【目的】アトピー性皮膚炎(AD)の発症要因として,免疫応答の異常や腸内細菌叢の乱れが報告されている。リンゴペクチンは,特定の腸内細菌のみを選択的に増殖させることや血中ヒスタミン濃度の降下作用が報告されていることから,アレルギー性疾患に対する予防効果が期待されている。しかし,ADの改善効果は明らかになっていない。本研究では,リンゴペクチンによるADの改善効果と腸内細菌叢に与える影響について検討した。【方法】NC/Ngaマウス(雄)の背中に塩化ピクリル溶液を塗布してADを発症させたAD群,0.4,4%リンゴペクチン溶液をAD発症マウスに35日間経口投与したものを0.4%P群,4%P群とした。35日後に,発症部位の皮膚と糞便を採取し,AD改善効果と腸内細菌叢の変化について検討した。【結果】ADを発症させた背部皮膚を比較すると,AD群では皮膚の乾燥や紅斑等がみられたが,全ペクチン群においてAD皮膚症状の改善がみられた。COX-2タンパク質発現量,IL-4,TSLP mRNA発現量は,Control群と比較してAD群で有意に増加(p<0.05)したが,AD群に比較して全ペクチン群で有意に低下した(p<.05)。Foxp3 mRNA発現量,およびTh1サイトカインであるIFN-γは,AD群に比較して全ペクチン群で有意に増加した(p<0.05)。腸内細菌については,AD群に比較して4%P群でFirmicutes門の減少傾向とBacteroides門の増加傾向が認められた(p=0.103)。【考察】リンゴペクチン投与は,AD発症部位でTh1/Th2バランスを改善し皮膚の炎症を抑えることおよび腸内細菌叢の組成を変化させることで,ADを改善することが示唆された。(著者抄録)

【目的】アトピー性皮膚炎(AD)はそう痒感や湿疹を主病変とする。ADは,2型ヘルパーT細胞(Th2)へ分化が偏ることで発症する。また制御性T細胞(Treg)が免疫応答を制御しADの発症を防いでいる。腸内フローラがADに深く関わると報告されていることから,プロバイオティクスの一つであるBifidobacterium breve strain Yakult(BY)に着目した。本研究では,ヒト様ADモデル(Nc/Nga)マウスを用いてBY経口投与による,AD改善効果について検討した。【方法】NC/Ngaマウスの背中に塩化ピクリル溶液を塗布しADを発症させ,0.2mL/日の水道水を経口投与した群をAD群,BY懸濁液(5×109CFU/0.2mL/日)を経口投与しADを発症させた群をBY群とした。ADを発症させずに水道水(0.2mL/日)の経口投与した群をControl群とした。飼育期間終了後,病変部位である背中の皮膚・小腸を採取し,検討した。【結果】背中の病変部位において,AD群と比較してBY群で病変の改善が見られた。また病変部位で,炎症のマーカーであるCOX-2タンパク質発現量が有意に減少した。そして,Th1・Th2発現がAD群と比較してBY群で減少し,Treg発現は増加した。小腸において,Th1,Treg発現がAD群と比較してBY群で増加し,Th2発現は減少した。【考察】病変部位ではBY投与により,Tregの発現を増加に伴いTh2の発現が抑制され,ADが改善された。また小腸でも,Tregの増加により免疫バランスが改善した。以上より,BY投与により腸管免疫が改善されることで,ADが改善することが示唆された。(著者抄録)

【目的】アトピー性皮膚炎(AD)は増悪と寛解を繰り返すそう痒のある湿疹を主病変とする慢性皮膚疾患であり,発症の原因には,免疫機能や皮膚バリア機能の異常が報告されている。近年,アトピー性皮膚炎の炎症,及び症状の進行と腸内細菌叢には深い関係が在ると考えられる。その背景には,AD患者では腸内細菌叢が偏った状態であるという報告や,プロバイオティクスの投与がADの予防や治療に有効であるという報告がある。本研究では,アレルギー疾患患者数が少ない滋賀県の特産品であるフナ寿司由来の乳酸菌,Lactiplantibacillus plantarum FKW200108(LB)をADモデルマウスに経口投与し,症状の改善効果を検討した。【方法】ヒトADモデルマウスであるNC/Ngaマウス(約30g/雄)を使用し,通常飼育を行ったものをControl群,背中に塩化ピクリルを用いてAD発症させたものをAD群,LB懸濁液を経口投与しAD発症させたものをLB群とした。飼育35日後に背中の皮膚と小腸を採取し検討した。【結果】マウスの背中の皮膚において,AD群と比較してLB群で損傷の改善が見られた。加えて,COX-2タンパク発現量は,背中の皮膚においてAD群に比較してLB群で有意に減少した(p<0.05)。また,IFN-γ mRNA発現量は,背中の皮膚でAD群に比較してLB群で有意に減少(p<0.05),小腸ではAD群に比較してLB群で増加傾向であった(p=0.09)。加えてIL-4mRNA発現量は,背中の皮膚および小腸でAD群に比較してLB群で有意に減少した(p<0.05)。さらに,FOXP3mRNA発現量は,背中の皮膚および小腸でAD群に比較してLB群で減少傾向であった(p=0.15,p=0.17)。【考察】LB懸濁液を投与することで腸内細菌叢の組成を改善し,免疫バランスを調整することで,AD症状が改善することが示唆された。(著者抄録)

The current main treatment for ulcerative colitis (UC) is induction therapy by long-term administration of 5-aminosalicylic acid (5-ASA), but various side effects have been reported. Therefore, a radical cure for UC is desired. A vitamin C (VC) has anti-inflammatory effects. Therefore, this study investigated whether a VC solution enema shortens induction of remission in colitis model rats. Wistar rats (6 wk old/male) were allowed to freely ingest a 1% dextran sulfate sodium (DSS) solution for 10 d and then switched to tap water for normal breeding for 10 d (UC group). At the time of switching to tap water, an enema was performed with a 5-ASA solution (40 mg/kg/d) or VC solution (460 mg/kg/d) for 10 d. The neutrophil number, COX-2, which is an index of inflammation, and type III collagen, which is an early healing marker, were significantly increased in the UC group. However, the VC group showed decreases compared with UC groups. Furthermore, compared with UC and 5-ASA groups, the VC group showed increased expression of type I collagen, which is expressed late in healing, and significant epithelial regeneration was observed in colon tissue. The VC solution enema shortened the induction of remission by directly suppressing inflammation of damaged large intestinal tissues and promoting mucosal healing.

Increased oxidative stress in the human brain is observed in neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD), and is considered to be a major cause of progression of these disease states. A very long-chain fatty acid, nervonic acid (NA), is the main fatty acid found in various sphingolipid species in the central nervous system. NA plays an important role in forming the plasma membrane's lipid bilayer and in maintaining normal myelin function. In this study, we examined the neuroprotective effect of NA against rat pheochromocytoma (PC-12) cells stimulated by 6-hydroxydopamine (6-OHDA), which served as a cell model of PD. PC-12 cells were pre-treated with different concentrations of NA for 48 h then subsequently co-treated with NA and 6-OHDA for 48 h to induce cellular oxidative stress. Cell viability was significantly increased by pre-treatment with a very low concentration of NA. The level of malondialdehyde, a marker of lipid peroxidation, was significantly decreased in NA-treated cells. The expression levels of superoxide dismutases (Mn SOD and Cu/Zn SOD) and γ-glutamylcysteine synthetase (GCLC), responsible for the synthesis of glutathione, were significantly increased, indicating that pre-treatment with NA activated the cellular antioxidant defense system. These results suggest that NA may play a role as a neuroprotective mediator in the brain.

BACKGROUND: Malignant melanoma is among the deadliest forms of skin cancers, and its incidence has been increasing over the past decades. In malignant melanoma, activation of the nuclear factor kappa B (NF-κB) promotes survival, migration, and invasion of cancer cells. Anti-NF-κB agents for treating metastatic melanoma would be beneficial, but no such drug is approved as either monotherapy or adjuvant therapy. Dimethyl fumarate (DMF) is an approved anti-inflammatory drug already in clinical use for psoriasis and multiple sclerosis. OBJECTIVE: We investigated the anti-tumour effect of DMF treatment in metastatic melanoma in vitro and in vivo. METHODS: The cell viability was assessed via trypan blue exclusion assay. The migration and invasion was analyzed in a Boyden chamber assay. The anti-metastatic effects and anti-tumour activity of DMF was determined in an in-vivo model. The expressions of NF-κB pathway and NF-κB regulatory proteins were detected via western blotting. RESULTS: DMF decreased the cell viability, migration and invasion in vitro. In addition, DMF inhibited spontaneous metastasis and tumour growth. Mechanistically, DMF prevented the nuclear translocation of NF-κB, whereas no changes were observed in the phosphorylation levels of inhibitor of kappa B (IκB). In addition, DMF inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs). Furthermore, DMF treatment decreased the expression of Survivin and Bcl-extra large (Bcl-XL) proteins. CONCLUSION: Our results suggest that DMF as a novel inhibitor of NF-κB may be a potential therapeutic agent for metastatic melanoma.

AIMS: Stroke-prone spontaneously hypertensive rats (SHRSP) show significantly lower body weight than normotensive Wistar-Kyoto rats (WKY). Our hypotheses are as follows: weight loss of the skeletal muscle is related to hypertension-related diseases, and muscle hypotrophy is useful as a therapeutic target for hypertension and hypertension-related diseases. In this study, we aimed to investigate the pathophysiological characteristics of muscle hypotrophy in SHRSP to determine the therapeutic target molecule(s). MAIN METHODS: The difference in skeletal muscles in the lower leg between WKY and SHRSP was evaluated mainly through weight/tibial length, histological, gene expression, and protein expression analyses. KEY FINDINGS: SHRSP had a significantly lower weight/tibial length in soleus and gastrocnemius, but not in plantaris and tibialis anterior, indicating that muscles consisting of a relatively high amount of slow muscle fiber were affected. This result was confirmed by the histological analysis of soleus, showing that type I fiber mainly decreased the fiber size. Microarray and protein expression analyses showed that the muscle-specific ubiquitin ligase, muscle RING finger 1 (MuRF1), but not atrogin-1, was highly expressed in soleus, but not in plantaris, in SHRSP. TNF-like weak inducer of apoptosis receptor (TWEAKR) was predicted as a MuRF1 up-regulator by Ingenuity Pathway Analysis and immunostained only in type II fiber in WKY but in both type I and II fibers in SHRSP. SIGNIFICANCE: TWEAKR is a type II-specific receptor in the skeletal muscle. Ectopic TWEAKR expression in type I fiber of SHRSP is most likely involved in slow muscle-specific hypotrophy through MuRF1 overexpression.

Anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis is the most common type of autoimmune encephalitis. The disease predominantly affects women (1:5-1:10), with only 3 reports of autopsy findings in women being published to date. The present study reports findings from the first autopsy performed on a man with anti-NMDAR encephalitis. The patient had some scattered lesions in the limbic system with neuronal loss, gliosis, and microglial activation. The temporal and frontal cortices showed additional patchy demyelination. T-lymphocyte infiltration was detectable in the fusiform gyrus lesion. These findings were partly similar to those reported in female patients. Although clinical differences based on the sex of the patient are reported for this disease, the observed pathological similarities potentially help to establish common therapeutic strategies for all patients. Severe testicular damage was additionally observed in the male patient in this study. Biopsy-proven severe testicular damage was also confirmed in another, previously fertile man who became azoospermic. Moreover, serum follicle-stimulating hormone levels, which often increased in response to disturbed spermatogenesis, were elevated, and testosterone/luteinizing hormone ratio reflecting Leydig cell function was low in all 5 male patients in this study. Overall, these findings suggest similar brain pathology in patients of both sexes and severe testicular damage in male patients.

Myelodysplastic/myeloproliferative neoplasm (MDS/MPN) with ring sideroblasts and thrombocytosis (MDS/MPN with RS-T), which exhibits both an increased number of marrow ring sideroblasts and thrombocytosis, is a rare disorder classified as one of the newly established forms of MDS/MPN in the WHO 2016 classification. A 77-year-old female with marked thrombocytosis of 1,024×109/L was tentatively diagnosed with essential thrombocythemia in 2011, and the thrombocytosis was controlled using hydroxycarbamide and low-dose busulfan. In 2016, the leukocyte count increased to a peak value of 68.8×109/L (86.6% mature neutrophils) during platelet-reduction therapy. Bone marrow aspirate exhibited hypercellularity with ring sideroblasts comprising 41.5% erythroblasts without excess myeloblasts. Cytogenetic examination demonstrated the JAK2 V617F mutation and chromosomal abnormality of 46,XX,del(20)(q1?). Furthermore, dysplastic features of erythroid and granuloid precursors, as well as many large atypical megakaryocytes, were observed. Further genetic examinations revealed the SF3B1 K700E mutation, but not amplification of the JAK2 gene or pathogenic mutations in the 13 other genes examined. A diagnosis of MDS/MPN with RS-T was established and hyperleukocytosis was controlled using a higher dose of hydroxycarbamide. Although the patient maintained a stable disease state, she became RBC transfusion-dependent. Hyperleukocytosis, regardless of chemotherapy, is rare and may be novel in this disorder.

Several autocrine soluble factors, including macrophage inflammatory protein-1α (MIP-1α), tumor necrosis factor-α, and hepatocyte growth factor, promote cell survival and growth in multiple myeloma (MM) cells. We hypothesized that inhibition of the MIP-1α autocrine loop may enhance the cytotoxic effect of anticancer drugs in MM cell lines. In the present study, an MIP-1α neutralizing antibody suppressed cell proliferation and enhanced the cytotoxic effect of melphalan or bortezomib on MM cells. In addition, melphalan resistance cells (RPMI8226/L-PAM and HS-sultan/L-PAM cells) secreted MIP-1α and neutralizing antibody of MIP-1α partially overcame melphalan resistance. Moreover, combination treatment with MIP-1α neutralizing antibody and melphalan or bortezomib inhibited extracellular signal regulated kinase 1/2 (ERK1/2), Akt, and mammalian target of rapamycin (mTOR) activation, Bcl-2, Bcl-xL, and Survivin expression, and upregulated the expression of Bim and cleaved Poly (ADP-ribose) polymerase (PARP). Treatment of IM9 cells with MIP-1α siRNA suppressed the activation of ERK1/2, Akt, and mTOR, and enhanced the cytotoxic effect of melphalan and bortezomib. These results indicate that MIP-1α neutralizing antibodies or MIP-1α siRNA enhance the cytotoxic effect of melphalan and bortezomib by suppressing the chemokine receptor/ERK and chemokine receptor/Akt/mTOR pathways. The inhibition of MIP-1α may thus provide a new therapeutic approach to control tumor progression and bone destruction in patients with MM.

Pioglitazone is an anti-diabetic agent that belongs to the thiazolidinedione class, which target peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor in the nuclear receptor family. Different cancer cells expressing high levels of PPARγ and PPARγ ligands induce cell cycle arrest, cell differentiation, and apoptosis. However, the mechanisms underlying these processes remain unknown. Here, we investigated the mechanism underlying pioglitazone-induced apoptosis in human cancer cells. We showed that at similar concentrations, pioglitazone induced death in cancer cells expressing high or low levels of PPARγ. Combined treatment of pioglitazone and GW9662, a PPARγ antagonist, did not rescue this cell death phenotype. Z-VAD-fmk, a pan-caspase inhibitor, did not reverse pioglitazone-induced apoptosis in cancer cells expressing PPARγ at high or low levels. Pioglitazone suppressed the activation of signal transducers and activator of transcription 3 (STAT3) and Survivin expression, and enhanced the apoptosis-inducing factor (AIF) levels in these cells. Furthermore, pioglitazone enhanced the cytotoxic effect of cisplatin and oxaliplatin by suppressing Survivin and increasing AIF expression. These results indicated that pioglitazone induced apoptosis via a PPARγ-independent pathway, thus describing pioglitazone as a potential therapeutic agent for controlling the progression of different cancers.

Bavachin is a phytoestrogen purified from natural herbal plants such as Psoralea corylifolia. In this study, we examined the effect of bavachin in multiple myeloma (MM) cell lines. We found that bavachin decreased the viability of MM cell lines, but was not cytotoxic towards normal cells. It inhibited the activation of nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3). Furthermore, bavachin increased the expression of p53 and NOXA, and decreased the expression of X-linked inhibitor of apoptosis protein (XIAP), survivin, B cell lymphoma-extra large (Bcl-xL), and Bcl-2. Additionally, bavachin induced apoptosis by the activation of caspase-3 and caspase-9, implicating the involvement of the mitochondrial pathway. Our results suggest that bavachin induces apoptosis through the inhibition of NF-κB and STAT3 activation in MM cell lines. Most importantly, few NF-κB and STAT3 inhibitors with high efficiency, specificity, and safety are currently available for clinical cancer therapy. Hence, bavachin, which targets NF-κB and STAT3, is a potential anticancer agent for the treatment of MM.

Oral mucositis is a common adverse effect of chemotherapy that limits the required dose of chemotherapeutic agents. Numerous attempts to mitigate chemotherapy-induced oral mucositis have failed to identify an appropriate treatment. Recently, it has been indicated that rebamipide prevents chemoradiotherapy-induced oral mucositis in patients. However, the details of the underlying mechanism involved in the cytoprotective effect of rebamipide remain obscure. In the present study, we investigated the mechanism behind rebamipide cytoprotective effect in the oral mucosa using primary normal human oral keratinocytes (NHOK cells). We found that rebamipide prevented 5-fluorouracil (5-FU)-induced cell death in NHOK cells. In addition, rebamipide increased the levels of phosphorylated Akt and mTOR, enhanced the Bcl-2 and Bcl-xL expressions, and suppressed the expression of Bax and Bim. This is in contrast to 5-FU-induced suppression of Akt and mTOR activation, Bcl-2 and Bcl-xL expressions, and the enhanced expression of Bax and Bim. These findings suggest that rebamipide can potentially be used for the protection of oral mucosa from chemotherapy-induced mucositis. This is the first study that elucidates the specific molecular pathway for the cytoprotective effect of rebamipide.

NEW FINDINGS: What is the central question of this study? An inverse correlation between circulating adiponectin and many diseases has been reported, but some studies have found no correlation. To evaluate this controversy, we investigated the relationship between heart-bound adiponectin and hypertension or cardiac hypertrophy, compared with serum adiponectin. What is the main finding and its importance? Using hypertensive and normotensive rats, we found that heart-bound adiponectin was inversely correlated with cardiac hypertrophy, suggesting that heart-bound adiponectin has a more important function in preventing cardiac hypertrophy than circulating adiponectin. Our study provides new insights regarding the role of adiponectin in diseases. The inverse correlation between circulating adiponectin concentration and hypertension or cardiac hypertrophy is still controversial. In addition to circulating adiponectin, adiponectin is also bound to tissues such as the heart and skeletal muscle. In this study, we investigated the relationship of serum adiponectin and heart-bound adiponectin with hypertension and cardiac hypertrophy. Four types of hypertensive rats presenting different blood pressure levels were used at different ages, as follows: normotensive Wistar-Kyoto rats (WKYs); two sub-strains (strains C and B2, having low and high blood pressure, respectively) of spontaneously hypertensive rats (SHRs); and stroke-prone SHRs (SHRSPs). Blood pressure, heart-to-body weight ratio, serum adiponectin and heart-bound adiponectin were determined. Histopathological analysis of the heart was carried out to evaluate the relationship with heart-bound adiponectin. Serum adiponectin concentration was not inversely correlated with blood pressure or heart-to-body weight ratio. In contrast, heart-bound adiponectin levels were significantly lower in SHRSPs than in other strains at respective ages. This resulted from a decrease in T-cadherin expression, which induced adiponectin binding to tissues. No significant difference in heart-bound adiponectin among WKYs and SHRs (C and B2) was detected, indicating that heart-bound adiponectin is not related to hypertension. In addition, differences in heart-bound adiponectin did not affect AMP-activated protein kinase in the traditional adiponectin activation cascade. Histopathological analysis revealed that heart-bound adiponectin was inversely correlated with cardiomyocyte hypertrophy and left ventricular wall thickness and, in part, with cardiac fibrosis. These results suggest that the decreased level of heart-bound adiponectin in SHRSPs is more related to their cardiac hypertrophy than circulating adiponectin.

