我々は、ビデオの製作と優秀な支援のためのジャンニシモーネに感謝しています。

HIVに感染した血液サンプルの取り扱いが重要な推奨事項が必要です。 血液のすべての標本は、堅牢な漏れ防止のコンテナで輸送する必要があります。コンテナの外装のと試料に付随する事務処理の汚染を避けるために標本を収集する際には注意が必要です。 感染した血液を処理するすべての者は、手袋を着用する必要があります。手袋が変更され、手は、検体処理の完了後に洗浄する必要があります。 HIV感染血液サンプルの処理は、クラスIIバイオハザードキャビネットのフードで行う必要があります。 機械式ピペットエイドを使用する必要があります。 針や他の鋭利（ガラスなどのピペットやキャピラリーチューブを含む）の使用は選択肢が存在しない状況に限定されている必要があります。 実験室の表面は血の流出後の適切な化学消毒剤で除染する必要があり、作業活動が完了しているとき。 使用される汚染された材料は再使用前に除染する必要がありますまたは臨床廃棄物の経路を経由して正しく廃棄する必要があります。 介入が効果的であることが速やかに開始しなければならないとして、潜在的に感染血液や体液（すなわち、それらの必要とする普遍的予防策）への職業ばく露のすべての事件は、医療上の緊急事態として扱う必要があります。 プロトコルは、その完成のための5日間が必要です。時間枠は、図1に詳述されています。 細菌種は、図2で説明した方法を用いて識別されます。 1。サンプルコレクション<ol><li>全血9mlのは、EDTAを含む試験管に描画されます。 </li><li>チューブは、プラズマを得るために、室温で10分間2000rpmで遠心分離されています。 </li><li>血漿は、無菌の2ミリリットルエッペンドルフチュー​​ブに回収される。 </li></ol> 2。血漿サンプルからのDNA抽出<ol><li>適切に無菌性を保証するために、実験に必要なフード、ピペットと材料を消毒する。 </li><li>少なくとも30分間UVライトの下にすべての材料を合わせます。 </li><li>消毒と手袋を拭きます。 </li><li>エタノールのいくつかの紙タオルを浸し、ボンネットの下に配置します。たびに先端がウェットペーパータオルの上でピペットを拭いて破棄されます。 </li></ol> DNAは、製造業者の指示（簡単DNAキット、Invitrogen社、カールスバッドカリフォルニア州、米国）以下の市販のキットを使用して抽出されます。 <ol start="5"><li>血漿350μLを滅菌した2ミリリットルエッペンドルフチュー​​ブに配置されます。 </li><li>フィルタ処理された超純水350μLを陰性コントロールとして使用されています。 </li><li>サンプルにリゾチーム（1mg/ml）を10μLを加える。 </li><li> 37℃で30分間インキュベート℃に</li><li>のLysis Solution 500μLを追加し、サンプルに軽く混合します。 </li><li> 65℃7分間インキュベート℃に</li><li>サンプルにクロロホルム900μLを加える。 </li><li>サンプルが均一に粘性になるまで激しくボルテックスする。 </li><li>降水量のソリューションと激しくボルテックス200μLを加える。 </li><li>相を分離し、インタフェースを形成するために室温で10分間、10500 rpmで試料を遠心分離します。 </li><li>エタノール1 mlの100％を含む新しい微量遠心管に上部の水相を転送します。 </li><li> 4℃で10分間、10500 rpmで試料を遠心℃に</li><li>エタノールを削除します。 </li><li> 70％エタノールを1mlを追加。 </li><li> 4℃で10分間、最高速度で試料を遠心℃に</li><li>エタノールを削除します。残留エタノールはpipettatorで削除する必要があります。 </li><li>超純水50μLでペレットを再懸濁する。 </li><li>分光光度計でDNA濃度を読み取る。 </li></ol> 3。 16S rRNA遺伝子のPCR PCR増幅は、前述の9として実行されます。 <ol><li> 10 × PCR緩衝液の10μL、25 mMのMgCl 2の5μL、2mMの合計のdNTPs 5μL、50μMのプライマーRW01（AACTGGAGGAAGGTGGGGAT）、50μMのプライマーDG74の1μLの1μLから成る100μL反応混合物中のDNAを増幅（AGGAGGTGATCCAACCGCA）、TaqポリメラーゼのH20および0.5μL（のAmpliTaqゴールド、アプライドバイオシステム、フォスターシティー、CA、米国）34μL。 </li><li>細菌のTaqポリメラーゼによって可能な汚染を避けるためにフィルタにPCRミックスを転送します。 </li><li> 4で500 rcfで30分間、遠心フィルター（マイクロコン、ミリポア、ビルリカ、MA、米国）℃ </li><li>アリコート（正と負のPCRコントロール、試料と抽出された水）、増幅する必要のあるサンプル数に応じてPCRミックス。 </li><li>以下のサーマルサイクラー条件使用：94℃で10分間、94℃1分ごとに時間の40倍° C、55 ° Cおよび72 ° C、および72℃で10分</li><li> 2％アガロースゲルでPCR産物を可視化する。 100 bpのDNAラダーを使用してください。 PCR産物のサイズは360 bpの（図3）についてです。 </li></ol> 4。 PCR産物の精製唯一のPCR陽性検体は精製されるべきである。精製は、使用して実行されますメーカーの指示（PureLink PCR microkit、Invitrogen社、カールスバッドカリフォルニア州、米国）以下の市販のキット。 <ol><li> PCR産物の1ボリュームあたりのイソプロパノールを含む結合緩衝液の4容量を追加。 </li><li>列への結合バッファーでPCR産物を転送する。 </li><li> RTで10000 RCFで1分間遠心します。 </li><li>エタノールを含有Wash Bufferでカラムを洗浄します。 </li><li> RTで10000 RCFで1分間遠心します。 </li><li>シリカメンブレンを乾燥し、エタノールで残存する洗浄バッファーを除去するために、室温で1分間最大速度で遠心する。 </li><li>超純水10μLを加える。 </li><li>室温で1分間インキュベートする。 </li><li>精製されたDNAを回収するために室温で2分間、最高速度で遠心する。 </li></ol> 5。クローニング溶原性ブロス（LB）プレートの準備<ol><li>水の約800 mlのLBミックスの25グラムを溶かす。 </li><li> 1リットルの最終容量をもたらす</li><li> Bactoagarの15グラムを追加。 </li><li> 20分間オ​​ートクレーブ。 </li><li>オートクレーブした後、精力的にスワールフラスコ内の溶液は、溶融寒天を混合する。 </li><li> 50℃にソリューションを冷却</li><li>アンピシリン（50μg/ mlの）と渦巻き、それが溶けるまでを追加。 </li><li>約3mmの深さに板を注ぐ。 </li><li>室温でプレートを残す。 </li><li> 37各LBプレートとインキュベートのスプレッドはX - Gal（40mg/ml）とIPTG（100 mM）を° Cの使用の準備ができるまで。 </li></ol>コンピテントセルの形質転換変換は、市販のキット以下のメーカーの指示に従って（TOPO TAクローニングキット、Invitrogen社、カールスバッドカリフォルニア州、米国）を使用して実行されます。 <ol><li>クローニング反応ミックスを（新鮮なPCR産物1-4μL、塩溶液の1μL、ベクトルと水の1μL必要に応じて）用意してください。 </li><li>穏やかに反応を混合し、室温で5〜10分間インキュベートする。 </li><li>氷上で反応を置きます。 </li><li>コンピテントセルのバイアルにクローニング反応の2μLを加え、穏やかに混ぜる。 </li><li> 30分間氷上でインキュベートする。 </li><li> 42℃で30秒間熱ショック細胞℃に</li><li>直ちに氷中にチューブを移す。 </li><li>培地250μLを加える。 </li><li> tighlyチューブにフタをし、37℃で水平にチューブを振る℃で1時間。 </li><li>予熱した選択プレート上の各変換から50μLを広げる。 </li><li> 37℃で一晩インキュベート℃、 </li></ol>インキュベーション後、白色と青色のコロニーがプレート上で開発しています。白いコロニーがPCR産物の挿入のために正であり、青色のコロニーは、PCR産物の挿入のために陰性である。 6。配列解析シークエンシング反応<ol><li> 2,5μlのDNA、1μlのプライマー（μL/ 5pmol）1,1μlのBigDye（アプライドバイオシステム、フォスターシティー、CA、米国）：ミックスは、これらの試薬を用いて行われます。 </li><li>サーマルサイクラーの条件：96℃で1分間、4分15秒と60℃で10秒、55℃に対して96℃の場合は25回。 </li></ol>カラム精製<ol><li>各反応について、あるマイクロスピンカラム（キアゲン、、ミラノ、イタリア）を準備する。 </li><li>樹脂を混合する列と渦を反転。 </li><li>カラムの底部を折ります、マイクロ遠心チューブに蓋¼回転、および位置の列を緩めます。 </li><li> 1分間3200 rpmで遠心する。 </li><li>きれいな微量遠心チューブにカラムを移し。 </li><li>慎重にピペット列の中心に全体のPCRシークエンス反応。 </li><li> 1分間3200 rpmで遠心する。 </li><li>精製したDNAは、チューブ（超純水20μl）にeluiteなります。 </li></ol>配列解析<ol><li> 96マルチウェルプレートに、精製サンプル（20μl）をロードする。 </li><li>シーケンサーにプレートをロードする。自動DNAシーケンサは、シーケンスの実行の結果を示す4つの色のクロマトグラムを生成する。 </li><li>公共シーケンスデータベースに対してクエリとしてヌクレオチド配列入力。検索は基本的なローカル配列検索ツール（BLAST）で、NCBIのデータベースとサーバー上で実行されます。 </li><li> 98から100パーセントの相同性と細菌だけを考えてみましょう。 </li></ol> 7。代表的な結果<img src="/files/ftp_upload/2830/2830fig1.jpg" alt="図1" /> 図1の手順のタイムライン。 <img src="/files/ftp_upload/2830/2830fig2.jpg" alt="図2" /> 図2。全体の細菌同定の手順のフローチャート。 <img src="/files/ftp_upload/2830/2830fig3.jpg" alt="図3" /> 図3。幅広い範囲の16S rRNA遺伝子のPCR産物を示す、2％アガロースゲル。レーン1は100ベーシスポイントのDNAラダーを含む、レーン2はPCR陽性コントロールが含まれている、レーン3は負のPCRコントロールを示しています。レーン4は、HIV陽性の患者からサンプルを示し、レーン5は水が含まれています。レーン6 SH健康な人と否定的なPCR反応からのOWSのサンプル、レーン7は、水を含んでいます。唯一のHIV陽性患者では、肯定的なPCR増幅が表示されます。抽出工程中に、ネガティブコントロールとして使用される超純水は、コンタミネーションが発生していないことを示します。 <img src="/files/ftp_upload/2830/2830fig4.jpg" alt="図4" /> プラスミドを示す図4。0.7％アガロースゲルでは、ミニプレップの手順で抽出した。レーン1は1キロベースのDNAラダーを含む、レーン2は3〜12は、白色のコロニーを含んでいる車線、青いコントロールコロニーが含まれています。青いコロニーからのプラスミドは、PCR産物のインサートが含まれていません。すべての10個の白色コロニーが正しいインサートを持つプラスミドが含まれています。 <img src="/files/ftp_upload/2830/2830fig5.jpg" alt="図5" /> 図5は、1つHIV陽性の個体からの血漿中の細菌のシークエンス解析の例を示します。我々の結果は、HIV感染症の微生物の転座は、細菌のトランスロケーションを制御する腸管免疫の実質的な障害を示唆するHIV陰性の被験者ではない、と思われているpolimicrobic植物を、含むことを示している。

