Nasu-Hakola disease with a splicing mutation of TREM2 in a Japanese family.

BACKGROUND: Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by a combination of progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of DAP12 and TREM2, which constitute a receptor/adapter signaling complex expressed on osteoclasts, dendritic cells, macrophages, and microglia. No Japanese patients with TREM2 mutations have been reported previously. METHODS: We reported three siblings affected with NHD in a Japanese family. Amongst them, two died of NHD during the fourth decade of life. The analysis of genomic DNA, cDNA cloning, and western blot of lymphocyte proteins was performed on samples of the living patient. The transcriptome was studied in the autopsied brain of one patient. RESULTS: We identified a homozygous conversion of a single nucleotide T to C at the second position of intron 3 in the splice-donor consensus site (c.482+2T>C) of the TREM2 gene, resulting in exon 3 skipping and aberrant expression of truncated proteins. We identified 136 upregulated genes involved in inflammatory response and immune cell trafficking and 188 downregulated genes including a battery of GABA receptor subunits and synaptic proteins in the patient's brain. CONCLUSIONS: This is the first report of a Japanese NHD family caused by a splicing mutation of TREM2 that induces both neuroinflammation and neurodegeneration.

Desflurane but not sevoflurane can increase lung resistance via tachykinin pathways.

BACKGROUND: Although there is evidence that the volatile anaesthetic desflurane directly relaxes preconstricted airway smooth muscle in vitro, the anaesthetic increases the lung resistance in vivo. The constrictive mechanisms of desflurane are, however, still unknown. This study was conducted to clarify the increasing mechanisms of desflurane on lung resistance by examining the vagal nerve reflexes in guinea pigs. METHODS: The effects of desflurane and sevoflurane on total lung resistance (R(L)) and dynamic lung compliance (C(Dyn)) were investigated in animals that were either untreated, pretreated with atropine or vagotomy, pretreated with the tachykinin receptor antagonists sendide or MEN-10376, or given chronic pretreatment with capsaicin. RESULTS: Desflurane biphasically and dose-dependently increased R(L) (by 180% and 230% at the first and second peaks, respectively, at 2 minimum alveolar concentration) concomitant with a decrease in C(Dyn). However, sevoflurane had little effect on either R(L) or C(Dyn). Although vagotomy partially inhibited the first peak of R(L) by 30%, neither atropine nor vagotomy had any effect on the other respiratory responses to desflurane. Antagonization of tachykinin receptors of airway smooth muscles completely diminished the increase in R(L) induced by desflurane. Desflurane also had little effect on respiratory parameters after the capsaicin pretreatment, in which tachykinin containing afferent C-fibres was desensitized. CONCLUSIONS: Desflurane but not sevoflurane increased R(L) concomitant with a decrease in C(Dyn) in guinea pigs. The increase in lung resistance by desflurane might be due to antidromic tachykinin release from afferent C-fibres but not acetylcholine release from parasympathetic efferent nerves.

Performance of four carbon dioxide absorbents in experimental and clinical settings.

To evaluate the performance of four kinds of carbon dioxide (CO(2)) absorbents (Medisorb GE Healthcare, Amsorb Plus Armstrong Medical, YabashiLime Yabashi Industries, and Sodasorb LF Grace Performance Chemicals), we measured their dust production, acceptability of colour indicator, and CO(2) absorption capacity in in vitro experimental settings and the concentration of compound A in an inspired anaesthetic circuit during in vivo clinical practice. In vitro, the order of the dust amount was Sodasorb LF > Medisorb > Amsorb Plus = YabashiLime both before and after shaking. The order of the color acceptability was similar: Sodasorb LF > Amsorb Plus = Medisorb > YabashiLime both initially and 16 h after CO(2) exhaustion. During exposure to 200 ml.min(-1) CO(2) in vitro, the period until 1 kg of fresh soda lime allowed inspired CO(2) to increase to 0.7 kPa (as a mark of utilisation of the absorbent) was longer with Medisorb (1978 min) than with the other absorbents (1270-1375 min). In vivo, compound A (1.0% inspired sevoflurane) was detected only when using Medisorb. While Medisorb has the best ability to absorb CO(2), it alone produces compound A.

Inhibitory effects of the alpha-2 adrenergic agonists clonidine and dexmedetomidine on enhanced airway tone in ovalbumin-sensitized guinea pigs.

