Although the role of interleukin(IL)-6 in inflammatory disease has been previously examined, its role in hemostasis, fibrinolysis, and coagulation during inflammation remins to be established. The present study reviewed the role of IL-6 in hemostatic and coagulatory changes during inflammation, with special emphasis on the inflammation induced by intraperitoneal administration of lipopolysaccharide in IL-6 null-/- mice and corresponding wild type(WT) mice. After LPS challenge, IL-6-/- mice revealed significant prolongation of prothrombin time and partial thromboplastin time and a significant decrease in platelet counts as compared with WT mice. LPS treatment induced marked pulmonary hemorrahage with neutrophilic inflammation in IL-6-/- mice, in contrast, only mild neutrophilic infiltration in WT mice confirmed by macroscopic and histological findings. The protein levels of proinflammatory mediators in the lungs were significantly greater in IL-6-/- mice than in WT mice after LPS challenge. Our results conclude that IL-6 is protective against coagulatory and hemostatic disturbance and subsequent pulmonary hemorrhage induced by bacterial endotoxin, at least party, via the modulation of proinflammatory processes. In addition, our studies suggest that anti-IL-6 therapy may not be efficient for systemic inflammatory disease such as disseminated intravascular coagulation, acute lung injury, and sepsis.
Although it was long thought that the damaged adult central nervous system (CNS) cannot regenerate, spinal cord injury (SCI) is now an important target in regenerative medicine. A series of studies on stem cell-based therapies for SCI has led us to believe that neural stem/progenitor cells (NSPCs) are a promising source for neural tissue replacement therapy after SCI, when applied appropriately. In this review, we summarize our previous findings on stem cell therapies for SCI and discuss the future directions of this work.
Happy New Year!At the beginning of 2006, allow me to recall and describe some of my experiences during the last year.In August and September 2005, I had the opportunity to visit 3 countries, Australia, South Korea, and the United States, within a 3-week period, and the trips provided me with a chance to think deeply about international collaboration as well as national policies regarding science and technology.Life science research is very attractive in all three countries, despite their different characteristics and cultural backgrounds.I believe that our country, Japan, has much to learn from their national policies regarding life science, in terms of both strong points and weak points.
BACKGROUND AND PURPOSE: The inflammatory bowel diseases(IBD) such as ulcerative colitis(UC) are intractable diseases of unknown origin. The mucosal injury of UC is presumed to be partly caused by reactive oxygen species(ROS) produced by neutrophils. Chronic inflammation of UC results in chronic anemia, to which iron is given. In this study, to evaluate the effect of sodium ferrous citrate(SFC) on neutrophil functions, we measured superoxide generation and migration of neutrophils treated with SFC in vitro.METHODS: Superoxide generation induced by phorbol myristate acetate(PMA), opsonized zymosan(OPZ) and N-formyl-menthionyl-leucyl-phenylalanine(fMLP) was determined based on the superoxide dismutaseinhibitable cytochrome c reduction. To further clarify the inhibitory mechanism of SFC, the membrane fraction was isolated and NADPH-dependent superoxide production was measured. Moreover, neutrophil migration was evaluated with a Boyden chamber assay using zymosan-activated serum (C5a) as a chemoattractant.RESULTS: SFC dose-dependently inhibited the superoxide production induced by all the stimuli examined. Similarly, SFC-treatment inhibited the activity of NADPH oxidase in the membrane fraction of neutrophils. In addition, SFC suppressed chemotaxis toward zymosan-activated serum in a dose-dependent fashion.CONCLUSIONS: In this study, we revealed that SFC could suppress neutrophil functions such as superoxide generation (NADPH oxidase activity) and migration. During episodes of IBD, stimulated neutrophils are accumulated in inflamed mucosa via the migration through vascular endothelium, where they release ROS such as superoxide. Thus, the present observations suggest that SFC may have a potential to reduce mucosal injury by suppressing migration and ROS generation by neutrophils in UC.
