Inhibiting NADPH oxidase protects against long-term memory impairment induced by neonatal sevoflurane exposure in mice.

BACKGROUND: Neonatal exposure to anaesthetics such as sevoflurane has been reported to result in behavioural deficits in rodents. However, while oxidative injury is thought to play an underlying pathological role, the mechanisms of neurotoxicity remain unclear. In the present study, we investigated whether the NADPH oxidase inhibitor apocynin protects against long-term memory impairment produced by neonatal sevoflurane exposure in mice. METHODS: Postnatal day six mice were divided into four groups; (1) non-anaesthesia, (2) intraperitoneal apocynin (50 mg kg(-1)) treatment, (3) 3% sevoflurane exposure for 6 h, and (4) apocynin treatment combined with sevoflurane exposure. Superoxide concentrations and NADPH oxidase expression in the brain were determined using dihydroethidium fluorescence and immunoblotting, respectively. Cleaved caspase-3 immunoblotting was used for the detection of apoptosis, and cytochrome c immunoblotting was performed to evaluate mitochondrial function. Long-term cognitive impairment was evaluated using the fear conditioning test in adulthood. RESULTS: Sevoflurane exposure increased concentrations of superoxide (109%) and the NADPH oxidase subunit p22phox (39%) in the brain, and apocynin abolished these increases. Neonatal sevoflurane exposure caused learning deficits in adulthood. Apocynin also maintained long-term memory function in mice given neonatal sevoflurane exposure, and it reduced apoptosis and decreased cytochrome c concentrations in the brains of these mice. CONCLUSIONS: Apocynin reduces neuronal apoptosis and protects against long-term memory impairment in mice, neonatally exposed to sevoflurane by reducing superoxide concentrations. These findings suggest that NADPH oxidase inhibitors may protect against cognitive dysfunction resulting from neonatal anaesthesia.

Permanent acceptance of mouse cardiac allografts with CD40 siRNA to induce regulatory myeloid cells by use of a novel polysaccharide siRNA delivery system.

The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-ß-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the treatment with siCD40/SPG targeting DCs could generate antigen-specific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. These findings have important implications for clarifying the mechanism underlying the induction of tolerance in DCs, and also highlight the potential of immunomodulation and the feasibility of siRNA-based clinical therapy in the transplantation field.

Dysgammaglobulinemia Associated With Glu349del, a Hypomorphic XIAP Mutation.

BACKGROUND: X-linked lymphoproliferative syndrome type 2 is a rare hereditary immunodeficiency caused by mutations in the XIAP gene. This immunodeficiency frequently results in hemophagocytic lymphohistiocytosis, although hypogammaglobulinemia and dysgammaglobulinemia are also common. OBJECTIVE: We identified 17 patients from 12 Japanese families with mutations in XIAP. The Glu349del mutation was observed in 3 patients, each from a different family. Interestingly, these patients exhibited dysgammaglobulinemia but not hemophagocytic lymphohistiocytosis. We conducted an immunological study of patients carrying Glu349del and other mutations to elucidate the pathogenic mechanisms of dysgammaglobulinemia in patients with mutations in the XIAP gene. PATIENTS AND METHODS: We performed an immunological study of 2 patients carrying the Glu349del mutation and 8 patients with other mutations. RESULTS: Flow cytometry showed that the percentage of memory B cells in patients with a mutation in XIAP was lower than that observed in the healthy controls. The patients with the Glu349del mutation had a lower percentage of memory B cells than those with other mutations. Ig production was reduced in patients with the Glu349del mutation. Increased susceptibility to apoptosis was observed in the patients with other mutations. Susceptibility to apoptosis was normal in patients with Glu349del. Microarray analysis indicated that expression of Ig-related genes was reduced in patients with the Glu349del mutation and that the pattern was different from that observed in the healthy controls or patients with other mutations in XIAP. CONCLUSIONS: Patients carrying the Glu349del mutation in the XIAP gene may have a clinically and immunologically distinct phenotype from patients with other XIAP mutations. The Glu349del mutation may be associated with dysgammaglobulinemia.

4ß-Hydroxywithanolide E isolated from Physalis pruinosa calyx decreases inflammatory responses by inhibiting the NF-κB signaling in diabetic mouse adipose tissue.