BACKGROUND AND OBJECTIVE: The alpha-2 adrenergic agonists clonidine and dexmedetomidine are used as an antihypertensive and a sedative, respectively. The aim of this study was to determine the effects of these agonists on ovalbumin-sensitized airway tone in guinea pigs. METHODS: The animals were divided into two groups: control and sensitized. The sensitized group received ovalbumin intraperitoneally and was boosted by exposure to aerosolized ovalbumin. The effects of the alpha-2 agonists were investigated by measuring (1) total lung resistance and (2) smooth muscle tension using a tracheal ring preparation. RESULTS: In the control group, acetylcholine significantly increased total lung resistance in a dose-dependent manner. In the sensitized animals, total lung resistance was significantly higher (by 95%) at 6 mug kg-1 acetylcholine than that in the control group. Both clonidine and dexmedetomidine had a slight but significant inhibitory effect on the response curve of lung resistance at higher concentrations of carbachol, a potent muscarinic receptor agonist. Similar to the data obtained in the control group, both clonidine and dexmedetomidine significantly decreased total lung resistance and the inhibitory effects of these alpha-2 agonists on lung resistance were significantly distinguishable. Similar direct inhibitory effects of the alpha-2 agonists on carbachol-induced muscle contraction were observed in both the control and sensitized groups, the inhibitory effects in the sensitized group being significantly greater. CONCLUSION: Both clonidine and dexmedetomidine can relax the airway even in the hyper-reactive state.

Analysis of the composition of 'original' and generic sevoflurane in routine use.

BACKGROUND: Original sevoflurane (Sevofrane) contains a small amount of water, which can inhibit the production of hydrofluoric acid. Hydrofluoric acid is highly pungent, and sevoflurane that contains a high concentration of hydrofluoric acid is not suitable for volatile induction of anaesthesia. Recently, generic sevoflurane (Sevoness) has become available in some countries. The generic product is produced by a different method and kept in a different kind of bottle. We questioned whether the original and generic sevoflurane differed in their composition and thus might differ in their resistance to degradation. METHODS: Sevoflurane from groups of three bottles of Sevofrane and three bottles of Sevoness was kept in the bottle at 24-37 degrees C for 2 weeks or in two kinds of vaporizer for 3 days, and the resulting contents measured by gas chromatography. RESULTS: Both products contained sevoflurane concentrations exceeding 99.998%. Fluoride ion concentration did not differ between the products (0.043 ppm). The original sevoflurane contained more (0.07% w/v) water than the generic anaesthetic (0.003% w/v). Original sevoflurane contained 5 ppm compound A, 10 ppm sevomethylether, and 5 ppm of unknown materials. Generic sevoflurane contained 32 ppm hexafluoroisopropanol and 12 ppm of unknown materials. While stored in a vaporizer for 3 days, the water content in the original sevoflurane decreased by two-thirds but the water in the generic sevoflurane increased by a factor of three-fold. CONCLUSIONS: Generic sevoflurane contains high-quality sevoflurane and only a small amount of fluoride ions, making it comparable with the original sevoflurane product.

Adult-onset Krabbe disease with homozygous T1853C mutation in the galactocerebrosidase gene. Unusual MRI findings of corticospinal tract demyelination.

A 51-year-old woman developed a slowly progressive spastic paraparesis and diminished vibration sense beginning at age 38. Intellectual capacity was normal. Krabbe disease was confirmed by markedly reduced leukocyte galactocerebrosidase (GALC) activity, typical inclusions in Schwann cell cytoplasm, and an identification of the homozygous point mutation T1835C (Leu618Ser) in the GALC gene. T2-weighted MRI of the brain showed symmetric high-signal-intensity lesions in the bilateral frontoparietal white matter, the centrum semiovale, and the posterior limb of the internal capsule with sparing of the periventricular white matter. This case is unusual because of the late onset, protracted clinical course, and MRI findings of demyelination confined to the corticospinal tracts.

Differential expression of gangliosides and galactolipids in fetal human oligodendrocytes and astrocytes in culture.