Resistance to the breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitor (TKI) imatinib poses a major problem when treating chronic myeloid leukemia (CML). Imatinib resistance often results from a secondary mutation in BCR-ABL1. However, in the absence of a mutation in BCR-ABL1, the basis of BCR-ABL1-independent resistance must be elucidated. To gain insight into the mechanisms of BCR-ABL1-independent imatinib resistance, we performed an array-based comparative genomic hybridization. We identified various resistance-related genes, and focused on MET. Treatment with a MET inhibitor resensitized K562/IR cells to BCR-ABL1 TKIs. Combined treatment of K562/IR cells with imatinib and a MET inhibitor suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) activation, but did not affect AKT activation. Our findings implicate the MET/ERK and MET/JNK pathways in conferring resistance to imatinib, providing new insights into the mechanisms of BCR-ABL1 TKI resistance in CML.

Statins induce apoptosis of tumour cells by inhibiting the prenylation of small G-proteins. However, the details of the apoptosis-inducing mechanisms remain poorly understood. The present study showed that the induction of apoptosis by statins in four different human head and neck squamous cell carcinoma ( HNSCC) cell lines, HSC-3, HEp-2, Ca9-22, and SAS cells was mediated by increased caspase-3 activity. Statins induced apoptosis by the suppression of geranylgeranyl pyrophosphate biosynthesis. Furthermore, statins decreased the levels of phosphorylated ERK and mTOR by inhibiting the membrane localization of Ras and enhancing Bim expression in HSC-3 and HEp-2 cells. We also found that in all the cell types analyzed, the IC50 values for fluvastatin and simvastatin were highest in HEp-2 cells. In addition, HSC-3, Ca9-22, and SAS cells had higher Ras expression and membrane localization, higher activation of ERK1/2 and mTOR, and lower levels of Bim expression than HEp-2 cells. Our results indicate that statins induce apoptosis by increasing the activation of caspase-3 and by enhancing Bim expression through inhibition of the Ras/ERK and Ras/mTOR pathways. Furthermore, the sensitivity of HNSCC cells to statin treatment was closely related to Ras expression and prenylation levels, indicating that statins may act more effectively against tumours with high Ras expression and Ras-variability. Therefore, our findings support the use of statins as potential anticancer agents.

Statins induce apoptosis of tumour cells by inhibiting the prenylation of small G-proteins. However, the details of the apoptosis-inducing mechanisms remain poorly understood. The present study showed that the induction of apoptosis by statins in four different human head and neck squamous cell carcinoma (HNSCC) cell lines, HSC-3, HEp-2, Ca9-22, and SAS cells was mediated by increased caspase-3 activity. Statins induced apoptosis by the suppression of geranylgeranyl pyrophosphate biosynthesis. Furthermore, statins decreased the levels of phosphorylated ERK and mTOR by inhibiting the membrane localization of Ras and enhancing Bim expression in HSC-3 and HEp-2 cells. We also found that in all the cell types analyzed, the IC50 values for fluvastatin and simvastatin were highest in HEp-2 cells. In addition, HSC-3, Ca9-22, and SAS cells had higher Ras expression and membrane localization, higher activation of ERK1/2 and mTOR, and lower levels of Bim expression than HEp-2 cells. Our results indicate that statins induce apoptosis by increasing the activation of caspase-3 and by enhancing Bim expression through inhibition of the Ras/ERK and Ras/mTOR pathways. Furthermore, the sensitivity of HNSCC cells to statin treatment was closely related to Ras expression and prenylation levels, indicating that statins may act more effectively against tumours with high Ras expression and Ras-variability. Therefore, our findings support the use of statins as potential anticancer agents.

Advanced metastatic melanoma, one of the most aggressive malignancies, is currently without reliable therapy. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone and exerts many beneficial biological activities. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we evaluated the effect of mangiferin on metastasis and tumor growth in a mouse metastatic melanoma model. We found that mangiferin inhibited spontaneous metastasis and tumor growth. Furthermore, mangiferin suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated NF-κB-inducing kinase (NIK), inhibitor of kappa B kinase (IKK), and inhibitor of kappa B (IκB) and increases the expression of IκB protein in vivo. In addition, we found that mangiferin inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs) in vivo. Mangiferin treatment also increased the expression of cleaved caspase-3, cleaved Poly ADP ribose polymerase-1 (PARP-1), p53 upregulated modulator of apoptosis (PUMA), p53, and phosphorylated p53 proteins, and decreased the expression of Survivin and Bcl-associated X (Bcl-xL) proteins in vivo. These results indicate that mangiferin selectivity suppresses the NF-κB pathway via inhibition of NIK activation, thereby inhibiting metastasis and tumor growth. Importantly, the number of reported NIK selective inhibitors is limited. Taken together, our data suggest that mangiferin may be a potential therapeutic agent with a new mechanism of targeting NIK for the treatment of metastatic melanoma.

CCR4 is a major chemokine receptor expressed by Treg cells and Th17 cells. While Treg cells are known to suppress antitumor immunity, Th17 cells have recently been shown to enhance the induction of antitumor cytotoxic T lymphocytes. Here, CCR4-deficient mice displayed enhanced tumor growth upon intradermal inoculation of B16-F10 melanoma cells. In CCR4-deficient mice, while IFN-γ+CD8+ effector T cells were decreased in tumor sites, IFN-γ+CD8+ T cells and Th17 cells were decreased in regional lymph nodes. In wild-type mice, CD4+IL-17A+ cells, which were identified as CCR4+CD44+ memory Th17, were found to be clustered around dendritic cells expressing MDC/CCL22, a ligand for CCR4, in regional lymph nodes. Compound 22, a CCR4 antagonist, also enhanced tumor growth and decreased Th17 cells in regional lymph nodes in tumor-bearing mice treated with Dacarbazine. In contrast, CCR6 deficiency did not affect the tumor growth and the numbers of Th17 cells in regional lymph nodes. These findings indicate that CCR4 is critically involved in regional lymph node DC-Th17 cell interactions that are necessary for Th17 cell-mediated induction of antitumor CD8+ effector T cells in mice bearing B16 melanoma.

Interaction between multiple myeloma (MM) cells and the bone marrow microenvironment plays a critical role in MM pathogenesis and the development of drug resistance. Recently, it has been reported that MM cells express the receptor activator of nuclear factor-κB (NF-κB) (RANK). However, the role of the RANK/RANK ligand (RANKL) system in drug resistance remains unclear. In this study, we demonstrated a novel function of the RANK/RANKL system in promoting drug resistance in MM. We found that RANKL treatment induced drug resistance in RANK-expressing but not RANK-negative cell lines. RANKL stimulation of RANK-expressing cells increased multidrug resistance protein 1 (MDR1), breast cancer resistance protein (BCRP), and lung resistance protein 1 (LRP1) expression and decreased Bim expression through various signaling molecules. RNA silencing of Bim expression induced drug resistance, but the RANKL-mediated drug resistance could not be overcome through the RNA silencing of MDR1, BCRP, and LRP1 expression. These results indicate that the RANK/RANKL system induces chemoresistance through the activation of multiple signal transduction pathways and by decreasing Bim expression in RANK-positive MM cells. These findings may prove to be useful in the development of cell adhesion-mediated drug resistance inhibitors in RANK-positive MM cells.

Multiple myeloma (MM) is still an incurable hematological malignancy with a 5-year survival rate of ~35%, despite the use of various treatment options. The nuclear factor κB (NF-κB) pathway plays a crucial role in the pathogenesis of MM. Thus, inhibition of the NF-κB pathway is a potential target for the treatment of MM. In a previous study, we showed that mangiferin suppressed the nuclear translocation of NF-κB. However, the treatment of MM involves a combination of two or three drugs. In this study, we examined the effect of the combination of mangiferin and conventional anticancer drugs in an MM cell line. We showed that the combination of mangiferin and an anticancer drug decreased the viability of MM cell lines in comparison with each drug used separately. The decrease in the combination of mangiferin and an anticancer drug induced cell viability was attributed to increase the expression of p53 and Noxa and decreases the expression of XIAP, survivin, and Bcl-xL proteins via inhibition of NF-κB pathway. In addition, the combination treatment caused the induction of apoptosis, activation of caspase-3 and the accumulation of the cells in the sub-G1 phase of the cell cycle. Our findings suggest that the combination of mangiferin and an anticancer drug could be used as a new regime for the treatment of MM.

Mangiferin is a naturally occurring glucosyl xanthone, which induces apoptosis in various cancer cells. However, the molecular mechanism underlying mangiferin-induced apoptosis has not been clarified thus far. Therefore, we examined the molecular mechanism underlying mangiferin-induced apoptosis in multiple myeloma (MM) cell lines. We found that mangiferin decreased the viability of MM cell lines in a concentration-dependent manner. We also observed an increased number of apoptotic cells, caspase-3 activation, and a decrease in the mitochondrial membrane potential. In addition, mangiferin inhibited the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated inhibitor kappa B (IκB) and increased the expression of IκB protein, whereas no changes were observed in the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase 1/2 (JNK1/2), and mammalian target of rapamycin (mTOR). The molecular mechanism responsible for mangiferin-induced inhibition of nuclear translocation of NF-κB was a decrease in the expression of phosphorylated NF-κB-inducing kinase (NIK). Moreover, mangiferin decreased the expression of X-linked inhibitor of apoptosis protein (XIAP), survivin, and Bcl-xL proteins. Knockdown of NIK expression showed results similar to those observed with mangiferin treatment. Our results suggest that mangiferin induces apoptosis through the inhibition of nuclear translocation of NF-κB by suppressing NIK activation in MM cell lines. Our results provide a new insight into the molecular mechanism of mangiferin-induced apoptosis. Importantly, since the number of reported NIK inhibitors is limited, mangiferin, which targets NIK, may be a potential anticancer agent for the treatment of MM.

Macrophage inflammatory protein-1alpha (MIP-1α) is detected at high concentrations in patients with multiple myeloma. It is thought to play an important role in the etiology of multiple myeloma and osteolysis. Thus, inhibiting MIP-1α expression may be useful in developing therapeutic treatments for multiple myeloma-induced osteolysis. In this study, we investigated the potential of statins to inhibit mRNA expression and secretion of MIP-1α in mouse myeloma cells (MOPC-31C). We found that statins inhibited the lipopolysaccharide (LPS)-induced MIP-1α mRNA expression and protein secretion in MOPC-31C cells. This inhibition was reversed when farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), intermediates of the mevalonate pathway, were combined with statins. Furthermore, statins reduced the GTP form of Ras, a phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated Akt. Our results indicate that statins inhibit biosynthesis of FPP and GGPP and thereby down regulate signal transduction of Ras/ERK and Ras/Akt pathways. The net effect suppresses LPS-induced MIP-1α mRNA expression and protein secretion in MOPC-31C cells. Thus, statins hold great promise for developing effective therapies against myeloma-induced osteolysis.

The Japanese school lunch program with milk was designed to supply 33-50% of the necessary nutrients per day and 50% of the recommended dietary allowance for calcium, which is difficult to obtain from Japanese meals. Although this program contributes to the mental and physical development of children, the effect of these meals on the bone growth in children remains unknown. Therefore, we compared the effect of school lunch with milk on bone growth between elementary school children attending schools that did not enforce the school lunch with milk program (box-lunch group) and those attending schools that did enforce the program (school-lunch group). The study subjects included fourth-grade children during the 2009-2013 school years, of whom 329 children were in the school-lunch group and 484 children in the box-lunch group. The bone area ratio of the right calcaneus was evaluated using quantitative ultrasound (Benus III). Dietary intakes were assessed using brief self-administered diet history questionnaires. The subjects were asked to record their activities for 3 d so that the mean physical activity intensity and the time spent sleeping could be estimated. The bone area ratios (%) were significantly higher in the school-lunch group than in the box-lunch group (males 31.0±0.3 vs. 30.3±0.2; females 30.6±0.2 vs. 29.7±0.2). This tendency did not change even after adjustment for confounding factors associated with bone growth. The results suggest that nutrients supplied by the Japanese school lunch program contributed to increased bone growth in elementary school children.

Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor β1 (TGF-β1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-β1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases.

AIM: Non-alcoholic steatohepatitis (NASH) is a globally recognized liver disease. A methionine- and choline-deficient diet is used to induce NASH in mice; however, this diet also causes severe body weight loss. To resolve this issue, we examined the effects of methionine content in a high-fat and choline-deficient (HFCD) diet on body weight and the development of NASH in mice. METHODS: C57BL/6J mice (male, 10 weeks of age) were fed an L-amino acid rodent (control) diet, high-fat (HF) diet, or HFCD diet containing various amounts of methionine (0.1-0.6% (w/w)) for 12 weeks. Plasma lipid levels, hepatic lipid content and inflammatory marker gene expression were measured, and a pathological analysis was conducted to evaluate NASH. RESULTS: The 0.1% methionine in HFCD diet suppressed body weight gain, which was lower than that with control diet. On the other hand, the 0.2% methionine in HFCD diet yielded similar body weight gains as the control diet, while more than 0.4% methionine showed the same body weight gains as the HF diet. Liver weights and hepatic lipid contents were the greatest with 0.1% methionine and decreased in a methionine dose-dependent manner. Pathological analysis, NAFLD activity scores and gene expression levels in the liver revealed that 0.1% and 0.2% methionine for 12 weeks induced NASH, whereas 0.4% and 0.6% methionine attenuated the induction of NASH by HFCD diet. However, the 0.2% methionine in HFCD diet did not induce insulin resistance, despite the body weight gain. CONCLUSIONS: The 0.2% methionine in HFCD diet for 12 weeks was able to induce NASH without weight loss.

Oxaliplatin is a key drug commonly used in colorectal cancer treatment. Despite high clinical efficacy, its therapeutic application is limited by common, dose-limiting occurrence of neuropathy. As usual symptomatic neuropathy treatments fail to improve the patients' condition, there is an urgent need to advance our understanding of the pathogenesis of neuropathy to propose effective therapy and ensure adequate pain management. Oxaliplatin-induced neuropathy was recently reported to be associated with protein kinase C (PKC) activation. It is unclear, however, whether PKC inhibition can prevent neuropathy. In our current studies, we found that a PKC inhibitor, tamoxifen, inhibited oxaliplatin-induced neuropathy via the PKC/extracellular signal-regulated kinase (ERK)/c-Fos pathway in lumbar spinal cords (lumbar segments 4-6). Additionally, tamoxifen was shown to act in synergy with oxaliplatin to inhibit growth in tumor cells-implanted mice. Moreover, mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, PD0325901, suppressed oxaliplatin-induced neuropathy and enhanced oxaliplatin efficacy. Our results indicate that oxaliplatin-induced neuropathy is associated with PKC/ERK/c-Fos pathway in lumbar spinal cord. Additionally, we demonstrate that disruption of this pathway by PKC and MEK inhibitors suppresses oxaliplatin-induced neuropathy, thereby suggesting that PKC and MEK inhibitors may be therapeutically useful in preventing oxaliplatin-induced neuropathy and could aid in combination antitumor pharmacotherapy.

Background and ObjectivesWe carried out a phase II trial to evaluate the feasibility, efficacy, and tolerability of perioperative chemotherapy including single intraperitoneal(IP) administration of paclitaxel(PTX) followed by intravenous(IV) administrations of PTX with S-1 in a neoadjuvant setting for serosa-positive gastric cancer. MethodsPatients with cT4a gastric cancer were enrolled. A laparoscopic survey was performed before study inclusion for the confirmation of serosal invasion, negative lavage cytology, and negative peritoneal metastasis. IP PTX (80mg/m(2)) was administered, followed by systemic chemotherapy. Surgery was performed after the completion of chemotherapy. The primary endpoint was the treatment completion rate. Results37 patients were recruited. The treatment completion rate was 67.6% (25/37; 90% CI, 52.8-80.1%), which was significantly higher than 50%; we set this as a threshold value (P=2.4% [one-sided]). 14 patients had target lesions; of these, 10 showed a partial response (71.4%), three had stable disease (21.4%), and one had progressive disease(7.2%). The response rate was 71.4% (10/14). All patients underwent gastrectomy with D2 lymph node dissection. The 3- and 5-year OS rates were 78.0 and 74.9%, respectively. ConclusionsPerioperative chemotherapy including neoadjuvant IP PTX followed by sequential IV PTX with S-1 for serosa-positive gastric cancer is feasible, safe, and efficient. J. Surg. Oncol. 2015 111:1041-1046. (c) 2015 Wiley Periodicals, Inc.

The acquisition of anti-cancer drug resistance is a major limitation of chemotherapy for multiple myeloma(MM) and it is thus important to identify the mechanisms by which MM cells develop such drug resistance. In a previous study, we showed that multidrug resistance (MDR) involves the overexpression of MDR1 and survivin in vincristine-resistant RPMI8226/VCR cells. However, the underlying mechanism of MDR remains unclear. In this study, we investigated the mechanism of MDR in RPMI8226/VCR cells, and found that RPMI8226/VCR cells exhibit increased levels of activated ERK1/2, Akt, and NF-kappa B, while the levels of activated mTOR, p38MAPK, and JNK do not differ between RPMI8226/VCR cells and their vincristine-susceptible counterparts. In addition, the inhibition of ERK1/2, Akt, or NF-kappa B by inhibitors reversed thedrug resistance of RPMI8226/VCR cells via the suppression of survivin expression, but did not affect MDR1 expression; RNA silencing of survivin expression completely reversed vincristine resistance, while MDR1 silencing only weakly suppressed vincristine resistance in RPMI8226/VCR cells. These results indicate that enhanced survivin expression via the activation of ERK1/2, Akt, and NF-kappa B plays a critical role in vincristine resistance in RPMI8226/VCR cells. Our findings suggest that ERK1/2, Akt, and NF-kappa B inhibitors are potentially useful as anti-MDR agents for the treatment of vincristine-resistant MM. (C) 2015 Elsevier Ltd. All rights reserved.

Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic inflammation of synovial joints, ultimately leading to a progressive and irreversible joint destruction. Activation of nuclear factor-kappa B (NF-κB) promotes production of proinflammatory cytokines in various inflammatory diseases including rheumatoid arthritis. Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside (C-glucosyl xanthone), is a naturally occurring polyphenol. Our previous results showed that mangiferin suppressed NF-κB activation. However, it is unclear, whether mangiferin can prevent rheumatoid arthritis through suppression of NF-κB activation and expression of various cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6), which play a critical role in the pathogenesis of rheumatoid arthritis. In the present study, we found that mangiferin suppressed the progression and incidence of CIA in DBA1/J mice. In CIA mice, mangiferin inhibited the mRNA expression of cytokine genes in thymus and spleen of CIA mie and led to decreased serum levels of IL-1β, IL-6, TNF-α, and receptor activator NF-κB ligand (RANKL) via inhibition of NF-κB and activation of extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, mangiferin markedly inhibited not only developing but also clinically evident CIA. These findings suggest that mangiferin has potential clinical applications for the treatment of rheumatoid arthritis.

Metastatic melanoma is a life-threatening disease for which no effective treatment is currently available. In melanoma cells, Rho overexpression promotes invasion and metastasis. However, the effect of statins on spontaneous metastasis and tumor growth remains unclear. In the present study, we investigated the mechanism of statin-mediated tumor growth and metastasis inhibition in an in vivo model. We found that statins significantly inhibited spontaneous metastasis and tumor growth. Statins inhibited the mRNA expression and enzymatic activities of matrix metalloproteinases (MMPs) in vivo and also suppressed the mRNA and protein expression of very late antigens (VLAs). Moreover, statins inhibited the prenylation of Rho as well as the phosphorylation of LIM kinase, serum response factor (SRF), and c-Fos downstream of the Rho signaling pathway. In addition, statins enhanced p53, p21, and p27 expression and reduced phosphorylation of cyclin-dependent kinase and expression of cyclin D1 and E2. These results indicate that statins suppress Rho signaling pathways, thereby inhibiting tumor metastasis and growth. Furthermore, statins markedly improved the survival rate in a metastasis model, suggesting that statins have potential clinical applications for the treatment of metastatic cancers.

