\ \ www.nhls.ac.zaとhttp://www.plgcd4：特別な感謝は、HIV関連のフロー診断サービスの全国的な組織のための南アフリカ共和国の国立衛生研究所サービス（NHLS）（HTTPによるものである。ネット）。特色の研究は、血液学および分子医学、ウィットウォーターズランド大学の学科で行われている（ヨハネスブルグ、南アフリカ、部長、教授ウェンディスティーブンスでサポート）。著者らは、民間企業や資金提供機関から、このパブリケーションに対する助成金を受け取っていません。意見は、必ずしもウィットウォータースランドや南アフリカNHLSの大学の機関の見解を反映せずに著者のものであり。

<ol>
	<li>Mugyenyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+versus+clinically+driven+laboratory+monitoring+of+HIV+antiretroviral+therapy+in+Africa+(DART):+a+randomized+non-inferiority+trial.">Routine versus clinically driven laboratory monitoring of HIV antiretroviral therapy in Africa (DART): a randomized non-inferiority trial.</a> Lancet. 375, 123-131 (2009).</li><li>Donnell, D., Kiarie, J., Thomas, K., Baeten, J., Lingappa, J., Cohen, C., Celum, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ART+and+risk+of+heterosexual+HIV-1+transmission+in+HIV-1+serodiscordant+African+couples:+A+Multinational+Prospective+Study.">ART and risk of heterosexual HIV-1 transmission in HIV-1 serodiscordant African couples: A Multinational Prospective Study.</a> , (2010).</li><li>Montaner, J., Wood, E., Kerr, T., Yip, B., Lima, V., Shannon, K., Harrigan, R., Hogg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Expanded+HAART+Coverage+with+a+Decrease+in+New+HIV+Diagnoses,+Particularly+among+Injection+Drug+Users+in+British+Columbia,+Canada.">Association of Expanded HAART Coverage with a Decrease in New HIV Diagnoses, Particularly among Injection Drug Users in British Columbia, Canada.</a> , (2010).</li><li>Petti, C. A., Polage, C. R., Quinn, T. C., Ronald, A. R., Sande, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+medicine+in+Africa:+a+barrier+to+effective+health+care.">Laboratory medicine in Africa: a barrier to effective health care.</a> Clin Infect Dis. 42, 377-382 (2006).</li><li>Brown, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+health:+Great+expectations.">Global health: Great expectations.</a> BMJ. 334, 874-876 (2007).</li><li>Crisp, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+the+World+Upside+Down.">Turning the World Upside Down.</a> Royal Society of Medicine Press. , (2009).</li><li>Hecht, R., Bollinger, L., Stover, J., McGreevey, W., Muhib, F., Madavo, C. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+optimal+lymphocyte+gates+for+immunophenotyping+by+flow+cytometry.">Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry.</a> Cytometry. 11, 453-459 (1990).</li><li>Glencross, D., Scott, L. E., Jani, I. V., Barnett, D., Janossy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD45-assisted+PanLeucogating+for+accurate,+cost-effective+dual-platform+CD4++T-cell+enumeration.">CD45-assisted PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration.</a> Cytometry. 50, 69-77 (2002).</li><li>Glencross, D. K., Janossy, G., Coetzee, L. M., Lawrie, D., Aggett, H. M., Scott, L. E., Sanne, I., McIntyre, J. 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T., Barnett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+rate+calibration:+a+new+approach+for+performing+absolute+cell+counts.">Flow rate calibration: a new approach for performing absolute cell counts.</a> Cytometry B Clin Cytom. 55, 1-7 (2003).</li><li>Coetzee, L., Lawrie, D., Stevens, W., Glencross, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+pipetting+methodology+to+assess+precision+and+accuracy+of+single+platform+PLG/CD4+enumeration.">Evaluation of pipetting methodology to assess precision and accuracy of single platform PLG/CD4 enumeration.</a> , (2006).</li><li>Borowitz, M. J., Guenther, K. L., Shults, K. E., Stelzer, G. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+of+CD45+patterns+in+the+PanLeucoGating+CD4+test+can+be+used+for+diagnosis+of+white+cell+abnormalities.">Interpretation of CD45 patterns in the PanLeucoGating CD4 test can be used for diagnosis of white cell abnormalities.</a> Poster presentation. , (2007).</li><li>Lawrie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wide-spread+clinical+uses+of+PanLeucoGating+technology+in+the+HIV+diagnosis+oriented+immunohaematology+laboratory.">The wide-spread clinical uses of PanLeucoGating technology in the HIV diagnosis oriented immunohaematology laboratory.</a> PhD Thesis, in preparation. , (2010).</li><li>Badri, M., Lawn, S. 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CD4方式のための国際的な特許権には、ここで説明する南アフリカNHLSで、現在はBCI、マイアミ、フロリダ州、米国へのライセンスの下で開催されます。