BACKGROUND: Chronic inflammation in adipose tissue together with obesity induces insulin resistance. Inhibitors of chronic inflammation in adipose tissue can be a potent candidate for the treatment of diabetes; however, only a few compounds have been discovered so far. The objective of this study was to find a novel inhibitor that can suppress the inflammatory response in adipose tissue and to elucidate the intracellular signaling mechanisms of the compound. METHODS: To find the active compounds, we established an assay system to evaluate the inhibition of induced MCP-1 production in adipocyte/macrophage coculture in a plant extract library. The active compound was isolated by performing high-performance liquid chromatography (HPLC) and was determined as 4ß-hydroxywithanolide E (4ßHWE) by nuclear magnetic resonance (NMR) and mass spectroscopy (MS) spectral analyses. The effect of 4ßHWE on inflammation in adipose tissue was assessed with adipocyte culture and db/db mice. RESULTS: During the screening process, Physalis pruinosa calyx extract was found to inhibit production of MCP-1 in coculture strongly. 4ßHWE belongs to the withanolide family of compounds, and it has the strongest MCP-1 production inhibitory effect and lowest toxicity than any other withanolides in coculture. Its anti-inflammatory effect was partially dependent on the attenuation of NF-κB signaling in adipocyte. Moreover, in vivo experiments showed that the oral administration of 4ßHWE to db/db mice resulted in the inhibition of macrophage invasion and cytokine expression in adipose tissue after 2 weeks of treatment; improved the plasma adiponectin, non-esterified fatty acids and MCP-1 concentrations; and increased glucose tolerance after 3 to 4 weeks of treatment. CONCLUSIONS: These results suggest that 4ßHWE has anti-inflammatory effect via inhibition of NF-κB activation in adipocyte. Moreover, the attenuation of inflammation in adipocyte has an effect on the inhibition of macrophage accumulation in obese adipose tissue. Consequently, 4ßHWE improves impaired glucose tolerance. Thus, 4ßHWE is a useful natural anti-inflammatory compound to attenuate progression of diabetes and obesity.

Diagnostic potential of antibody titres against Candida cell wall ß-glucan in Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis syndrome of unknown aetiology in children. The administration of Candida cell wall antigens induced KD-like coronary vasculitis in mice. However, the responses of KD patients to Candida cell wall antigen are unknown. In this study, we examined the response of KD patients to ß-glucan (BG), one of the major fungal cell wall antigens, by measuring the anti-BG titre. In KD patients, the anti-C. albicans cell wall BG titre was higher than that in normal children. The anti-BG titre was also higher in KD patients compared to children who served as control subjects. The efficacy of intravenous immunoglobulin (IVIG) therapy in KD is well established. We categorized the KD patients into three groups according to the therapeutic efficacy of intravenous immunoglobulin (IVIG) and compared the anti-BG titre among these groups. Anti-BG titres were similar in the control group and the non-responsive group. In the fully responsive group, the anti-BG titre showed higher values than those in the normal children. This study demonstrated clinically that KD patients have high antibody titres to Candida cell wall BG, and suggested the involvement of Candida cell wall BG in the pathogenesis of KD. The relationship between IVIG therapy and anti-BG titre was also shown. These results provide valuable insights into the therapy and diagnosis of KD.

Cedar and cypress pollen counts are associated with the prevalence of allergic diseases in Japanese schoolchildren.

BACKGROUND: Patients allergic to pollen have been known to become more symptomatic during pollen season compared with the nonpollen season. However, there are few studies regarding whether higher exposure to pollen might increase the prevalence of allergic diseases. METHODS: An ecological analysis was conducted to evaluate whether pollen exposure is associated with the prevalence of allergic diseases in schoolchildren. Pollen count data of Japanese cedar (Cryptomeria japonica) and Japanese cypress (Chamaecyparis obtusa), which are the major pollen allergens in Japan, were obtained from each prefecture. The prevalence of allergic diseases in schoolchildren in each prefecture was based on a nationwide cross-sectional survey using the International Study of Asthma and Allergies in Childhood questionnaire. RESULTS: After omitting three prefectures where pollen data were not available, data of 44 prefectures were analysed. The prevalence of allergic rhinoconjunctivitis in children aged 6-7 years was positively associated with both cedar and cypress pollen counts (P = 0.01, both), whereas the prevalence of allergic rhinoconjunctivitis in children aged 13-14 years was positively associated with only cypress pollen counts (P = 0.003). Furthermore, the prevalence of asthma was positively associated with cedar pollen counts in 6- to 7-year-old children (P = 0.003) but not cypress pollen counts in either age group. CONCLUSIONS: There are ecological associations between pollen counts and the prevalence of allergic diseases in Japanese schoolchildren. Further studies are needed to determine whether the difference between the effects of cedar and cypress pollens is attributable to pollen counts or allergenicity.