INTRODUCTION: Mesalazine(Pentasa) is widely used for ulcerative colitis(UC). However, the anti-inflammatory actions of Mesalazine are not fully understood. In this study, we evaluated the actions of Mesalazine in vitro on the functions of neutrophil, a major inflammatory cell. Furthermore, we evaluated the combined effects of Mesalazine and sodium ferrous citrate(SFC) on neutrophil functions, since SFC is clinically administered to UC patients together with Mesalazine for treatment of chronic anemia associated with UC. METHODS: Superoxide production by neutrophils was measured on the basis of superoxide dismutaseinhibitable cytochrome c reduction. Preincubated neutrophils were stimulated with fMLP, PMA or complement-opsonized zymosan particles. Cytochrome c reduction was calculated by the absorbance difference at 550 nm. Neutrophil migration was measured by a Boyden chamber assay with using zymosan-activated serum (C5a) as a chemotactic factor. Neutrophils were preincubated in the presence of 0.01-0.5mM Mesalazine and then incubated with fMLP. The cells were further incubated with phycoerythrin (PE)-labeled anti-CD11b monoclonal antibody, and the expression of CD11b was analyzed by flow cytometry. Neutrophils were preincubated with 5μg/ml SFC and 0.01-0.1mM Mesalazine, and superoxide generation and migration were measured.RESULTS: Mesalazine dose-dependently inhibited the superoxide production induced by PMA and fMLP at > 0.05 mM. Furthermore, Mesalazine inhibited chemotaxis toward zymosan-activated serum in a dosedependent fashion. Moreover, 0.5mM Mesalazine inhibited the fMLP-induced up-regulation of CD11b expression. Interestingly, Mesalazine combined with SFC inhibited the superoxide generation and migration by neutrophils, compared with Mesalazine alone. Thus, the present observations suggest that a combined administration of Mesalazine and SFC may have a potential to reduce mucosal injury by suppressing neutrophil functions (migration, ROS generation and expression of adhesion molecule) in UC.
Human umbilical cord blood (UCB) cells have many advantages as a source of cell transplantation because of their immaturity compared with adult cells. Here, with a view to utilize UCB for cell transplantation into injured liver, we investigated whether UCB cells have a potential for differentiation into functional hepatocyte-like cells (HLCs).UCB cells were cultured in the primary culture system supplemented with a combination of FGF-1, FGF-2, LIF, SCF and HGF. About 50% of the adherent UCB cells expressed albumin (ALB) and hepatocyte-lineage markers were co-expressed in the ALB-positive cells. Then, in the model for cell transplantation into NOD/SCID mice with transient liver injury, human UCB-derived HLCs were detected in the recipient-livers. And, the UCB-derived HLCs secreted human ALB into the recipient-sera. Moreover, in the model for cell transplantation into SCID mice with chronic liver damage, the engrafted UCB cells appeared at higher frequencies than the model with transient damage, and human ALB was abundantly expressed in UCB-derived HLCs in the recipient-liver.This study provides the evidence that UCB can supply functional HLCs. UCB could be a potential source of cell transplantation for liver injury.
Prostaglandin(PG) D2 and PGE2 are major cyclooxygenase metabolites of arachidonic acid produced during allergic reactions including asthma. However, the role of PGD2 and PGE2 in allergic inflammation has long been ambiguous. This is partly because non-steroidal anti-inflammatory drugs that inhibit prostanoid synthesis are generally ineffective in allergic disorders. Both PGs exert their actions by acting on G-proteincoupled receptors; PGD2 acts at the PGD receptor(DP), PGE2 acts at four subtypes of PGE receptor, EP1 to EP4. To dissect the roles of PGD2-DP pathway and each PGE2-EP pathway in allergic reactions, we subject mice deficient in DP, EP1, EP2, EP3 and EP4 receptor individually to ovalbumin-induced allergic asthma as a model of type I allergy. These studies have revealed that there are opposing actions between two prostanoid pathways in allergic reactions; PGD2-DP pathway and PGE2-EP3 pathway. We found that the PGD2 is an important mediator of allergic responses and that PGE2-EP3 signaling negatively regulates progression of allergic inflammation. This review focuses on the opposite roles of these prostanoid pathways in allergic reactions obtained by our studies and other studies. These findings suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of allergic diseases, such as bronchial asthma, allergic rhinitis and conjunctivitis.