Osteolytic bone disease in multiple myeloma (MM) is associated with upregulated osteoclast activity. Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is crucially involved in the development of osteolytic bone lesions in MM. We previously reported that minodronate inhibited lipopolysaccharide-induced MIP-1 alpha secretion in mouse myeloma cells. However, it remains unknown whether bisphosphonates and statins inhibit MIP-1 alpha secretion by human MM cells. In present study, we investigated whether bisphosphonates and statins had any inhibitory effect on MIP-1 alpha secretion by human myeloma cells and the mechanism underlying this effect. In this study, we found that bisphosphonates and statins inhibited MIP-1 alpha mRNA and MIP-1 alpha secretion and suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation by inhibiting Ras prenylation. Moreover, bisphosphonates and statins suppressed the expression of acute myeloid leukemia-1 alpha (AML-1A) mRNA, a MIP-1 alpha transcription factor. These results indicate that bisphosphonates and statins suppress the Ras/mitogen-activated protein kinase kinase/ERK/AML-1A and Ras/phosphatidylinositol-3 kinase/Akt/AML-1A pathways, thereby inhibiting MIP-1 alpha secretion by MM cells. Therefore, use of MIP-1 alpha expression inhibitors such as bisphosphonates and statins may provide a new therapeutic approach to inhibiting tumour progression and bone destruction in MM patients.

Aim: Resveratrol has been shown to mimic the beneficial effects of dietary restriction (DR). We previously reported that DR delays stroke onset and extends the lifespan in Stroke-Prone Spontaneously Hypertensive rats (SHRSP). Therefore, we examined whether resveratrol mimics DR and delays stroke onset in SHRSP. Methods: Cerebrovascular endothelial cells (CVECs) from SHRSP were treated with resveratrol, and the inflammatory gene expression levels and NF kappa B protein levels were measured. In order to address the effects of resveratrol in vivo, SHRSP (male, 10 weeks of age) were fed an experimental diet containing several doses of resveratrol (0 - 0.05% (w/w)), after which we measured the plasma cytokine levels and examined the stroke onset and lifespan. Results: Treatment with resveratrol (100 mu M, 24 hours) in CVECs from SHRSP significantly decreased the interleukin (IL)-1 beta-induced monocyte chemoattractant protein-1 (MCP-1) mRNA expression levels and p50 and p65 protein levels in the nuclear fraction. When the SHRSP were fed a diet containing resveratrol for one week, the resveratrol treatment did not affect the plasma lipid and glucose levels, body weight or weight of each tissue. Resveratrol slightly, but not significantly, decreased the plasma levels of IL-1 beta and MCP-1 compared with that observed in the control group. In addition, resveratrol decreased the IL-1 beta and MCP-1 mRNA expression levels in the brain versus the control animals. However, no doses of resveratrol delayed stroke onset or extended the lifespan in SHRSP. Conclusions: In this study, resveratrol did not delay stroke onset in SHRSP, although it partially suppressed systemic and cerebral inflammation. These results suggest that resveratrol does not mimic the beneficial effects of DR on stroke in vivo.

Dimethyl fumarate (DMF) is a fumaric acid ester that is used to treat psoriasis and multiple sclerosis. Recently, DMF was found to exhibit anti-tumor effects. However, the molecular mechanisms underlying these effects have not been elucidated. In this study, we investigated the mechanism of DMF-induced apoptosis in different human hematopoietic tumor cell lines. We found that DMF induced apoptosis in different human hematopoietic tumor cell lines but it did not affect the normal human B lymphocyte cell line RPMI 1788. We also observed a concurrent increase in caspase-3 activity and in the number of Annexin-V-positive cells. Furthermore, an examination of the survival signals, which are activated by apoptotic stimuli, revealed that DMF significantly inhibited nuclear factor-kappa B (NF-kappa B) p65 nuclear translocation. In addition, DMF suppressed B-cell lymphoma extra-large (Bcl-xL) and X-linked inhibitor of apoptosis (XIAP) expression whereas Bcl-2, survivin, Bcl-2-associated X protein (Bax), and Bim levels did not change. These results indicated that DMF induced apoptosis by suppressing NF-kappa B activation, and Bcl-xL and XIAP expression. These findings suggested that DMF might have potential as an anticancer agent that could be used in combination therapy with other anticancer drugs for the treatment of human hematopoietic tumors. (C) 2014 Elsevier Masson SAS. All rights reserved.

Background: Bisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Recent studies have shown that nitrogen-containing bisphosphonates induced apoptosis in rabbit osteoclasts and prevented prenylated small GTPase. However, whether bisphosphonates inhibit osteoclast formation has not been determined. In the present study, we investigated the inhibitory effect of minodronate and alendronate on the osteoclast formation and clarified the mechanism involved in a mouse macrophage-like cell lines C7 and RAW264.7. Results: It was found that minodronate and alendronate inhibited the osteoclast formation of C7 cells induced by receptor activator of NF kB ligand and macrophage colony stimulating factor, which are inhibited by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. It was also found that minodronate and alendronate inhibited the osteoclast formation of RAW264.7 cells induced by receptor activator of NF-kappa B ligand. Furthermore, minodronate and alendornate decreased phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; similarly, U0126, a mitogen protein kinase kinase 1/2 (MEK1/2) inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited osteoclast formation. Conclusions: This indicates that minodronate and alendronate inhibit GGPP biosynthesis in the mevalonate pathway and then signal transduction in the MEK/ERK and PI3K/Akt pathways, thereby inhibiting osteoclast formation. These results suggest a novel effect of bisphosphonates that could be effective in the treatment of bone metabolic diseases, such as osteoporosis.

The calcium channel blocker verapamil inhibits the transport function of multidrug resistance protein 1 (MDR1). Although verapamil acts to reverse MDR in cancer cells, the underlying mechanism remains unclear. In the present study, we investigated the mechanism of reversing MDR by verapamil in anticancer drug-resistant multiple myeloma (MM) cell lines. We found that verapamil suppresses MDR1 and survivin expressions and increases Bim expression via suppression of Src activation. Furthermore, dasatinib reversed the drug-resistance of the drug-resistant cell lines. These findings suggest that Src inhibitors are potentially useful as an anti-MDR agent for the treatment of malignant tumor cells. (C) 2013 Elsevier Ltd. All rights reserved.

Intraperitoneally administrated epithelial cellular adhesion molecule (EpCAM) monoclonal antibody is a therapeutic agent in patients with malignant effusion in several types of carcinoma. However, the role of EpCAM in peritoneal metastasis (PM) lesions and primary lesions of gastric cancer (GC) is still unclear. Therefore, in this study, we investigated EpCAM expression in GC patients with PM. We investigated the expression of EpCAM in 35PM lesions and 104 biopsy samples as primary lesions. Immunohistochemical staining was performed using the Ventana Benchmark XT (Roche Diagnostics) system. EpCAM expression was evaluated by calculating the total immunostaining score, which is the product of the proportion score and the intensity score. Overexpression was defined as a total score greater than 4. All PM specimens showed overexpression of EpCAM, and GC cells in both the surface layer and the deep layer of the PM showed a high expression of EpCAM. Meanwhile, in the biopsy sample, the expression of EpCAM ranged from none to strong. The EpCAM score results for PM specimens and biopsy samples were 11.0 ± 2.0 and 6.9 ± 3.9, respectively. The difference between the scores was statistically significant (P < 0.05). The intraperitoneally administrated EpCAM antibody might have a anti-cancer effect in PM lesions of GC. Additionally, it can be assumed that only GC cells which express a high level of EpCAM might metastasize to the peritoneum. © 2012 Springer-Verlag France.

Several autocrine soluble factors, including macrophage inflammatory protein-1 alpha and tumour necrosis factor-alpha (TNF-alpha), promote the survival and growth of multiple myeloma (MM) cells. We hypothesised that inhibition of the TNF-alpha autocrine loop may enhance the cytotoxic effect of anticancer drugs in MM cell lines. In the present study, a TNF-alpha-neutralizing antibody suppressed cell proliferation and enhanced the cytotoxic effect of anticancer drugs onMMcells. In addition, combination treatment with the TNF-alpha-neutralizing antibody and the chemotherapy agent melphalan inhibited nuclear factor kappa B (NF-kappa B) p65 nuclear translocation and mammalian target of rapamycin (mTOR) activation and upregulated the expression of Bax and Bim. Treatment of ARH-77 cells with the NF-kappa B inhibitor dimethyl fumarate or the mTOR inhibitor rapamycin suppressed NF-kappa B p65 nuclear translocation and enhanced the cytotoxic effect of melphalan. Furthermore, infliximab, a monoclonal antibody against TNF-alpha, also enhanced the cytotoxic effect of anticancer drugs in ARH-77 cells. These results indicated that TNF-alpha-neutralizing antibodies or infliximab enhanced the cytotoxic effect of anticancer drugs by suppressing the TNF receptor/mTOR/NF-kappa B pathways. The inhibition of TNF-alpha may thus provide a new therapeutic approach to control tumour progression and bone destruction in MM patients. (C) 2013 Elsevier Ltd. All rights reserved.

Our previous study indicated that consuming (-)-epigallocatechin gallate (EGCG) before or after traumatic brain injury (TBI) eliminated free radical generation in rats, resulting in inhibition of neuronal degeneration and apoptotic death, and improvement of cognitive impairment. Here we investigated the effects of administering EGCG at various times pre- and post-TBI on cerebral function and morphology. Wistar rats were divided into five groups and were allowed access to (1) normal drinking water, (2) EGCG pre-TBI, (3) EGCG pre- and post-TBI, (4) EGCG post-TBI, and (5) sham-operated group with access to normal drinking water. TBI was induced with a pneumatic controlled injury device at 10 weeks of age. Immunohistochemistry and lipid peroxidation studies revealed that at 1, 3, and 7 days post-TBI, the number of 8-Hydroxy-2'-deoxyguanosine-, 4-Hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells, and levels of malondialdehyde around the damaged area were significantly decreased in all EGCG treatment groups compared with the water group (P < 0.05). Although there was a significant increase in the number of surviving neurons after TBI in each EGCG treatment group compared with the water group (P < 0.05), significant improvement of cognitive impairment after TBI was only observed in the groups with continuous and post-TBI access to EGCG (P < 0.05). These results indicate that EGCG inhibits free radical-induced neuronal degeneration and apoptotic death around the area damaged by TBI. Importantly, continuous and post-TBI access to EGCG improved cerebral function following TBI. In summary, consumption of green tea may be an effective therapy for TBI patients.

Aim: Patients with scirrhous carcinoma of the gastrointestinal tract frequently develop peritoneal carcinomatosis particularly of the peritoneal extension type (PET), which has a bad prognosis. We developed a novel animal model, suitable for testing treatments for PET. Material and Methods: In order to develop the model, we scraped the entire peritoneum of Fischer 344 rats with sterile cotton swabs and injected 1x10(6) cells of the RCN-9 cell type into the peritoneal cavity. Results: In the novel experimental model, RCN-9 cells adhered only to the exposed basement membrane. The submesothelial layer and fibroblasts in the submesothelial layer grew and increased to a maximum at day 7, then decreased during late-phase peritoneal carcinomatosis. At day 14, RCN-9 cells coated the peritoneum in a manner similar to PET. Conclusion: We successfully established a novel animal model of peritoneal carcinomatosis that mimics clinicopathological features of PET. Fibroblasts in the submesothelial layer potentially play an important role in peritoneal carcinomatosis.

We have previously reported free radical production after traumatic brain injury (TBI), which induces neural stem cell (NSC) degeneration and death. However, the effects of aging on NSC proliferation around the damaged area following TBI have not been investigated. Therefore, in this study, we used 10-week (young group) and 24-month-old (aged group) rat TBI models to investigate the effects of aging on NSC proliferation around damaged tissue using immunohistochemical and ex vivo techniques. Young and aged rats received TBI. At 1, 3 and 7 days after TBI, immunohistochemical and lipid peroxidation studies were performed. Immunohistochemistry revealed that the number of nestin-positive cells around the damaged area after TBI in the aged group decreased significantly when compared with those in the young group (P < 0.01). However, the number of 8-hydroxy-2'-deoxyguanosine-, 4-hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells and the level of peroxidation around the damaged area after TBI significantly increased in the aged group, compared with those in the young group (P < 0.01). Furthermore, almost all ssDNA-positive cells in young and aged groups co-localized with NeuN and nestin staining. Ex vivo studies revealed that neurospheres, which differentiated into neurons and glia in culture, could only be isolated from injured brain tissue in young and aged groups at 3 days after TBI. These results indicate that, although there were fewer NSCs that have the potential to differentiate into neurons and glia, these NSCs escaped free radical-induced degeneration around the damaged area after TBI in the aged rat brain.

Nitrogen-containing bisphosphonates (N-Bps) induce apoptosis in tumor cells by inhibiting the prenylation of small G-proteins. However, the details of the apoptosis-inducing mechanism remain obscure. The present study showed that the induction of apoptosis by N-Bps in hematopoietic tumor cells is mediated by mitochondrial apoptotic signaling pathways, which are activated by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. Furthermore, N-BPs decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK) and mTOR via suppression of Ras prenylation and enhanced Bim expression. The present results indicated that N-BPs induce apoptosis by decreasing the mitochondrial transmembrane potential, increasing the activation of caspase-9 and caspase-3, and enhancing Bim expression through inhibition of the Ras/MEK/ERK and Ras/mTOR pathways. The accumulation of N-BPs in bones suggests that they may act more effectively on tumors that have spread to bones or on Ras-variable tumors. This is the first study to show that the specific molecular pathways of N-BP-induced apoptosis. (C) 2012 Elsevier Inc. All rights reserved.

Background: Increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT), which occurs during cancer progression and metastasis. Recent studies have indicated the expression of receptor activator of nuclear factor-κB (RANK) in various solid tumors, including breast cancer. Although activation of the RANK ligand (RANKL)/RANK system promotes cell migration, metastasis, and anchorage-independent growth of tumor-initiating cells, it remains to be investigated if RANKL induces EMT in breast cancer cells. In this study, we investigated whether RANKL induces EMT in normal breast mammary epithelial cells and breast cancer cells, and the mechanism underlying such induction. Methods. Expression levels of vimentin, N-cadherin, E-cadherin, Snail, Slug, and Twist were examined by real-time polymerase chain reaction. Cell migration and invasion were assessed using Boyden chamber and invasion assays, respectively. The effects of RANKL on signal transduction molecules were determined by western blot analyses. Results: We found that stimulation by RANKL altered the cell morphology to the mesenchymal phenotype in normal breast epithelial and breast cancer cells. In addition, RANKL increased the expression levels of vimentin, N-cadherin, Snail, and Twist and decreased the expression of E-cadherin. We also found that RANKL activated nuclear factor-κB (NF-κB), but not extracellular signal-regulated kinase 1/2, Akt, mammalian target of rapamycin, c-Jun N-terminal kinase, and signal transducer and activator of transcription 3. Moreover, dimethyl fumarate, a NF-κB inhibitor, inhibited RANKL-induced EMT, cell migration, and invasion, and upregulated the expressions of Snail, Twist, vimentin, and N-cadherin. Conclusions: The results indicate that RANKL induces EMT by activating the NF-κB pathway and enhancing Snail and Twist expression. These findings suggest that the RANKL/RANK system promotes tumor cell migration, invasion, and metastasis via the induction of EMT. © 2013 Tsubaki et al. licensee BioMed Central Ltd.

We conducted a phase II study involving a single administration of intraperitoneal chemotherapy with paclitaxel followed by sequential systemic chemotherapy with S-1+ paclitaxel for advanced gastric cancer patients with peritoneal metastasis. Gastric cancer patients with peritoneal metastasis were enrolled. Paclitaxel (80 mg/m(2)) was administered intraperitoneally at staging laparoscopy. Within 7 days, patients received systemic chemotherapy with S-1 (80 mg/m(2)/day on days 1-14) plus paclitaxel (50 mg/m(2) on days 1 and 8), followed by 7-days rest. The responders to this chemotherapy underwent second-look laparoscopy, and gastrectomy with D2 lymph node dissection was performed in patients when the disappearance of peritoneal metastasis had been confirmed. The primary endpoint of the study was overall survival rate. Thirty-five patients were enrolled. All patients were confirmed as having localized peritoneal metastasis by staging laparoscopy. Eventually, gastrectomy was performed in 22 patients. The median survival time of the total patient population and those patients in which gastrectomy was performed was 21.3 and 29.8 months, respectively. The overall response rate was 65.7 % for all patients. The frequent grade 3/4 toxic effects included neutropenia and leukopenia. Sequential intraperitoneal and intravenous paclitaxel plus S-1 was well tolerated in gastric cancer patients with peritoneal metastasis.

The prognosis of gastric cancer patients with peritoneal metastasis is very poor. Recent findings suggest that use of trastuzumab, a monoclonal antibody-based agent that targets human epidermal growth factor receptor 2 (HER2), may improve the prognosis of gastric cancer patients with HER2 overexpression and/or gene amplification. However, whether these mechanisms of HER2 upregulation are present in gastric cancer patients with peritoneal metastasis is unclear. The status of HER2 expression in a cohort of samples obtained from 35 gastric cancer patients with peritoneal metastasis was investigated using immunohistochemistry and fluorescence in situ hybridization. In 18 cases, we also investigated the influence of induction chemotherapy on HER2 overexpression. The frequency of HER2 overexpression and gene amplification was 2.9 % (1/35) in peritoneal metastatic lesions. There was concurrence in HER2 status in the samples examined prior to and following induction of chemotherapy. Most samples from the gastric cancer patients with peritoneal metastasis did not show HER2 amplification and/or overexpression. Although our study size was small, these results suggest that trastuzumab, which is critically dependent on HER2 expression, might not be an effective agent for these patients. Consequently, other therapeutic approaches for these patients must be developed.

Multidrug resistance represents a major obstacle for the chemotherapy of a wide variety of human tumors. To investigate the underlying mechanisms associated with resistance to anti-cancer drugs, we established anti-cancer drug-resistant multiple myeloma (MM) cell lines RPMI8226/ADM, RPMI8226/VCR, RPMI8226/DEX, and RPMI8226/L-PAM, the 50% inhibitory concentration values of which were 77-, 58-, 79-, and 30-fold higher than their parental cell lines, respectively. The resistant cell lines overexpressed MDR1 and survivin, or showed decreased Bim expression. These results indicated that regulating these factors with inhibitors might be a viable approach to increasing the susceptibility of quiescent MM cells to chemotherapy. (C) 2012 Elsevier Ltd. All rights reserved.

Osteoclast differentiation is influenced by receptor activator of the NF-kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and CD9, which are expressed on bone marrow stromal cells and osteoblasts. In addition, osteoprotegerin (OPG) is known as an osteoclastogenesis inhibitory factor. In this study, we investigated whether bisphosphonates and statins increase OPG expression and inhibit the expression of CD9, M-CSF, and RANKL in the bone marrow-derived stromal cell line ST2. We found that bisphosphonates and statins enhanced OPG mRNA expression and inhibited the expression of CD9, M-CSF, and RANKL mRNA. Futhermore, bisphosphonates and statins decreased the membrane localization of Ras and phosphorylated ERK1/2, and activated the p38MAPK. This indicates that bisphosphonates and statins enhanced OPG expression, and inhibited the expression of CD9. M-CSF, and RANKL through blocking the Ras/ERK pathway and activating p38MAPK. Accordingly, we believe that its clinical applications will be investigated in the future for the development of osteoporosis therapy. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

Aim: A preliminary study with the aim of evaluating the safety and efficacy of a single intraperitoneal administration of paclitaxel, combined with intravenous administration of paclitaxel plus S-1, was carried out in gastric cancer patients with peritoneal metastasis. Patients and Methods: Paclitaxel was administered intraperitoneally at 80 mg/m(2). After one to two weeks, S-1 was administered at 80 mg/m(2)/day for 14 consecutive days, followed by seven days' rest. Paclitaxel was administered intravenously at 50 mg/m(2) on days 1 and 8. The safety, pharmacokinetic analysis and efficacy of this therapy were investigated. Results: Fifteen patients were enrolled in this study. The toxic effects of the intraperitoneal chemotherapy were mild. The toxic effects with the systemic chemotherapy were acceptable. The ratio of (AUC peri)/(AUC pla) was 1065:1 in the pharmacokinetic analysis. The one-year overall survival rate was 10/15 (66.7%). Conclusion: A single intraperitoneal administration of paclitaxel combined with intravenous administration of paclitaxel plus S-1 is a well-tolerated and feasible treatment for patients with gastric cancer with peritoneal metastasis.

