伊藤 弘康

In this study, we demonstrated that lipopolysaccharide (LPS) markedly increased nitric oxide (NO) production and indoleamine 2,3-dioxygenase (IDO) activity in mouse peritoneal cells in the presence of activated Vα14 natural killer T cells. Moreover, LPS-induced NO production in peritoneal cells from IDO-knockout (KO) mice was more increased than that from wild-type mice. However, there was no significant difference in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein between the wild-type and IDO-KO mice. No significant difference was also observed in the ratio of CD3- and DX5-positive cells and F4/80- and TLR4-positive cells in peritoneal cells between the wild-type and IDO-KO mice. Since the IDO activity was enhanced by an NO inhibitor, NO may be post-translationally consumed by inhibiting the IDO activity. IDO is well known to play an important role in immunosuppression during inflammatory disease. Therefore, the inhibition of IDO by NO may exacerbate inflammation in the peritoneal cavity. © 2009 Elsevier Inc. All rights reserved.

自動分析装置の2項目分析機能を利用することによりLDL-CとT-CHOの同時測定を可能とした試薬、"デュアルCHO T&L「生研」"の基礎的性能評価を行った。日常の臨床検査として提出された患者検体270例を対象とした。同時再現性、日差再現性を検討し、LDL-C、T-CHOともに各濃度において変動係数(CV%)は0.33〜1.63%と良好な再現性であった。LDL-C;612mg/dL、T-CHO;781mg/dLまで原点を通る直線性を確認した。最小検出実行感度は、LDL-C、T-CHOともに1mg/dL付近であった。患者検体140例について従来法との相関を検討し、良好な相関が得られた。高トリグリセライド(TG)血症検体130例において、少なくとも1270mg/dLまでTGの影響がないことを確認した。

Cellulomonas denverensis is a small and thin gram-positive rod-shaped bacterium that was proposed as a new species in 2005. Here we report a female case of acute cholecystitis and sepsis in which C. denverensis was determined to be causative.

Diabetes mellitus is becoming a major cause of premature disability in Japan, and peripheral neuropathy is a common complication of diabetes. The aim of this study was to evaluate the relationship between the results of nerve conduction studies (NCS) and the size of the nerve determined by sonography in diabetic patients.Twenty diabetic patients (mean age +/- SD, 57.1 +/- 13.6 years) and 20 healthy volunteers (mean, 61.1 +/- 8.9 years) were enrolled in this study. Patients'wrists that had symptoms of carpal tunnel syndrome were not included in the study; those that were included had negative Phalen test results. We then divided the patients into 2 groups (patients with and without diabetic symmetric polyneuropathy [DPN]). The cross-sectional area (CSA) was measured in the carpal tunnel 5 cm proximal to the wrist and elbow joint of the median nerve.There was a significant increase in the CSA in patients with DPN in the carpal tunnel compared with the control participants (P<.01) and patients without DPN (P<.01). The CSA in the carpal tunnel showed a significant correlation with the motor nerve conduction velocity (r = -0.473).The CSA of the median nerve in the carpal tunnel of pati

Human hepatocytes are known to express an array of inflammatory cytokines and chemokines. In this study, we examined the potential roles of hepatocytes in regulating immune responses in the liver, by assessing the induction of Th1- or Th2-specific chemokines in HepG2 cells after various inflammatory stimulations. HepG2 cells were stimulated with IL-1 alpha, IFN-gamma, IL-4, IL-10, and/or CCL2, harvested at several time points, and served for the analyses of cytokine/chemokine mRNA expressions by semi-quantitative RT-PCR. (i) IL-1 alpha up-regulated mRNA levels of CXCL8, CXCL10, and CCL2. IFN-gamma increased those of CXCL9, CXCL10, and CCL5, while IL-4 or IL-10 had no effect. (ii) Addition of IL-4 to the culture of IFN-gamma-stimulated cells, down-regulated CXCL9 and CXCL10 mRNA levels. (iii) Addition of IFN-gamma to the culture of IL-1 alpha-stimulated cells, further up-regulated CXCL9 and CXCL10 mRNA levels. Addition of IL-4 decreased CXCL8 and CXCL10 levels, and increased CCL2 level in IL-1 alpha-stimulated cells. (iv) CCL2 induced IL-4 mRNA expression. IFN-gamma augmented mRNA expression of Th1-specific chemokines (CXCL9 and CXCL10) in HepG2 cells. IL-4 had no effect on those of Th2-spesific chemokines (CCL17 and CCL22); however, it was supposed to augment Th2 response indirectly through the induction of CCL2 under the inflammatory condition. The findings suggest that hepatocytes have ability to promote immune responses in the liver toward the direction, initially determined by the cytokine balances in the local inflammatory region.