Triggering receptor expressed on myeloid cells(TREM)-1 is a cell surface molecule that has been identified on monocytes and neutrophils and is implicated in the early inflammatory response induced by microbes. However, it has been remained unclear how the expression of TREM-1 is regulated in non-microbial inflammatory diseases. This study was undertaken to evaluate the biologic role of TREM-1 in the initiation of acute attacks of gout. We investigated TREM-1 expression by monosodium urate monohydrate(MSU) crystal-stimulated resident murine peritoneal macrophages(RPM), and assessed whether TREM-1 signaling amplified response to MSU crystals. TREM-1 expression by RPM stimulated with various inflammatory agents was determined by quantitative real-time PCR and western blot analysis. Cytokine production by RPM cultured with both anti-TREM-1 agonist antibody and MSU crystals was assayed by ELISA. TREM-1 expression was significantly induced in RPM by MSU crystals. The peaks of TREM-1 transcript and product occurred rapidly after 1 and 4 hours of MSU crystal stimulation, respectively. The level of TREM-1 transcript induced was consistent with the result obtained using LPS, a potent TREM-1 inducer. Induction of TREM-1 by anti-TREM-1 agonist Ab and MSU crystals synergistically increased production of IL-1 β and MCP-1 by RPM compared with MSU crystals alone. These findings indicate that rapid induction of TREM-1 expression in RPM by MSU crystals may contribute to the onset of acute gouty arthritis, followed by induction of proinflammatory cytokines.
Grepafloxacin is an asymmetric fluoroquinolone derivative, which possesses high tissue penetrability as well as strong, broad-spectrum antimicrobial activities. Human neutrophils actively internalize fluoroquinolones, especially grepafloxacin, and grepafloxacin induces a priming effect on neutrophil respiratory burst produced by fMLP. However the precise mechanism of the uptake and the priming effect are not fully understood. We show here that mitogen activated protein kinase (MAPK) plays a role in the uptake and in the priming effect in neutrophils. Our results strongly suggest that grepafloxacin negatively regulates its uptake in neutrophils, and p38 MAPK activation is involved in this down-regulation of grepafloxacin uptake. The ciprofloxacin uptake is positively regulated by the activation of PKC, and p44/42 MAPK activation is involved in this up-regulation. Neither PKC, nor p38 or p44/42 MAPK is involved in the regulation of ofloxacin uptake. Grepafloxacin stereospecifically primes neutrophil respiratory burst. Stereospecific phosphorylation of p38MAPK, and translocation of p47 and p67 phox proteins are closely related to grepafloxacin priming.
Clostridium difficile (C. difficile) is a major causative agent of antibiotics-associated diarrhea and pseudomembranous colitis in humans. Pathogenic strains of C. difficile release toxin A, which is referred as enterotoxin due to its capacity to disrupt intestinal epithelial structure. Toxin A causes tissue damages directly by glucosylation of Rho and indirectly by inducing massive infiltration of neutrophils. We previously observed that interferon (IFN)-γ-deficient mice exhibited less neutrophil infiltration and tissue damages in several types of acute inflammation in liver. These observations prompted us to explore the roles of IFN-γ in toxin A-induced acute enteritis model. Injection of toxin A into ileal loops caused massive fluid secretion, disruption of epithelial structure, and massive neutrophil infiltration in wild-type (WT) mice, accompanied with increases in IFN-γ mRNA expression and protein contents in the intestine. A double-color immunofluorescence analysis detected IFN-γ protein in infiltrating neutrophils and to a lesser degree, CD3-positive lymphocytes. On the contrary, toxin A failed to induce fluid secretion, disruption of intestinal epithelial structure, and neutrophil infiltration in IFN-γ-deficient mice. Similarly, pretreatment of neutralizing anti-IFN-γ antibody prevented toxin A-induced enteritis. These observations suggest that IFN-γ is a good molecular target for the control of C. difficile-associated pseudomembranous colitis.