Parallel phase-shifting digital holography is capable of three-dimensional measurement of a dynamically moving object with a single-shot recording. In this letter, we demonstrated a parallel phase-shifting digital holography using a single femtosecond light pulse whose central wavelength and temporal duration were 800 nm and 96 fs, respectively. As an object, we set spark discharge in atmospheric pressure air induced by applying a high voltage to between two electrodes. The instantaneous change in phase caused by the spark discharge was clearly reconstructed. The reconstructed phase image shows the change of refractive index of air was -3.7 x 10(-4). (C) 2012 Optical Society of America

A major component of green tea is (-)-epigallocatechin gallate (EGCG), which has strong antioxidant properties. Here, we investigated the effect of EGCG on neural stem cell (NSC) proliferation around the damaged area following traumatic brain injury (TBI). In this study, male Wistar rats that had access to normal drinking water, or water containing 0.1% (w/v) EGCG, ad libitum received TBI at 10 weeks of age. Immunohistochemistry revealed that the number of nestin-positive cells around the damaged area after TBI in the EGCG treatment group increased significantly compared with the normal water group (P < 0.05). However, the number of 8-hydroxy-2'-deoxyguanosine-, 4-hydroxy-2-nonenal-, single-stranded DNA (ssDNA)-positive cells and the level of peroxidation around the damaged area after TBI significantly decreased in the EGCG treatment group when compared with the water group (P < 0.05). Furthermore, in contrast to the EGCG group, almost all ssDNA-positive cells in the water group co-localized with NeuN and nestin-staining. Ex vivo studies revealed that spheres could only be isolated from injured brain tissue in the water group at 3 days following TBI. However, in the EGCG group, spheres could be isolated at both 3 and 7 days following TBI. A greater number of spheres could be isolated from the EGCG group, which differentiated into neurons and glia in culture without basic fibroblast growth factor. These results indicate that consumption of water containing EGCG pre- and post-TBI inhibits free radical-induced degradation of NSCs, which have the potential to differentiate into neurons and glia around the area of damage following TBI.

Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Some patients continue to have a poor prognosis, because of the metastatic disease. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. YM529/ONO-5920 has recently been reported to induce apoptosis in various tumors including osteosarcoma. However, the mode of metastasis suppression in osteosarcoma by YM529/ONO-5920 is unclear. In the present study, we investigated whether YM529/ONO-5920 inhibited tumor cell migration, invasion, adhesion, or metastasis in the LM8 mouse osteosarcoma cell line. We found that YM529/ONO-5920 significantly inhibited metastasis, cell migration, invasion, and adhesion at concentrations that did not have antiproliferative effects on LM8 cells. YM529/ONO-5920 also inhibited the mRNA expression and protein activities of matrix metalloproteinases (MMPs). In addition, YM529/ONO-5920 suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine/threonine protein kinase B (Akt) by the inhibition of Ras prenylation. Moreover, U0126, a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, also inhibited LM8 cell migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP. The results indicated that YM529/ONO-5920 suppressed the Ras/MEK/ERK and Ras/PI3K/Akt pathways, thereby inhibiting LM8 cell migration, invasion, adhesion, and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumor cell metastasis in osteosarcoma. (C) 2012 Elsevier Inc. All rights reserved.

Background and Purpose-There is an inverse association between dairy food consumption and the incidence of stroke in observational studies. However, it is unknown whether the relationship is causal or, if so, what components in milk are responsible for reducing the incidence of stroke. Methods-Stroke-prone spontaneously hypertensive rats were fed diets comprising amino acids, proteins from different sources (casein, whey, soybean, or egg white), or fats from different sources (butter, beef tallow, or cocoa butter) and the onset of stroke and lifespan were examined. Results-Increasing the amount of dietary casein (5% to 55% of caloric intake) markedly delayed the onset of stroke. However, when stroke-prone spontaneously hypertensive rats were fed diets containing 55% of caloric intake as protein, rats fed casein or whey protein, a major component of milk, displayed a delayed onset of stroke compared with rats fed soybean or egg white protein. Rats fed an amino acids diet containing the same amino acids composition as casein did not have a delay in the onset of stroke. Increasing dietary fats, including butter as well as beef tallow and cocoa butter, did not affect the onset of stroke. All diets did not affect blood pressure in the early stage. Conclusions-These data suggest that the inverse association between dairy food consumption and incidence of stroke in epidemiological studies is causal and that peptides in milk protein, but not fat, might be responsible for this effect. (Stroke. 2012;43:470-477.)

High blood levels of inflammatory biomarkers and immune cells in stroke lesions have been recognized as results of stroke. However, recent studies have suggested that inflammation occurs prior to stroke onset. In this study, we aimed to clarify the role of inflammation in stroke onset among stroke-prone spontaneously hypertensive rats (SHRSP). At 4 weeks of age (before stroke onset), the plasma level of IL-1 beta was significantly higher in SHRSP (153.0 +/- 49.7 pg/ml) than in Wistar Kyoto rats (WKY) (7.7 +/- 3.4 pg/ml, P < 0.001 versus SHRSP) or spontaneously hypertensive rats (SHR) (28.0 +/- 9.1 pg/ml, P < 0.001 versus SHRSP) (n = 6 per strain). Stimulated IL-1 beta signal was also observed in cerebrovascular endothelial cells of SHRSP. Gene expressions of IL-1 beta, IL-1 receptors, caspase-1, and downstream genes (MCP-1 and ICAM-1), which associated with immune cell recruitment, were significantly greater in SHRSP than in WKY or SHR, coincident with greater NF kappa B protein levels in SHRSP compared to WKY or SHR. In addition, continuous administration of IL-1 beta (2 mu g/day) using an osmotic pump slightly increased the incidence of stroke in SHR (P = 0.046) and significantly accelerated the onset of stroke in SHRSP (P = 0.006) compared to each control (n = 10 per group). These results suggest that a stimulated IL-1 beta signal might be a cause of stroke onset when concomitant with severe hypertension.

Exercise enhances neuronal stem cell (NSC) proliferation and neurogenesis. However, the effect of exercise on NSC proliferationsurrounding the area of damage after traumatic brain injury (TBI) isunknown. Here, we investigate the effect of running on NSCproliferation following TBI in the rat. Wistar rats received TBI andwere randomly divided into two groups: (1) non-exercise group and(2) exercise group. The exercise group ran on a treadmill for 30min/day at 22 m/min for 7 consecutive days. Immunohistochemistry wasused to monitor NSC proliferation around the damaged area and ex vivo techniques were used to isolate NSCs from the damaged region in both groups. The number of nestin-and Ki-67-positive cells observed at 3 and 7 days after TBI was significantly greater in the exercise group than in the non-exercise group (P < 0.01). Furthermore, most nestin-positive cells in the exercise group co-localized with Ki-67-positive cells. In ex vivo studies, spheres could be isolated from injured brain tissue from the exercise group at 3 and 7 days following TBI, but at only 3 days in the non-exercise group. The number of spheres isolated from injured brain tissue was greater in the exercise group than in the non-exercise group. Spheres were immunopositive for nestin and comprised of NSCs that could differentiate into neurons and glia. Exercise increases the proliferation of NSCs around the damaged area following TBI. Therefore, exercise therapy (rehabilitation) in the early phase following TBI is important for recuperation from cerebral dysfunction induced by TBI.

Stroke-prone spontaneously hypertensive rats (SHRSP) are the only animal model that suffers from spontaneous cerebral stroke. In this study, we investigated the appearance of neural stem cells (NSCs) and new neurons in the penumbra and the subventricular zone (SVZ) after cerebral stroke in SHRSP. SHRSP before cerebral stroke were intraperitoneally injected with 5-bromo-2′-deoxyuridine (BrdU). SHRSP were divided into acute and chronic phase groups after cerebral stroke. Brain sections from both groups were studied with cellspecific markers such as BrdU, a cell division and proliferation marker, SOX2, a marker of NSCs, nestin, an NSC and immature astrocyte marker, doublecortin (DCX), an immature new neuron marker, and NeuN, a marker of mature neurons. NSCs and new neurons appeared in the penumbra in the early stages after cerebral stroke, and these cells differentiated into mature neurons in the chronic phase. Furthermore, soon after being affected by a cerebral stroke, there were many new neurons and immature cells, which appear to be NSCs, in the ipsilateral SVZ. The findings of the study indicate that immature cells and new neurons from the ipsilateral SVZ might migrate into the penumbra after cerebral stroke.

Background The aim of this study was to examine the safety, pharmacokinetics, and cytological efficacy against free intraperitoneal cancer cells of intraperitoneal chemotherapy (IPC) with paclitaxel after gastrectomy with en-bloc D2 lymph node dissection (GD2) in cases of gastric cancer with peritoneal carcinomatosis (PC) and/or positive cytological findings in peritoneal washings (CFPW). Methods: Twenty-one patients with gastric cancer with PC and/or positive CFPW who underwent GD2 were treated with early, post-operative, intraperitoneal paclitaxel. Intra-chemotherapeutic toxicity and operative complication were measured using the common toxicity criteria of the National Cancer Institute, version 3.0. Intraperitoneal and plasma paclitaxel concentrations were measured using a high-performance liquid chromatography assay. Results: Grade 3 anemia occurred in two patients (9.5%) and neutropenia was observed in three patients (14.3%). No grade 4 toxicity was observed. A grade 2 operative complication was a superficial surgical site infection (4.8%) that was treated with antibiotics. Cytologically, no viable cancer cells were observed in the intra-abdominal fluid 24 hr after intraperitoneal administration of paclitaxel. The intraperitoneal/plasma area under the drug concentration-time curve (AUC) ratio was 596.9:1. Conclusion: IPC with paclitaxel after GD2 is a safe and cytologically effective treatment modality for free intraperitoneal cancer cells. However, additional data are required to determine the effect on survival. J. Surg. Oncol. 2012;105:43-47. (C) 2011 Wiley Periodicals, Inc.

A major component of green tea, a widely consumed beverage, is (-)-epigallocatechin gallate (EGCG), which has strong antioxidant properties. Our previous study has indicated that free radical production following rat traumatic brain injury (TBI) induces neural degeneration. In this study, we investigated the effects of EGCG on cerebral function and morphology following TBI. Six-week-old male Wistar rats that had access to normal drinking water, or water containing 0.1% (w/v) EGCG ad libitum, received TBI with a pneumatic controlled injury device at 10 weeks of age. Immunohistochemistry and lipid peroxidation studies revealed that at 1, 3 and 7 days post-TBI, the number of 8-hydroxy-2'-deoxyguanosine-, 4-hydroxy-2-nonenal-and single-stranded DNA (ssDNA)-positive cells, and the levels of malondialdehyde (MDA) around the damaged area after TBI, significantly decreased in the EGCG treatment group compared with the water group (P < 0.05). Most ssDNA-positive cells in the water group co-localized with neuronal cells. However, in the EGCG treatment group, few ssDNA-positive cells co-localized with neurons. In addition, there was a significant increase in the number of surviving neuronal cells and an improvement in cerebral dysfunction after TBI in the EGCG treatment group compared with the water group (P < 0.05). These results indicate that consumption of water containing EGCG pre- and post-TBI inhibits free radical-induced neuronal degeneration and apoptotic cell death around the damaged area, resulting in the improvement of cerebral function following TBI. In summary, consumption of green tea may be an effective therapy for TBI patients.

Even after radical surgery for stage II and stage III colorectal cancer, metachronous liver metastasis is frequently observed. The aim of this study was to identify the risk of metachronous liver metastasis with retrospective clinicopathological study. Immunohistochemistry was performed to evaluate the expression of Osteopontin (OPN), CD-68, and CD105 in 41 cases of stage II and stage III colorectal cancer tissue. Stage II and stage III colorectal cancer patients who had undergone R0 resection were classified into two groups: with metachronous liver metastasis (m-LM; n = 17) and without liver metastases (control; n = 24). Additionally, double-immunofluorescence staining was performed using antibodies to OPN and CD68. OPN-positive cells were frequently colocalized with CD68 immunoreactivity. OPN and microvascular density expression in the central area were significantly higher in the m-LM (OPN; control 4.3 +/- 0.56, m-LV 10.8 +/- 1.48, P < 0.05; microvascular density control 18.5 +/- 2.86, m-LV 31.4 +/- 4.39, P < 0.05), while CD68 expression in the invasive margin was significantly higher in the control group (control 98.9 +/- 7.31, m-LV 28.2 +/- 3.18, P < 0.05). These results suggest that the risk of metachronous liver metastasis could be well predicted by immunohistochemical staining of OPN in the central areas, and CD68 in the invasive margins of tumors.

Exercise is reported to inhibit neuronal apoptotic cell death in the hippocampus and improve learning and memory. However, the effect of exercise on inhibition of neuronal apoptosis surrounding the area of damage after traumatic brain injury (TBI) and the improvement of cerebral dysfunction following TBI are unknown. Here, we investigate the effect of exercise on morphology and cerebral function following TBI in rats. Wistar rats received TBI by a pneumatic controlled injury device were randomly divided into two groups: (1) non-exercise group and (2) exercise group. The exercise group ran on a treadmill for 30 min/day at 22 m/min for seven consecutive days. Immunohistochemical and behavioral studies were performed following TBI. The number of single-stranded DNA (ssDNA)-positive cells around the damaged area early after TBI was significantly reduced in the exercise group compared with the non-exercise group (P < 0.05). Furthermore, most ssDNA-positive cells in the non-exercise group co-localized with neuronal cells. However, in the exercise group, a few ssDNA-positive cells co-localized with neurons. In addition, there was a significant increase in neuronal cell number and improvement in cerebral dysfunction after TBI in the exercise group compared with the non-exercise group (P < 0.05). These results indicate that exercise following TBI inhibits neuronal degeneration and apoptotic cell death around the damaged area, which results in improvement of cerebral dysfunction. In summary, treadmill running improved cerebral dysfunction following TBI, indicating its potential as an effective clinical therapy. Therefore, exercise therapy (rehabilitation) in the early phase following TBI is important for recuperation from cerebral dysfunction.

Background: Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. The inhibition of this key enzyme in the mevalonate pathway leads to suppression of cell proliferation and induction of apoptosis. However, the molecular mechanism of apoptosis induction by statins is not well understood in glioblastoma. In the present study, we attempted to elucidate the mechanism by which statins induce apoptosis in C6 glioma cells. Methods: The cytotoxicity of statins toward the C6 glioma cells were evaluated using a cell viability assay. The enzyme activity of caspase-3 was determined using activity assay kits. The effects of statins on signal transduction molecules were determined by western blot analyses. Results: We found that statins inhibited cell proliferation and induced apoptosis in these cells. We also observed an increase in caspase-3 activity. The apoptosis induced by statins was not inhibited by the addition of farnesyl pyrophosphate, squalene, ubiquinone, and isopentenyladenine, but by geranylgeranyl-pyrophosphate (GGPP). Furthermore, statins decreased the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. Conclusions: These results suggest that statins induce apoptosis when GGPP biosynthesis is inhibited and consequently decreases the level of phosphorylated ERK1/2 and Akt. The results of this study also indicate that statins could be used as anticancer agents in glioblastoma.

The tumor microenvironment plays a critical role in modulating malignant behavior and can dramatically influence cancer treatment strategies. We investigated whether statins inhibit the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and transforming growth factor-beta (TGF-beta) mRNA in the mouse osteosarcoma cell line LM8. We found that statins significantly inhibited mRNA expressions of bFGF, HGF, and TGF-beta, and bFGF. HGF, and TGF-beta secretions at concentrations that did not have antiproliferative effects on LM8 cells, but had no effect on the mRNA expression and secretion of VEGF. The inhibition of bFGF, HGF, and TGF-beta mRNA expression, and bFGF, HGF, TGF-beta secretions was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination with statins. Furthermore. statins reduced the membrane localization of K-Ras, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylatecl Ala. Our research indicates that statins inhibit GGPP biosynthesis in the mevalonate pathway, and then inhibit signal transduction in the Ras/ERK and Ras/Akt pathways, thereby inhibiting bFGF, HGF, TGF-beta expression in LM8 cells. These results suggest that statins are potentially useful as anti-angiogenic agents for the treatment of osteosarcoma. (C) 2011 Elsevier Ltd. All rights reserved.

Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-beta-D-glucoside (C-glucosylxanthone), is a xanthone derivative that is widely distributed in higher plants. Recently, mangiferin was found to exhibit potential antitumor effects. However, the molecular mechanisms of this effect have not been elucidated. In the present study, we attempt to clarify the mechanism of mangiferin-induced apoptosis in the human acute myeloid leukemia cell line HL-60; mangiferin was found to induce apoptosis. We also observed a concurrent increase in caspase-3 activity and DNA fragmentation. Furthermore, on examining the survival signals expressed during apoptotic induction, we observed that mangiferin caused a remarkable decrease in the nuclear entry of NF-kappa B p65. However, there were no changes in the expression of other survival signals, such as extracellular signal-regulated kinase 1/2, protein kinase B, and p38 mitogen-activated protein kinase. In addition, mangiferin suppressed the expressions of Bcl-xL and XIAP; however, we did not note any changes in the levels of Bcl-2, Bax, and Bim. These results indicate that mangiferin induces apoptosis by suppressing NF-kappa B activation and expressions of Bcl-xL and XAIP. These findings suggest that mangiferin may be useful as an anticancer agent and can be used in combination therapy with other anticancer drugs for the treatment of acute myeloid leukemia.

Exercise enhances neuronal stem cell (NSC) proliferation and neurogenesis. However, the effect of exercise on NSC proliferation surrounding the area of damage after traumatic brain injury (TBI) is unknown. Here, we investigate the effect of running on NSC proliferation following TBI in the rat. Wistar rats received TBI and were randomly divided into two groups: (1) non-exercise group and (2) exercise group. The exercise group ran on a treadmill for 30 min/day at 22 m/min for 7 consecutive days. Immunohistochemistry was used to monitor NSC proliferation around the damaged area, and ex vivo techniques were used to isolate NSCs from the damaged region in both groups. The number of nestin- and Ki67-positive cells observed at 3 and 7 days after TBI was significantly greater in the exercise group than in the non-exercise group (P < 0.01). Furthermore, most nestin-positive cells in the exercise group co-localized with Ki67-positive cells. In ex vivo studies, spheres could be isolated from injured brain tissue from the exercise group at 3 and 7 days following TBI, but at only 3 days in the non-exercise group. The number of spheres isolated from injured brain tissue was greater in the exercise group than in the non-exercise group. Spheres were immunopositive for nestin and comprised NSCs that could differentiate into neurons and glia. Exercise increases the proliferation of NSCs around the damaged area following TBI. Therefore, exercise therapy (rehabilitation) in the early phase following TBI is important for recuperation from cerebral dysfunction induced by TBI.

脳の発達、動物による脳の違い、男女による脳の違い、脳の伝達物質、食品栄養による脳の発達や神経疾患にについて概説した。

This paper presents a new method for suppressing circulation currents in a motor simulator system. In conventional system a large transformer at grid frequency has to be used to avoid circulation currents between the motor simulator and a test inverter further a regenerative converter is required too. In the proposed system, the high frequency components of the circulation current are suppressed by means of a common mode choke, and the low frequency components are suppressed by controlling the zero-phase current. Furthermore, a small medium frequency common mode choke is used instead of both a regenerative converter and the grid frequency transformer. In addition, the proposed system can be used to simulate the transient response of the motor. The proposed method is validated on the basis of simulation and experimental results. The primary current waveforms with distortions due to voltage errors caused the dead time agree well in the case of the motor simulator and the actual motor. Further, the low frequency component of the circulation current is suppressed to a value less than 1 % of the fundamental component in the proposed system. © 2011 The Institute of Electrical Engineers of Japan.

