وأيد هذا العمل من المنح المقدمة من المعاهد الوطنية للصحة (AI079442 AI091918 و) منحت لSEFD

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+CD8+T+cell+responses+with+Listeria+monocytogenes+infection.">Probing CD8 T cell responses with Listeria monocytogenes infection.</a> Advances in Immunology. 113, 51-80 (2012).</li><li>Disson, O., 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=Modeling+human+listeriosis+in+natural+and+genetically+engineered+animals.">Modeling human listeriosis in natural and genetically engineered animals.</a> Nat Protoc. 4, 799-810 (2009).</li><li>Czuprynski, C. J., Faith, N. G., Steinberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A/J+mice+are+susceptible+and+C57BL/6+mice+are+resistant+to+Listeria+monocytogenes+infection+by+intragastric+inoculation.">A/J mice are susceptible and C57BL/6 mice are resistant to Listeria monocytogenes infection by intragastric inoculation.</a> Infect. Immun. 71, 682-689 (2003).</li><li>Gajendran, N., 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=Regional+IFNgamma+expression+is+insufficient+for+efficacious+control+of+food-borne+bacterial+pathogens+at+the+gut+epithelial+barrier.">Regional IFNgamma expression is insufficient for efficacious control of food-borne bacterial pathogens at the gut epithelial barrier.</a> Int. Immunol. 19, 1075-1081 (2007).</li><li>Lecuit, M., 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+transgenic+model+for+listeriosis:+role+of+internalin+in+crossing+the+intestinal+barrier.">A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.</a> Science. 292, 1722-1725 (2001).</li><li>Pron, 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=Comprehensive+study+of+the+intestinal+stage+of+listeriosis+in+a+rat+ligated+ileal+loop+system.">Comprehensive study of the intestinal stage of listeriosis in a rat ligated ileal loop system.</a> Infect. Immun. 66, 747-755 (1998).</li><li>Wollert, T., 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=Extending+the+host+range+of+Listeria+monocytogenes+by+rational+protein+design.">Extending the host range of Listeria monocytogenes by rational protein design.</a> Cell. 129, 891-902 (2007).</li><li>Ghanem, B. o. u., N, E., 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=InlA+promotes+dissemination+of+Listeria+monocytogenes+to+the+mesenteric+lymph+nodes+during+food+borne+infection+of+mice.">InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice.</a> PLoS Pathogens. , (2012).</li><li>Kowal, M., Buda-Lewandowska, D., Plytycz, B., Styrna, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Day/night+food+consumption+in+mice+is+strain+and+age-dependent.">Day/night food consumption in mice is strain and age-dependent.</a> Folia Biol. (Krakow). 50, 1-3 (2002).</li><li>Freitag, N. E., Port, G. C., Miner, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+-+from+saprophyte+to+intracellular+pathogen.">Listeria monocytogenes - from saprophyte to intracellular pathogen.</a> Nature reviews. Microbiology. 7, 623-628 (2009).</li><li>Camejo, 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=The+arsenal+of+virulence+factors+deployed+by+Listeria+monocytogenes+to+promote+its+cell+infection+cycle.">The arsenal of virulence factors deployed by Listeria monocytogenes to promote its cell infection cycle.</a> Virulence. 2, 379-394 (2011).</li></ol>

يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة. أجريت جميع التجارب وصفها هنا في الامتثال مع مكتب مختبر رعاية الحيوان (OLAW) مع موافقة ICAUC في جامعة كنتاكي.

الفئران الفطرية ليست تقبلا موحد لتغذية في جميع الأوقات من اليوم، وسوف استعدادها لنأكل الخبز الملوثة تعتمد على حد سواء على نوع السلالة والعمر من الفئران 17. في تجربتنا، 6-9 أسبوع من العمر B6 الفئران هي تقبلا للتغذية في أي وقت من اليوم، ولكن سوف الفئران BALB لا يأكل با?...