Indoleamine 2,3-dioxygenase (IDO), which catabolizes L-tryptophan (L-TRP) to L-kynurenine (L-KYN), is an immunoregulatory factor that is up-regulated via an interferon-gamma (IFN-gamma)-dependent and/or independent mechanism. In this study, we investigated the localization of IDO and whether induction of IDO expression is an IFN-gamma-dependent and/or -independent mechanism in the CNS after cerebral ischemia. The expressions of IDO protein and mRNA were investigated at different time points following cerebral ischemia using immunohistochemistry, immunofluorescence and RT-PCR. Hippocampal neuron IDO mRNA and immunohistochemical staining were significantly up-regulated 72 h after transient global ischemia. Although IFN-gamma is a dominant inducer of IDO, hippocampal neuron IDO was clearly up-regulated in IFN-gamma KO mice. In summary, this is the first finding that up-regulation of IDO in hippocampal neurons after transient global ischemia occurs via INF-gamma-independent mechanisms. (C) 2009 Published by Elsevier Ireland Ltd and the Japan Neuroscience Society.

Chronic Kidney Disease (CKD) is an important risk factor of the End Stage Renal Disease (ESRD). In this study, we investigated whether the protein to creatinine ratio (the ratio of P/C) determined by the semiquantitative urinary stick test and urinary sediments are useful for theearly detection of CKD. One hundred sixty patients were classified to four or five groups by P/C ratio and various biochemical markers were analyzed. As a result, the 300 mg/g x Cr of P/C group showed a significantly increased serum cystatin C level. The positive rate of the P/C ratio in CKD stage was significantly increased compared with the conventional protein qualitative analysis. Further, the amounts of urinary sediments in CKD stage 1 to 2 were increased, such as hyaline cast, and pathological casts were increased in CKD stage 3 to 5. Thus, our present study suggests that the ratio of P/C and urinary sediments are useful for the screening of CKD.

Indoleamine-2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme inducing suppression of T-cell function and immune tolerance. In hepatitis B virus (HBV) transgenic (Tg) mice, the adoptive transfer of HBV-specific cytotoxic T lymphocytes (CTL) causes a necroinflammatory liver disease that is histologically similar to acute viral hepatitis in man. The present study aimed to determine IDO expression in the liver and hepatocytes during an acute hepatitis model.Serum l-kynurenine (l-Kyn) concentration in HBV Tg mice administered with HBV-specific CTL was measured over time, together with serum levels of alanine aminotransferase (ALT). Furthermore, we examined the expression of IDO in the total liver and isolated hepatocytes of HBV Tg mice after CTL injection using immunohistochemical analysis and reverse-transcription polymerase chain reaction (PCR).In HBV Tg mice, HBV-specific CTL induced, over the course of several days, a chronic increase in serum l-Kyn levels, which was associated with a sustained enhancement of liver IDO activity. In particular, IDO expression was enhanced in the liver parenchymal cells (hepatocytes) after HBV-specific CTLinjection both in immunohistochemical

Vα14 natural killer T (Vα14 NKT) cells activated by α-galactosylceramide (α-GalCer) secrete a large amount of Th1 and Th2 cytokines. IFN-γ plays a crucial role in the inflammation response, and is also known as an activator of nitric oxide (NO) production. We previously reported that lipopolysaccharide (LPS)-induced NO production is augmented by α-GalCer in mouse peritoneal cells. Since the liver is susceptible to LPS stimulation via the portal vein, we examined the effect of α-GalCer on LPS-induced NO production in murine intra-hepatic lymphocytes (IHLs). Although IHLs augmented LPS-induced NO production by α-GalCer administration, such an augmentation was not observed in non-treated mice. Furthermore, α-GalCer did not augment LPS-induced NO production in IHLs from IFN-γ knockout mice. In flow cytometry analysis of IHLs from α-GalCer-treated mice, the ratio and number of F4/80- and TLR4-positive cells rose as compared with non-treated mice. The liver injury may be induced by LPS and NO under the condition where Vα14 NKT cells were activated. © 2008 Elsevier Inc. All rights reserved.