The phenotypic expression of gangliosides and galactolipids was investigated using primary cultures of fetal human oligodendrocytes and astrocytes. These glial cells were isolated from fetal human brains of 12-18 weeks' gestation. Expression of gangliosides and galactolipids in oligodendrocytes and astrocytes was investigated by double labeling immunocytochemistry using rabbit antibodies specific for galactocerebroside (GalC, a cell type-specific marker for oligodendrocyte) and glial fibrillary acidic protein (GFAP, a cell type-specific marker for astrocyte) in combination with a panel of mouse monoclonal antibodies which react with specific gangliosides or galactolipids. A considerable number of GalC+ oligodendrocytes expressed intense immunoreactivities specific for GM3 (19%) and GM2 (45%) gangliosides. Approximately 11% of GalC+ oligodendrocytes expressed GM4 immunoreactivity, and smaller numbers of GalC+ oligodendrocytes expressed GD3 (4%), GD2 (1%), GT1b (5%) and A2B5 (3%) immunoreactivities. However, GalC+ oligodendrocytes did not express GM1, GD1a, GT1b or GQ1c. Major populations of GalC+ oligodendrocytes immunolabeled by rabbit anti-GalC antibody reacted with anti-GalC mAb (Ranscht mAb, 81%) or by anti-sulfatide mAb (O4 mAb, 91%). A considerable number of GFAP+ astrocytes expressed intense GM2 (26%) and GD2 (15%) immunoreactivities, while a smaller population expressed intense GM3 (3%), GD3 (6%) and GM4 (4%) immunoreactivities. Weak immunoreactions specific for GD1b, A2B5 and sulfatide were found in less than 1% each of GFAP+ astrocytes, while GFAP+ astrocytes did not express GM1, GD1a, GT1a, GT1b or GQ1b. These results indicate that GM3, GM2 and sulfatide are expressed in a major population of GalC+ oligodendrocytes, while GM3, GM2, GD3, GD2, and GM4 are expressed in a small but distinctive population of GFAP+ astrocytes. Our results suggest that GM4, GM1 and GD3, which are utilized as markers for adult human oligodendrocytes and myelin, are not the major ganglioside constituents in cultured fetal human oligodendrocytes.

Alpha-synuclein expression is up-regulated in NTera2 cells during neuronal differentiation but unaffected by exposure to cytokines and neurotrophic factors.

Increasing evidence has indicated that proinflammatory cytokines such as TNF-alpha and IL-1beta, produced by activated microglia and astrocytes, play a key role in progressive degeneration of the nigrostriatal dopaminergic neurons in Parkinson's disease (PD). Since alpha-synuclein is a major component of Lewy bodies in PD brains, we studied the constitutive and cytokine/neurotrophic factor-regulated expression of alpha-synuclein in cultured human neurons by Northern blot and Western blot analyses. The constitutive expression of alpha-synuclein mRNA was identified in a variety of human neural and non-neural cell lines. The levels of alpha-synuclein expression were elevated markedly in NTera2 teratocarcinoma cells following retinoic acid-induced neuronal differentiation, accompanied with an increased expression of synphilin-1, while they were unaltered in NTera2-derived differentiated neurons by exposure to TNF-alpha, IL-1beta, BDNF or GDNF. These results indicate that alpha-synuclein expression in human neurons is up-regulated during differentiation, but is unaffected by a panel of cytokines and neurotrophic factors which are supposed to be involved in the nigral neuronal death and survival.

Molecular network of the comprehensive multiple sclerosis brain-lesion proteome.

BACKGROUND: A recent proteomics study of multiple sclerosis (MS) lesion-specific proteome profiling clearly revealed a pivotal role of coagulation cascade proteins in chronic active demyelination. However, among thousands of proteins examined, nearly all of remaining proteins are yet to be characterized in terms of their implications in MS brain-lesion development. METHODS: By the systems biology approach using four different pathway analysis tools of bioinformatics, we studied molecular networks and pathways of the proteome dataset of acute plaques, chronic active plaques (CAP), and chronic plaques (CP). RESULTS: The database search on Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein analysis through evolutionary relationships (PANTHER) indicated the relevance of extracellular matrix (ECM)-mediated focal adhesion and integrin signaling to CAP and CP proteome. KeyMolnet disclosed a central role of the complex interaction among diverse cytokine signaling pathways in brain-lesion development at all disease stages, as well as a role of integrin signaling in CAP and CP. Ingenuity pathway analysis (IPA) identified the network constructed with a wide range of ECM components, such as collagen, type I alpha1, type I alpha2, type VI alpha2, type VI alpha3, fibronectin 1, fibulin 2, laminin alpha1, vitronectin, and heparan sulfate proteoglycan, as one of the networks highly relevant to CAP proteome. CONCLUSIONS: Although four distinct platforms produced diverse results, they commonly suggested a role of ECM and integrin signaling in development of chronic lesions of MS. These in silico observations indicate that the selective blockade of the interaction between ECM and integrins in brain lesions in situ would be a target for therapeutic intervention in MS.

Detection of anti-Nogo receptor autoantibody in the serum of multiple sclerosis and controls.

