ويود المؤلفون أن يعترفوا بجائزة فيرتكس فارماسيوتيكالز وجائزة الابتكار في مجال التليف الكيسي للتمويل R. كوين والمعاهد الوطنية للصحة / نييد لمنحة التمويل 1 U01 AI124316-01، وهو نهج بيولوجيا النظم لمعالجة مسببات الأمراض المقاومة للأدوية المتعددة. ونود أيضا أن نشكر قسم الهندسة الميكانيكية والفضاء في جامعة كاليفورنيا أوسسد في الهندسة المعمارية العليا دورة التصميم لتسهيل التعاون مع الجوانب الهندسية لهذا العمل.

<ol>
	<li>Quinn, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Winogradsky-based+culture+system+shows+an+association+between+microbial+fermentation+and+cystic+fibrosis+exacerbation.">A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation.</a> ISME J . 9, 1024-1038 (2015).</li><li>Quinton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis:+impaired+bicarbonate+secretion+and+mucoviscidosis.">Cystic fibrosis: impaired bicarbonate secretion and mucoviscidosis.</a> Lancet. 372 (9636), 415-417 (2008).</li><li>Harrison, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+ecology+of+the+cystic+fibrosis+lung.">Microbial ecology of the cystic fibrosis lung.</a> Microbiology. 153 (Pt 4), 917-923 (2007).</li><li>Caverly, L. J., Zhao, J., LiPuma, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+lung+microbiome:+Opportunities+to+reconsider+management+of+airway+infection.">Cystic fibrosis lung microbiome: Opportunities to reconsider management of airway infection.</a> Pediatr pulmonol. 50, S31-S38 (2015).</li><li>Blainey, P. C., Milla, C. E., Cornfield, D. N., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+the+human+airway+microbial+ecology+reveals+a+pervasive+signature+for+cystic+fibrosis.">Quantitative analysis of the human airway microbial ecology reveals a pervasive signature for cystic fibrosis.</a> Sci Transl Med. 4 (153), 153ra130 (2012).</li><li>Willner, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+distribution+of+microbial+communities+in+the+cystic+fibrosis+lung.">Spatial distribution of microbial communities in the cystic fibrosis lung.</a> ISME J. 6 (2), 471-474 (2012).</li><li>Delhaes, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+airway+microbiota+in+cystic+fibrosis:+a+complex+fungal+and+bacterial+community--implications+for+therapeutic+management.">The airway microbiota in cystic fibrosis: a complex fungal and bacterial community--implications for therapeutic management.</a> PloS one. 7 (4), e36313 (2012).</li><li>Rogers, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D.+Bacterial+diversity+in+cases+of+lung+infection+in+cystic+fibrosis+patients:+16S+ribosomal+DNA+(rDNA)+length+heterogeneity+PCR+and+16S+rDNA+terminal+restriction+fragment+length+polymorphism+profiling.">D. Bacterial diversity in cases of lung infection in cystic fibrosis patients: 16S ribosomal DNA (rDNA) length heterogeneity PCR and 16S rDNA terminal restriction fragment length polymorphism profiling.</a> J clin microbiol. 41 (8), 3548-3558 (2003).</li><li>Stenbit, A. E., Flume, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+exacerbations+in+cystic+fibrosis.">Pulmonary exacerbations in cystic fibrosis.</a> Curr Opin Pulm Med. 17 (6), 442-447 (2011).</li><li>Twomey, K. 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Soc. 10 (3), 179-187 (2013).</li><li>Worlitzsch, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reduced+mucus+oxygen+concentration+in+airway+Pseudomonas+infections+of+cystic+fibrosis+patients.">Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.</a> J. Clin. Invest. 109 (3), 317-325 (2002).</li><li>Cowley, E. S., Kopf, S. H., LaRiviere, A., Ziebis, W., Newman, 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=Pediatric+Cystic+Fibrosis+Sputum+Can+Be+Chemically+Dynamic,+Anoxic,+and+Extremely+Reduced+Due+to+Hydrogen+Sulfide+Formation.">Pediatric Cystic Fibrosis Sputum Can Be Chemically Dynamic, Anoxic, and Extremely Reduced Due to Hydrogen Sulfide Formation.</a> mBio. 6 (4), e00767-e00715 (2015).</li><li>Sriramulu, D. D., L&#252;nsdorf, H., Lam, J. S., R&#246;mling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcolony+formation:+a+novel+biofilm+model+of+Pseudomonas+aeruginosa+for+the+cystic+fibrosis+lung.">Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung.</a> J. Med. Microbiol. 54 (Pt 7), 667-676 (2005).</li><li>Quinn, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogeochemical+forces+shape+the+composition+and+physiology+of+polymicrobial+communities+in+the+cystic+fibrosis+lung.">Biogeochemical forces shape the composition and physiology of polymicrobial communities in the cystic fibrosis lung.</a> mBio. 5 (2), (2014).</li></ol>

الكتاب ليس لديهم ما يكشف.