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T-cell lymphotropic virus type I (HTLV-1). Indoleamine 2,3-dioxygenase (IDO), the L-tryptophan (L-TRP)-degrading enzyme, plays a key role in the powerful immunomodulatory effects of several different types of immune cells. In this study, we investigated the IDO expression in ATLL cells and the effect of chemotherapy on IDO-initiating L-TRP catabolism in patients with ATLL. Serum L-kynurenine (L-KYN) concentrations, L-KYN/L-TRP ratio, and the level of IDO mRNA expression in ATLL cells were significantly increased in ATLL patients compared to those in healthy and HTLV-positive carrier subjects. On the other hand, L-TRP level was significantly decreased in ATLL patients compared to that in healthy subjects. In the immunohistochemical staining, IDO was strongly expressed in cytoplasm of ATLL cells. Interestingly, serum L-KYN as well as soluble IL-2 receptor concentrations was significantly reduced, and L-TRP concentrations were significantly increased after chemotherapy. These data provide evidence that IDO is highly expressed in ATLL cells, and that IDO-initiating L-TRP catabolism changes with chemotherapy. (C) 2008 Elsevier Ltd. All rights reserved.

In previous studies, the mechanisms of acute liver injury and virus exclusion have been examined using a model wherein HBsAgspecific CTL are injected into HBsAg transgenic (Tg) mice. The importance of the role of TNF-α in virus exclusion was shown, but its role in liver injury was unclear. We crossed the TNF-α knockout mouse and HBsAg-Tg mouse to establish the HBsAg-Tg/TNF-αKO mouse, and examined the influence of TNF-αon liver injury. The severity of liver damage, as determined by serum alanine aminotransferase activity, was ∼100 times greater in HBsAg-Tg/TNF-α+/+ than in HBsAg-Tg/TNF-α-/- mice after i.v. administration of 5 × 106 CTLs. This liver damage reached the peak of its severity within 24-48 h, and was restored 7 days later. Histopathological examination showed hepatocellular necrosis and inflammatory cell infiltrate 24 h after the CTL injection in HBsAg-Tg/TNF-α+/+ mice but not in HBsAg-Tg/TNF-α -/- mice. The liver damage was fatal for all HBsAg-Tg/TNF- α+/+ mice that received 1.5 × 107 CTLs. In contrast, 1.5 × 107 CTLs could not kill the HBsAg-Tg/TNF-α+/+ mice. The TNF-α production level was enhanced after the CTL injection in not only intrahepatic macrophages but also other types of mononuclear cells from non-HBsAg-Tg/TNF-α+/+ mice. An adoptive transfer examination revealed that severe liver damage occurred in HBsAg-Tg/ TNF-α-/- mice that had received mononuclear cells from TNF-α+/+ mice. In conclusion, the present study provides evidence that TNF-α produced by intrahepatic non-Ag-specific inflammatory cells is critical in the development of lethal necroinflammatory liver disease. Copyright © 2008 by The American Association of Immunologists, Inc.

Statins are effective in lowering cholesterol levels, but cause fatal rhabdomyolysis in susceptible individuals. Because it has been hypothesized that muscle damage could result from alterations in Ca(2+) homeostasis in muscle cells, we tested whether measuring statin-induced changes in intracellular calcium ([Ca(2+)](i)) is useful for predicting susceptibility to statin-muscle damage, using human CD19+ primary B lymphocytes.Statin-induced alterations in [Ca(2+)](i) were studied using the human THP-1 cell line and CD19+ primary B lymphocytes. Changes in [Ca(2+)](i) were measured directly in fluo-3- loaded cells using either singleor dual-color flow cytometry.The Ca(2+) release study suggested that statin-induced changes in [Ca(2+)](i) were due to Ca(2+) release from ryanodine-sensitive Ca(2+) stores and mitochondrial compartments. Further, statin users who experienced elevated creatine kinase (n=8) exhibited significantly greater statin-induced Ca(2+) release in B cells than healthy volunteers (n=45) and statin users without elevated creatine kinase (n=16), while no difference was seen between the latter two groups.Statin-induced Ca(2+) release from ryanodine-sensitive stores and m