OBJECTIVES: A myelin-associated neurite outgrowth inhibitor Nogo-A plays a key role in inhibition of axonal regeneration. Axonal damage beginning at the early stage of multiple sclerosis (MS) is responsible for permanent neurological deficits, although its molecular mechanism remains unknown. The aim was to study the prevalence of autoantibodies against Nogo-A and Nogo receptor (NgR) in the serum of MS. METHODS: The antibodies were identified in the serum of 30 MS patients, 22 patients with non-MS other neurological diseases (OND), and 22 healthy control (HC) subjects by Western blot using recombinant human Nogo-A-specific segment (NAS), the shared segment of Nogo-A and -B (NAB), Nogo-66 (N66), the non-glycosylated form of NgR, the glycosylated NgR (NgR-Fc), and myelin oligodendrocyte glycoprotein (MOG). RESULTS: None showed immunoglobulin G (IgG) antibodies against NAS or NAB. In contrast, 30% of MS, 23% of OND and 32% of HC subjects exhibited anti-N66 IgG, while 27% of MS, 27% of OND and 18% of HC showed anti-MOG IgG. None of HC but 33% of MS and 14% of OND showed anti-non-glycosylated NgR IgG. Furthermore, 60% of MS, 18% of OND and 14% of HC showed anti-NgR-Fc IgG. CONCLUSIONS: Because IgG autoantibodies against N66, NgR and MOG are often detected in the serum of MS and controls, they do not serve as an MS-specific marker.

Cytokines and neurotrophic factors fail to affect Nogo-A mRNA expression in differentiated human neurones: implications for inflammation-related axonal regeneration in the central nervous system.

Nogo is a novel myelin-associated inhibitor of neurite outgrowth which regulates stable neuronal connections during axonal regeneration following injury in the adult mammalian central nervous system (CNS). Because cytokines and neurotrophic factors play a key role in inflammation-related axonal regeneration, we investigated: (i) the constitutive expression of Nogo and the Nogo receptor (NgR) mRNA in human neural cell lines; (ii) Nogo and NgR mRNA levels in the NTera2 human teratocarcinoma cell line during retinoic acid (RA)-induced neuronal differentiation; and (iii) their regulation in NTera2-derived differentiated neurones (NTera2-N) after exposure to a battery of cytokines and growth factors potentially produced by activated glial cells at post-traumatic inflammatory lesions in the CNS. By reverse transcriptase-polymerase chain reaction analysis, the constitutive expression of Nogo-A, the longest isoform of three distinct Nogo transcripts and NgR mRNA was identified in a wide variety of human neural and non-neural cell lines. By Northern blot analysis, the levels of Nogo-A mRNA were elevated markedly in NTera2 cells following RA-induced neuronal differentiation, accompanied by an increased expression of the neurite growth-associated protein GAP-43 mRNA. In contrast, Nogo-A, Nogo-B, NgR and GAP-43 mRNA levels were unaltered in NTera2-N cells by exposure to basic fibroblast growth factor, brain-derived neurotrophic factor, glia-derived neurotrophic factor, tumour necrosis factor-alpha, interleukin-1beta, dibutyryl cyclic AMP or phorbol 12-myristate 13-acetate. These results indicate that both Nogo-A and NgR mRNA are coexpressed in various human cell types, including differentiated neurones, where their expression is unaffected by exposure to a panel of cytokines and neurotrophic factors which might be involved in inflammation-related axonal regeneration in the CNS.

Differential expression of heat shock protein HSP27 in human neurons and glial cells in culture.

HSP27 expression was investigated in cultured neurons and glial cells isolated from fetal human brains using immunoblotting and immunocytochemistry. Under unstressed conditions, HSP27 was identified at a high level in astrocytes (> 99%), at a low level in neurons (7%), and at a minimally detectable level in microglia (< 1%), whereas it was undetectable in oligodendrocytes. Under these conditions, HSP27 was located in the cytoplasm, fractionated into the Triton X-100-soluble phase, and composed chiefly of the basic isoform (HSP27a). After exposure to heat stress (43 degrees C/90 min), the level of HSP27 expression was not altered in astrocytes but was elevated significantly in neurons (11-21%) and microglia (4-7%) during 8-48 hr postrecovery periods, while it remained undetectable in oligodendrocytes. In addition, various human neural cell lines exhibited differential patterns of HSP27 expression under unstressed and heat-stressed conditions. Following heat shock treatment (45 degrees C/30 min), granular aggregates of HSP27 were identified in the cytoplasm of astrocytes. Under heat-stressed conditions, HSP27 was distributed within the Triton X-100-insoluble fraction associated with an increase in two more acidic isoforms (HSP27b and HSP27c). HSP27 and alpha B-crystallin were coexpressed in astrocytes under unstressed and heat-stressed conditions. When astrocytes were exposed to known HSP27 inducers, hydrogen peroxide and cysteamine reduced the synthesis of HSP27, while estradiol showed no effects. The differential patterns of constitutive and heat-induced expression of HSP27 in cultured human neurons and glial cells suggest that the cellular mechanisms by which HSP27 expression is regulated are different among various cell types in the human central nervous system.