تحتوي التركيبة الميكروبيولوجية للرئة مع سف على مجموعة كبيرة ومتنوعة من الكائنات الحية، ولكن الظروف داخل الرئة على الأرجح لها تأثير كبير على أنواع الميكروبات التي يمكن أن تعيش وتزدهر 13 ، 15 . آليات محددة من خلالها تغير هذه الظروف والآثا...

وتسمى الطريقة الموصوفة في هذه المخطوطة نظام وينف 1 . الهدف العام من وينف هو توفير الإعداد التجريبية قادرة على محاكاة البيئة من الشعب الهوائية الرئة مليئة المخاط المخاط. هذا سوف يسمح لنظام قابل للدراسة لدراسة مسببات الأمراض الميكروبية من أمراض الرئة مع النمط الظاهري فرط المخاط بما في ذلك التليف الكيسي (سف)، مرض الانسداد الرئوي المزمن (كوبد)، والربو وغيرها. تم تصميم هذا الإجراء خصيصا لدراسة سف، والتي تتميز الطفرات التي تسبب إفرازات الرئة لتصبح سميكة وصعبة واضحة، في نهاية المطاف ملء القصبات وغيرها من الممرات الصغيرة مع المخاط 2 . هذا الانسداد في الرئة تمنع تبادل الغاز لأن الهواء المستنشق لم يعد قادرا على الوصول إلى العديد من الحويصلات الهوائية وأيضا توفير موطن للاستعمار الجرثومي 3 ، 4 . عدم القدرة على منع نمو الميكروبات فيالإفراط في مخاط رئة يؤدي في النهاية إلى تطور العدوى المزمنة المعقدة لمجرى الهواء. هذه المجتمعات تحتوي على مجموعة متنوعة من الكائنات الحية، بما في ذلك الفيروسات والفطريات والبكتيريا مثل الزائفة الزنجارية، وكلها تتفاعل مع بعضها البعض 5 و 6 و 7 و 8. ويعتقد أن نشاط microbiome CF الرئة أن تشارك في مشاعل الأعراض تسمى التفاقم الرئوية 1 و 9 و 10 و 11. WinCF يمكن دراسة سلوك المجتمع الميكروبي حول هذه التفاقم ويجري الآن توسيعها لتكون بمثابة نظام تجريبي قاعدة لدراسة الرئة الميكروبية البيئة. تقليديا، وقد تم دراسة التفاقم من خلال التحليل المباشر للعينات المأخوذة من الرئة. العديد من العوامل المربكة تجعل التحليل المباشر للب الميكروبيةehavior في الرئتين الصعبة، مع نظام WinCF، وكثير من هذه العوامل يتم إزالتها وسلوك microbiome الرئة يمكن دراستها بصورة مباشرة، مما يسمح للتحليل أدق النشاط الجرثومي في قصيبة-توصيل المخاط. ويوفر النظام WinCF طريقة لتنمو وتحليل البكتيريا بطريقة يحاكي البيئة بشكل فعال الرئة. الطرق التقليدية لزراعة البكتيريا الرئة عينات زراعة في كثير من الأحيان المعنية على لوحات أجار التقليدية. هذه الأساليب تترك العينات مفتوحة للأكسجين في الغلاف الجوي، إهمال لحساب ميتة وغالبا الظروف الأكسجين الموجود في القصيبات الرئوية توصيله مع مخاط 12 و 13. زراعة على أجار تحت الظروف الهوائية شيء مثل بيئة CF الرئة ويمكن تضليل الأطباء والباحثين بشأن سلوك مسببات الأمراض التي يحاولون علاج. بالإضافة إلى ذلك، العناصر الغذائية المتاحة للبكتيريا على لوحات أجارلا يختلف عن تلك المتوفرة في البلغم الفعلي، الذي استأثرت في WinCF من خلال الاستفادة من وسائل الإعلام البلغم الاصطناعي (ASM). كما يتضح من الثقافات الزائفة في Sriramulu وآخرون. 14، ويشمل ASM مجموعة محددة من المكونات التي يحاكي الموارد المتاحة للميكروبات البلغم ويعيد التناسق البدني من البلغم أيضا. لأن الرئة المريضة لديها microbiome معين، يجب دراسة هذه الكائنات الدقيقة من الناحية المثالية أن تتم في الظروف المحددة في الرئة كذلك. النظام WinCF يمكن تحليل سريع والتلاعب سهل من الظروف التجريبية لمراقبة التغيرات الميكروبية مماثلة إلى الكيفية التي يمكن أن تحدث في قصيبة الرئة الفعلية. هذا الأسلوب يسمح للتلقيح عدد لا يحصى من أنواع عينة ذات الصلة بما في ذلك البلغم واللعاب وغيرها من إفرازات الجسم والثقافات البكتيرية نقية أو مختلطة. طبيعة الإعداد التجريبية تسمح التفسير البصري المباشر للتم تصميم سلوك المجتمع الميكروبي ولتمكين التطبيق المصب سهل من العديد من الإجراءات الميكروبيولوجية وOMICS. مثل هذه الدراسات مهمة لتغيير تركيبة المجتمع البكتيرية بناء على الظروف الفيزيائية بيئتهم. مع WinCF الظروف الكيميائية وسائل الإعلام يمكن التلاعب بها لتحليل الآثار المترتبة على النشاط البكتيري. على سبيل المثال، الحموضة وسائل الإعلام يمكن أن تتغير قبل التلقيح مع عينة. بعد مرور فترة الحضانة، والنشاط الجرثومي في كل من هذه الشروط يمكن مقارنة مباشرة، ويمكن استخلاص استنتاجات حول كيف تتصرف البكتيريا في تلك العينات البلغم ردا على تفاوت درجة الحموضة. هنا، ونحن الخطوط العريضة لإجراءات تطبيق نظام WinCF وأمثلة لكيفية الكيمياء وسائل الاعلام يمكن التلاعب بها لدراسة الآثار المترتبة على microbiome الرئة.