Inthe present study, we used bone marrow transplanted mice and revealed the role of bone marrow derived cells in liver regeneration after partial hepatectomy (PH). Irradiated wild type (WT) mice received a bone marrow transplant from either WT, TNF (tumor necrosis factor)-alpha knockout (KO), or interleukin (IL)-6 KO donors. Both TNF-alpha KO- and IL-6 KO-transplanted mice compared with WT-transplanted mice showed decreased hepatocyte DNA synthesis after PH. TNF-alpha KO-transplanted mice showed no nuclear factor kappa B (NF-kappaB) and signal transducer and activator of transcription (STAT) 3 binding after PH, while IL-6 KO-transplanted mice showed NF-kappaB, but not STAT3, binding. Lack of AP-1 or C/EBP binding or expression of c-jun or c-myc mRNA after PH was unrelated to the timing and amount of DNA replication. In conclusion, The TNF-alpha and IL-6 signals from the blood are necessary for liver regeneration and NF-kappaB and STAT3 binding are activated via TNF-alpha and IL-6 signal pathways.

CTLs are thought to be major effectors for clearing viruses in acute infections including hepatitis B virus (HBV). Persistent HBV infection is characterized by a lack of or a weak CTL response to HBV, which is thought to reflect tolerance to HBV antigens. In the present study, we found that alpha-galactosylceramide (α-GalCer), a ligand for Vα14-positive NKT cells, strongly enhanced the induction and proliferation of HBV-specific CTLs by HBsAg. In HBsAg transgenic mice, which are thought to be tolerant to HBV-encoded antigens, administration of HBsAg or α-GalCer alone failed to induce HBsAg-specific CTLs, but they were induced by co-administration of both compounds. Furthermore, by limiting dilution analysis, we confirmed the existence of HBsAg-specific CTL precursors in the HBsAg transgenic mice immunized with HBsAg and α-GalCer. A blocking experiment using antibodies to cytokines and CD40 ligand showed that IL-2 and CD40-CD40L interaction mediate the enhancement of CTL induction caused by α-GalCer through NKT cell activation. Our results may open up a new method for clearing the virus from patients with persistent HBV infection. © The Japanese Society for Immunology. 2008. All rights reserved.

Background: To expand the criteria of immune thrombocytopenic purpura (ITP), which is a diagnosis of exclusion, we analyzed proteins separated by 1-dimensional gel electrophoresis of the retained fraction in a Con A-Sepharose column from ITP patients' sera. Methods: Serum samples were from 19 adult patients with chronic ITP, 9 patients with thrombocytopenia of decreased production, and 20 healthy controls. Samples were applied to a Con A-Sepharose column, and the retained fraction was subjected to 10% SDS-PAGE. The % area of each densitometric protein peak was compared between the two groups and proteins in each band were identified using LC-MS/MS. Results: Eleven protein bands were distinctly separated by 1-dimensional electrophoresis. The percent area of bands #2 and #3 were significantly higher in ITP patients than in controls. The percent area of band #2 (p<0.01) was significantly higher in ITP patients than in non-ITP patients. We identified alpha(2)-macroglobulin, ceruloplasmin (Cp), and C3 in band #2 and complement factor B in #3 band. Serum concentrations Of alpha(2)-macroglobulin and Cp were significantly higher in ITP patients than in controls. Serum concentrations of Cp were significantly higher in ITP patients than in non-ITP patients (p = 0.0005). Serum complement factor B concentrations were significantly higher in ITP patients and non-ITP patients than in controls. ROC analysis showed that the total percent area of bands #2 and #3, and Cp had higher diagnosis availability for ITP patients when compared with controls and non-ITP patients, respectively. Conclusions: Measurement of Cp separated by the present method could be useful for the diagnosis of ITP in the presence of thrombocytopenia and a non- or low-inflammatory state. (C) 2007 Elsevier B.V. All rights reserved.