<ol>
	<li>Hooper, L. V., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+adaptations+that+maintain+homeostasis+with+the+intestinal+microbiota.">Immune adaptations that maintain homeostasis with the intestinal microbiota.</a> Nat Rev Immunol. 10, 159-169 (2010).</li><li>Macpherson, A. J., Harris, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+commensal+intestinal+bacteria+and+the+immune+system.">Interactions between commensal intestinal bacteria and the immune system.</a> Nat Rev Immunol. 4, 478-485 (2004).</li><li>Kanauchi, O., Mitsuyama, K., Araki, Y., Andoh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+intestinal+flora+in+the+treatment+of+inflammatory+bowel+disease.">Modification of intestinal flora in the treatment of inflammatory bowel disease.</a> Curr Pharm Des. 9, 333-346 (2003).</li><li>Brenchley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+translocation+is+a+cause+of+systemic+immune+activation+in+chronic+HIV+infection.">Microbial translocation is a cause of systemic immune activation in chronic HIV infection.</a> Nat Med. 12, 1365-1371 (2006).</li><li>Brenchley, J. M., Price, D. A., Douek, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+disease:+fallout+from+a+mucosal+catastrophe.">HIV disease: fallout from a mucosal catastrophe.</a> Nat Immunol. 7, 235-239 (2006).</li><li>Jiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+levels+of+bacterial+DNA+correlate+with+immune+activation+and+the+magnitude+of+immune+restoration+in+persons+with+antiretroviral-treated+HIV+infection.">Plasma levels of bacterial DNA correlate with immune activation and the magnitude of immune restoration in persons with antiretroviral-treated HIV infection.</a> J Infect Dis. 199, 1177-1185 (2009).</li><li>Marchetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+translocation+is+associated+with+sustained+failure+in+CD4++T-cell+reconstitution+in+HIV-infected+patients+on+long-term+highly+active+antiretroviral+therapy.">Microbial translocation is associated with sustained failure in CD4+ T-cell reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy.</a> AIDS. 22, 2035-2038 (2008).</li><li>Marchetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Microbial+Translocation+and+Immune+Hyperactivation+in+Disease+Progression+of+HIV++Patients+with+Preserved+CD4+Count+in+the+Absence+of+ART.">Role of Microbial Translocation and Immune Hyperactivation in Disease Progression of HIV+ Patients with Preserved CD4 Count in the Absence of ART.</a> , (2010).</li><li>Greisen, K., Loeffelholz, M., Purohit, A., Leong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+primers+and+probes+for+the+16S+rRNA+gene+of+most+species+of+pathogenic+bacteria,+including+bacteria+found+in+cerebrospinal+fluid.">PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.</a> J Clin Microbiol. 32, 335-351 (1994).</li></ol>