近年、慈善団体主導と助成金に割り当てられたプログラムは、高いHIV負担と地球の資源限られた地域で、いくつかの優れた臨床実践の確立を支援してきました。 ARTプログラムは死亡率を減らすことに成功していると、今より高いCD4で開始されているが2,3をカウントします。これらの新たな対策は、HIV -特異的なワクチンが不足しているにもかかわらず、HIV感染4,5の広がりを減少することを示す兆候はすでにあります。 ARTの患者を監視する際に、これらのテクニックが良く集中し、より費用対効果の高い西洋研究所（リスト1）で実行ルーチン実際に観察されたものよりなることができること私たちのビデオでは、文書。 このようなアフリカ主導のアプローチは、当技術分野で治療されたHIV感染は、慢性疾患になってきている時に検査サービスのアップグレードをサポートするために不可欠な場合があります。 ARTのようなモニタリングは、カウント正確なCD4とウイルス量、負荷に関連する技術、より要求の厳しいタスクは、単にART 16開始についての決定を支援するために任意のグループに患者を配置するためのCD4カウントを実行する必要があります。これらの診断の助けを借りて、すなわちCD8/CD38テストを使用して、それがART 16,19の患者の&gt; 60％で、VL -関連の監視コストを低減することができる。それは薬を服用でVLリバウンドおよび/ ​​または患者のコンプライアンスの欠如の初期兆候を拾う可能性が高いCD4数37、CD8/CD38テスト16,17であり、ないことを強調することも重要です。 協調費用対効果の高い中央のサービスの本質的な要素は、操作の技術的および医学的側面に精通している専門家や学術アドバイザーがサポートしている大規模な国や地域のネットワークの可用性です。これは、小規模なセンターのために励まし、教育と質の評価を提供し、これらはまた、&quot;ポイントオブケア&quot;サービスも含む南アフリカNHLSのネットワークの活動とCD4 - PLG.netの訓練スキーム33、36で示されている。 重要なのは、中央およびポイントオブケア研究所の業務は相補的である。前者は費用対効果の高い大規模サービスの操作だけでなく、周辺の研究室33,38のための基本的な/高度な訓練を提供する二重の役割を持っている、後者は、HIVを募集してARTの広がりを確認するために貢献しながら、+の対象となる患者を農村部の診療所など、大規模拠点からのART。ポイントオブケア診断がはるかに高価なことができると、自動的に精度が38を保証するものではありませんので、ここではいくつかの注意が必要です。それは、2層システムを維持するために、どちらかのアームが細るできるようにすることなく、2つの層の間の連携を促進することが賢明です。 結論として、感染症の臨床管理の診断モニタリングの分野で必要なコスト削減が大幅に改善品質13,39で安価、よりうまく調整近代的な技術を使用してAPTサービスによって達成することができます。実質的にすべての助成金はすぐにこれらの入念な事前にせずに豊かな国々から輸入されるとして、高価および/ ​​または未経験の方法で無駄になっているときによく組織国家（および地域）診断サービスの規定は、より良いシナリオを回避することによって6-8をサポートする必要がありますプリエンプティブ建設的な精査。同様に、地元の医師や医療従事者の完全な関与と臨床治療試験のより柔軟な設計は不必要に頻繁におよび/ ​​または実験室試験3 -野心以上の削減につながるはずだ。このポリシーは、ARTを受けている患者のコーディネート臨床/臨床検査による評価で利用可能な正確な診断結果をより深く利用するために多くのチャンスを与えるだろう。これらのスマートな診断には、資源の乏しい国のために結核と既存の馴染みのフローサイトメトリー、プラットフォーム40を使用して白血病の分野で8を開拓することができます。

comprehensive &amp; cost effective laboratory monitoring of hiv/aids: an african role model