小学4〜6年の県内トップレベル柔道強化選手48名(男児・女児各24名)を対象に、超音波検査による脂肪肝所見とメタボリックシンドローム(MS)関連因子について検討した。脂肪肝所見の有無により脂肪肝群と正常群の2群に分け、身体計測、血液生化学検査、血圧値を比較した。身体計測では身長、体重に関しては両者間に有意差を認めなかったがBMI、肥満度、体脂肪率(%FAT)、ウエスト径、ウエスト/ヒップ比(W/Hi)、ウエスト/身長比(W/He)において全て脂肪肝群が有意に高値を示した。肝機能、糖代謝関連においても全ての項目で脂肪肝群が高値を示した。血液検査ではWBCで脂肪肝群が有意に高値を示したが他項目では有意差を認めなかった。血圧では両者間に有意差を認めなかった。身体計測値ではFLI(Fatty Liver Index)とBMIがr=0.741、肥満度はr=0.782、%FATではr=0.710と非常に高い相関関係を示した。

Murine acquired immunodeficiency syndrome (MAIDS) induced by LP-BM5 murine leukemia virus is used as a model of human immunodeficiency virus (HIV)-related neurologic dysfunction. Mice infected with LP-BM5 have mnemonic abnormalities (i.e., spontaneous alternation behavior in the Y-maze and performance in the Morris water maze) and biochemical alternations (i.e., cytokines, platelet- activating factor, quinolinate, glutamate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) that produce neurologic symptoms similar to those observed in HIV-related neurologic dysfunction. To identify proteins associated with dysmnesia in the MAIDS model, we examined the expression of neuronal proteins in LP-BM5-infected mice using two-dimensional polyacrylamide gel electrophoresis (2-DE). Neuronal protein expression in LP-BM5-infected mice was compared with that in non-infected mice using the Image Master 2D. We detected approximately 800 protein spots, of which 35 were distinguishable between non-infected and LP-BM5-infected mice. Most of these spots were downregulated in LP-BM5-infected mice. Three of the spots were identified as 14-3-3 protein zeta/delta, synapsin 2 and protein disulfide isomerase using a capillary nanoliquid chromatography tandem mass spectrometric system. We verified the expression levels of these proteins by Western blot. Analysis of these 35 spots could provide insight into mechanisms of dysmnesia in the MAIDS model of HIV-related neuronal dysfunction. (c) 2007 Published by Elsevier Ireland Ltd.

The effect of 5-fluorouracil (5-FU) on the production of nitric oxide ( NO) in macrophages was examined by using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. 5-FU at non-toxic concentrations significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition by 5-FU was mediated by attenuated expression of an inducible NO synthase protein and mRNA. 5-FU inhibited the activation of nuclear factor (NF)-kB and the subsequent nuclear translocation. Furthermore, 5-FU inhibited the phosphorylation of Akt, an upstream molecule of NF-kB signaling. 5-FU did not affect a series of mitogen-activated protein kinases. Therefore, 5-FU was suggested to inhibit the LPS-induced NO production in activated macrophages through preventing Akt-dependent NF-kB activation.

Many studies indicate that Chlamydia pneumoniae infection is a crucial risk factor in atherogenesis. The most relevant cell type for the pathogenesis is the macrophage, which possesses classical scavenger receptors that uptake oxidized low-density lipoprotein (LDL). Here, a direct involvement of vascular endothelial cells in atherogenesis was examined employing in vitro infection of human umbilical vein endothelial cells (HUVEC) with C. pneumoniae. Chlamydia pneumoniae infection greatly enhanced the uptake of oxidized LDL, but not of acetylated LDL, by HUVEC. Among the scavenger receptors analyzed, LOX-1 transcription, which prefers oxidized LDL to acetylated LDL, was significantly amplified.