利害の衝突は宣言されません。

<p class="jove_content"&gt;我々はここにHIV感染者の末梢血中の細菌の転座を特徴づけるためにPCR /シーケンシングプロトコールを示す。</p&gt;<p class="jove_content"EDTA含有チューブに集められた血漿を使用するときに&gt;最も信頼性の高い結果が得られる。採血後、血漿が溶血し、DNA断片の劣化を避けるために、2 / 3時間以内に遠心分離によって分離されるべきである。サンプルは-80℃で、約5日間と℃、-20℃で最初に格納さ...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>イースティ- DNAキット</td><td>インビトロジェン</td><td> K 180001 </td><td></td></tr><tr><td>クロロホルム</td><td>シグマアルドリッチ</td><td> C2432 </td><td></td></tr><tr><td>エタノール</td><td>シグマアルドリッチ</td><td> E7203 </td><td></td><tr/><tr><td>超高純度Waer </td><td>インビトロジェン</td><td> 10977049 </td><td></td></tr><tr><td>リゾチーム</td><td>フルカ</td><td> 62970 </td><td>蒸留水で10μg/ mLの</td></tr><tr><td>のAmpliTaqゴールド</td><td>アプライドバイオシステム</td><td> 26478701 </td><td></td></tr><tr><td>マイクロコントローラ100 </td><td>ミリポア</td><td> 42413 </td><td></td></tr><tr><td> PCRプライマー</td><td>インビトロジェン</td><td></td><td></td></tr><tr><td>アガロース</td><td>エッペンドルフ</td><td> C1343 </td><td></td></tr><tr><td> DNAラダー</td><td>インビトロジェン</td><td> 15628019 </td><td></td></tr><tr><td> Purelink PCRマイクロキット</td><td>インビトロジェン</td><td> K310050 </td><td></td></tr><tr><td> LB寒天、粉</td><td>インビトロジェン</td><td> 22700025 </td><td></td></tr><tr><td> Bactoagar </td><td>インビトロジェン</td><td></td><td></td></tr><tr><td>アンピシリン</td><td>インビトロジェン</td><td> 11593027 </td><td>蒸留水に10 mg / mLの</td></tr><tr><td> X - galを</td><td>インビトロジェン</td><td> 15520034 </td><td> DMFで40mg/mL </td></tr><tr><td> IPTG </td><td>インビトロジェン</td><td> 15529019 </td><td>蒸留水に100mMの</td></tr><tr><td> TOPO TAクローニングキット</td><td>インビトロジェン</td><td> K450002 </td><td></td></tr><tr><td> Purelinkクイックプラスミドミニプレップキット</td><td>インビトロジェン</td><td> K210010 </td><td></td></tr><tr><td>ビッグ染料</td><td>アプライドバイオシステム</td><td> 4337455 </td><td></td></tr><tr><td> DyeEx 2.0スピンキット</td><td>キアゲン</td><td> 63204 </td><td></td></tr></tbody></table>