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Color-Coded Capillary Tubes</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">22-260943</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cha-seal Tube Sealing Compound</td>
			<td style="width:93px;">Kimble-Chase</td>
			<td align="right" style="width:119px;">43510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mucin from porcine stomach</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">M1778</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ferritin, cationized from horse spleen</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">F7879</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Salmon sperm DNA Sodium salt (sonified)</td>
			<td style="width:93px;">AppliChem Panreac</td>
			<td>A2159</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MEM Nonessential Amino Acids</td>
			<td style="width:93px;">Corning cellgro</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MEM Amino Acids</td>
			<td style="width:93px;">Cellgro</td>
			<td style="width:119px;">25-030-CI</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Egg Yolk Emulsion, 50%</td>
			<td style="width:93px;">Dalynn Biologicals</td>
			<td style="width:119px;">VE30-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Chloride</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">P2157500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Chloride</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">S271500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">15mL centriguge tubes with Printed Graduations and Flat Caps</td>
			<td style="width:93px;">VWR</td>
			<td style="width:119px;">89039-666</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50mL centrifuge tubes with Printed Graduations and Flat Caps</td>
			<td style="width:93px;">VWR</td>
			<td style="width:119px;">89039-656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5mL microcentrifuge tubes</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">MCT-150-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2.0mL microcentrifuge tubes</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">MCT-200-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

the wincf model - an inexpensive and tractable microcosm of a mucus plugged bronchiole to study the microbiology of lung infections

