J Clin Invest. 2005; 115: 1607–1615.

Bacterial programmed cell death of cerebral endothelial cells involves dual death pathways
Daniela Bermpohl,1 Annett Halle,1 Dorette Freyer,1 Emilie Dagand,1 Johann S. Braun,1 Ingo Bechmann,2 Nicolas W.J. Schröder,3 and Joerg R. Weber1
1Department of Neurology, 2Department of Cell Biology and Neurobiology, Center of Anatomy, and 3Institut für Mikrobiologie und Hygiene, Charité — Universitätsmedizin Berlin, Berlin, Germany.
Address correspondence to: Joerg R. Weber, Department of Neurology, Charité — Universitätsmedizin Berlin, Schumannstr. 20/21, 10117 Berlin, Germany. Phone: 49-30-450-560104; Fax: 49-30-450-560942; E-mail: joerg.weber@charite.de.


Major barriers separating the blood from tissue compartments in the body are composed of endothelial cells. Interaction of bacteria with such barriers defines the course of invasive infections, and meningitis has served as a model system to study endothelial cell injury. Here we report the impressive ability of Streptococcus pneumoniae, clinically one of the most important pathogens, to induce 2 morphologically distinct forms of programmed cell death (PCD) in brain-derived endothelial cells. Pneumococci and the major cytotoxins H202 and pneumolysin induce apoptosis-like PCD independent of TLR2 and TLR4. On the other hand, pneumococcal cell wall, a major proinflammatory component, causes caspase-driven classical apoptosis that is mediated through TLR2. These findings broaden the scope of bacterial-induced PCD, link these effects to innate immune TLRs, and provide insight into the acute and persistent phases of damage during meningitis.

急性肺傷害レビュー Lancet. 2007;369:1553-64

Wheeler AP, Bernard GR.
Acute lung injury and the acute respiratory distress syndrome: a clinical review.
Lancet. 2007 May 5;369(9572):1553-64. Review.

急性肺傷害の総説です。
是非，読まれてみてください。

培養細胞からの細胞質タンパクおよび細胞膜タンパクの抽出

培養細胞からの細胞質タンパクおよび細胞膜タンパクの抽出（内御堂 亮・松田直之）

【準　備】

１． 作業はすべて氷上で行うため，十分量の氷を用意する。
２． －80℃で凍結していた細胞を4℃冷蔵庫で解凍する。
３． プロテアーゼインヒビタカクテル（SIGMA）を室温で解凍する。（時間がかかるので，まず，解凍を優先する。一度，解凍したものは10µLずつPCR用サンプリングチューブに小分けし，-20℃で専用ボックス内に保存する。）
４． 遠心機の温度を4℃に設定しておく。（温度が下がるのに時間がかかる可能性があるので，まず，やっておくこと。）
５． 十分量のHEPSバッファー（pH 7.5）を氷の中で冷やす。
６． ダウンス型ホモゲナイザを超純水で洗い，氷上で冷やしておく。
７． 廃液用のバッド，および，洗いに使う超純水ボトルを用意する。
８． ホモゲナイズのために用いる氷入りビーカーを用意する。（ホモゲナイザーが入る背の高い300~500 mLレベルの大きさのビーカーを選ぶとよい。）
９． ホモゲナイズしたサンプルを回収するエッペンドルフチューブを，サンプル群数の2倍数用意し，最終的保存に用いるチューブにはテプラⓡで作成した保存用のラベルを張る。（テプラⓡ：3行記載推奨；サンプル内容，群の特徴，作成日）（例：HMVEC，Glu 200 mg/dL 24h，07/06/12）

【全タンパク抽出】
作業はすべて氷上4℃で行うことを厳守すること。

１． 解凍した細胞が入ったマイクロチューブにHEPSバッファを300 μＬ入れる。
２． 続けてプロテアーゼインヒビターカクテルを3 μＬ入れる。
３． マイクロチューブ内の細胞をピペッティングする。
４． ピペッティングした細胞をピペットで回収し，ダウンス型ホモゲナイザに入れる。
５． ダウンス型ホモゲナイザで，上下20回，細胞をホモゲナイズする。すりつぶす感覚で行う。
６． 泡がおさまるまで，氷上でしばらく待つ。
７． 泡がおさまったら内容物をピペットで回収し，用意していたマイクロチューブに入れる。
８． ホモゲナイザーを超純水で洗う。（壷は超純水で最低3回は洗い，棒は一度キムワイプで付着物をふき取ってから，超純水でよく洗う）
９． 新しいサンプルで，2～8の肯定を繰り返す。
１０． ホモゲナイズされたサンプルを以下の条件で，遠心分離する。条件：4℃，600 g，10分間。松田研究グループは，チューブのヒンジを外側にするよう統一している。
１１． 遠心の間に，上清を回収するためのマイクロチューブを氷上に用意する。
１２． ヒンジの下の沈殿物を確認し，上清を回収し，全タンパクとする。沈殿物は核分画と未破砕細胞として，-80℃に保存する，あるいは，核タンパク回収系に持ち込む。