sequencing of bacterial microflora in peripheral blood: our experience with hiv-infected patients

健康な消化管は、生理的体液性および細胞性粘膜免疫系の1,2の開発に影響を与える共生微生物の多種多様によって植民地化されている。 微生物叢は、強力な粘膜関門を介した免疫系から遮蔽されている。感染症と抗生物質は、通常の消化管の障壁と3つの可能な免疫異常につながることが常駐細菌の構成、両方を変更することが知られています。 HIVは、全身免疫活性化4-7を貢献する、リポ多糖と細菌のDNA断片のような細菌のバイオ製品、、の全身循環への粘膜免疫と漏れの進行性破壊と消化管バリアの侵害を引き起こします。微生物の転座は、HIV /エイズ免疫病原性と治療4,8への応答に関与している。 我々は、HIV感染患者の末梢血中に転位細菌の組成を特徴づけることを目的とした。私たちの目的を追求するために我々は、シークエンス解析に続いてpanbacteric 16S ribosomial遺伝子のPCR反応をセットアップします。 簡単に言えば、両方のHIV感染と健常人全血が使用されます。健常者が正常な腸内恒常性を提示することを考えるとフローラのは転座は、これらの患者に期待されていません。静脈穿刺および血漿分離によって全血採取に続いて、DNAは血漿から抽出し、panbacteric 16S ribosomial遺伝子9用の幅広い範囲のPCR反応を実行するために使用されます。以下のPCR産物の精製、クローニングとシークエンシング解析を実施しています。

Watch this Scientific Journal Video about Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients at JoVE.com

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

私たちの実験では、HIV陽性患者の末梢血中に転位細菌種のシークエンス解析を実行する方法を説明します。

HIV感染患者との経験：末梢血中の細菌叢のシーケンシング

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

sequencing-bacterial-microflora-peripheral-blood-our-experience-with

Research

JoVE Journal

Medicine

12.2K ビュー.  San Paolo Hospital University of Milan, Italy. 私たちの実験では、HIV陽性患者の末梢血中に転位細菌種のシークエンス解析を実行する方法を説明します。

ビデオ: Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

マウスにおけるモデリング脳出血：自己血または細菌性コラゲナーゼの注入

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

医学

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

HIV感染者でもろさを測定する。虚弱患者の同定は、もろさの改善と逆転への第一歩です。

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

間歇性跛行患者における姿勢制御評価のためのコンピュータ化されたダイナミックPosturography

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

慢性腎臓病患者における血管機能の評価

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

II-45同系ラット中皮腫モデルにおける同所移植と末梢免疫細胞の監視

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

重症患者における全血エンドトキシア血症の検出に関するエンドトキシン活性アッセイ

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Digital Handwriting Analysis of Characters in Chinese Patients with Mild Cognitive Impairment

An increasing amount of evidence shows that cognitive deficits and movement dysfunctions are not separated. Patients with mild cognitive impairment (MCI) can manifest fine motor disorders of the upper extremities. Handwriting is a complex and unique human activity involving both motor and cognitive coordination. Researchers from western countries have discovered that patients with MCI have abnormal handwriting features. However, no relevant studies have been conducted in the Chinese population. Owing to the cross-culture phenomenon of handwriting, the aim of this study is to find new handwriting tasks to demonstrate the differences in handwriting features between elderly patients with MCI and age-matched healthy individuals.

This study proposed a digital handwriting analysis of characters in individuals with mild cognitive impairment to find more information than is revealed by traditional pencil&#8211;paper handwriting analysis.