Background: There is no standard treatment available for gastric cancer patients whose sole 'non-curative factor' is positive cytological findings in peritoneal washings (CFPW). The aim of this study was to examine the safety, pharmacokinetics and efficacy for free intraperitoneal cancer cells of intraperitoneal chemotherapy with paclitaxel after gastrectomy with en bloc D2 lymph node dissection in cases of gastric cancer with positive CFPW. Methods: Ten patients with gastric cancer who underwent gastrectomy and systemic lymphadenectomy with D2 dissection, without any other non-curative factors besides positive CFPW, were treated with early postoperative intraperitoneal paclitaxel. Intra-chemotherapeutic toxicity and operative complications were measured using NCI-CTC version 3.0. Intraperitoneal and plasma paclitaxel concentrations were measured using a high-performance liquid chromatographic assay. Results: Grade 3/4 toxic effects included anemia (20%) and neutropenia (10%) that required no treatment. Operative complications were, for example, superficial surgical site infections (10%) that were treated with antibiotics. No viable cancer cells were observed in the intra-abdominal fluid 24 h after intraperitoneal administration of paclitaxel. The intraperitoneal/plasma area under the drug concentration-time curve ratio was 2,003.3: 1. Conclusion: Intraperitoneal chemotherapy with paclitaxel is a safe and effective treatment modality for free intraperitoneal cancer cells. Copyright (C) 2011 S. Karger AG, Basel

Multiple myeloma (MM) is a bone disease that affects many individuals. It was recently reported that macrophage inflammatory protein (MIP)-1 alpha is constitutively secreted by MM cells. MIP-1 alpha causes bone destruction through the formation of osteoclasts (OCs). However, the molecular mechanism underlying MIP-1 alpha-induced OC formation is not well understood. In the present study, we attempted to clarify the mechanism whereby MIP-1 alpha induces OC formation in a mouse macrophage-like cell line comprising C7 cells. We found that MIP-1 alpha augmented OC formation in a concentration-dependent manner; moreover, it inhibited IFN-beta and ISGF3 gamma mRNA expression, and IFN-beta secretion. MIP-1 alpha increased the expressions of phosphorylated ERK1/2 and c-Fos and decreased those of phosphorylated p38MAPK and IRF-3. We found that the MEK1/2 inhibitor U0126 inhibited OC formation by suppressing the MEK/ERK/c-Fos pathway. SB203580 induced OC formation by upregulating c-fos mRNA expression, and SB203580 was found to inhibit IFN-beta and IRF-3 mRNA expressions. The results indicate that MIP-1 alpha induces OC formation by activating and inhibiting the MEK/ERK/c-Fos and p38MAPK/IRF-3 pathways, respectively, and suppressing IFN-beta expression. These findings may be useful in the development of an OC inhibitor that targets intracellular signaling factors. J. Cell. Biochem. 111: 1661-1672, 2010. (C) 2010 Wiley-Liss, Inc.

The small GTPases of the Ras and Rho families are widely involved in tumorigenesis and metastasis. We recently showed that YM529/ONO-5920, a new developed bisphosphonate, inhibits the mevalonate pathway, is required for the prenylation of the small GTPases. In this study, we investigated whether YM529/ONO-5920 inhibits tumor cell migration, invasion, adhesion, and metastasis in B16BL6 cells, a mouse melanoma cell line. It was found that YM529/ONO-5920 significantly inhibited lung metastasis, cell migration, invasion, and adhesion at concentrations that did not show anti-proliferative effects on B16BL6 cells. YM529/ONO-5920 also inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs). Furthermore, YM529/ONO-5920 suppressed Rho activation, but not activation of Ras. The results indicate that YM529/ONO-5920 suppresses the Rho/ROCK pathways, thereby inhibiting B16BL6 cell migration, invasion, adhesion and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumour cell metastasis.

Background: We performed short-term neoadjuvant chemotherapy (s-NAC) to examine whether anticancer drugs can change the proliferative ability of cancer cells in gastric cancer patients. Methods: Chemotherapy was performed for 72 h before gastrectomy in 63 gastric cancer patients. Patients were classed into four groups: Group F, 16 cases who received a single administration of 5-fluorouracil (5-FU); Group C, 15 cases who received a single administration of cis-diamminedichloroplatinum (CDDP; cisplatin); Group FC, 16 cases who received both 5-FU+CDDP; and a Control group, 16 cases who did not receive chemotherapy. We reviewed neoadjuvant biopsy tissue and gastric cancer tissue delivered by operation in these cases. The TUNEL method and immunohistochemistry with an anti-MIB-1 antibody were used to evaluate cellular apoptosis and proliferative ability, respectively. The apoptotic index (Al) and an MIB-1 index (MI) were also calculated. Results: There were no differences in Al or MI in biopsy tissue between the groups. The Al of gastric cancer tissue in Group FC was significantly higher than in the other groups (P < 0.01). The MI of Group FC was significantly lower than in the other groups (P < 0.05). In addition, after s-NAC operation there was a significant inhibition of proliferative potency and an induction of apoptosis in Group FC. Conclusion: Combination of CDDP and 5-FU reduced proliferative potency and increased cellular apoptosis in gastric cancer cells. (C) 2010 Elsevier Ltd. All rights reserved.

The macrophages that infiltrate the tumor stroma are termed tumor-associated macrophages (TAMs). TAMs contribute to hematogenous spread of cancer cells especially liver metastasis. Osteopontin (OPN) is also related to tumor metastasis and proliferation of tumors. OPN is mainly expressed in macrophages of stroma other than that of tumor cells. The aim of the present study was to investigate differences in OPN-positive TAMs between cases of colorectal cancer with synchronous liver metastasis and those without liver metastasis. A total of 54 subjects who had undergone resection of a primary tumor of advanced colorectal cancer were classified into two groups: synchronous colorectal liver metastasis group (s-CLM group; n = 30) and no liver metastasis group (controls; n = 24). The number of OPN- and CD68-positive cells and the microvascular density (MVD) were determined using the CD105 antibody in the stroma of the invasive margin of the tumor and in the stroma of the central area. There was no difference in the patient profiles between the two groups. OPN and MVD expression in the central area were significantly higher in the s-CLM group (OPN: control 4.3 +/- A 1.42, s-CML 12.1 +/- A 1.42, P < 0.05; MVD: control 18.5 +/- A 2.86, s-CML 27.5 +/- A 2.94, P < 0.05), whereas CD68 expression in the invasive margin was significantly higher in the control group (control 98.9 +/- A 7.31, s-CML 29.0 +/- A 4.44, P < 0.05). These data suggest that OPN in the central area may have induced high microvascular density, which led to liver metastasis. Thus, OPN might be a potential target for novel antiangiogenesis therapy for treating colorectal cancer.

Stroke-prone spontaneously hypertensive rats (SHRSP) are the only animal model that suffers from spontaneous cerebral stroke. In this study, we investigated the appearance of neural stem cells (NSCs) and new neurons in the penumbra and the subventricular zone (SVZ) after cerebral stroke in SHRSP. SHRSP before cerebral stroke were intraperitoneally injected with 5-bromo-2'-deoxyuridine (BrdU). SHRSP were divided into acute and chronic phase groups after cerebral stroke. Brain sections from both groups were studied with cell-specific markers such as BrdU, a cell division and proliferation marker, sex-determining region Y-box 2, a marker of NSCs, nestin, an NSC and immature astrocyte marker, doublecortin, an immature new neuron marker, and neuron-specific nuclear protein, a marker of mature neurons. NSCs and new neurons appeared in the penumbra in the early stages after cerebral stroke, and these cells differentiated into mature neurons in the chronic phase. Furthermore, soon after being affected by a cerebral stroke, there were many new neurons and immature cells, which appear to be NSCs, in the ipsilateral SVZ. Immature cells and new neurons from the ipsilateral SVZ might migrate into the penumbra after cerebral stroke, and this is the first report of their observation after a spontaneous cerebral stroke.

Edaravone is a novel free radical scavenger used clinically in patients with acute cerebral infarction; however, it has not been assessed in traumatic brain injury (TBI). We investigated the effects of edaravone on cerebral function and morphology following TBI. Rats received TBI with a pneumatic controlled injury device. Edaravone (3 mg/kg) or physiological saline was administered intravenously following TBI. Numbers of 8-OHdG-, 4-HNE-, and ssDNA-positive cells around the damaged area after TBI were significantly decreased in the edaravone group compared with the saline group (P < 0.01). There was a significant increase in neuronal cell number and improvement in cerebral dysfunction after TBI in the edaravone group compared with the saline group (P < 0.01). Edaravone administration following TBI inhibited free radical-induced neuronal degeneration and apoptotic cell death around the damaged area. In summary, edaravone treatment improved cerebral dysfunction following TBI, suggesting its potential as an effective clinical therapy.

Background. Melanomas are highly malignant and have high metastatic potential hence, there is a need for new therapeutic strategies to prevent cell metastasis. In the present study, we investigated whether statins inhibit tumor cell migration, invasion, adhesion, and metastasis in the B16BL6 mouse melanoma cell line. Methods. The cytotoxicity of statins toward the B16BL6 cells were evaluated using a cell viability assay. As an experimental model, B16BL6 cells were intravenously injected into C57BL/6 mice. Cell migration and invasion were assessed using Boyden chamber assays. Cell adhesion analysis was performed using type I collagen-, type IV collagen-, fibronectin-, and laminin-coated plates. The mRNA levels, enzyme activities and protein levels of matrix metalloproteinases (MMPs) were determined using RT-PCR, activity assay kits, and Western blot analysis, respectively the mRNA and protein levels of vary late antigens (VLAs) were also determined. The effects of statins on signal transduction molecules were determined by western blot analyses. Results. We found that statins significantly inhibited lung metastasis, cell migration, invasion, and adhesion at concentrations that did not have cytotoxic effects on B16BL6 cells. Statins also inhibited the mRNA expressions and enzymatic activities of matrix metalloproteinases (MMPs). Moreover, they suppressed the mRNA and protein expressions of integrin 2, integrin 4, and integrin 5and decreased the membrane localization of Rho, and phosphorylated LIM kinase (LIMK) and myosin light chain (MLC). Conclusions. The results indicated that statins suppressed the Rho/Rho-associated coiled-coil-containing protein kinase (ROCK) pathways, thereby inhibiting B16BL6 cell migration, invasion, adhesion, and metastasis. Furthermore, they markedly inhibited clinically evident metastasis. Thus, these findings suggest that statins have potential clinical applications for the treatment of tumor cell metastasis. © 2010 Kidera et al licensee BioMed Central Ltd.

Mucin glycoproteins from the gallbladder epithelium are thought to contribute to the matrix or nucleus of gallstones and other biomineralization systems. The involved acidic glycoproteins have been reported in bile and gallstones. In addition, osteopontin (Opn) is a noncollagenous acidic bone matrix glycoprotein that possesses calcium-binding properties. To investigate the role of Opn in pigment gallstone formation, the involvement of Opn in pigment gallstone formation was studied immunohistochemically in the gallbladder wall and in the stones. Staining for Opn was strongly positive in the epithelium of stone-laden gallbladders and in their stones. The stone-laden gallbladders were infiltrated by macrophages, which intensely stained for Opn. Sections of the pigment stones, under low magnification, showed a lamellar pattern of Opn immunolabeling and showed a reticular pattern under high magnification. Our results indicate that Opn, an acidic glycoprotein from the gallbladder epithelium, seems to be involved in lithiasis. Opn from macrophages and/or the epithelium seems to help form the matrix protein.

Edaravone is a novel free radical scavenger that is clinically employed in patients with acute cerebral infarction, but has not previously been used to treat traumatic brain injury (TBI). In this study, we investigated the effect of edaravone administration on rat TBI. In particular, we used immunohistochemistry to monitor neural stem cell (NSC) proliferation around the area damaged by TBI. Two separate groups of rats were administered saline or edaravone (3 mg/kg) after TBI and then killed chronologically. We also used ex vivo techniques to isolate NSCs from the damaged region and observed nestin-positive cells at 1, 3, and 7 days following TBI in both saline- and edaravone-treated groups. At 3 days following TBI in both groups, there were many large cells that morphologically resembled astrocytes. At 1 and 7 days following TBI in the saline group, there were a few small nestin-positive cells. However, in the edaravone group, there were many large nestin-positive cells at 7 days following TBI. At 3 and 7 days following TBI, the number of nestin-positive cells in the edaravone group increased significantly compared with the saline group. There were many single-stranded DNA-, 8-hydroxy-2'-deoxyguanosine-, and 4-hydroxy-2-nonenal-positive cells in the saline group following TBI, but only a few such cells in the edaravone group following TBI. Furthermore, almost all ssDNA-positive cells in the saline group co-localized with Hu, nestin, and glial fibrillary acidic protein (GFAP) staining, but not in the edaravone group. In the ex vivo study, spheres could only be isolated from injured brain tissue in the saline group at 3 days following TBI. However, in the edaravone group, spheres could be isolated from injured brain tissue at both 3 and 7 days following TBI. The number of spheres isolated from injured brain tissue in the edaravone group showed a significant increase compared with the saline group. The spheres isolated from both saline and edaravone groups were immunopositive for nestin, but not Tuj1 or vimentin. Moreover, the spheres differentiated into Tuj1-, GFAP-, and O4-positive cells after 4 days in culture without bFGF. This result indicated that the spheres were neurospheres composed of NSCs that could differentiate into neurons and glia. Edaravone administration inhibited production of free radicals known to induce neuronal degeneration and cell death after brain injury, and protected nestin-positive cells, including NSCs, with the potential to differentiate into neurons and glia around the area damaged by TBI.

NF-kappa B acts as a signal transducer during tumor progression, cell invasion, and metastasis. Dimethylfumarate (DMF) is reported to inhibit tumor necrosis factor-alpha-induced nuclear entry of NF-kappa B/p65. However, only a few reports suggest that DMF inhibits tumor metastasis; also the molecular mechanisms underlying the inhibition of metastasis are poorly understood. We investigated the inhibition of tumor invasion and metastasis by DMF in a melanoma cell line, B16BL6. DMF inhibited B16BL6 cell invasion and metastasis by suppressing the expression and activities of MMPs. DMF also inhibited the nuclear entry of NF-kappa B/p65, thus inhibiting B16BL6 cell invasion and metastasis. These results suggest that DMF is potentially useful as an anti-metastatic agent for the treatment of malignant melanoma. (C) 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Osteopontin (OPN) is significantly overexpressed in a variety of malignancies. However, little is known concerning the significance of OPN expression in human cancers. Thus, the aim of this study was to determine the relationship between the degree of OPN expression, the proliferative activity of cancer cells, and the clinicopathological findings for surgically resected gastric cancer. We evaluated the immunohistochemical expression of OPN in 85 specimens of cancer. Additionally, we investigated a cancer cell proliferative index using an anti-MIB-1 antibody and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling staining. Levels of OPN expression in gastric cancers were classified into three groups. To compare the relationship between OPN expression and clinicopathological findings, the features of cancer lesions were classified using the TNM Classification of Malignant Tumors, 6th Edition. Immunohistochemical examination of OPN expression in gastric cancer revealed diffuse granular staining in the cytoplasm. High OPN expression was observed in 37 of 85 carcinomas. Strong OPN expression was significantly associated with a low apoptotic index, a high proliferative index, depth of invasion, lymphatic invasion, and venous invasion. Pathologically, intestinal type carcinoma showed strong expression of OPN. These data suggested that OPN may play an important role in the invasiveness and the progressive nature of gastric cancer.

Background and Purpose-Previously, an inverse association has been found between the dietary proportion of protein or fat and incidence of intracerebral hemorrhage. A positive association has been found with respect to carbohydrate intake. To examine what changes in macronutrient intake are causative, animal studies were conducted. Methods-Stroke-prone spontaneously hypertensive rats (SHRSP) were fed diets with varying ratios of macronutrients ad libitum, and the onset of stroke was examined. Results-When 10% of calories were from fat, rats fed a high-protein/low-carbohydrate diet (55% calories from protein) had a delayed onset of stroke, whereas rats fed a low-protein/high-carbohydrate diet (5% calories from protein) had an accelerated onset of stroke. When 30% of calories were from carbohydrate, a marked delay in the onset of stroke was observed when the diet was high in protein. When 85% of calories were from carbohydrate, rats fed 7.5% of calories as protein displayed an accelerated onset of stroke. When 20% of calories were from protein, increased fat content did not affect the onset of stroke. However, with a fat-free diet, when 20% of calories were from protein, the onset of stroke was delayed, whereas when 10% of calories were from protein, the onset of stroke was accelerated. Conclusions-The amount of protein, but not of carbohydrate and fat, is a primary determinant of the onset of stroke. However, when calories from protein are relatively low in the diet (10%), fat is necessary to delay the onset of stroke in SHRSP. (Stroke. 2009; 40: 2828-2835.)

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKC alpha and PKC delta phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis. (C) 2009 Elsevier Inc. All rights reserved.

We previously demonstrated the increased amyloid precursor protein (APP) immunoreactivity around the site of damage after traumatic brain injury (TBI). However, the function of APP after TBI has not been evaluated. In this study, we investigated the effects of direct infusion of an anti-APP antibody into the damaged brain region on cerebral function and morphological changes following TBI in rats. Three days after TBI, there were many TUNEL-positive neurons and astrocytes around the damaged region and a significantly greater number of TUNEL-positive cells in the PBS group compared with the anti-APP group found. Seven days after TBI, there were significantly a greater number of large glial fibrillary acidic protein-positive cells, long elongated projections, and microtubule-associated protein-2-positive cells around the damaged region in the anti-APP group compared with the PBS group found. Seven days after TBI, the region of brain damage was significantly smaller and the time to arrival at a platform was significantly shorter in the anti-APP group compared with the PBS group. Furthermore, after TBI in the anti-APP group, the time to arrival at the platform recovered to that observed in uninjured sham operation group rats. These data suggest that the overproduction of APP after TBI inhibits astrocyte activity and reduces neural cell survival around the damaged brain region, which speculatively may be related to the induction of Alzheimer disease-type dementia after TBI.

Objective: The actual relationship between neural stem cells and SDF-1 alpha/CXCR4 after brain injury has not yet been elucidated, although recent studies have speculated that stromal cell-derived factor-1 alpha (SDF-1 alpha) and its receptor, CXCR4, could contribute to neural stem cells migration after brain injury. In the present study, the temporal relationship between neural stem cells (NSCs) and SDF-1 alpha/CXCR4 around a damaged area was investigated using a rat traumatic brain injury (TBI) model. Methods: We used molecular biology techniques and immunohistochemistry to investigate the relationship between SDF-1 alpha/CXCR4 expression and NSCs existence around a damaged area after TBI in the rat brain. Results: SDF-1 alpha mRNA expression and SDF-1 alpha protein synthesis did not increase after TBI. However, SDF-1 alpha leaked from the injured area and diffused into the cortex 1-3 days after TBI. Subsequently, the levels of CXCR4 mRNA expression and CXCR4 protein synthesis increased significantly. Many small cells with a nestin-positive cytoplasm and fibers also showed immunopositivity for both CXCR4 and SOX-2, but not for GFAP, 3-7 days after TBI. Moreover, a proportion of the CXCR4-positive cells and fibers also showed immunostaining for neurofilaments. Discussion: These results suggest that the leaked SDF-1 alpha attracted CXCR4-positive NSCs as well as elongated nerve fibers. It is considered that the SDF-1 alpha/CXCR4 system in the brain contributes to neural stem cells appearance and maturation after TBI. Therefore, exploitation of the SDF-1 alpha/CXCR4 system around a damaged area may improve the brain dysfunction after TBI. [Neurol Res 2009; 31: 90-102]

Objective: Previous reports have demonstrated that some focal brain injuries increase amyloid precursor protein (APP) immunoreactivity in the region surrounding the injury in the cerebral cortex. However, the chronologic changes in APP expression have not been evaluated after traumatic brain injury (TBI). Methods: In this study, we immunohistochemically and biologically investigated chronologic changes in cellular sources and levels of APP production after rat TBI. Results: In the present report, we show that traumatic brain injury increased the expression of APP in the neuronal perikarya and in damaged dystrophic neurites from 1 to 90 days after injury. Moreover, 7 days after injury, some macrophages/microglia also were co-localized with APP, which was overproduced by the neuronal perikarya and APP-positive dystrophic neurites after injury and then APP were phagocytosed by macrophages/microglia during this phase. However, astroglia did not express APP immunopositivity after brain injury. Discussion: These results suggested that long-term overexpression of APP was confirmed by immunohistochemical and biologic technique after TBI. This may be related to the induction of Alzheimer type dementia and it is a very important risk factor for this disease. [Neurol Res 2009; 31: 103-109]

This paper presents a new suppression method of circulation current for a motor simulator system. A conventional system requires a large transformer at grid frequency to avoid a circulation current between the motor simulator and a test inverter, besides a regenerative converter is required too. In the proposed topology, the high frequency components of the circulation current are suppressed by a common mode transformer, and the low frequency components are suppressed by zero phase current control. Furthermore, a small size medium frequency common mode transformer is used instead of the regenerative converter and the grid frequency transformer. In addition, the proposed system can simulate the transient condition of the motor. The proposed method is validated based on the simulation and experimental results. The motor simulator primary current waveform agrees well with that of the actual motor, including the distortion due to a voltage error by a dead time period. Also the low frequency component of the circulation current was suppressed to less than 1% of the fundamental component by using the proposed method.