Heme oxygenase(HO)-1 is an enzyme, an inducible form of HO, which catalyzes heme into carbon monoxide(CO), Fe2+, and biliverdin. CO suppresses apoptosis and macrophage activation, whereas biliverdin is converted into bilirubin, an antioxidant. Fe2+ stimulates the production of ferritin, a protective protein. Various noxious stimuli such as hypoxia or heavy metal loading induce expression of HO-1, which possesses cytoprotective effects mediated by the heme degradation products. Indeed, forced or chemically induced expression of HO-1 has beneficial effects on respiratory, inflammatory, renal diseases, and inflammatory disorders in animal models. On the other hand, HO-1 deficiency leads to systemic inflammation in mice and a patient. HO-1 is involved in pathogenesis of various diseases. Some of malignant tumors express abundant HO-1, resulting in suppressed apoptosis of tumor cells, whereas reduced expression of HO-1 is implicated in degenerative diseases such as Alzheimer dementia. Our preliminary results showed increased expression of HO-1 in joint lesions of rheumatoid arthritis and circulating leukocytes from patients with adult-onset Still's disease and hemophagocytic syndrome, though the role of HO-1 remains unknown in these diseases. Excessively expressed HO-1 appears toxic in some conditions. HO-1 induction by auranofin and statins has been shown to contribute to the pharmacological effects at least in part. To optimize HO-1 expression may lead to development of novel therapeutic strategies in various diseases including inflammatory disorders.
Vioxx (rofecoxib) was withdrawn from the market because of a higher rate of cardiovascular events. An increased risk of cardiovascular events has also been reported with valdecoxib in patients with coronary artery bypass grafting and in a recent colon cancer trial of high-dose celecoxib.The reason for the propensity of COX-2 inhibitors to facilitate cardiovascular events remains unclear. One possible mechanism is an imbalance between prostacyclin (PGI2, antithrombotic) and thromboxane (TXA2, prothrombotic) since PGI2 production mainly depends on COX-2 activity. However, recent reports demonstrated that low-dose aspirin did not mitigate cardiovascular risk associated with COX-2 inhibitors. Co-administration of aspirin with a COX-2 inhibitor may restore the balance between PGI2 and TXA2 because low-dose aspirin selectively inhibits TXA2 production in platelets. Thus, the failure of low-dose aspirin to attenuate the cardiovascular risk associated with COX-2 inhibitors implies that the COX-2 inhibitor increases the risk of cardiovascular events, at least in part, by mechanisms unrelated to TXA2 production.The heart has a remarkable ability to adapt to various stresses, such as ischemia, hypoxia, and oxidative stress. This is exemplified by ischemic preconditioning (IPC), the phenomenon whereby a sublethal ischemic stress greatly enhances the tolerance of the myocardium to subsequent ischemia. The occurrence of IPC has been shown in almost all mammalian tissues and has also been confirmed in humans, where it may play an important role in protecting the heart from coronary artery disease. We demonstrated that induction of COX-2 plays an obligatory role in the development of IPC, most likely via the production of cardioprotective prostanoids such PGI2 and PGE2. Since then, mounting evidence has accumulated which supports the concept that COX-2 is a cardioprotective protein that alleviates myocardial ischemia/reperfusion injury. We have suggested that induction of COX-2 is a fundamental mechanism whereby the heart adapts to stress. Importantly, low-dose aspirin does not interfere either with COX-2 or with IPC.We propose another mechanism for the detrimental effects of COX-2 inhibitors, namely, that, in addition to their prothrombotic actions, these drugs may deprive the heart of its natural ability to adapt to ischemic stress (i.e., IPC), thereby increasing the susceptibility of the myocardium to infarction and stunning. We suggest that the biological effects of COX-2 may differ depending on the degree of its expression, cellular types where it is expressed (e.g., infiltrating cells vs. cardiac myocytes), and the ability of cells to metabolize COX-2-derived PGH2 into cytoprotective prostanoids.