Ubiquitin C-terminal hydrolase-L1 (PGP9.5) expression in human neural cell lines following induction of neuronal differentiation and exposure to cytokines, neurotrophic factors or heat stress.

Dysfunction of the ubiquitin-dependent proteolytic pathway contributes to progressive accumulation of ubiquitinated protein inclusions in neurodegenerative disorders, such as Parkinson's disease (PD). Ubiquitin C-terminal hydrolase-L1 (UCH-L1), alternatively designated protein gene product 9.5 (PGP9.5), is a neural deubiquitinating enzyme which is identified as a principal constituent of Lewy bodies. To clarify the regulatory mechanism of UCH-L1 expression in human neural cells, we studied the constitutive, cytokine/neurotrophic factor-regulated, and heat stress-induced expression of UCH-L1 in cultured human neural cell lines by Western blot analysis. The constitutive expression of UCH-L1 was identified in SK-N-SH neuroblastoma cells, IMR-32 neuroblastoma cells, U-373MG astrocytoma cells, and NTera2 teratocarcinoma-derived differentiated neurones (NTera2-N). The levels of UCH-L1 expression were unaltered in these cell lines following treatment with TNF-alpha, IL-1beta, BDNF, GDNF, dibutyryl cyclic AMP, or phorbol 12-myristate 13-acetate, and remained unchanged by exposure to heat stress. In contrast, its levels were elevated substantially in NTera2 teratocarcinoma cells following retinoic acid-induced neuronal differentiation, accompanied with an increased expression of alpha-synuclein and synaptophysin. These results indicate that UCH-L1 is expressed constitutively in human neual cell lines, where it is upregulated following induction of neuronal differentiation, but unaffected by exposure to heat stress, cytokines, or growth/differentiation factors which are supposed to be invloved in the nigral neuronal death and survival in PD.

A putative polymorphic Val44Ala variation in the synphilin-1 gene is undetectable in Japanese sporadic Parkinson's disease patients.

Recently, a novel protein-interaction partner of alpha-synuclein, designated synphilin-1, is identified as a constituent of Lewy bodies (LB) in Parkinson's disease (PD) brains. To investigate an involvement of genetic variations of synphilin-1 in development of sporadic PD, a possible single nucleotide polymorphism (SNP) of T131C corresponding to a valine (Val) to alanine (Ala) substitution at codon 44 in exon 3 of the synphilin-1 gene was studied in a Japanese population of 55 patients with sporadic PD and 61 patients with non-PD by direct sequencing analysis. All 116 subjects showed a homozygosity of Val at codon 44 in the synphilin-1 gene, suggesting that this SNP is unlikely to affect genetic susceptibility to sporadic PD in the Japanese population.

Three-dimensional imaging of the temporal bone using a helical CT scan and its application in patients with cochlear implantation.

Recent advanced techniques for the reconstruction of three-dimensional (3D) images allow further visual recognition of the fine structures of the temporal bone. We demonstrate the advantages of reconstruction 3D imaging in helical CT scans of the normal temporal bone and in patients who have received cochlear implants. After the temporal bone was scanned in the axial plane in the helical scan mode, the data were transferred to a workstation and 3D reconstruction was performed. The normal temporal bone structures were well recognized on these 3D images. The spatial relationship between the lymphatic space of the inner ear and the electrode array is visible. This method provides a detailed anatomy of the insertion of the electrodes into the cochlea and provides precise images of electrode routes by means of varying the angles of view on the computer display. Individual electrodes could not be distinguished. The information from 3D images may be useful not only for pre-but also postoperative investigations in cochlear implantation.

Spinocerebellar ataxia type 6: MRI of three Japanese patients.

We describe the MRI findings in three Japanese patients with spinocerebellar ataxia type 6 (SCA6) in which a polymorphic CAG repeat was identified in the gene encoding the alpha 1A voltage-dependent P/Q-type Ca2+ channel subunit (CACNL1A4). All showed slowly progressive cerebellar ataxia and mild pyramidal signs. Neuroradiologically, they had moderate cerebellar atrophy, most prominently in the superior vermis, whereas the brain stem appeared to be spared. No abnormal signal intensity was identified.