A role for the US3 protein kinase of herpes simplex virus (HSV) in regulating virus-induced neuronal apoptosis was investigated in an experimental mouse system, in which wild-type HSV invades the central nervous system (CNS) via the olfactory and vomeronasal systems upon intranasal infection. Wild-type HSV-2 strain 186 infected a fraction of olfactory and vomeronasal chemosensory neurons without inducing apoptosis and was transmitted to the CNS, precipitating lethal encephalitis. In sharp contrast, an US3-disrupted mutant, L1BR1, induced neuronal apoptosis in these peripheral conduits upon infection, blocking viral transmission to the CNS and causing no signs of disease. An US3-repaired mutant, LIB(-)11, behaved similarly to the wild-type virus. Only 5 p.f.u. of L1BR1 was sufficient to compromise mice when the mutant virus was introduced directly into the olfactory bulb, a viral entry site of the CNS. These results suggest that the US3 protein kinase of HSV regulates virus-induced neuronal apoptosis in peripheral conduits and determines the neuroinvasive phenotype of HSV. Furthermore, virus-induced neuronal apoptosis of peripheral nervous system cells may be a protective host response that blocks viral transmission to the CNS. (c) 2006 Elsevier SAS. All rights reserved.

To understand the pathogenesis of chronic hepatitis B virus (HBV) infection, we examined the serum levels of IL-10, TNF-alpha IL-12 p70, and IL-12 p40 in 77 patients chronically infected with HBV and 19 controls. The patients were classified into four groups: asymptomatic carriers (ASC), patients with chronic hepatitis (CH), patients with liver cirrhosis (LC), and patients with hepatocellular carcinomas (HCC). The cytokine values among these groups were compared and their relations to clinical parameters were investigated. All these cytokine values were higher in the patient groups than in controls. IL-10 and TNF-alpha became higher in accordance with the progress of the disease phases, from ASC to LC, and lowest when the patients had HCC. IL-12 p40 was also lowest in HCC, however, the group with highest levels was CH. IL- 12 p70 was unchanged among ASC, CH, and LC, but were raised in HCC. Serial analyses for the cytokine values in the same patients showed the similar tendencies. Regression analysis showed the significant correlations between ALT and IL-10. Serum cytokine values well reflected the pathological differences of the individual disease phases, and may become useful indices to understand the pathogenesis of chronic HBV infection. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

The effect of alpha-galactosylceramide (alpha-GalCer) on lipopolysaccharide (LPS)-mediated lethality was examined. Administration of LPS killed all mice pretreated with alpha-GalCer, but not untreated control mice. The lethal shock in alpha-GalCer-sensitized mice was accompanied by severe pulmonary lesions with marked infiltration of inflammatory cells and massive cell death. On the other hand, hepatic lesions were focal and mild. A number of cells in pulmonary and hepatic lesions underwent apoptotic cell death. alpha-GalCer sensitization was ineffective for the development of the systemic lethal shock in V alpha 14-positive natural killer T cell-deficient mice. Sensitization with alpha-GalCer led to the circulation of a high level of interferon (IFN)-gamma and further augmented the production of tumor necrosis factor (TNF)-alpha in response to LPS. The lethal shock was abolished by the administration of anti-IFN-gamma or TNF-alpha antibody. Further, the lethal shock did not occur in TNF-alpha-deficient mice. Taken together, alpha-GalCer sensitization rendered mice very susceptible to LPS-mediated lethal shock, and IFN-gamma and TNF-alpha were found to play a critical role in the preparation and execution of the systemic lethal shock, respectively. The LPS-mediated lethal shock using alpha-GalCer sensitization might be useful for researchers employing experimental models of sepsis and septic shock.

The effect of lipopolysaccharide (LPS) on the cell death induced by endoplasmic reticulum (ER) stress agents in RAW 264.7 cells was studied. LPS prevented the cell death by brefeldin A, but not thapsigargin and tunicamycin. CpG DNA as well as LPS prevented brefeldin A-induced cell death whereas tumor necrosis factor-alpha or interferon-gamma did not. Brefeldin A-induced cell death was mediated with apoptotic cell death and it was significantly inhibited by LPS. LPS abolished the activation of ER stress-related caspases, such as caspases 1, 3, and 4. LPS prevented brefeldin A-induced morphological changes in RAW 264.7 cells. Further, LPS prevented brefeldin A-induced Golgi dispersion. Therefore, LPS was suggested to diminish the stress of ER/Golgi complexes induced by brefeldin A and inhibit apoptosis. The preventive action of LPS on brefeldin A-induced apoptosis is discussed. (c) 2005 Elsevier Inc. All rights reserved.