軽度認知障害を有する中国患者における文字のデジタル手書き分析

An increasing amount of evidence shows that cognitive deficits and movement dysfunctions are not separated. Patients with mild cognitive impairment (MCI) ...

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

重度卵巣機能障害患者における不妊治療の保存

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...

Evaluation of a Point-of-Care Testing Analyzer for Measuring Peripheral Blood Leukocytes

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At present, most primary medical institutions in China have a poor percentage of adoption of blood-testing technology, and a hematology detection system with a high price to performance ratio and easy operation is urgently needed in primary healthcare centers. This paper introduces the principle and operation procedures of a point-of-care testing (POCT) card-based leukocyte analyzer (evaluated system), which was used to detect WBC indexes such as neutrophils, lymphocytes, and intermediate group cells (including eosinophils, basophils, and monocytes) in whole blood. The results from the evaluated system were compared to those from two commercial automatic hematology analyzers (reference system). The correlation and consistency between the evaluated system and the commercial reference systems were analyzed. The results showed that WBC count and number of granulocytes detected by the evaluated and reference systems showed a strong positive correlation (rs = 0.972 and 0.973, respectively), while the number of lymphocytes showed a relatively low correlation (rs = 0.851). A Bland-Altman plot showed that the major difference between the values detected by the evaluated system and the reference systems is within 95% limits of agreement (LoA), indicating that the two systems are in good agreement. In conclusion, the evaluated system has an excellent correlation, robust consistency, and a reliable comparison with the results of the widely used automatic hematology analyzers. It is ideal for WBC detection in primary medical institutions where a full-automatic five-category hematology analyzer is unavailable, especially during the COVID-19 normalized prevention and control period.

A protocol to measure peripheral blood leukocytes using a POCT card-based leukocyte analyzer is presented here. Same blood samples were tested by two automated hematology analyzers to evaluate the consistency and accuracy of the results. The results showed that the evaluated analyzer had a good correlation with the reference system.

末梢血白血球を測定するためのポイントオブケア検査分析装置の評価

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At ...

Cox-Maze IV Procedure Concomitant with Valvular Surgery In Situs Inversus Dextrocardia: A Single-Center Experience in China

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The use of ablation technologies made the Cox-Maze IV procedure (CMP-IV) technically easier, faster, becoming the gold standard for the surgical treatment of AF. However, the efficacy and safety of CMP-IV in situs inversus dextrocardia are largely unknown. This paper summarizes the CMP-IV procedure performed concomitantly with valvular surgery in patients with situs inversus dextrocardia at this institution.
From February 2016 to September 2020, three dextrocardia patients with persistent AF and valvular diseases were referred to this institution for valvular and CMP-IV surgery. CMP-IV was performed using either cryoablation with a nitrous oxide (N2O)-based cryoprobe or a bipolar radiofrequency clamp and bipolar radiofrequency pen. Mechanical valve replacement or mitral vavuloplasty was performed in another patient in addition to tricuspid annuloplasty. Transmurality of the ablated atrial tissues was evaluated by electron microscopy. Heart function was assessed by transthoracic echocardiography. Cardiac rhythm was monitored by 24 h Holter at 3, 6, 12, 18, 24, and 48 months follow-up. 
All the AF was successfully eliminated in the ablation procedure without recurrence or other complications during hospitalization. The mean bypass and crossclamp times were similar in all the patients. The postoperative ventilator support time, the duration of stay in the ICU, and postoperative residence time were also not significantly different among the patients. Transmural atrial necrosis was detected in the ablated atrial tissues. Sinus rhythm maintenance was achieved at 3, 6, 12, 18, 24, and 48 months follow-up in all the patients. All valve protheses switched freely; no tricuspid regurgitation was observed. The results of the present study demonstrate that the CMP-IV is safe and effective in eliminating AF in dextrocardia patients concomitant with valvular surgery.

We summarize the Cox-Maze IV procedure concomitant with valvular surgery performed in patients with situs inversus dextrocardia at this institution.

上皮内対右心不全の弁膜手術に伴うCox-MazeIV手順:中国での単一施設の経験

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The use of ablation technologies made the Cox-Maze IV procedure (CMP-IV) technically ...