المستوحدة الليستيريا هي اختياري مسببات الأمراض البكتيرية بين الخلايا التي تسبب الالتهابات المنقولة عن طريق الأغذية في البشر. البكتيريا المقاومة لكثير من العمليات المستخدمة لحماية امدادات الغذاء لدينا، مثل التجفيف والتمليح، أو ترتبط 1،2 التبريد والالتهابات عادة إلى معالجة، &quot;جاهزة للأكل&quot; الأطعمة التي لا يتم تسخينها قبل الاستهلاك . في عدة حالات سابقة، مصدر L. تم تحديد المواد الغذائية المستوحدة الملوثة، وتم رصد مجموعة محدودة من الأفراد المعرضين بشكل وثيق 3-6. في هذه الأمثلة، وأمراض السريرية في الأفراد الأصحاء تراوحت بين خفيفة، التهاب المعدة والأمعاء محدودة ذاتيا إلى الالتهابات المعوية والجهازية أكثر حدة التي تتطلب العلاج في المستشفيات. L. وقد ارتبطت الالتهابات المستوحدة في مأمن الأفراد للخطر، بما في ذلك حديثي الولادة وكبار السن، مع معدل وفيات مرتفع (25-30٪)، وحتى مع العلاج بالمضادات الحيوية <sتصل&gt; 7،8. ولا يعرف الكثير عن الجرعة المعدية لL. المستوحدة، أو العوامل المضيفة التي تحكم التعرض للعدوى بعد انتقال عن طريق الفم، ويرجع ذلك أساسا إلى عدم وجود نموذج حيواني الصغيرة التي يلخص هذه مجموعة واسعة من نتائج العدوى. النموذج الأكثر استخداما على نطاق واسع من الليستريات هو عن طريق الوريد (IV) تلقيح الفئران. النموذج الرابع هو تكرار للغاية، وكانت مفيدة للغاية لدراسة استجابات كل من السذاجة والذاكرة خلية T خلال العدوى 9،10. العيب من النموذج الرابع هو أنه يتجاوز تماما المرحلة المعوية العدوى. بعد انتقال تنقلها الأغذية، الغشاء المخاطي الأمعاء يوفر الحاجز الذي يفترض أن يؤدي إلى إبطاء ويحد من عدد من البكتيريا التي يمكن أن تنشر باستمرار إلى الأنسجة الطرفية. في المقابل، فإن اللقاح بأكملها يمكن العثور عليها في الطحال والكبد في غضون دقائق من الإدارة الرابع، وهذا بلعة كبيرة من الكائنات الحية قد تطغى الفطرية د المناعيefenses في هذه الأنسجة. يستخدم العدوى عن طريق الفم من الفئران عن طريق التغذية الأنبوبية أقل شيوعا، وذلك لأن الجرعات الكبيرة (10 -10 9 11 كفو) عادة ما تكون مطلوبة لتحقيق الاستعمار المعوية 10. أيضا، المعدة (IG) التلقيح بإبرة التغذية لا تولد الفترة استنساخه من عدوى الجهاز الهضمي قبل انتشار النظامية. وأفادت بعض المختبرات التي L. المستوحدة وصول إلى الطحال والكبد في غضون 4-12 ساعة بعد العدوى (HPI)، في حين أظهر البعض الآخر لا انتشار النظامية حتى 48 HPI 11-15. قد يكون هذا الاختلاف مختبر إلى مختبر نتيجة للطبيعة الغازية من IG التلقيح، والذي يمكن أن يؤدي إلى صدمة طفيفة على بطانة المريء، وتعزيز غزو مجرى الدم مباشرة من البكتيريا. وضعنا مؤخرا نموذج الفأر الرواية الشفوية من L. عدوى المستوحدة الذي يحاكي عن كثب جميع مراحل المرض الإنسان 16. تحدث العدوى عندما يبتلع قطعة من الفئران تابعaminated الغذائي، وهي عملية غير مؤلمة، و لا يتطلب مهارات متخصصة من قبل المحققين المختبر. لفترة من الوقت منفصلة (36-48 ساعة)، L. المستوحدة استعمار بتكاثر فقط الجهاز الهضمي، مما يسمح التحقيق في الآليات المستخدمة من قبل الممرضة الليستيريا لنقل من عبر الغشاء المخاطي للأمعاء ونشرها إلى الأنسجة الطرفية. الأهم من ذلك أن نموذج يمكن استخدامه لدراسة الاختلافات في المضيف المقاومة الفطرية للإصابة. في دراسة سابقة، أظهرت لنا أن BALB / ج / عن طريق / J الفئران كانت عرضة للغذاء الحامل بالعدوى المنقولة، مع النسخ المتماثل الأسي من L. المستوحدة التي تحدث في الأمعاء والطحال، والكبد، والمرارة 16. في المقابل، كانت C57BL / 6 الفئران المقاومة للعدوى المنقولة عن طريق الأغذية، مع فقط الاستعمار عابرة من L. المستوحدة التي تحدث في كل من هذه الأنسجة. ميزة إضافية من طراز تنتقل عن طريق الأغذية الذي يحاكي بشكل وثيق الأمراض التي تصيب البشر هي أن نشر الطبيعيلوقعت في الدماغ خلال مراحل لاحقة من عدوى (5-7 نقطة في البوصة).

<table border="1">
	<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Brain Heart Infusion Agar</td>
 <td>Difco</td>
 <td>BD-241830</td>
 <td></td>
 </tr>
 <tr>
 <td>Lithium chloride</td>
 <td>Sigma</td>
 <td>L9650</td>
 <td ></td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>Omnipur</td>
 <td>4840</td>
 <td ></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Gibco</td>
 <td>15575-038</td>
 <td ></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Sigma</td>
 <td>D5545</td>
 <td ></td>
 </tr>
 <tr>
 <td>Collagenase, type IV</td>
 <td>Worthington</td>
 <td>LS004089</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAse I</td>
 <td>Worthington</td>
 <td>LS002007</td>
 <td></td>
 </tr>
 <tr>
 <td>Diff-Quik</td>
 <td>Dade-Behring</td>
 <td>B4132-1A</td>
 <td></td>
 </tr>
 <tr>
 <td>PowerGen 1000 homogenizer</td>
 <td>Fisher</td>
 <td>14-261-06</td>
 <td></td>
 </tr>
 <tr>
 <td>stainless steel type 304 mesh #80</td>
 <td>Small Parts, Inc.</td>
 <td>CX-0080-C</td>
 <td></td>
 </tr>
 <tr>
 <td>Cytospin</td>
 <td>Statspin</td>
 <td>M801-22</td>
 <td></td>
 </tr>
	 </tbody>
 </table>

oral transmission of listeria monocytogenes in mice via ingestion of contaminated food