経済的な大規模な診断テストのための南 - アフリカの役割モデルを表す - 我々は、aptラボサービスによって抗レトロウイルス療法（ART）の支援に関するビデオを提示する。時病理サービスの質は全体的にまだできない状況でも - 安価なARTがHIV血清陽性患者の生存率の見通しを変えたが、ARTのとどのようなコストでの検査によるモニタリングの必要性があるかどうか疑問がある低所得国における非常に低いです。適切な答えは、より良い調整と欧米諸国に見られるよりも厳しい社内品質評価と経済的に健全なサービスを確立することです。ウィットウォータースランド大学、ヨハネスブルグ、南アフリカ、で国立衛生研究所サービス（NHLS - SA）の位置で撮影したこの映像は、オリジナルの2色CD4 - CD45 PanLeucoGating戦略（PLG）を拡大するような協調スキームを提供します。したがって、ビデオプレゼンテーションの6つのモジュールは、シングルチューブで、血液学的免疫学的およびウイルス学関連のテストを組み合わせて4色フローサイトメトリーアッセイのシンプルさを明らかにする。これらのビデオモジュールは、（i）セットアップの楽器のこと、（ii）サンプルの調製、（ⅲ）ビーズカウントレートで各サンプルの絶対数と監視の品質をテストすること、（iv）ホワイトセルのheamatological CD45テストカウントと格差、（v）のCD4数、および（vi）CD8の活性化+ CD38ディスプレイにより測定されたT細胞は、ウイルス量に関連するパラメータ。潜在的なコスト削減には目を見張るものがある。この配置では、西部の研究室では、ともNHLS - SAのネットワーク内の他の研究所への技術移転に適用されるよりも厳格な品質管理と一日あたりの&gt; 800から1000のテストを行うの実現可能性のための典型的な例です。現代の医療技術を導入することについて決定を下す義務を運ぶ専門のアドバイザーが、実験室の管理者や政策立案者は、作業負荷の大きな負担を持つ大規模な地域の研究室で行わ最新の技術的な詳細を確認する立場に頻繁ではありません。したがって、このビデオでは、これらの新たな展開の詳細が表示されます。

Watch this Scientific Journal Video about Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model at JoVE.com

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

HIV /エイズの治療に抗レトロウイルス療法は、大規模に南アフリカで監視されます。フローサイトメトリーは、血液（CD45）、免疫（CD4）とウイルス負荷リンクされているCD38アッセイのために結合されます。 NHLS - SA研究所、ヨハネスブルグで記録された、これらの近代的な方法は、リソースが限られた診断のためのロールモデルとして、高まる地元の内部品質管理とコスト面で効率的です。

HIV / AIDSの包括的＆コスト効率に優れた研究室の監視：アフリカのロールモデル

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

comprehensive-cost-effective-laboratory-monitoring-hivaids-an-african

Research

JoVE Journal

Immunology and Infection

16.6K ビュー.  National Health Laboratory Services (NHLS-SA). HIV /エイズの治療に抗レトロウイルス療法は、大規模に南アフリカで監視されます。フローサイトメトリーは、血液（CD45）、免疫（CD4）とウイルス負荷リンクされているCD38アッセイのために結合されます。 NHLS - SA研究所、ヨハネスブルグで記録された、これらの近代的な方法は、リソースが限られた診断のためのロールモデルとして、高まる地元の内部...

ビデオ: Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

のための免疫担当マウスにおける漿液性卵巣癌の同所性モデル in vivoで</em腫瘍免疫応答の>腫瘍イメージングとモニタリング

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

免疫・感染学

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

オーラルバイオフィルムの包括的、生化学的構造とトランスクリプトーム評価のための分析ツールボックスは、ミュータンス連鎖球菌によって仲介

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3の人口ベースのシーケンシングを用いたHIV - 1トロピズムの遺伝子型推論

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

特定のヒトと動物のウイルスを用いた微生物ソースのトラッキングのための費用対効果の高い方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植片対宿主病の誘導および<emインビボで&gt;</emMHCマッチしたマウスモデルを使用する&gt; T細胞のモニタリング

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

1.バイオセーフティレベル4戦士研究所スイートの入り口と出口の手順：（A）BSL-4実験室での安全上のご注意と操作手順

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

（A）BSL-4実験室での安全上のご注意と操作手順：3.空中生物学

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

アフリカトリパノソーマ症の後期感染を検出するための生物発光イメージング

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

臨床的に関連のセフォペラゾン処理されたマウスモデル<em&gt;クロストリジウム・ディフィシル</em&gt;ひずみR20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...