Biological activities of lipopolysaccharide (LPS) from Brucella melitensis 16M were characterized in comparison with LPS from Escherichia coli O55. LPS extracted from B. melitensis was smooth type by electrophoretic analysis with silver staining. The endotoxin-specific Limulus activity of B. melitensis LPS was lower than that of E. coli LPS. There was no significant production of tumor necrosis factor-alpha and nitric oxide in RAW 264.7 macrophage cells stimulated with B. melitensis LPS, although E coli LPS definitely induced their production. On the other hand, B. melitensis LPS exhibited a higher anti-complement activity than E coli LPS. B. melitensis LPS as well as E. coli LPS exhibited a strong adjuvant action on antibody response to bovine serum. The characteristic biological activities of B. melitensis are discussed.

Previously, we found that mouse TH2.52 cells possess the characteristic of CD5(+) B1 cells and proliferate in response to lipopolysaccharide (LPS). The effect of LPS on cytokine production by TH2.52 B1 cells was studied. TH2.52 cells constitutively produced a small amount of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and TNF-alpha and IL-6 production was markedly enhanced by LPS stimulation. Although interferon (IFN)-y caused the production of various cytokines, such as IL-2, IL-4, IL-6 and TNF-a in TH2.52 cells, LPS did not cause the production of such cytokines. LPS did not induce IFN-P production in TH2.52 cells and TH2.52 cells lacked the expression of several molecules participating in the MyD88-independent pathway in LPS signaling. Defective responsiveness of TH2.52 B1 cells to LPS in cytokine production might be responsible for the failure of IFN-P production due to the lack of molecules participating in the MyD88-independent pathway.

Herpes simplex virus (HSV), a neurotropic virus, establishes life-long and, although rare, life-threatening infection in humans, and it may precipitate substantial medical and psychosocial morbidity. Here we show that HSV-1 strain HF clone 10 (HF 10) exhibits impaired neuroinvasiveness in peripheral olfactory, vomeronasal and trigeminal conduits following intranasal as well as corneal inoculation. HF10 attenuation likely arises from multiple defects of HSV genes, so that HF10 will not revert to a virulent phenotype. Intranasal vaccination of mice with HF 10 conferred significant protection against lethal challenge with HSV-1 and HSV-2 via the intranasal and intravaginal routes. Thus, we propose that HF10 explicitly meets the prerequisites for a candidate live attenuated HSV vaccine. (C) 2005 Elsevier SAS. All rights reserved.

Using beta-amyloid precursor protein immunolabeling, we have detected axonal injury in experimental herpes simplex encephalitis. amyloid precursor protein-specific signals were found in the mouse brain as either puncta or axon-like structures. They appeared where infected neurons were undergoing apoptosis and Iba1-immunopositive microglia transformed themselves into macrophages. These results show, for the first time, that axonal injury, i.e., functional disturbance of the fast axonal transport, can take place during the course of acute viral encephalitis. (c) 2005 Elsevier B.V All rights reserved.