المستوحدة L. هي اختياري مسببات الأمراض البكتيرية بين الخلايا التي تسبب الالتهابات المنقولة عن طريق الأغذية في البشر. القليل جدا هو المعروف عن المرحلة الهضمي من الليستريات نظرا لعدم وجود نموذج حيواني صغير الذي يحاكي بشكل وثيق الأمراض التي تصيب البشر. تصف هذه الورقة نموذج الفأر رواية لنقل الشفوي L. المستوحدة. باستخدام هذا النموذج، الفئران التي غذيت L. المستوحدة الملوثة الخبز يكون مرحلة منفصلة من عدوى الجهاز الهضمي، تليها بدرجات متفاوتة من انتشار النظامية في سلالات الفئران عرضة (BALB / ج / عن طريق / J) أو مقاومة (C57BL / 6). خلال مراحل لاحقة من عدوى، لوحظ نشرها على المرارة والدماغ. نموذج تنتقل عن طريق الأغذية من الليستريات هو تكرار للغاية، لا يتطلب مهارات متخصصة، ويمكن استخدامها مع مجموعة واسعة من العزلات البكتيرية وسلالات الفئران المختبرية. على هذا النحو، هو نموذج مثالي لدراسة كل الاستراتيجيات الفوعة المستخدمة من قبل L. المستوحدة </em&gt; لتعزيز الاستعمار الأمعاء، وكذلك استجابة المضيف في الغذاء الغازية تتحمل عدوى بكتيرية.

سوف L. المستوحدة المستعمرات تكون مرئية على BHI / L + G لوحات بعد 36-48 ساعة الحضانة عند 37 درجة مئوية. المستعمرات لديها، على شكل قبة مظهر أبيض دسم ناعمة (الشكل 1A). وأن النمو يتأثر سلبا بالنسبة للغالبية من مجهريات البقعة المعوية، ولكن من الشائع أن نرى بعض المستعمر?...

Watch this Scientific Journal Video about Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food at JoVE.com

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

تصف هذه الورقة طريقة جديدة للعدوى عن طريق الفم من الفئران باستخدام<em&gt; المستوحدة الليستيريا</em&gt;-الأغذية الملوثة. يمكن بسهولة أن تتكيف بروتوكول للاستخدام مع غيرها من المواد الغذائية مسببات الأمراض البكتيرية التي تنتقل عن طريق.

النقل الشفوي لل<em&gt; المستوحدة الليستيريا</em&gt; في الفئران عن طريق ابتلاع الطعام الملوث

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

oral-transmission-listeria-monocytogenes-mice-via-ingestion

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Immunology and Infection

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

19.1K Views.  University of Kentucky. تصف هذه الورقة طريقة جديدة للعدوى عن طريق الفم من الفئران باستخدام المستوحدة الليستيريا-الأغذية الملوثة. يمكن بسهولة أن تتكيف بروتوكول للاستخدام مع غيرها من المواد الغذائية مسببات الأمراض البكتيرية التي تنتقل عن طريق.

Video: Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

قياس الحمولة الجرثومية والاستجابات المناعية في الفئران المصابة المستوحدة الليستيريا</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

التصوير InlC إفراز المعنية بالتحقيق في العدوى البكتيرية الخلوية من قبل الممرض<em&gt; المستوحدة الليستيريا</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

تقييم الغزو البكتيري لخلايا القلب في الثقافة والاستعمار القلب في الفئران المصابة باستخدام monocytogenes الليستيريا

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

التحول من الخميرة بروبيوتيك وشفائهم من الجهاز الهضمي المناعي الأنسجة بعد بالتزقيم عن طريق الفم في الفئران

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA تنقية من داخل الخلية المزروعة<em&gt; المستوحدة الليستيريا</em&gt; في خلايا البلاعم

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

العدوى التجريبية مع<em&gt; المستوحدة الليستيريا</em&gt; نموذجا لدراسة المضيف الردود انترفيرون γ

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

ل<em&gt; في المختبر</em&gt; كاسيم ملزمة الفحص الذي يتنبأ اختراق المخدرات في الآفات السل

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

الليستريه المستوحدة التهاب الدماغ

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

تصوير إنتاج غاما للإنترفيرون في الموقع في طحال الفأر بعد عدوى الليستيريا أحادية الخلايا

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.