中枢神経における神経細胞の損傷と修復に関して、教室のデータを中心に概説した。高血圧による神経細胞傷害にはアンジオテンシンIIにより活性化された好中球で産生されるNO ラジカルが重要な役割を演じていること、また損傷後の神経細胞の修復は局所で産生されるケモカインによってもたらされることなど、実験モデルにおける結果を総括した。

Objective: Protein-free extracts from the inflamed skin of rabbits inoculated with vaccinia virus (Rosemorgen(R) and Neurotropin(R)) are widely employed to combat chronic pain and treat allergic conditions in human subjects in Japan. However, the pharmacologic mechanisms of Rosemorgen(R) and Neurotropin(R) remain unclear. Methods: In this study, we examined the effects of Rosemorgen(R) on L-glutamic acid (Glu)-induced cell death in N18-RE-105 neural cell line, which only possessed non-N-methyl-Daspartate (NMDA)-type receptors. Results: There were many large cytoplasmic cells and elongation of fivers in phosphate-buffered saline (PBS) additional group without Glu. In PBS and Glu simultaneous additional group, the survival ratio was decrease significantly compared with PBS alone group. Moreover, there were dead cells which did not have cytoplasm and aggregated nucleus. The Glu-induced cell death of N18-RE-105 cells was inhibited by both pre-treatment (24 hours before Glu treatment) and simultaneous treatment with Rosemorgen(R). There were many large cytoplasmic cells and elongation of fivers in Rosemorgen(R) group. Discussion: From this finding in N18-RE-105 cells, Rosemorgen(R) was concluded to inhibit Glu-induced cell death via non-NMDA type receptors. One of the pharmacologic mechanisms of Rosemorgen(R) has been clear. These results suggest that Rosemorgen(R) depresses allodynia and chronic pain through interaction with non-NMDA type receptors.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is detected at high concentrations in patients with multiple myeloma, and it is thought to play an important role in the etiology of multiple myeloma and osteolysis. Thus, we investigated whether or not YM529/ONO-5920, a new bisphosphonate, inhibited MIP-1 alpha mRNA expression in, and MIP-1 alpha secretion from, mouse myeloma cells. When the cells were stimulated by lipopolysaccharide, increased MIP-1 alpha mRNA expression and MIP-1 alpha secretion were observed. YM529/ONO-5920 inhibited MIP-1 alpha mRNA expression and MIP-1 alpha secretion in a concentration-dependent manner. A transient increase in the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) and Akt was observed after lipopolysaccharide stimulation. After YM529/ONO-5920 was given, there was no transient increase in the phosphorylation of ERK1/2 or Akt. These results indicated that YM529/ONO-5920 inhibited the expression and secretion of MIP-1 alpha through blocking the signaling pathway of the Ras/mitogen-activated protein kinase kinase/ERK and Ras/phosphatidylinositol-3 kinase/Akt. Accordingly, YM529/ONO-5920 appears to have promise for use in effective future therapy for osteolysis and myeloma cell growth that depends on MIP-1 alpha.

Osteolytic lesions are rapidly progressive during the terminal stages of myeloma, and the bone pain or bone fracture that occurs at these lesions decreases the patients' quality of life to a notable degree. In relation to the etiology of this bone destruction, it has been reported recently that MIP-1 alpha, produced in large amounts in myeloma patients, acts indirectly on osteoclastic precursor cells, and activates osteoclasts by way of bone-marrow stromal cells or osteoblasts, although the details of this process remain obscure. In the present study, our group investigated the mechanism by which RANKL expression is induced by MIP-1 alpha and the effects of MIP-1 alpha on the activation of osteoclasts. RANKL mRNA and RANKL protein expressions increased in both ST2 cells and MC3T3-E1 cells in a MIP-1 alpha concentration-dependent manner. RANKL mRNA expression began to increase at 1 h after the addition of MIP-1 alpha; the increase became remarkable at 2 h, and continuous expression was observed subsequently. Both ST2 and MC3T3-E1 cells showed similar levels of increased RANKL protein expression at 1, 2, and 3 days after the addition of MIP-1 alpha. After the addition of MIP-1 alpha, the amount of phosphorylated ERK1/2 and Akt protein expressions showed an increase, as compared to the corresponding amount in the control group. On the other hand, the amount of phosphorylated p38MAPK protein expression showed a decrease from the amount in the control group after the addition of MIP-1 alpha. U0126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor) was added to ST2 and MC3T3-E1 cells, and was found to inhibit RANKL mRNA and RANKL protein expression in these cells. When SB203580, a p38MAPK inhibitor, was added, RANKL mRNA and RANKL protein expression were increased in these cells. MIP-1 alpha was found to promote osteoclastic differentiation of C7 cells, an osteoclastic precursor cell line, in a MIP-1 alpha concentration-dependent manner. MIP-1 alpha promoted differentiation into osteoclasts more extensively in C7 cells incubated together with ST2 and MC3T3-E1 cells than in C7 cells incubated alone. These results suggested that MIP-1 alpha directly acts on the osteoclastic precursor cells and induces osteoclastic differentiation. This substance also indirectly induces osteoclastic differentiation through the promotion of RANKL expression in bone-marrow stromal cells and osteoblasts. The findings of this investigation suggested that activation of the MEK/ERK and the PI3K/Akt pathways and inhibition of p38MAPK pathway were involved in RANKL expression induced by MIP-1 alpha in bone-marrow stromal cells and osteoblasts. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.

Protein kinase C (PKC) has been shown to be a signal transducer during tumorigenesis, tumor cell invasion, and metastasis. Recent studies have reported that the PKC inhibitor, 7-hydroxystaurosporine, inhibits tumor cell invasion. However, the molecular mechanisms of this inhibition of invasion and metastasis are not well understood. In the present study, we attempt to clarify the mechanism by which H7, a PKC inhibitor, inhibits tumor cell invasion and metastasis in the melanoma cell line B16BL6. It was found that H7 inhibits B16BL6 cell invasion and metastasis. We also observed that H7 inhibits the mRNA expression and protein activities of matrix metalloproteinase (MMP)-1, -2, -9 and MT1-MMP. Furthermore, H7 suppresses phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). However, other signal transduction factors, such as p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase 1/2 (JNK1/2), were unaffected. Moreover, U0126, a MEK1/2 inhibitor, also inhibited B16BL6 cell invasion and metastasis, as well as the mRNA expression and protein activities of MMP-1, -2, -9 and MT1-MMP. This indicates that H7 inhibits signal transduction through the PKC/MEK/ERK pathway, thereby inhibiting B16BL6 cell invasion and metastasis. These results suggest that PKC inhibitors have potential clinical applications in the treatment of tumor cell metastasis.

Objectives: Glial scars around a damaged area after brain injury inhibit neurite elongation from surviving neurons and axonal plasticity, and thus prevent neural network regeneration. However, the generation, differentiation and maturation of neural stem cells (NSCs) among glial scars after brain injury have not yet been reported. Methods: In the present study, we investigated the chronological relationship between gliosis and maturation of new neurons around a damaged area using a rat traumatic brain injury (TBI) model. Results: Between 1 and 7 days after injury, many nestin-positive cells were observed around the damaged area. Three days after injury, many small nestin-positive cells showed an astrocytic morphology. Between 1 and 30 days after injury, doublecortin (DCX)-positive cells were present around the damaged area. Three and 7 days after injury, a small number of nestin-positive cells were immunopositive for glial fibrillary acidic protein (GFAP). Seven days after injury, there were DCX-positive cells in the gliosis occurring in the lesion. Thirty days after injury, DCX-positive cells were observed near and among the glial scars and a small number of these cells were immunopositive for NeuN. Discussion: These results suggest that DCX-positive cells were present near and among the glial scars after brain injury, and that these cells changed from immature to mature neurons. It is considered that promotion of the maturation and differentiation of newly formed immature neurons near and among glial scars after injury may improve the brain dysfunction induced by glial scars after brain injury.

Previously, we reported the occurrence of neural stem cells (NSCs) around an area of damage after rat traumatic brain injury (TBI); but it was unclear if this was due to blastgenesis in astrocytes, or to NSCs migrating from the subventricular zone (SVZ). In this study, NSCs were isolated and cultured from cultured type I astrocytes taken from newborn rat cortex in which the subventricular zone, and hippocampus had been discarded. All cultured type 1 astrocytes showed glial fibrillary acidic protein (GFAP) immunopositivity. Nestin immunopositive spheres were isolated from type 1 astrocytes and cultured in the presence of bFGF and EGF in the medium. Neurospheres differentiated into Tuj1-, GFAP- and A2B5-positive cells after 4 days of culture without bFGF and EGF. These results indicate that isolated neurospheres from brain cortex astrocytes can differentiate into neurons and glia and might contribute to neurogenesis and neuroplasticity.

Stromal cell-derived factor-1α (SDF-1α) has been considerd a important chemokine for neurogenesis during development. In this study, we investigated the temporal changes on SDF-1α around a damaged area of cerebral cortex using a rat traumatic brain injury (TBI) model, because the relationship between neurogenesis and SDF-1α after TBI has not yet been elucidated. SDF-1α protein synthesis in cerebral cortex did not increase although SDF-1α in spinal fluid increased due to proteinous analysis and SDF-1α immunopositive area increased due to immunohistochemical examination at 1 to 7 days after TBI. These results indicated that SDF-1α leaked from the injured area and diffused into the cortex after TBI. It has pointed that SDF-1α induces the neural stem cells migration and guides nerve fibers elongation of mature neurons. It is suggested the leaked SDF-1α facilitated neural stem cells migration as well as nerve fibers elongation to the damaged area of cerebral cortex after TBI.

Miki Nakajima, Masahiro Itoh, Hiroyuki Yamanaka, Tatsuki Fukami, Shogo Tokudome, Yasuhiko Yamamoto, Hiroshi Yamamoto, Tsuyoshi Yokoi, 2006, 'Isoflavones Inhibit NicotineC-Oxidation Catalyzed by Human CYP2A6', <i>The Journal of Clinical Pharmacology</i>, vol. 46, no. 3

脳外傷において、損傷周囲で損傷後に神経幹細胞が存在するか報告されていない。今回、ラット脳外傷モデルを用いて大脳皮質に直接、脳損傷を与えた後、経時的にnestin陽性細胞の変化と損傷部位での神経幹細胞の分離、培養を行った。さらに分離した細胞を形態学的に調べた。脳損傷後、損傷周囲で損傷後1日で少数のnestin陽性細胞が見られた。損傷後3日では細胞質および突起にnestin陽性の大型の細胞が多数見られた。7日後では突起にnestin陽性を示す細胞が少数見られた。nestin陽性細胞数は損傷後３日で最大であった。損傷後3日の損傷周囲の組織のみからsphereの単離、培養が可能であった。そのsphereはnestinに陽性でTuj1とGFAPには陰性でありneurosphereと考えられた。Neurosphere の分化誘導実験ではTuj1、GFAP、vimentin、S-100とO4に陽性を示した。これらのことから単離されたneurosphereは神経細胞、アストロサイトとオリゴデンドロサイトへの分化が示された。損傷後、損傷周囲では神経細胞やグリア

ラット脳外傷において、損傷後、早期に損傷周囲で発現するnestin陽性細胞の単離はされていない。さらにそのnestin陽性細胞が神経再生に関与しているかどうかも調べられていない。今回、ラット脳外傷モデルを用いて大脳皮質に直接、脳損傷を与えた後、経時的にnestin陽性細胞の変化とその細胞を分離、培養を行い神経細胞、アストロサイト、オリゴデンドロサイトへの分化について調べた。脳損傷後、損傷周囲で1日、3日及び7日後でnestin陽性細胞が認められた。損傷後3日では細胞質及び突起にnestinを認める陽性細胞が多数見られた。さらにnestin陽性細胞数が最大であった。無処置および損傷後1日、7日後では損傷周囲の組織からnestin陽性細胞の単離、培養はできなかった。しかし損傷後3日からsphereの単離ができた。単離されたsphereはnestin陽性であった。得られたneurosphere の培養液からbFGF及びEGFを取り除いた後、更に4日間培養した分化誘導実験ではTuj1、GFAP、vimentin、S-100蛋白、O4に陽性を

Nestin-positive cells were seen around the damaged area at 24 h, 72 In and 7 days after rat traumatic brain injury. Tissue was isolated from around the damaged area at 72 h after injury and spheres were cultured with basic fibroblast growth factor and epidermal growth factor. These spheres could not be isolated at 24 h and 7 days after injury. Isolated spheres consisted of nestin-positive neural stem cells. Neurospheres differentiated into Tujl-positive, glial fibrillary acidic protein-positive and O4-positive cells after 4 days in culture without basic fibroblast growth factor and epidermal growth factor. These results indicate that isolated and cultured neurospheres can differentiate into neurons and glia. An increase in nestin-positive cells around a cerebral cortical damaged area might contribute to neurogenesis and neuroplasticity.

Objectives: The effect of growth factors on the three-dimensional culture of neural stem cells has not been reported. We studied the effect of basic fibroblast growth factor (bFGF) on cultured rat neural stem cells in a three-dimensional culture. Methods: We cultured rat neural stem cells in collagen gel matrix for three-dimensional culture and examined the effect of bFGF under such culture conditions. Results: After 4 days culture, the cell density in the bFGF treatment roup was 12 times that of the non-treatment group, reaching a significantly high value. In the bFGF treatment group, microtubule associate protein (MAP)-2-positive cell aggregation occurred, although in the bFGF non-treatment group there was no MAP-2-positive cell aggregation and few of the cells were sparsely distributed. Also, in the bFGF treatment group, MAP-2-positive cell aggregation had a luminal structure similar to neural rosettes. There was elongation of MAP-2-positive neurites from the cell aggregation to the circumference in the bFGF treatment group. Discussion: bFGF is known to induce the proliferation, but not the differentiation of neural stem cells in two-dimensional cultures. However, in the three-dimensional culture, bFCF induced both the proliferation and differentiation of neural stem cells. The three-dimensional culture is, therefore, considered a useful method for predicting the response of neural stem cells to cytokines or biologically active substances in vivo.

Purpose. The purpose of this study was to investigate the vascularity of primary gastric cancer lesions using color Doppler ultrasonography. Methods. We used color Doppler ultrasonography to study 78 patients with gastric cancer detected on B-mode ultrasonographic examination and 14 patients without gastric tumors but with a slightly thickened gastric wall that was also detected on B-mode ultrasound. The color Doppler signals of the gastric lesions were graded as (-), no color signals (+), slight increase in number of color signals and (++), an obvious increase in number of color signals. The vessel area outside the tumor area in the microscopic pathological specimens was also calculated. Results. The color signals of 13 (18%) of the 71 gastric cancer patients were graded (-) those of 14 (20%) patients were graded (+) and those of 44 (62%) patients were graded (++). The color signals for 9 (65%) of 14 patients without gastric tumors were graded (-) those of 4 (28%) patients were graded (+), and those of 1 patient (7%) were graded (++). These differences were significant (P = 0.0002). The vessel count ratio in the microscopic pathologic specimens was also significantly higher in patients with an increased number of color signals than in those without an increased number of color signals (P = 0.002). Conclusion. Color Doppler ultrasound showed increased vascularity in the gastric cancers in most of the subjects (82%, 58/71). Furthermore, color Doppler ultrasound also showed no increase in vascularity in most subjects (65%, 9/14) whose B-mode ultrasonograms showed thickened gastric walls but who did not have gastric cancer. Thus, color Doppler imaging may prove useful as a screening modality for gastric cancer. © The Japan Society of Ultrasonics in Medicine 2005.

脳損傷後、APPが損傷周囲のアストロサイトの活性を抑制し、さらに神経細胞に対して障害性に働くことにより、APPが脳機能の低下をもたらしていると考えられた。（英文）

APPは外傷後長期にわたり神経細胞に強く発現していた。長期にわたるAPPの過剰発現は結果的に Aβの沈着を促進する可能性が考えられることから外傷がアルツハイマー型痴呆の危険因子になる原因と考えられた。（英文）

脳虚血実験モデルでTNFαの神経保護作用が指摘されており、今回の検討から脳損傷後に神経細胞が産生するTNFαは、マイクログリア、マクロファージが産生するTNFαと異なり神経細胞に対して保護的に働くと考えられた。（英文）

脳外傷におけるTNF-αの役割についての報告例は少ない。実験的脳損傷後に形成されるcavity、その周囲でのTNF-αの時間的な細胞分布の変化と蛋白量の変化を免疫組織染色、ELISA法にて形態計測学的、生化学的に解析した。（英文）

７日間のテトラベナジン(TBZ)反復投与はTBZの単回投与とは異なり投与終了後も行動量は完全には回復せず，中脳黒質緻密層では不可逆性の神経細胞数の減少，萎縮をもたらした。TBZの反復投与がパーキンソン病の慢性モデルになる可能性が示唆された。(英文)

ラットの実験的脳損傷モデルを作成し、灰白質および白質での細胞反応の違いをbFGF,vimentin,GFAPを用いて検討した

We examined the effect of FPF-1070 (Cerebrolysin(R)) on neurite outgrowth in explant cultures of dorsal root ganglia (DRG), sympathetic trunks (ST), and ciliary ganglia (CG) from 10- to 11-day chicken embryos. FPF-1070 significantly promoted neurite outgrowth in DRG and ST neurons at all concentrations examined, in comparison with phosphate buffered saline-treated negative controls; however, this effect on neurite outgrowth was not as significant as that observed for nerve growth factor-treated positive controls on DRG and ST neurons. Additionally, FPF-1070 exhibited an inverted U relationship between concentration and effectiveness in DRG and ST neurons. In contrast, FPF-1070 did not affect neurite outgrowth in CG neurons although ciliary neurotrophic factor-treated positive controls showed striking neurite outgrowth. Our results demonstrate that FPF-1070 has different neurotrophic effects depending on the subpopulation of neurons. This study clarifies a role for neurotrophic activity in the mechanism of action of FPF-1070.