Treatment of defects of articullar cartilage remains a problem in orthopedic surgery because cartilage has little self-healing capability. Recently a new approach for repair of articular cartilage has appeared using tissue engineering to create cartilage-like tissues in a three-dimensional scaffolding with autologous chondrocytes. However, the number of autologous chondrocytes for autograft is limited. To solve this problem, mesenchymal stem cells (MSCs) sourced from bone marrow have been investigated as candidates for producing chondrocytes. One of the possible sources may be the placenta thrown away as a medical waste after the collection of cord blood. In this report, we determined the potential of chondrogenic differentiation of mesenchymal progenitor cells derived from chorionic villi of fetal human placenta (PDMSCs). Our results show that PDMSCs include cells which have chondrogenic differentiation potential and may serve as an alternative source of cells for repair of articular cartilage.
It has been increasingly recognized that Chlamydia pneumoniae may be involved in the pathogenesis of atherosclerosis; accordingly, detection of this bacterium in blood from atherosclerotic patients may play an important role in diagnosis of possible complications of atherosclerosis including stroke. However, there is little understanding about the detection incidence of C. pneumoniae in the blood of stroke patients. In the present study, we performed the PCR specific for C. pneumoniae 16S rRNA to determine the C. pneumoniae DNA in peripheral blood mononuclear cells (PBMCs) obtained from patients with atherothrombotic infarction. C. pneumoniae DNA was detected in 5 out of 13 blood samples from the patients with atherothrombotic infarction. These results indicate a possible correlation between C. pneumoniae infection and atherothrombotic infarction attributable to atherosclerosis. Rec.5/24/2004, Acc.9/6/2004, pp55-59 Departments of 1)General Internal Medicine, 2)Neurology and 3)Microbiology, St. Marianna University School of Medicine 4)Division of Molecular Microbiology, Department of Basic Laboratory Science, Osaka University Graduate School of Medicine *Present address: Marine Biology and Ecology Research Program, Japan Agency for Marine-Earth Science and Technology
再生医療の臨床応用の中でも角膜培養移植はその最先端を行っており,東京歯科大学眼科,京 都府立医科大学,愛媛大学,大阪大学などすでに国内数施設で実施されている.細胞のリソース として片眼性の場合は健眼から採取が可能で,自己による細胞のため拒絶反応の問題はない.一 方,両眼が障害を受けた場合は,アイバンクによるアロのドナー角膜や親類縁者からの細胞提供 によったが,免疫抑制剤やステロイドの長期使用が必須であり,眼圧上昇などの合併症の問題が あった.これに対して2004年には京都府立医科大学,大阪大学と両施設において口腔粘膜上皮細 胞から角膜様の上皮細胞に分化させて眼表面の治療に成功し,特に大阪大学Nishida らのNew England Journal of Medicineに掲載された“温度応答性培養上皮シート”は大きな話題を呼んだ. 口腔粘膜上皮から角膜上皮細胞様へ細胞の性質が変化するtrans differentiationの可能性としても興 味深いとともに,現実的な応用価値が高いからである. 炎症関連でも最近角膜において大きな進歩があった.従来角膜移植の成功率が高く,拒絶反応 が起きないのは,角膜はimmuno previledged siteであるからだ,と説明されてきた.その一つの理 由として,角膜は無血管組織であり,ランゲルハンス細胞などの抗原提示細胞が存在しないため である.そうだとすると,移植された角膜は必ずドナーの抗原提示細胞によって抗原提示される ことになる.ところがレシピエントの抗原提示細胞が存在しないと理屈に合わない現象もあり混 乱を招いていた.ハーバード大学のDanaらは,角膜にはHLAクラスII抗原が陽性の典型的なラ ンゲルハンス細胞は存在しないものの,HLAクラスII抗原の発現していない特殊なタイプの抗原 提示細胞が存在することを発見した.この抗原提示細胞は炎症によってクラスIIを発現するよう になり,通常のランゲルハンス細胞と同様の抗原提示を行えるようになる.角膜移植は通常拒絶 反応が起きないが,炎症があると抗原提示細胞が働き始め,同時に免疫寛容が失われる.風邪を ひいたり,流行性角結膜炎やヘルペス角膜炎に罹患すると,とつぜん角膜移植後拒絶反応が起き るのはこのためだったのである. 再生医療によって臓器,組織が再生されたとしても長期のサバイバルのためには炎症のコント ロールはどうしても欠かせない.アロの組織を用いた場合は前述のような拒絶反応も大きなファ クターだが,自己の細胞を使った場合においても炎症のコントロールは欠かせない.体はもとよ り,組織の老化(エイジング)は炎症に密接に結びついていることがわかってきたからだ.現在 加齢と最も関連の深いといわれる動脈硬化,II型糖尿病,アルツハイマー病,パーキンソン病な どは遷延した炎症によって引き起こされると考えられている.正常の組織でさえ,炎症で障害を