العديد من أمراض مجرى الهواء المزمن تؤدي إلى انسداد المخاط من الشعب الهوائية. الرئتين من فرد مع التليف الكيسي هي حالة مثالية حيث القصيبات متصلة المخاط خلق الموئل مواتية للاستعمار الميكروبي. العديد من مسببات الأمراض تزدهر في هذه البيئة تتفاعل مع بعضها البعض، والقيادة العديد من الأعراض المرتبطة مرض سف. ومثل أي مجتمع ميكروبي، فإن الظروف الكيميائية لموئلها لها تأثير كبير على هيكل المجتمع ودينامياته. على سبيل المثال، الكائنات الحية الدقيقة المختلفة تزدهر في مستويات مختلفة من الأوكسجين أو غيرها من تركيزات المذاب. هذا صحيح أيضا في الرئة سف، حيث يعتقد أن تركيزات الأكسجين لدفع علم وظائف الأعضاء المجتمع وهيكل. الطرق الموصوفة هنا مصممة لتقليد بيئة الرئة وتنمو مسببات الأمراض بطريقة أكثر مماثلة لتلك التي تسبب المرض. ثم يستخدم التلاعب بالمحيط الكيميائي لهذه الميكروبات لدراسة كيفية كيميستري من التهابات الرئة يحكم البيئة الميكروبية. ويستند هذا الأسلوب، ودعا نظام وينف، على البلغم الاصطناعي وأنابيب الشعرية الضيقة التي تهدف إلى توفير التدرج الأكسجين مماثلة لتلك التي توجد في القصيبات توصيل المخاط. يسمح التعامل مع الظروف الكيميائية، مثل درجة الحموضة لوسائل الإعلام من البلغم أو ضغط المضادات الحيوية، لتصور الاختلافات الميكروبيولوجية في تلك العينات باستخدام مؤشرات ملونة، ومشاهدة لإنتاج الغاز أو بيوفيلم، أو استخراج وتسلسل محتويات الحمض النووي من كل عينة.

النمو الميكروبيولوجي في مختلف الظروف الكيميائية التي يسببها داخل عينات تختلف بشكل كبير في بعض الحالات وأكثر بمهارة في مناطق أخرى. وكانت العديد من التغيرات في النشاط المرئية في الطبيعة، ويجري بادية للعيان فور انتهاء فترة الحضانة. في المثال التلاعب ...

Watch this Scientific Journal Video about The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections at JoVE.com

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

إن المسالك الهوائية الموصولة بالمخاط من التليف الكيسي (سف) هي بيئة مثالية لمسببات الأمراض الميكروبية لتزدهر. تصف المخطوطة طريقة جديدة لدراسة ميكروبيوم الرئة سف في بيئة تحاكي حيث تسبب المرض وكيف أن التغيرات في الظروف الكيميائية يمكن أن تدفع الديناميات الميكروبية.

نموذج وينف - غير مكلفة و تراكتابل مصغر من المخاط توصيل القصبات لدراسة الأحياء الدقيقة من التهابات الرئة

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

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Lower Respiratory Disorders

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9.0K Views.  University of California, San Diego.  إن المسالك الهوائية الموصولة بالمخاط من التليف الكيسي (سف) هي بيئة مثالية لمسببات الأمراض الميكروبية لتزدهر. تصف المخطوطة طريقة جديدة لدراسة ميكروبيوم الرئة سف في بيئة تحاكي حيث تسبب المرض وكيف أن التغيرات في الظروف الكيميائية يمكن أن تدف...

Video: The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

لين العريكة التهابات خلايا الثدييات مع طفيل وحيد الخلية، تستعد البكتيريا

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

على<em&gt; في المختبر</em&gt; نموذج لدراسة عدم تجانس التمايز بلعم الإنسان والاستقطاب

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

نموذج تجريبي لدراسة الملاريا والسل عدوى مرافقة على انتقال الطبيعية السل المتفطرة و المتصورة berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

أنسنة نموذج الفأر لدراسة الالتهابات البكتيرية استهداف الأوعية الدموية الدقيقة

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

الثقافات المشيمة وساقطي الجهاز الإنسان لدراسة العدوى في واجهة الأم والجنين

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

تطوير التهاب القولون نموذج مدفوعة مستضد لدراسة عرض المستضدات التي كتبها مستضد الخلايا إلى خلايا T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

منهجية لدراسة نقل أفقي جين في<em&gt; المكورات العنقودية الذهبية</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

تأتي إلى "جانب الضوء": "في فيفو" الرصد الزائفة الزّنجاريّة بيوفيلم الالتهابات في "الجروح المزمنة" في "نموذج مورين أصلع السكري"

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

اختبار تحمل الجلوكوز داخل، قياس وظيفة الرئة، والتثبيت في الرئة لدراسة تأثير السمنة والايض البصر على النتائج الرئوية

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

التهابات المفصليات كفحص أدوات للتعرف على العوامل المضادة للفيروسات إيمونومودولاتورس والمضيف الفيروسية

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...