discussions on the nosology of the syndrome.

PURPOSE: The aim of this study is to elucidate the clinical and neurophysiological characteristics of the myoclonic, myoclonic-astatic, or astatic seizures in patients with myoclonic-astatic epilepsy (MAE) of early childhood, and to discuss on the nosology of this unique epileptic syndrome. SUBJECTS: The subjects included 30 patients, who fulfilled the following modified International League Against Epilepsy (ILAE) criteria for MAE, and whose main seizures were captured by video-electroencephalographs (EEG) or polygraphs. The modified ILAE criteria includes: (1) normal development before onset of epilepsy and absence of organic cerebral abnormalities; (2) onset of myoclonic, myoclonic-astatic or astatic seizures between 7 months and 6 years of age; (3) presence of generalized spike- or polyspike-wave EEG discharges at 2-3 Hz, without focal spike discharges; and (4) exclusion of severe and benign myoclonic epilepsy (SME, BME) in infants and cryptogenic Lennox-Gastaut syndrome based on the ILAE definitions. RESULTS: The seizures were investigated precisely by video-EEG (n=5), polygraph (n=2), and video-polygraph (n=23), which identified myoclonic seizures in 16 cases (myoclonic group), atonic seizures, with or without preceding minor myoclonus, in 11 cases (atonic group), and myoclonic-atonic seizures in three cases. All patients had a history of drop attacks, apart from ten patients with myoclonic seizures. Myoclonic seizures, involving mainly the axial muscles were classified into those with mild intensity not sufficient to cause the patients to fall (n=10) and those that are stronger and sufficient to cause astatic falling due to flexion of the waist or extension of the trunk (n=6). Patients in the atonic group fell straight downward, landed on their buttocks, and recovered immediately. Analysis of the ictal EEGs showed that all attacks corresponded to the generalized spike or polyspikes-and-wave complexes. In the atonic form, the spike-and-wave morphology was characterized by a positive-negative-deep-positive wave followed by a large negative slow wave. In two patients, the intensity of the atonia appeared to correspond to the depth of the positive component of the spike-and-wave complexes. We did not detect any significant differences in the clinical and EEG features and prognosis, between the atonic and myoclonic groups. CONCLUSIONS: Although the determination of exact seizure type is a prerequisite for diagnosing an epileptic syndrome, the strict differentiation of seizure type into either a myoclonic or atonic form, does not appear to have a significant impact on the outcome or in delineating this unique epileptic syndrome. At present, we consider it better to follow the current International Classification of Epileptic Syndromes and Epilepsies until a more appropriate system than the clinico-electrical approach for classifying patients with MAE is available.

History of clinical identification of West syndrome--in quest after the classic.

The West syndrome (WS) is a distinct age-dependent global encephalopathy which encompasses manifold problems of developing brain, and because of this, WS stands out as a symbolic syndrome for child neurology as a whole. It is unanimously recognized that this syndrome was first described by Dr W.J. West of Tunbridge, UK in 1841. In the following 100 years, however, the disease remained in the dark of neglect and misconception. An extensive literature survey carried out by Gastaut et al. revealed that only a few articles followed after West; about one article per decade between 1840 and 1920, and 18 per decade between 1920-1950. Among those, most distinguished contributions were detailed clinical observations made by Asal and Moro (1925) and Zellweger (1948), according to the author's opinion. An explosion of scientific interest at the world level was triggered by the discovery of hypsarhythmia on EEG by Gibbs and Gibbs in 1952 and of dramatic therapeutic effect of ACTH by Sorel and Dusaucy-Bauloye in 1958. In Japan, Katsutaka Takagi first reported four cases of apparent WS in 1903. An extensive search for Japanese classic literatures conducted by the author revealed 13 highly probable WS cases scattered in eight papers by 1945. A great confusion in terms of a concept of the disease had been prevailed for 50 years after Takagi until 1957, when the author first reported clinical and EEG findings in 99 cases, together with a 16 mm film demonstration of typical spasms in three cases at the Japan Pediatric Society meeting. Needless to say, however, WS turned out to be one of the most popular targets for clinical investigation of child neurologists in Japan afterwards, and nowadays, about 30 to 40 reports continue to be either published or orally presented at the meeting each year.

[Validity of various indices of obesity calculated from height and weight data for adult males use of the underwater-weighing method as a reference].