Efficient gene transfer into stem cells which are able to self-renew and differenciate into certain type of cell is essential for not only difining the precise molecular mechanism of self-renewal and differenciation, but the supplying of the cells for regenerative medicine. In this paper, we review our approach to the efficient gene transfer into hematopoietic stem cell, mesenchymal stem cell, and ES cell by modified adenovirus vectors.
Embryonic stem(ES) cells have a potential to differentiate into various progenitor cells. In this review we investigated the capacity of mouse ES cells to differentiate into renal tubular cells. After stably transfecting Wnt4 cDNA to mouse ES cells(Wnt4-ES cells) by electroporation, the ES cells were incubated by the hanging drop culture method without leukemia inhibitory factor(LIF) to induce differentiation to embryoid bodies(EBs).During the culture of the EBs derived from the Wnt4-ES cells, aquaporin-2(AQP2) mRNA and protein were expressed within 15-20 days after the removal of LIF. The expression of AQP2 in Wnt4-EBs was enhanced in the presence of hepatocyte growth factor(HGF) and activin A. We observed AQP2 positive tubular-like formation from Wnt4-EBs in three-dimensional culture. Our result shows that two new findings: firstly, that cultured Wnt4-EBs have an ability to differentiate into renal tubular-like cells; and secondly, that Wnt4, HGF, and activin A may promote the differentiation of ES cells to renal tubular-like cells.
The neurosphere method is widely used to examine self-renewal activity and the multipotency of neural stem cells (NSCs). We developed an efficient method for introducing a transgene into neurospheres using a retroviral vector and isolating a transgenic subpopulation of NSCs labeled with green fluorescent protein (GFP) using fluorescence-activated cell sorting, enabling these cells to be used in subsequent biological assays. This method is expected to be useful for studying the function of genes in NSCs.
It is important to prepare the cell culture devices with functional polymer surfaces. Notch ligand delta-1 is considered as one of the important membrane proteins for self-renewal of hematopoietic stem cells. In this study, a polymer matrix immobilized with Notch ligand delta-1 was prepared by photo-immobilization technique for culture the stem cells. It has been reported that photo-reactive polymers bearing azidophenyl groups could immobilize functional proteins without losing their biological activity. Therefore, a novel photo-reactive phospholipid polymer, 2-methacryloyloxyethyl phosphorylcholine polymer bearing azidophenyl groups, was applied as the matrix to conjugate with Notch ligand delta-1. A leukemia cell line, TMD7, was cultured on the bioconjugated polymer surface. The polymer surface immobilized with Notch ligand delta-1 was recognized by TMD7, and the cells efficiently grow on the phospholipid polar group concentrated surface (PC surface) with protein. It was considered that the PC surface provided a suitable environment around the membrane proteins without denaturation. The photo-reactive phospholipid polymer was expected to constitute on in vitro niche to culture hematopoietic stem cells.