基礎研究 当教室におけるzVAD-fmkに関する敗血症研究における留意点

カスパーゼ阻害薬の使用によって，アポトーシスに代わる予備の細胞死プログラムの存在が明らかになってきている。広域スペクトルのカスパーゼ阻害薬zVAD-fmkは，3種類の主要な細胞死を調節するが，zVAD-fmkの添加により，アポトーシス性細胞死が阻害され，細胞の壊死性細胞死に対する感受性が高まり，自己貪食性細胞死が誘発される。いくつかの研究により，RIP1およびアデノシンヌクレオチド輸送体（ANT）-サイクロフィリンD（CypD）複合体が壊死性細胞死に重要な役割を果たすことが示されている。zVAD-fmkを介する壊死性細胞死の感作のもとになる機構には，カスパーゼ-8を介するRIP1の分解の抑制と，ANT-CypD相互作用の阻害が関与している。RIP1は，自己貪食性細胞死に関与していることが知られている。壊死性および自己貪食性細胞死の双方でRIP1は正の役割，カスパーゼ-8は負の役割を果たすことから，これら2種類の細胞死経路は相互に関連があると推測されている。壊死性細胞死は，ミトコンドリアの活性酸素種（ROS）産生，アデノシン三リン酸濃度の低下などの細胞傷害に関わる迅速な細胞応答を意味する。一方で，自己貪食性細胞死の主体は，ROSに傷害されたミトコンドリアを除去することによって生き残ろうとする試みとして始まる。しかし，この過程が過剰に起こると，自己貪食そのものが細胞傷害性となり，最終的には自己貪食性細胞死に至る。これらの代替的な細胞死経路の分子機構をさらに解明することによって，虚血-再灌流障害，感染症，神経変性疾患などに伴う細胞死に対して有効な治療手段が得られる可能性があり，細胞傷害性の新たな治療戦略の開発が進む可能性がある。

Sci. STKE, 24 October 2006
Vol. 2006, Issue 358, p. pe44
[DOI: 10.1126/stke.3582006pe44]

PERSPECTIVES
Caspase Inhibitors Promote Alternative Cell Death Pathways
Peter Vandenabeele*, Tom Vanden Berghe, and Nele Festjens

Molecular Signalling and Cell Death Unit, Department for Molecular Biomedical Research, Flanders Interuniversity Institute of Biotechnology (VIB) and Ghent University, Fiers-Schell-Van Montagu Building, Technologiepark 927, B-9052 Ghent, Belgium.

Abstract: The use of caspase inhibitors has revealed the existence of alternative backup cell death programs for apoptosis. The broad-spectrum caspase inhibitor zVAD-fmk modulates the three major types of cell death. Addition of zVAD-fmk blocks apoptotic cell death, sensitizes cells to necrotic cell death, and induces autophagic cell death. Several studies have shown a crucial role for the kinase RIP1 and the adenosine nucleotide translocator (ANT)–cyclophilin D (CypD) complex in necrotic cell death. The underlying mechanism of zVAD-fmk–mediated sensitization to necrotic cell death involves the inhibition of caspase-8–mediated proteolysis of RIP1 and disturbance of the ANT-CypD interaction. RIP1 is also involved in autophagic cell death. Caspase inhibitors and knockdown studies have revealed negative roles for catalase and caspase-8 in autophagic cell death. The positive role of RIP1 and the negative role of caspase-8 in both necrotic and autophagic cell death suggest that the pathways of these two types of cell death are interconnected. Necrotic cell death represents a rapid cellular response involving mitochondrial reactive oxygen species (ROS) production, decreased adenosine triphosphate concentration, and other cellular insults, whereas autophagic cell death first starts as a survival attempt by cleaning up ROS-damaged mitochondria. However, when this process occurs in excess, autophagy itself becomes cytotoxic and eventually leads to autophagic cell death. A better understanding of the molecular mechanisms of these alternative cell death pathways may provide therapeutic tools to combat cell death associated with neurodegenerative diseases, ischemia-reperfusion pathologies, and infectious diseases, and may also facilitate the development of alternative cytotoxic strategies in cancer treatment.

Circulation. 2007;115:1371-1375

Glucose Levels Predict Hospitalization for Congestive Heart Failure in Patients at High Cardiovascular Risk
C. Held, MD, PhD; H.C. Gerstein, MD, MSc; S. Yusuf, MD, DPhil; F. Zhao, MSc; L. Hilbrich, MD; C. Anderson, MBBS, PhD, FRACP; P. Sleight, MD; K. Teo, MD, PhD, for the ONTARGET/TRANSCEND Investigators
From the Karolinska Institutet, Department of Medicine, Unit of Cardiology, Karolinska University Hospital, Stockholm, Sweden (C.H.); Population Health Research Institute, Hamilton General Hospital, McMaster Clinic, Hamilton, Ontario, Canada (C.H., H.C.G., S.Y., F.Z., K.T.); Boehringer Ingelheim Pharma GmbH & Co KG, Ingelheim am Rhein, Germany (L.H.); The George Institute, Royal Prince Alfred Hospital, Sydney, Australia (C.A.); and John Radcliffe Hospital, Oxford, UK (P.S.).