アルツハイマー病の老人斑を形成するβアミロイド蛋白の前駆体であるamyloid precursor protein(APP)は脳外傷により長期にわたり発現する。しかしながら脳外傷後におけるAPPの機能は不明な点が多い。そこで今回、我々はAPPの影響を検討するためにラット実験的脳損傷後の損傷部位に抗APP抗体を直接投与し、脳機能評価と形態学的変化を検討した。抗APP抗体投与により脳機能評価で有意な改善が認められた。抗APP抗体投与により損傷部位の面積に有意な減少を示した。抗APP抗体投与により多数の大型のアストロサイトとGFAP陽性面積の有意な増加が認められた。抗APP抗体投与によりMAP-2陽性細胞数が有意に増加した。これらのことから脳損傷後、APPが損傷周囲のアストロサイトや神経細胞に対して障害性に働くと考えられた。(英文）

脳外傷では脳に直接的機械的な侵襲が加わり、障害を受けた中枢神経組織では神経細胞死や変性そして神経軸索の断裂や軸索の変性が引き起こされと考えられている。中枢神経で神経細胞の細胞死や変性により神経細胞が減少しても再生は起こらないとされていた。しかし、最近の研究ではヒト中枢神経において神経細胞やグリア細胞に分化することができる神経幹細胞がthe subventricular zone(SVZ)やthe subgranular zone(SGZ) at the dentate gyrus(DG)-hilus interfaceに存在し、神経組織が障害を受けたときに神経幹細胞（NSC）が増殖、分化することにより神経細胞死や変性した神経細胞におきかわることが報告された。しかしながら、脳外傷後の損傷周囲についての神経幹細胞の増殖、分化そしてneurogenesisについて不明な点が多く残されている。我々はラット脳外傷モデルを用いて大脳皮質に直接、脳損傷を与えた後、経時的に神経幹細胞のマーカーであるnestinの陽性細胞数の変化とそのnestin陽性細胞を分離、

目的；脳卒中自然発症stroke-prone spontaneously hypertensive rats (SHRSP)を使って、病変周囲組織と脳室周囲において神経幹細胞と新生神経細胞が出現することを報告した。さらにstromal cell-derived factor-1α (SDF-1α) / CXCR4が脳外傷後の神経幹細胞の出現に関与していることを報告した。しかしながら脳梗塞後の神経幹細胞の出現とSDF-1α/CXCR4についての報告はない。 方法； SHRSPを使って脳卒中発症後の病変周囲組織において神経幹細胞の出現とSDF-1α/CXCR4系について免疫組織学的及び生化学的に調べた。結果；発症周囲組織においてSDF-1α mRNAと蛋白合成の発現増加は認められなかった。発症後、発症周囲組織からSDF-1αが漏出し、周囲組織への拡散が見られ、脳脊髄液中でSDF-1α量の有意な増加が認められた。発症周囲組織ではCXCR4 mRNAと蛋白合成の発現の有意な増加が認められた。発症周囲組織ではnestin陽性細胞像とCXCR4陽性細胞像が一致した。 結論；このことから脳卒中発症後に障害を受けた細胞より逸

目的；脳卒中自然発症stroke-prone spontaneously hypertensive rats (SHRSP)を使って、病変周囲組織と脳室周囲において神経幹細胞と新生神経細胞が出現することを報告した。さらにstromal cell-derived factor-1α (SDF-1α) / CXCR4が脳外傷後の神経幹細胞の出現に関与していることを報告した。しかしながら脳梗塞後の神経幹細胞の出現とSDF-1α/CXCR4についての報告はない。方法； SHRSPを使って脳卒中発症後の病変周囲組織において神経幹細胞の出現とSDF-1α/CXCR4系について免疫組織学的及び生化学的に調べた。結果；発症周囲組織においてSDF-1α mRNAと蛋白合成の発現増加は認められなかった。発症後、発症周囲組織からSDF-1αが漏出し、周囲組織への拡散が見られ、脳脊髄液中でSDF-1α量の有意な増加が認められた。発症周囲組織ではCXCR4 mRNAと蛋白合成の発現の有意な増加が認められた。発症周囲組織ではnestin陽性細胞像とCXCR4陽性細胞像が一致した。結論；このことから脳卒中発症後に障害を受けた細胞より逸脱した

ラジカルスカベンジャーであるedaravoneは、臨床的に脳梗塞の治療薬として使用されているが外傷での適応は現在のところ無い。外傷的脳損傷に対するedaravoneの影響を今回実験的に検討した。特に損傷周囲に出現する神経幹細胞に注目し免疫組織化学的exvivo的方法により検討した。ラット脳外傷後にedaravoneを投与し群とsalineを投与した群を作製し経時的に屠殺した。免疫組織学的な検討に加えて、損傷周囲組織からの神経幹細胞の単離を試みた。脳損傷後、saline群において損傷周囲で１日、３日及び7日後でnestin陽性細胞が認められた。損傷後３日では形態的にアストロサイトに似た大型のnestin陽性細胞が多数見られた。１及び７日は小型のnestin陽性細胞であった。edaravone群でも同様にnestin陽性細胞が観察されるが損傷後７日でも大型のnestin陽性細胞が多数見られた。saline群に比較しedaravone群は損傷後３日及び７日でnestin陽性細胞数の有意な増加を示した。exvivoの実験においてsaline群では損傷

脳の病態とその病理及び機序について解説した。

背景：脳卒中易発症性高血圧自然発症ラット(SHRSP)は脳卒中を自然発症する唯一の動物であり、脳卒中発症後の脳病変周囲の神経細胞は変性脱落することが報告されている。しかしながら脳病変周囲組織の神経細胞の再生についての報告は見あたらない。そこで今回、SHRSPを用いて脳卒中発症後の脳病変周囲組織での神経再生について検討した。 方法：脳卒中発症前の16週齢雄性SHRSPにBrdUを連続二週間投与し、SHRSPを脳卒中発症直後の急性期群と発症約一ヶ月の慢性期群の二群に分けて実験を行った。急性期群および慢性期群のラット脳切片を分裂細胞のマーカーであるBrdU、神経幹細胞のマーカーであるSOX-2、神経幹細胞および幼弱なアストロサイトのマーカーであるnestin、幼弱な神経細胞のマーカーであるdoublecortin (DCX)および神経細胞のマーカーであるNeuNの免疫組織染色を行って、神経幹細胞の出現およびその神経細胞への分化の評価を行った。 結果：急性期群の脳病変周囲組織ではWK

我々は神経発生時に細胞の移動に関与することが指摘されているstromal cell-derived factor-1α(SDF1α)とそのレセプターであるCXCR4が外傷的脳損傷後の局所においても作用しており，SDF1αおよびCXCR4のアンタゴニストAMD投与により損傷局所に出現する神経幹細胞の数に変化が生じることを報告した。今回，神経幹細胞の移動を阻害すると想定される薬物を損傷局所に投与しその影響を検討した。pneumatic control injury deviceを用いてwistar rats脳に脳外傷を加えた。損傷直後より損傷局所に滲透圧ポンプ（Alzet）を用いてK252a, Cytochalasins-Bを２週間投与した。また，損傷直後より腹腔内にBrdUを投与した。損傷後1週間後および１ヶ月後にラットを潅流固定した。固定後脳を取り出し，薄切切片を作製し染色に用いた。染色は一次抗体として抗nestin，DCX, BrdU，glial fibrillary acidic protein（GFAP）他を用い免疫染色を試み観察した。損傷辺縁部のNestin陽性面積測定とDCX, BrdU陽性細胞数を計数した。K252投与群では損傷局所に

stromal cell-derived factor-1α(SDF1α）とそのレセプターであるCXCR4は神経発生時に細胞の移動に関与することが指摘されている。SDF1αとそのCXCR4のアンタゴニストであるAMD3100の脳損傷局所への投与がもたらす影響を神経幹細胞およびその分化に注目して検討した。pneumatic control injury deviceを用いてwistar rats脳に外傷を加えた。損傷直後より損傷局所に滲透圧ポンプ（Alzet）を用いてSDF1α(50ng/ml)あるいはAMD3100(5mg/ml)を２週間投与した。損傷直後より，尾静脈からBrdUを投与した。２週間後および１ヶ月後にラットを潅流固定した。固定後脳を取り出し，薄切切片を作製し染色に用いた。一次抗体として抗 nestin, DCX, BrdU, Neu N抗体等を用い免疫染色を試みた。損傷辺縁部のnestin, DCX, BrdU陽性細胞数を計数した。SDF1α投与群では損傷局所におけるnestin，DCX，Neu N，BrdU陽性細胞数が有意に増加がした。AMD3100投与群ではnestin，DCX，Neu N, BrdU陽性細胞数は有意に減少した。SDF1α，AMD3100投与によりnestin陽性細胞数，幼若

目的；脳外傷によるfree radicalが神経細胞障害を引き起こすことは知られている。radical scavengerであるedaravoneは脳梗塞の治療薬として使用されているが外傷での適応は無い。今回、外傷的脳損傷に対するedaravoneの影響を脳損傷周囲部位に出現する神経幹細胞に注目し免疫組織化学的及び生化学的に検討した。 方法；10週齢ratを用いて脳外傷受傷ratを作製し、受傷直後にedaravone及びsalineを投与した。Sham operate処置でsalineを投与した群をsham群とした。受傷1, 3, 7日後に屠殺し、神経幹細胞のmarkerであるnestinによる免疫染色と脳内のmalondialdehyde(MDA)量の測定を行った。 結果；Edaravone及びsaline群においてnestin陽性細胞が見られ、edaravone群では損傷3, 7日後に大型のnestin陽性細胞が多数見られた。さらにnestin陽性細胞数の有意な増加を認めた。Saline群と比較しedaravone群でMDA量の有意な減少が認められた。Edaravoneとsham群ではMDA量には差は無かった。 考察；脳外傷後のedaravoneの投与は脳外傷によるfree rad

背景：脳卒中易発症性高血圧自然発症ラット(SHRSP)は脳卒中を自然発症する唯一の動物であり、脳卒中発症後の脳病変周囲の神経細胞は変性脱落することが報告されている。しかしながら脳病変周囲組織の神経細胞の再生についての報告は見あたらない。そこで今回、SHRSPを用いて脳卒中発症後の脳病変周囲組織での神経幹細胞の出現とその分化について検討した。 方法：脳卒中発症前の16週齢雄性SHRSPにBrdUを連続二週間投与し、SHRSPを脳卒中発症直後の急性期群と発症約一ヶ月の慢性期群の二群に分けて実験を行った。急性期群および慢性期群のラット脳切片を分裂細胞のマーカーであるBrdU、神経幹細胞のマーカーであるSOX-2、神経幹細胞および幼弱なアストロサイトのマーカーであるnestin、幼弱な神経細胞のマーカーであるdoublecortin (DCX)および神経細胞のマーカーであるNeuNの免疫組織染色を行って、神経幹細胞の出現およびその神経細胞への分化の評価を行った。 結果：急性期群の

背景：脳卒中易発症性高血圧自然発症ラット(SHRSP)は脳卒中を自然発症する唯一の動物であり、脳卒中発症後の脳病変周囲の神経細胞は変性脱落することが報告されている。しかしながら脳病変周囲組織の神経細胞の再生についての報告は見あたらない。そこで今回、SHRSPを用いて脳卒中発症後の脳病変周囲組織での神経幹細胞の出現とその分化について検討した。 方法：脳卒中発症前の16週齢雄性SHRSPにBrdUを連続二週間投与し、SHRSPを脳卒中発症直後の急性期群と発症約一ヶ月の慢性期群の二群に分けて実験を行った。急性期群および慢性期群のラット脳切片を分裂細胞のマーカーであるBrdU、神経幹細胞のマーカーであるSOX-2、神経幹細胞および幼弱なアストロサイトのマーカーであるnestin、幼弱な神経細胞のマーカーであるdoublecortin (DCX)および神経細胞のマーカーであるNeuNの免疫組織染色を行って、神経幹細胞の出現およびその神経細胞への分化の評価を行った。 結果：急性期群の

［目的］stromal cell-derived factor-1α(SDF1α）とそのレセプターであるCXCR4はともに神経発生時に細胞の移動に関与することが指摘されている。我々はSDF1αおよびCXCR4が外傷的脳損傷後の局所においても作用していることを先に報告した。今回，実験的ラット脳外傷モデルの損傷局所にSDF1α投与およびCXCR4のアンタゴニスト投与を試みたので報告する。［方法］pneumatic control injury deviceを用いてwistar rats脳に脳外傷を加えた。損傷直後より損傷局所に滲透圧ポンプ（Alzet）を用いてSDF1α(50ng/ml)あるいはCXCR4のアンタゴニストであるAMD3100(5mg/ml)を２週間投与した。また，損傷直後より，尾静脈からBrdUを投与した。２週間後および１ヶ月後にラットを潅流固定した。固定後脳を取り出し，薄切切片を作製し染色に用いた。染色は一次抗体として抗SDF1α, CXCR4，nestin， DCX, BrdU， microtubule associated protein 2 ( MAP-2)，neurofilament(NF)，glial fibrillary acidic protein（GFAP)を用い免疫染色を試み観察した。損傷辺縁部のNestin

背景：脳卒中易発症性高血圧自然発症ラット(SHRSP)は脳卒中を自然発症する唯一の動物であり、脳卒中発症後の脳病変周囲の神経細胞は変性脱落することが報告されている。しかしながら脳病変周囲組織の神経細胞の再生についての報告は見あたらない。そこで今回、SHRSPを用いて脳卒中発症後の脳病変周囲組織での神経幹細胞の出現とその分化について検討した。 方法：脳卒中発症前の16週齢雄性SHRSPにBrdUを連続二週間投与し、分裂細胞のマーカーであるBrdU、神経幹細胞のマーカーであるSOX-2、神経幹細胞および幼弱なアストロサイトのマーカーであるnestin、幼弱な神経細胞のマーカーであるdoublecortin (DCX)および神経細胞のマーカーであるNeuNを用いて、免疫組織染色を経時的に行い、神経再生の評価を行った。 結果：脳卒中発症直後ではSOX-2+ / nestin+ 細胞及びBrdU+ / DCX+ 細胞が認められた。Subventricular zone(SVZ)ではSOX-2+ / nestin+ 細胞とDCX陽性細胞が多数認められた。発症約一ヶ月後ではBrdU+

ラット脳外傷において、損傷後、早期に損傷周囲で発現するnestin陽性細胞の単離はされていない。さらにそのnestin陽性細胞が神経再生に関与しているかどうかも調べられていない。今回、ラット脳外傷モデルを用いて大脳皮質に直接、脳損傷を与えた後、経時的にnestin陽性細胞の変化とその細胞を分離、培養を行い神経細胞やアストロサイトへの分化について調べた。脳損傷後、損傷周囲で24時間、72時間及び7日後でnestin陽性細胞が認められた。損傷後72時間では形態的にアストロサイトに似た大型のnestin陽性細胞が多数見られた。さらにnestin陽性面積が最大であった。損傷後24時間、7日後では損傷周囲の組織ではnestin陽性細胞の単離、培養はできなかった。しかし損傷後72時間ではsphereの単離ができた。単離されたsphereはnestin陽性でTuj1とVimentinは陰性でありsphereはneurosphereと考えられた。得られたneurosphere の培養液からbFGF及びEGFを取り除いた後、更に4日間培養すると培養細胞はTuj1とGFAPに陽

背景：脳卒中易発症性高血圧自然発症ラット(SHRSP)は脳卒中を自然発症する唯一の動物であり、脳卒中発症後の脳病変周囲の神経細胞は変性脱落することが報告されている。しかしながら脳病変周囲組織の神経細胞の再生についての報告は見あたらない。そこで今回、SHRSPを用いて脳卒中発症後の脳病変周囲組織での神経幹細胞の出現とその分化について検討した。 方法：脳卒中発症前の16週齢雄性SHRSPにBrdUを連続二週間投与し、SHRSPを脳卒中発症直後の急性期群と発症約一ヶ月の慢性期群の二群に分けて実験を行った。急性期群および慢性期群のラット脳切片を分裂細胞のマーカーであるBrdU、神経幹細胞のマーカーであるSOX-2、神経幹細胞および幼弱なアストロサイトのマーカーであるnestin、幼弱な神経細胞のマーカーであるdoublecortin (DCX)および神経細胞のマーカーであるNeuNの免疫組織染色を行って、神経幹細胞の出現およびその神経細胞への分化の評価を行った。 結果：急性期群の

［目的］stromal cell-derived factor-1α(SDF1α）とそのレセプターであるCXCR4はともに神経発生時に細胞の移動に関与することが指摘されている。今回，我々は実験的ラット脳外傷モデルにおけるこれらの発現に関して検討した。［方法］pneumatic control injury deviceを用いてwistar rats脳に脳外傷を加えた。損傷後1日，３日，７日で屠殺した。一次抗体として抗SDF1α, CXCR4，nestin，microtubule associated protein 2 ( MAP-2)，neurofilament(NF)，glial fibrillary acidic protein（GFAP)を用い免疫染色を試みた。SDF1α陽性面積を測定した。CXCR4およびNestin陽性細胞数を計数した。損傷周囲大脳を採取し，SDF1αとCXCR4に関してRT-PCRを行った。損傷周囲大脳の脳組織および脳脊髄液のSDF1αの蛋白量をELISAにて測定し，さらにwestern blot analysisにて損傷周囲大脳組織のSDF1αとCXCR4の蛋白量測定を行った。［結果］損傷後１日から７日において損傷周囲にSDF1α陽性像が認められ，陽性面積は損傷後１日でピークを示した。nestin陽性細胞の出現が損傷辺

［目的］近年，成熟脳においても神経幹細胞が生理的にも存在することが明らかにされた。我々は実験的脳外傷モデルでの脳損傷局所に神経幹細胞が出現することを報告してきたがそれら神経幹細胞の由来に関してはなお不明な点が多い。今回，我々は，Type1アストロサイト（AS）の純粋培養を試みそこからの神経幹細胞の分離を試みた。［方法］生後0-1日のWistarラット大脳皮質から，MacCarthyらの方法に準じてType1ASの純粋培養を行った。それにより得られた細胞をWeissらの方法に準じて，bFGFおよびEGFを加えた培養液にて培養する神経幹細胞選択培養法に従い12-13日培養したところ球状細胞塊が得られた。その細胞塊に対して分化誘導を試みた。球状の細胞塊および分化誘導後の細胞を用いて免疫組織学的にNestin,Tuj-1,GFAP,A2B5に関して検討した。［結果］球状細胞塊はNestin陽性を示した。分化誘導後,Tuj-1,GFAP,A2B5に関して各々陽性細胞が認められた。［考察］培養Type1ASから神経幹細胞が誘導され

背景：脳卒中易発症性高血圧自然発症ラット(SHRSP)は脳卒中を自然発症する唯一の動物であり、脳卒中発症後の脳病変周囲の神経細胞は変性脱落することが報告されている。しかしながら脳病変周囲組織の神経細胞の再生についての報告は見あたらない。そこで今回、SHRSPを用いて脳卒中発症後の脳病変周囲組織での神経再生について検討した。 方法：脳卒中発症前の16週齢雄性SHRSPにBrdUを連続二週間投与し、BrdU、幼弱な神経細胞のマーカーであるDCXおよび神経細胞のマーカーであるNeuNの免疫染色を経時的に行って神経再生の評価を行った。 結果：脳卒中発症直後ではDCX陽性細胞が多数見られ一部ではBrdUとDCX共に陽性を示す細胞が認められた。さらに発症約一ヶ月後ではBrdUとNeuN陽性の細胞が少数認められた。 考察：脳病変周囲で発症早期から新生神経細胞（DCX陽性）が出現し、成熟神経細胞（NeuN陽性）へ分化することが示された。このことから新生神経細胞をコントロールするこ

Stromal cell-derived factor-1α (SDF-1α) は神経発生時における重要なケモカインである。本研究は外傷的神経損傷ラットモデルにおける損傷周囲におけるSDF-1αの経時的変化に関して免疫組織化学，ELISA,RT-PCRを用いて検討した。RT-PCRの結果より損傷後SDF-1αの新規合成は生じていなかった。髄液中のSDF-1αの蛋白濃度のELISAによる解析より損傷後濃度の上昇がみられ，SDF-1αの脳組織の損傷周囲部で免疫組織学的陽性面積の増加が認められた。これらの結果より損傷後SDF-1αは破壊された脳組織より漏出し，損傷部周囲の脳組織に広がるものと考えられた。SDF-1αは神経幹細胞の移動を誘導し，成熟神経細胞の線維伸長をもたらすとされており，損傷後に漏出するSDF-1αが損傷部および損傷周囲の神経再生に多いに関与する可能性が示唆された。