PURPOSE: We evaluated associations between excess body fat (%Fat) and various indices of obesity calculated from height and weight data. METHODS: In 147 adult males, %Fat was measured by the underwater-weighing method, and obesity indices were generated by the following 5 approaches: the Broca-Katsura (Katsura method), the Kato-Wataya (Kato method), Japan Society for the Study of Obesity (BMI method; based on the body weight at which the BMI is 22), and the Meiji Life Insurance Co. methods, and the Tables and Figures for Assessment of Obesity and Leanness published by the Ministry of Health and Welfare (MHW method). RESULTS: %Fat was 20% or more (obese) in 67 males (45.6%), 15-20% in 39 (26.5%), 10-15% in 35 (23.8%), and less than 10% in 6 (4.1%). The correlation coefficients between the obesity indices and %Fat were 0.612 for the Katsura method, 0.590 for the Kato-method, 0.611 for the BMI method, 0.612 for the Meiji Life Insurance Co. method, and 0.550 for the MHW method, being significant in each case (P < 0.01). When the cut-off point was set as 110% for each obesity index, sensitivity was highest with the Kato-method (82.1%), and specificity was highest with the Meiji Life Insurance Co. method (93.8%). With the MHW method, the receiver operating characteristic (ROC) curve was slightly farther from the point of sensitivity of 100% and 1-specificity of 0% than the others. CONCLUSION: Excess fat accumulation can not be accurately assessed by obesity indices calculated from body height and weight data. Validity was similar among obesity indices examined.

Merosin-positive congenital muscular dystrophy with mental retardation, microcephaly and central nervous system abnormalities unlinked to the Fukuyama muscular dystrophy and muscular-eye-brain loci: report of three siblings.

Classical merosin (2 laminin)-positive congenital muscular dystrophy is a heterogeneous subgroup of disorders; a few cases characterized by severe mental retardation, brain involvement and no ocular abnormalities were called Fukuyama-like congenital muscular dystrophy. We report a family of healthy non-consanguineous parents, with four affected siblings, of which one died at the age of 7 months due to an intercurrent illness, who presented congenital hypotonia, severe mental retardation, microcephaly, delayed psychomotor development, generalized muscular wasting and weakness with mild facial involvement, calf pseudohypertrophy, joint contractures and areflexia. Muscle biopsy disclosed severe muscular dystrophy. Immunostaining for laminin 2 80 kDa and clone Mer3/22B2 monoclonal antibodies, 1 and 1 chain was preserved. Magnetic resonance imaging findings were consistent with pontocerebellar hypoplasia, bilateral opercular abnormalities and focal cortical dysplasia as well as minute periventricular white matter changes. Clusters of small T2-weighted focal hyperintensities in both cerebellar hemispheres consistent with cysts were observed in two of the three siblings studied with magnetic resonance imaging. Ophthalmologic and cardiologic examination was normal. Haplotype analysis using microsatellite markers excluded the Fukuyama congenital muscular dystrophy, LAMA2 and muscle-eye-brain disease loci. Thus, a wider spectrum of phenotypes, gene defects and protein deficiencies might be involved in congenital muscular dystrophy with brain abnormalities.

Aberrant methylation and simian virus 40 tag sequences in malignant mesothelioma.

Aberrant promoter methylation and resultant silencing of several genes plays an important role in the pathogenesis of many tumor types. We compared the methylation profile of 66 malignant mesotheliomas (MMs) and 40 lung adenocarcinomas using methylation-specific PCR for seven genes frequently methylated in lung cancer. We also compared the methylation frequencies of these genes as well as the methylation index, a reflection of all of the gene frequencies, with the presence of SV40 large T-antigen (Tag) sequences, histological subtype, and patient survival. Our major findings are: (a) with the exception of the RASSF1A promoter of the RASSF1 gene, frequencies of aberrant methylation were significantly lower in MMs than in adenocarcinomas; (b) the frequency of RASSF1A aberrant methylation and the value of the methylation index were significantly higher in SV40 sequence positive MM than in negative MM; and (c) the methylation index was higher in epithelial MM than in sarcomatous/mixed MM. Our results demonstrate a relationship between SV40 and aberrant methylation in MMs.

Triterpene and flavanone glycoside from Rhododendron simsii.

Antioxidative substances were isolated from the leaves of Rhododendron simsii. These were a triterpene and flavanone glycoside, together with the known matteucinol and two known benzoic acid derivatives. Their structures were characterized as 19,24-dihydroxyurs-12-en-3-one-28-oic acid and 7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranosylmatteucinol by spectroscopic analysis.

Structure and neurotrophic activity of seco-prezizaane-type sesquiterpenes from Illicium merrillianum.

An extract of the pericarps of Illicium merrillianum has yielded four new sesquiterpenes: 3 alpha-hydroxycycloparvifloralone (1), 1,2-dehydrocycloparvifloralone (2), (11) 7,14-ortholactone-3 alpha-hydroxyfloridanolide (3), and 11-O-debenzoyltashironin (4) along with cycloparvifloralone (5), merrillianone (6), and tashironin (7). The structures of 1--4 were determined on the basis of spectroscopic analyses. 11-O-Debenzoyltashironin (4) showed neurotrophic activity in primary culture of rat cortical neurons at 0.1--10 microM. However, cycloparvifloralone-type sesquiterpenes (1, 2, 5, and 6) and tashironin (7) had no neurotrophic activity at these concentrations.