Microstructure and cleavage in lath martensitic steels.

In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni) steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified 'classic' lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov-Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage.

Dehydroxymethylepoxyquinomicin (DHMEQ) can suppress tumour necrosis factor-α production in lipopolysaccharide-injected mice, resulting in rescuing mice from death in vivo.

Dehydroxymethylepoxyquinomicin (DHMEQ), a new nuclear factor (NF)-κB inhibitor, has several beneficial effects, including the suppression of tumour growth and anti-inflammatory effects. DHMEQ can also suppress the production of tumour necrosis factor (TNF)-α induced by lipopolysaccharide (LPS) in vitro. In the present study, we examine the effects of DHMEQ on TNF-α production in vivo and on the survival of mice injected with LPS. When DHMEQ was injected into mice 2 h before LPS injection, the survival of the LPS-injected mice was prolonged. When DHMEQ was injected twice (2 h before LPS injection and the day after LPS injection), all the mice were rescued. The injection of DHMEQ 1 h after LPS injection and the day after LPS injection also resulted in the rescue of all mice. The serum levels of TNF-α in the mice that received both LPS and DHMEQ were suppressed compared to the mice that received only LPS. These results suggest that DHMEQ can be utilized for the prevention and treatment of endotoxin shock.

Comparison of the surgical outcomes of thoracoscopic lobectomy, segmentectomy, and wedge resection for clinical stage I non-small cell lung cancer.

BACKGROUND: Video-assisted thoracoscopic surgery (VATS) for clinical stage I non-small cell lung cancer (NSCLC) has been widely used as a less invasive surgical procedure, but the resection method is still controversial. We retrospectively compared the surgical outcomes of lobectomy, segmentectomy and wedge resection. PATIENTS AND METHODS: A total of 411 patients with clinical stage I NSCLC who underwent VATS (218 males and 193 females, aged 69.3 years; 345 adenocarcinomas, 57 squamous cell carcinomas, and 9 others) were investigated. The surgical procedure was lobectomy in 289, segmentectomy in 38, and wedge resection in 84. Surgical outcomes were compared among these 3 groups. RESULTS: Demographic characteristics showed that the rate of elderly and male patients was higher in the wedge resection group. The 5-year survival rates for the lobectomy, segmentectomy, and wedge resection groups were 82.1, 87.2, and 55.4%, respectively. In the wedge resection group, the 5-year survival rate was 83.3% in patients undergoing intentional low-risk operations for small tumors with ground glass opacity, and 41.1% in those undergoing conservative high-risk operations because of comorbidities. Using Cox's proportional multivariate analysis and sex differences, histology, and tumor size as co-influential factors, the surgical procedure was found to be a significantly poor prognostic factor, and the hazard ratio of wedge resection relative to lobectomy was 4.30. CONCLUSION: The outcomes of VATS lobectomy and segmentectomy procedures for clinical stage I NSCLC were equivalent, while the outcome for VATS wedge resection was inferior. VATS wedge resection for clinical stage I NSCLC should be carefully indicated and requires adequate patient selection.

Functional analyses of thymic CD5+ B cells. Responsiveness to major histocompatibility complex class II-restricted T blasts but not to lipopolysaccharide or anti-IgM plus interleukin 4.

The function of thymic B cells in several standard in vitro assays was investigated. Thymic B cells, 75% of which were CD5+, showed a poor responsiveness to the mitogens LPS or anti-mu plus IL-4. Both proliferation and antibody formation were much lower in thymic than splenic B cell cultures. However, CD5- B cells purified using a cell sorter responded well to B cell stimulants, whereas purified CD5+ thymic B cells did not, indicating that CD5+ thymic B cells were unresponsive to B cell growth factor or LPS. Thymic B cells could be activated polyclonally by direct interaction with alloreactive T blasts, as manifested by DNA synthesis and antibody formation. These findings indicate that CD5+ thymic B cells may not be stimulated via sIg and IL-4, but require instead direct interaction with T blasts.