The present study was conducted to determine effects of 00126, a specific inhibitor of mitogen-activated kinase kinase 1/2, on production of nitric oxide (NO) in RAW264.7 macrophage cells. 00126 significantly enhanced NO production in lipopolysaccharide (LPS) but not CpG DNA or interferon-gamma-stimulated RAW264.7 cells. In contrast, 00124, a negative control for 00126, did not affect LPS-induced NO production. Further, a series of inhibitors of p38, phosphatidyl-inositol 3-kinase and Janus tyrosine kinase rather caused suppression in LPS-stimulated RAW264.7 cells. 00126 was found to definitely inhibit phosphorylation of extracellular signal-regulated kinase (Erk) 1/2 and augment the levels of inducible type of NO synthase. Antisense oligonucleotides of Erk1/2 also augmented LPS-induced NO production. Inactivation of Erk1/2 by 00126 furthermore inhibited LPS-induced activating protein-1 activation, but not nuclear factor-kappa B activation. The results suggest that Erk1/2 might negatively regulate NO production in LPS-stimulated RAW264.7 cells. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The effect of lipopolysaccharide on doxorubicin-induced cell death was studied by using mouse RAW 264.7 macrophage cells. Pretreatment with lipopolysaccharide at 10 ng/mL prevented doxorubicin-induced cell death and the inhibition was roughly dependent on the concentration of lipopolysaccharide. Posttreatment with lipopolysaccharide for 1 hour also prevented doxorubicin-induced cell death. Lipopolysaccharide inhibited DNA fragmentation and caspase-3 activation in doxorubicin-treated RAW 264.7 cells, suggesting the prevention of doxorubicin-induced apoptosis. Lipopolysaccharide did not significantly inhibit doxorubicin-induced DNA damage detected by single-cell gel electrophoresis (comet) assay. Lipopolysaccharide definitely inhibited the stabilization and nuclear translocation of p53 in doxorubicin-treated RAW 264.7 cells. Lipopolysaccharide, as well as being an inhibitor of p53, abolished doxorubicin-induced apoptosis. Therefore, p53 was suggested to play a pivotal role in the prevention of doxorubicin-induced apoptosis in RAW 264.7 cells by lipopolysaccharide.

The US11 gene product of herpes simplex virus is an RNA-binding protein with a C-terminal arginine-X-proline (RXP) repeating motif. We found that the RXP repeat mediates intercellular trafficking activity and accumulation in neuronal nuclei following in vivo transfection with the US11 gene, direct injection of the purified RXP-repeat fusion protein, or infection with herpes simplex virus. We discuss a possible therapeutic application of the US11 protein RXP repeat as a tactic against neurodegenerative disorders of the central nervous system. (c) 2005 Elsevier B.V. All rights reserved.

We have investigated the potential of neurotropic microbes to invade the central nervous system (CNS) via the peripheral nervous system. Herpes simplex virus type 1 (HSV-1) strain KH6 and herpes simplex virus type 2 (HSV-2) strain 186 were found to infect chemosensory neurons in the vomeronasal organ (the pheromone detector) following intranasal inoculation of mice. HSV-1 strain KH6 infection was further transmitted to the accessory olfactory bulb (first relay), the medial amygdala (second relay), and the bed nucleus of the stria terminalis and the ventromedial hypothalamus (third relay). HSV-1 strain KH6 also targeted the olfactory and trigeminal systems. HSV-2 strain 186 predominantly attacked the brainstem including the trigeminal system. While both viruses did not induce apoptosis in infected chemosensory neurons, they did in infected brain tissue. These results suggest that neurotropic viruses can invade the brain by infecting vomeronasal chemosensory neurons and that the restrained induction of apoptosis in the infected neurons may facilitate viral transmission to the CNS. (c) 2005 Elsevier Inc. All rights reserved.

The effect of inhibition of mitogen and stress-activated protein kinases 1/2 (MSK1/2) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was investigated. Pretreatment with Ro 31-8220, an inhibitor of MSK1/2, induced cell death in LPS-stimulated RAW 264.7 cells. In contrast, calphostin C, another inhibitor of protein kinase C, did not cause cell death. Cell death was not mediated by the release of pro-inflammatory mediators from LPS-stimulated RAW 264.7 cells. Cell death was accompanied by DNA fragmentation and annexin V binding, suggesting apoptotic cell death. Further, several caspase inhibitors did not prevent LPS-induced cell death of Ro 31-8220-pretreated RAW 264.7 cells. Nuclear translocation of apoptosis-inducing factor (AIF) was detected in Ro 31-8220-pretreated cells after LPS stimulation. Cell death was due to mitochondrial damage. Ro 318220 exclusively inhibited the phosphorylation. of cAMP-responsive element binding protein (CREB), a substrate of MSK1/2. RAW 264.7 cells transfected with the dominant-negative MSK1 clones underwent cell death in response to LPS. Hence, it was suggested that MSK1/2 might play a critical role in the survival of LPS-stimulated RAW 264.7 cells. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.