Correspondence to Claes Held, Karolinska Institutet, Department of Medicine, Unit of Cardiology, Karolinska University Hospital, 17176 Stockholm, Sweden.



Background— Patients with diabetes mellitus (DM) are at high risk of developing congestive heart failure (CHF). However, the relationships between glucose levels and CHF in people with or without a history of DM have not been well characterized.

Methods and Results— We evaluated the associations between fasting plasma glucose and risk of hospitalization for CHF during follow-up in patients at high cardiovascular risk and without CHF enrolled in a large-scale clinical trials program. Baseline fasting plasma glucose levels were assessed in 31 546 high-risk subjects with 1 coronary, peripheral, or cerebrovascular disease or DM with end-organ damage who are participating in 2 ongoing parallel trials evaluating the effects of telmisartan, ramipril, or their combination (Ongoing Telmisartan Alone and in Combination With Ramipril Global Endpoint Trial [ONTARGET]; n=25 620) and the effects of telmisartan against placebo in angiotensin-converting enzyme–intolerant patients (Telmisartan Randomized Assessment Study in ACE Intolerant Subjects With Cardiovascular Disease [TRANSCEND]; n=5926). Interim analyses blinded for randomized treatment were performed to compare baseline fasting plasma glucose with the adjusted CHF event rate at a mean follow-up of 886 days. Multivariable Cox regression models were performed, and associations were reported as hazard ratios and 95% confidence intervals. Among all subjects (mean age, 67 years; 69% men), of whom 11 708 (37%) had known DM and 1006 (3.2%) had newly diagnosed DM at baseline, 668 patients were hospitalized for CHF during follow-up. After adjustment for age and sex, a 1-mmol/L-higher fasting plasma glucose was associated with a 1.10-fold-increased risk of CHF hospitalization (95% confidence interval, 1.08 to 1.12; P<0.0001). The association persisted after adjustment for age, sex, smoking, previous myocardial infarction, hypertension, waist-to-hip ratio, creatinine, DM, and use of aspirin, ß-blockers, or statins (hazard ratio, 1.05; 95% confidence interval, 1.02 to 1.08; P<0.001). Conclusions— Fasting plasma glucose is an independent predictor of hospitalization for CHF in high-risk subjects. These data provide theoretical support for potential direct beneficial effects of glucose lowering in reducing the risk of CHF and suggests the need for specific studies targeted at this issue.


Kaplan-Meier estimates of the proportion of patients with hospitalization for CHF divided into classes of glycemia at baseline (log rank P<0.001). IFG indicates impaired fasting glucose.

PRKAG2

PRKAG2 cardiomyopathy in the context of glycogen metabolism and the other muscle glycogenoses. Glycogen is a branched glucose polymer containing 93% 1-4 bonds and 7% 1-6 bonds and contains a protein core. It constitutes an immediate reserve of glucose for glycolysis under intense muscle activity or reduced energy supply. Glycogen undergoes a constant turnover: new units are being added by glycogen synthase and brancher enzymes, or being broken down by glycogen phosphorylase and debrancher enzyme. Aged glycogen is degraded in the lysosome. Classic glycogen storage disorders result from deficiency in glycogen-degrading enzymes and are inherited as autosomal recessive or X-linked trait. Prominent cardiac involvement is associated with most glycogenoses (red). Phosphorylase deficiency, phosphorylase kinase deficiency, and debrancher enzyme deficiency cause cytoplasmic glycogen accumulation and often manifest on exercise as myalgia and myoglobinuria resulting from inability to metabolize glycogen. In brancher enzyme deficiency, an abnormal nonsoluble glycogen polymer is created, leading to polyglucosan deposits. Lysosomal glycogen storage diseases cause insidious accumulation and may manifest as cardiac hypertrophy often associated with electrophysiological abnormalities as well as myopathy. Lysosomal acid maltase deficiency causes Pompe disease. Lysosomal-associated membrane protein II defects lead to glycogen deposits in the context of generalized lysosomal dysfunction. PRKAG2 cardiomyopathy is characterized by cytoplasmic glycogen accumulation attributable to dysregulated metabolism. Unlike other diseases, there is no enzyme deficiency and the heart can reuse this glycogen on appropriate stimulation. The glycogen stored is less branched, having some features reminiscent of polyglucosan.109 The model is reproduced by permission of the Massachusetts Medical Society from Arad M, Maron BJ, Gorham JM, Johnson WH Jr, Saul JP, Perez-Atayde AR, Spirito P, Wright GB, Kanter RJ, Seidman CE, Seidman JG. Glycogen storage diseases presenting as hypertrophic cardiomyopathy. N Engl J Med. 2005;352:362–372.