脳外傷において、ケモカインの一つであるstromal cell-derived factor-1α (SDF-1α)とそのレセプターであるCXCR4がneurogenesisと関係しているのではないかと指摘されている。しかしながら脳外傷後におけるneurogenesisとSDF-1α / CXCR4に関する報告はない。今回、ラット脳外傷モデルを用いて大脳皮質に直接、脳損傷を与えた後、経時的にSDF-1α / CXCR4の発現とneurogenesisについて調べた。損傷後、障害組織からSDF-1αが漏出し、周囲組織に損傷後3日まで拡散することが認められた。損傷早期からnestin陽性細胞とCXCR4陽性細胞が多数認められ、nestin陽性細胞像とCXCR4陽性細胞像が一致した。さらにCXCR4陽性細胞像の一部はneurofilament陽性細胞像とも一致した。このことから脳外傷後に傷害を受けた組織より漏出したSDF-1αが神経幹細胞や損傷神経細胞のCXCR4発現を促進させ、神経幹細胞や神経突起を損傷周囲へと誘導し、神経再生や神経の再構築に関与したと考えられた。損傷局所に存在するSDF1α / CXCR4をコントロールす

［目的］近年，中枢神経組織が傷害を受けたときに神経幹細胞の増殖，分化による神経再生の可能性が報告された。今回，損傷周辺部のグリア増生部での未熟な神経細胞の出現とその動態に注目し免疫組織学的に検討した。［方法］pneumatic control injury deviceを用いてwistar rats脳に脳外傷を加えた。損傷後２４時間，７２時間，７日，３０日で４％パラホルムアルデハイドで還流固定し，脳を取り出し，Micro slicerを用いて損傷最大径の部分より厚さ５０μmの連続切片を作製した。１）nestinの免疫染色を行いnestin陽性細胞数を計数した。２）doublecortin(DCX)の免疫染色を行い，DCX陽性細胞数を計数した。３）DCXとglial fibrillary acid protein(GFAP)の二重染色をした。４）DCXとNeuNの二重染色をした。５）nestinとGFAPの二重染色をした。［結果］nestin陽性細胞は損傷後24時間で損傷周囲に認められ（3.6±2.4）その数は72時間で最大数（69.2±25.8）を示し７日で減少（2.2±3.3）した。DCX陽性細胞は24時間，72時間では少

ラット脳外傷において、損傷後、早期に損傷周囲で発現するnestin陽性細胞の単離はされていない。さらにそのnestin陽性細胞が神経再生に関与しているかどうかも調べられていない。今回、ラット脳外傷モデルを用いて大脳皮質に直接、脳損傷を与えた後、経時的にnestin陽性細胞の変化とその細胞を分離、培養を行い神経細胞、アストロサイト、オリゴデンドロサイトへの分化について調べた。脳損傷後、損傷周囲で1日、3日及び7日後でnestin陽性細胞が認められた。損傷後3日では細胞質及び突起にnestinを認める陽性細胞が多数見られた。さらにnestin陽性細胞数が最大であった。無処置および損傷後1日、7日後では損傷周囲の組織からnestin陽性細胞の単離、培養はできなかった。しかし損傷後3日からsphereの単離ができた。単離されたsphereはnestin陽性であった。得られたneurosphere の培養液からbFGF及びEGFを取り除いた後、更に4日間培養した分化誘導実験ではTuj1、GFAP、vimentin、S-100蛋白、O4に陽性を

「目的」我々はすでに高血圧性脳微小血管障害の発生に好中球が重要な役割を演じていることを明らかにしてきた。好中球の遊走に関しては多くのchemokineが関与していることが知られているが、中でもstromal cell drived factor-1(SDF-1)とそのレセプターCXCR4は中枢神経においても発現しており、近年、中枢神経の発生・発達やAIDS脳症の病理発生に関与していることが報告されている。そこで、脳血管障害あるいはその修復に対するchemokineの関与を検討するために、SHRSPを用いてSDF1とCXCR4の発現を神経幹細胞との関連において検索した。 「方法」胎令18日および生後20週令のSHRSPとWKYの大脳を用いて、SDF1とCXCR4の発現をRT-PCRおよびwestern blotting法により比較・検討した。また幹細胞のマーカーとしてnestinの発現をRT-PCRにより検討した。幹細胞におけるnestinとchemokineの関係を明らかにするために、培養幹細胞における両者の発現を免疫組織学的に比較検討した。 「結果」SHRSPでもWKYでも、大脳皮質にお

脳損傷後、APPが損傷周囲のアストロサイトの活性を抑制し、さらに神経細胞に対して障害性に働くことにより、APPが脳機能の低下をもたらしていると考えられた。

加齢により軸索に異常が生じた結果APPが神経線維内で貯留した状態が出現していると考えられた。その結果としてAbの沈着が誘導される可能性が示唆された。それを基にして老人斑の形成が生じ、記憶・学習に変化をきたすと考えられた。（英文）

Edaravone投与により損傷範囲が小さく神経細胞や神経ネットワークの損傷が軽度であり脳外傷後におけるエダラボン投与は脳機能の改善に有効であると考えられた。

脳外傷はアルツハイマー型痴呆（AD）の危険因子である。脳外傷とADとの関係を検討するために実験的脳損傷後のラット脳でのamyloid precursor protein(APP)の発現を形態学的，分子生物学的に検討した。方法；10周齢雄wistarラットの硬膜を露出し，pneumatic control injury deviceを用いて硬膜上より外力を与えた。受傷ラットを経時的に，watre mazeにて記憶学習能力の測定後，免疫組織染色用，遺伝子発現解析用，蛋白量測定用の三群に分け，屠殺した。結果；記憶学習は受傷後1日より対照群に比較して有意に低下し60日でも有意に低下を示した。APPは蛋白レベルでは受傷後６時間から12時間で有意な増加を示し60まで続いた。APPのRT-PCRでは受傷後1時間から6時間で有意な増加が認められ，60日まで続いた。免疫染色ではAPPは受傷直後は変性線維に一致して染色された。７日になると神経細胞の胞体に一致した染色性を示し一部ミクログリアにも陽性像が認められた。まとめ；受傷後APPの蛋白，RNAレベルでの増

目的：従来、中枢神経障害の形態学的な検索は、免疫組織化学、通常の透過電顕(TEM)あるいは共焦点レーザー顕微鏡で行われているが、我々は超高圧電子顕微鏡（UHVEM)を用いて厚い切片を観察し、電顕トモグラフィーによる細胞レベルでの3次元解析を試みている。方法：脳卒中病変を発症した雄のSHRSPの大脳を、型のごとく電子顕微鏡用に固定・包埋し3uの切片を作製した。UHVEM（日立H-300）を用いて加速電圧2000KVで観察し、同一部位から傾斜を変えて70枚の写真を撮影した。コンピュータ上で再構成し、電顕トモグラフィーによる検索を行った。結果・考察：通常のTEMによる観察では、浮腫性病変部では単に神経組織の変性・融解が認められる以外には情報が得られなかったが、UHVEMによる観察では個々の神経線維の走行や病変を観察が可能であった。神経線維の変性・断裂部では、断裂した突起の先端近くに多数の水泡状構造が認められ、細胞膜の障害による変化が強く示唆された。UHVEMに

APPは外傷後長期にわたり神経細胞に強く発現していた。長期にわたるAPPの過剰発現は結果的に Aβの沈着を促進する可能性が考えられることから外傷がアルツハイマー型痴呆の危険因子になる原因と考えられた。

Aβ40の脳実質への沈着は個体差や別の因子が脳実質への沈着に関与し、Aβ42 、Aβ43は年齢やscoreと相関を示したことからヒトと同様、加齢とともに脳実質に長鎖のAβ42,43が早期から沈着し、老人斑を形成するのではないかと考えられた。（英文）

脳損傷後に生じるアストロサイト（AS)の反応とbFGFの関係を検討した。損傷後早い時期ではbFGFがASのglial fibrillary acidic protein誘導に作用した。2日より生じるbFGFの高濃度状態はvimentinを誘導し，脳内の線維化を促進すると考えられた。

臨床的にも病理学的にも鑑別診断に苦慮した6歳女児の後腹膜黄色肉芽腫症の1例を経験した。病変は肝下面に癒着、上行結腸、右腎尿管を巻き込んで増生していた。後腹膜黄色肉芽腫症、黄色肉芽腫性腎盂腎炎、悪性線維性組織球腫が鑑別として問題となった。

テトラベナジン反復投与では単回投与と異なり行動学的、形態学的に不可逆な変化が認められ、長期間のドーパミンの代謝異常が神経の変性、萎縮を誘導したと考えられた。その変化は慢性で経時的に悪化傾向を示しヒトPDにより似たモデルと考えられた。

脳損傷後のマイクログリア、マクロファージ由来のTNFαは細胞障害性に働き神経細胞及び膠細胞のapoptosisを誘導する可能性が示唆され、脳損傷後の神経細胞により合成されたTNFαは神経細胞に対して保護的に働く可能性が示唆された。

老齢犬においてもAbの沈着と加齢および記憶・学習の変化は相関することが示され、初期のAbの沈着はすでに神経組織の機能の低下をあらわし、老齢犬脳の検討が人アルツハイマー病の初期像を理解する上で有用であると考えられた。（英文）

APPの代謝過程によりAbの沈着がもたらされると考えられるがその代謝系に関係するユビキチンの機能低下がAbの沈着をもたらす可能性が示唆され、ユビキチンシステムの破綻が神経細胞の変性、記憶・学習の障害と強く関係している可能性が示唆された。（英文）

ラットの脳損傷後にbasic fibroblast growth factor（bFGF）がどのように変化するかをELISA法，および免疫組織化学的方法にて検討した。損傷部から発生するbFGFのグリア細胞への取り込みがグリア瘢痕形成のinitiatorとして作用する可能性が示唆された。

脳外傷におけるTNF-αの役割についての報告例は少ない。実験的脳損傷後に形成されるcavity、その周囲でのTNF-αの時間的な細胞分布の変化と蛋白量の変化を免疫組織染色、ELISA法にて形態計測学的、生化学的に解析した。

ABの沈着程度は加齢による記憶･学習の変化と相関し，犬のABはびまん型でヒトでいう初期の沈着像であり，アストログリアやマイクログリアの発現とタウリン酸化も認められないことからも老齢犬脳はヒトアルツハイマー病の初期モデルに相当すると考えられた。

bFGFは二次元培養では神経幹細胞の細胞増殖に働き，三次元培養ではMAP2陽性細胞数の増加及びrosette形成と神経細胞への分化誘導を行い立体構造が重要である。In vivoでの細胞増殖因子の作用を検討する上で有用である。

高速液体クロマトグラフィと電気化学検出器を用いてテトラベナジン(TBZ)反復投与したラットの脳のドーパミン(DA)及びその代謝産物を測定した。TBZ反復投与群はDAの減少，3,4-dihydroxyphenyl acetic acidの増加を示し，細胞内DAの代謝亢進が示された。

臨床的に脊髄炎と脊髄腫瘍との鑑別に苦慮した小児剖検例；急速な上行性麻痺、呼吸停止より急性脊髄炎が疑われた。MRI像（脊髄広範囲瀰漫性腫脹）と症状からは炎症と腫瘍の鑑別困難。剖検で広範囲髄液播種を伴った胸髄原発glioblastomaと確定。

パーキンソン病と現在の治療薬との問題点から我々が開発した新規パーキンソン病薬MAO-Bインヒビターの違い及びその作用機序について

ABの沈着の程度は加齢による記憶・学習の変化と関係した。AB、Bcl2、Baxの関係はABの沈着がアポトーシスを誘導する可能性を示唆した。老齢犬脳におけるABの沈着はAPPのupregulationなしに生じる可能性が年齢、APP陽性細胞数、ABの沈着の関係より示唆された。

外傷による脳損傷修復の過程で重要な役割を果たすと考えられているastrocytesの動態を，bFGF染色及びVimentin染色等の免疫組織染色を用いて形態計測学的に解析した。これら蛋白の経時的変動発現がglia scar形成に重要に関わっている事が示唆された。（英文）

Tetrabenazineのラットへの７日間の投与により，中脳の黒質緻密層に形態学的変化をもたらすことを明らかにしてきたが，さらに，tyrosine hydrokinase等の免疫組織学的検討を加えた。中脳黒質緻密層におけるDopaminergic neuronの変化が明らかとなった。（英文）

49歳，男性．以前から存在した耳介の脂腺母斑から，多彩な組織像を示す二次性腫瘍が発症した稀な1例を報告した．脂腺，汗腺，毛包，表皮への分化を示す良性，悪性の上皮性腫瘍と肉腫様の腫瘍が認められた．

７日間のテトラベナジン(TBZ)連続投与により，ラット中脳黒質の緻密層の神経細胞に萎縮と数的減少が認められた。それらの変化はTBZの単回投与とは異なり，非可逆的変化であった。TBZ連続投与がヒトパーキンソン病の動物モデルとして使用できると考えられた。

本研究の最終的な目標は，緑茶カテキン（エピガロカテキンガレート、EGCG）飲料を用いて脳外傷後に出現する神経幹細胞の神経再生及び修復に関わる因子を網羅的に調べ，高次脳機能障害発症抑制関連因子を明らかにすることである。脳外傷後の神経再生・修復を行う上で神経幹細胞の生存維持は重要である。脳外傷ラットモデルでのEGCG水溶液摂取（EGCG飲水）群と非摂取（非EGCG飲水）群との行動学的な形質の差異を明らかにし，行動学的表現型と神経幹細胞の生存，分化との関係について解析を行う。 本年度は、申請者らはpneumatic control injury deviceを用いて10週齢ラットオスを用いて緑茶カテキン（エピガロカテキンガレート、EGCG）投与群、Contorol群に分け、脳に外傷を与えたラット脳外傷モデルを作製した。Control群とEGCG群での脳外傷部位で1，3，7，30日後の経時的に神経再生及び神経新生を確認するために切片を作製し、nestin抗体及びDCX抗、GFAP抗体を使用し、免疫染色を行った。損傷周囲部分では損傷後1日後からnestin陽性の神経幹細胞と考えられるnestin陽性細胞が増加し、3日後に最大数を示し、7日後まで確認された。EGCG群がどの日数においてもControl群に比較し、有意な増加が認められた。また、新生神経細胞と考えられるDCX陽性の細胞が外傷7日後より増加し30日で最大数であった。さらにnestin陽性細胞の変化と同様にDCX陽性細胞においてもControl群に比較し、EGCG群で有意な増加が見られた。gliosisの指標であるGFAP陽性細胞はEGCG群に比較し、Control群で有意に増加し、EGCG群でgliosisが抑制されていることが示された。

From our current study, we propose that the exercise in the early following traumatic brain injury increases the number of nestin-positive cells-including NSCs- with the potential to differentiate into neurons and glia around the area damaged by TBI, and enhanced proliferation activity of neural stem cells (NSCs) around damaged area after traumatic brain injury(TBI). Therefore, the exercise therapy (rehabilitation) in the early phase following TBI is very important for recuperation of induced cerebral dysfunction following TBI.

A major component of green tea, a widely consumed beverage, is EGCG, which has strong antioxidant properties. In this study, we investigated the effects of EGCG on cerebral function and morphology following TBI. 24-month-old rats male Wistar rats that had access to normal drinking water, or water containing 0.1% (w/v) EGCG ad libitum, received TBI with a pneumatic controlled injury device for 4weeks. Immunohistochemistry and lipid peroxidation studies revealed that at 1, 3 and 7 days post-TBI, the number of 8-OHdG-, 4-HNE- and ssDNA-positive cells, and the levels of MDA around the damaged area after TBI, significantly decreased in the EGCG treatment group compared with the water group. In addition, there was a significant increase in the number of surviving neuronal cells and an improvement in cerebral dysfunction after TBI in the EGCG treatment group compared with the water group. In summary, consumption of green tea may be an effective therapy for TBIaged patients.

In this study, we investigated pregnancy-induced hypertension and developmental disease. In the elevated plus maze, SHRSP showed less anxiety-related behaviour than WKY rats. A treatment by antihypertensive drug to pregnancy-induced hypertension prevent less anxiety-related behaviour. A treatment by antihypertensive agent to pregnancy-induced hypertension may prevent developmental disease of a child.

We investigate the effect of exercise on omorphology and cerebral function following traumatic brain injury(TBI) in aged (two years old) rats. Aged wistar rats received TBI by a pneumatic controled injury device and were randomly divided into two groups :1)non-exercise group and 2) exercise group.The exercise groupran on a treadmill.Immunohistochemical and behavioral studied were permormed follwowing TBI. The number of apoptosis cells early after TBI was significantly rediced in the aged exercise group. Furthermore, most apoptosis cells in the non-exercise group indicated neuronal cells. However, in the exercise group, few apoptosis cells shown. In addition, there was a significant inmprovement in cerebral dysfunction after TBI in the exercise group. These resuls indicate that aged person exercise follwoing TBI inhibits neuronal apoptotic neuronal cell death, which results in an improvement of cerebral dysfunction.

A major component of green tea is (-)-epigallocatechin gallate (EGCG), which has strong antioxidant properties. we investigated the effect of EGCG on neural stem cell (NSC) proliferation following traumatic brain injury (TBI). Male Wistar rats that had access to normal drinking water, or water containing 0.1% (w/v) EGCG, ad libitum. Immunohistochemistry revealed that the number of nestin-positive cells after TBI in the EGCG group increased significantly. The number of single-stranded DNA (ssDNA)-positive cells after TBI significantly decreased in the EGCG group. Almost all ssDNA-positive cells in the water group co-localized with NeuN and nestin-staining. Spheres could only be isolated in the water group at 3 days.In the EGCG group, spheres could be isolated at all days following TBI. Spheres differentiated into neurons and glia. These results indicate that EGCG inhibits degradation of NSCs, which have the potential to differentiate into neurons and glia following TBI.

we investigate the effect of exercise on morphology and cerebral function following TBI in rats. Wistar rats received TBI by a pneumatic controlled injury device and were randomly divided into two groups: 1) non-exercise group and 2) exercise group. The exercise group ran on a treadmill. Immunohistochemical and behavioral studies were performed following TBI. The number of ssDNA-positive cells early after TBI was significantly reduced in the exercise group. Furthermore, most ssDNA-positive cells in the non-exercise group co-localized with neuronal cells. However, in the exercise group, few ssDNA-positive cells co-localized. In addition, there was a significant increase in neuronal cell number and improvement in cerebral dysfunction after TBI in the exercise group. These results indicate that exercise following TBI inhibits neuronal apoptotic cell death, which results in an improvement of cerebral dysfunction.

A major component of green tea, a widely consumed beverage, is(-)-epigallocatechin gallate(EGCG), which has strong antioxidant properties. In this study, we investigated the effects of EGCG on cerebral function and morphology following traumatic brain injury(TBI). Six-week-old male Wistar rats that had access to normal drinking water, or water containing 0.1%(w/v) EGCG ad libitum, received TBI with a pneumatic controlled injury device at 10 weeks of age. Immunohistochemistry and lipid peroxidation studies revealed that at 1, 3 and 7 days post-TBI, the number of 8-OHdG-, 4-HNE-and ssDNA-positive cells, and the levels of MDA around the damaged area after TBI, significantly decreased in the EGCG treatment group compared with the water group. In addition, there was a significant increase in the number of surviving neuronal cells and an improvement in cerebral dysfunction after TBI in the EGCG treatment group compared with the water group. In summary, consumption of green tea may be an effective therapy for TBI patients.

Wistar rats received traumatic brain injury (TBI) and were randomly divided into two groups: 1) non-exercise group and 2) exercise group. The exercise group ran on a treadmill for 30 min/day at 22 m/min for 7 consecutive days. Immunohistochemistry was used to monitor neural stem cell (NSC) proliferation around the damaged area and ex vivo techniques were used to isolate NSCs from the damaged region in both groups. The number of nestin as a maker of NSC- and Ki67 as a marker of proliferation cell-positive cells observed at 3 and 7 days after TBI was significantly greater in the exercise group than in the non-exercise group (P<0.01). Furthermore, most nestin-positive cells in the exercise group co-localized with Ki67-positive cells. In ex vivo studies, spheres could be isolated from injured brain tissue from the exercise group at 3 and 7 days following TBI, but at only 3 days in the non-exercise group. The number of spheres isolated from injured brain tissue was greater in the exercise group than in the non-exercise group. Spheres were immunopositive for nestin and comprised of NSCs that could differentiate into neurons and glia. Exercise increases the proliferation of NSCs around the damaged area following TBI. Therefore, exercise therapy (rehabilitation) in the early phase following TBI is important for recuperation from cerebral dysfunction induced by TBI.