Extrathymic development of V alpha 14-positive T cells.

It is known that rearrangement of the T cell antigen receptor (TCR) gene occurs in the thymus during T cell development and consequently results both in the deletion of DNA between the variable (V) and diversity/joining segments and in the formation of a circular DNA with recombination signal sequences. Here, we provide evidence that V alpha 14+ TCR gene rearrangements take place in extrathymic sites, such as bone marrow, liver, and intestine, but not in spleen, because we were able to detect frequent productive and nonproductive V alpha 14+ coding and signal sequences as a result of TCR rearrangements in extrathymic sites. Similar findings were also detected in athymic mice. Quantitative analysis shows that the relative amounts of V alpha 14 gene-mediated signal sequences in extrathymic tissues are higher than those in thymus. On the contrary, TCR rearrangements of V alpha 1.1 T cells, which are known to develop in the thymus, were mainly detected in the thymus, Peyer's patch, and spleen, but not in other extrathymic tissues, showing patterns distinct from V alpha 14 TCR rearrangements. These findings are evidence of extrathymic development of V alpha 14+ T cells. Differential characteristic TCR rearrangement patterns also indicate that distinct TCR repertoires are generated in different lymphoid tissues.

A case of G-CSF-producing invasive apical cancer resected following preoperative adjuvant therapy.

The patient was a 47-year-old male with the chief complaints of right shoulder pain and fever. A 10-cm mass was found to be present in the right pulmonary apical region on chest X-ray examination. The mass was diagnosed as a G-CSF-producing invasive apical cancer and the patient underwent chemoradiotherapy followed by right upper lobectomy with combined resection of the chest wall, and lymph node dissection. A very small nodule was noted in the right frontal lobe on brain MRI before surgery, and the final diagnosis was pT3N0M1, stage IV lung adenocarcinoma. For the brain metastasis, gamma knife treatment and chemotherapy were performed after surgery, and there was no recurrence during 16 months after surgery. The prognosis for G-CSF-producing lung cancer and invasive apical cancer is poor. This was a rare and difficult case of lung cancer showing both characteristics of lung cancer.

Mediastinal tuberculous lymphadenitis with intractable fistula to the neck.

A 62-year-old female consulted our hospital due to an ulceration on the skin of her neck and a mediastinal tumor. Chest CT scan and MRI revealed a mediastinal tumor-like lesion with direct progression to the neck skin lesion. En bloc resection of the tumor including the neck skin ulcer was performed under a median sternotomy. Mediastinal tuberculous lymphadenitis was diagnosed histologically. This is a case of a very rare type of mediastinal tuberculous lymphadenitis that progressed to the neck with an intractable fistula.

Abnormal expression of the genes involved in cytokine networks and mitochondrial function in systemic juvenile idiopathic arthritis identified by DNA microarray analysis.

OBJECTIVES: Systemic juvenile idiopathic arthritis (sJIA) is a rheumatic disease in childhood characterised by systemic symptoms and a relatively poor prognosis. Peripheral leukocytes are thought to play a pathological role in sJIA although the exact cause of the disease is still obscure. In this study, we aimed to clarify cellular functional abnormalities in sJIA. METHODS: We analysed the gene expression profile in peripheral leukocytes from 51 patients with sJIA, 6 patients with polyarticular type JIA (polyJIA) and 8 healthy children utilising DNA microarrays. Gene ontology analysis and network analysis were performed on the genes differentially expressed in sJIA to clarify the cellular functional abnormalities. RESULT: A total of 3491 genes were differentially expressed in patients with sJIA compared to healthy individuals. They were functionally categorised mainly into a defence response group and a metabolism group according to gene ontology, suggesting the possible abnormalities in these functions. In the defence response group, molecules predominantly constituting interferon (IFN)gamma and tumour necrosis factor (TNF) network cascades were upregulated. In the metabolism group, oxidative phosphorylation-related genes were downregulated, suggesting a mitochondrial disorder. Expression of mitochondrial DNA-encoded genes including cytochrome c oxidase subunit 1(MT-CO1) and MT-CO2 were suppressed in patients with sJIA but not in patients with polyJIA or healthy children. However, nuclear DNA-encoded cytochrome c oxidases were intact. CONCLUSION: Our findings suggest that sJIA is not only an immunological disease but also a metabolic disease involving mitochondria disorder.

Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A.

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S-phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.

Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crm1+ which encodes a 115-kD protein preferentially localized in the nucleus and its periphery.

We isolated a novel class of Schizosaccharomyces pombe cold-sensitive mutants with deformed nuclear chromosome domains consisting of thread- or rodlike condensed segments at restrictive temperature. Their mutations were mapped in a novel, identical locus designated crm1 (chromosomal region maintenance). The crm1 mutants also show the following phenotypes. DNA, RNA, and protein syntheses diminish at restrictive temperature. At permissive temperature, the amount of one particular protein, p25, greatly increases. The mutant growth is hypersensitive to Ca2+ and resistant to protein kinase inhibitors. We cloned the 4.1-kb-long crm1+ gene that rescued the above phenotypes by transformation and determined its nucleotide sequence, which predicts a 1,077-residue protein. Affinity-purified antiserum raised against the crm1+ polypeptide expressed in Escherichia coli detected a 115-kD protein in S. pombe extracts. Genomic Southern hybridization and immunoblotting suggested that the crm1+ product might be highly conserved in distant organisms. Through immunofluorescence microscopy, the crm1+ protein appeared to be principally localized within the nucleus and also at its periphery. We speculate that the crm1+ protein might be one of those nuclear components that modify the chromosome structures or regulate the nuclear environment required for maintaining higher order chromosome structures.

Cytokine induction by a linear 1,3-glucan, curdlan-oligo, in mouse leukocytes in vitro.

OBJECTIVE: Curdlan, an extracellular bacterial polysaccharide, is a linear beta-1,3-glucan. Previously, we developed Curdlan-oligo (CRDO). We investigated its effect on the production of cytokines in leukocytes from mice, and compared its activity with that of SCG, a 6-branched 1,3-beta-glucan. METHODS: Splenocytes from DBA/2 mice were cultured with CRDO or SCG (0, 1, 10 or 100 microg/ml) in vitro, and then the supernatants were collected to measure cytokines. Bone marrow-derived dendritic cells (BMDCs) were cultured with CRDO (0, 1, 10 or 100 ng/ml) in vitro, and then the supernatant was collected to measure cytokines. RESULTS: SCG stimulated splenocytes in DBA/2 mice to produce GM-CSF, IFN-gamma and TNF-alpha. CRDO induced production of GM-CSF and IFN-gamma, but not TNF-alpha. The amounts of GM-CSF and IFN-gamma were small compared with those produced in response to SCG. The effect of SCG on TNF-alpha production was partially inhibited by CRDO. In bone marrow-derived dendritic cells, CRDO induced production of TNF-alpha and IL-6. CONCLUSION: Taken together, these results suggest that CRDO stimulated mouse leukocytes to induce the production of cytokines, and the mechanism of the effect of CRDO on leukocytes is different from that of SCG.

Pulmonary exposure to soluble cell wall beta-(1, 3)-glucan of aspergillus induces proinflammatory response in mice.

Compared to the significant immunomodulation of cell wall component(s) of bacterium such as lipopolysaccharide (E. Coli), that of pathogenic fungi has not been well elucidated, especially in vivo. Furthermore, although it has been implied that beta-(1, 3)-glucan of fungi possesses various biological activities, the impacts of the component have not been properly clarified, possibly due to its insolubility in water and alkali solutions. Previously, we isolated a soluble type of beta-(1, 3) -glucan from Aspergillus (referred to as ASBG). The present study investigated the effects of a single pulmonary exposure to ASBG on the immune (proinflammatory) responses in naïve mice. ASBG (12.5-100micorg/animal) exposure Induced neutrophilic lung inflammation with an enhanced local expression of proinflammatory cytokines such as interleukin (IL)-1beta and chemokines such as macrophage inflammatory protein -1a, and keratinocyte-derived chemoattractant in a dose-dependent fashion with overall trends. On the other hand, ASBG at relatively lower doses significantly amplified the lung expression of IL-2, IL-6, and IL-12 as compared with vehicle. ASBG significantly induced pulmonary edema. Furthermore, ASBG augmented the nuclear translocation of nuclear factor (NF)-kB and its binding capacity to the promoter site of DNA in the lung homogenate. These results suggest that pulmonary exposure to ASBG confers lung inflammation, at least partly, via the enhanced local expression of proinflammatory cytokines, likely through NF-kB-dependent pathway.