وأيد هذا العمل من قبل المعاهد الوطنية للحساسية والأمراض المعدية منح P01 A1078894 إلى CAG

<ol>
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تعلن الكتاب أنه ليس لديهم المصالح المالية المتنافسة.

وP. يوفر اللثوية نموذج العدوى عن طريق الفم أداة قيمة لدراسة مسببات الأمراض التي يسببها التهاب مزمن في المواقع المحلية والنظامية. يسمح هذا النموذج الفريد توصيف كل من المضيف والممرض آليات محددة تسهم في التهاب مزمن والباثولوجيا المناعية. وبالإضافة إلى ذلك، فإ...

الالتهاب المزمن هو المحرك الرئيسي للتلف الأنسجة المرضية وصفة توحيد العديد من الأمراض المزمنة في البشر. وتشمل هذه الأمراض الورمية، المناعة الذاتية والأمراض الالتهابية المزمنة 1. مسببات العديد من الأمراض المزمنة غير واضح ولكن من المفهوم أن تكون معقدة ومتعددة العوامل، تشمل كلا من الاستعداد الوراثي وإدخال العوامل البيئية. في حين أن الدوام للالتهاب لا تزال بعيدة المنال، لمحات الخلوية والجزيئية لتنشيط جهاز المناعة تتداخل إلى حد كبير مع تلك الأنماط التي لوحظت في استجابات المضيف لمسببات الأمراض 2. أدلة متزايدة على تورط العدوى مع مسببات الأمراض الميكروبية في تطور وتقدم من التهاب مزمن ومظاهره السريرية المتنوعة 2،3. يمكن أن تحفز مسببات الأمراض والحفاظ التهاب مزمن مباشرة من تخريب الجهاز المناعي المضيف ووضع العدوى المستمرة <sup&gt; 4. في غياب استمرار الجراثيم، يمكن يعجل عدوى التهاب مزمن من ردود فعل المناعة الذاتية الناجمة عن المحاكاة الجزيئية لمستضدات الذات، والتغيرات في المستضدات الذاتية التي تجعلها مناعة، أو الضرر الذي يطلق مستضدات المضيف ملثمين سابقا. ولكن نادرا ما يكون تم تحديد مسببات محددة كسبب العالمي لمرض مزمن معين. بدلا من ذلك، فإن غالبية البيانات المتاحة تشير إلى أن مسببات الأمراض تستخدم آليات متميزة للحصول التهاب مزمن مع طيف واسع من المظاهر السريرية ونتائج المرض في المضيف عرضة وراثيا 3. وبالتالي، فهم مفصل من الآليات التي تحفز مسببات الأمراض المحددة التهاب مزمن قد يكون لها آثار كبيرة على الصحة العامة، فضلا عن العلاج والوقاية من العديد من الأمراض المزمنة. على الرغم من أن آليات المضيف والممرض محددة تساهم في تحريض وصيانة التهاب مزمن وغير مفهومة، بدأت التقدم في النمذجة التي يسببها الممرض التهاب مزمن إلى زيادة فهمنا لهذه العمليات. وP. اللثوية نموذج العدوى عن طريق الفم هو فريد من نوعه، يتميز نموذج الفأر جيدا من العوامل المسببة للأمراض التي يسببها الالتهاب المزمن الذي يسمح للتحليل آليات المضيف والممرض محددة تساهم في التهاب مزمن في المحلية (فقدان العظام عن طريق الفم) ومواقع النظامية (تصلب الشرايين) 5،6. P. اللثوية هو، الممرض عن طريق الفم اللاهوائية سلبية الغرام المتورطين في أمراض اللثة البشري، وهو مرض التهابي مزمن يحركها العدوى يتميز تدمير الأنسجة الداعمة الأسنان 7. بالإضافة إلى أمراض في الموقع الأولي للعدوى، تراكم الأدلة تورط P. اللثوية يسببها التهاب مزمن في تطور وتقدم من الأمراض الجهازية بما في ذلك تصلب الشرايين 5، وهو مرض يتميز التهاب مزمنن من جدار الوعاء الدموي الشرياني. العدوى عن طريق الفم من معين الممرض الفئران الحرة مع P. اللثوية يؤدي الى الاستجابة المحلية للالتهابات التي تؤدي إلى تدمير الأسنان العظم السنخي الداعم 8. P. اللثوية يمكن استردادها من أفواه الفئران المصابة تصل إلى 42 أيام بعد الإصابة 8 والفئران تطوير مستويات عالية من الممرض محددة التتر الأجسام المضادة في مصل الدم 9. في نموذج الفأر أنشئت من تصلب الشرايين باستخدام ئي-E - / - الفئران (APOE - / -)، والعدوى عن طريق الفم مع P. اللثوية يحرض الالتهاب المزمن الذي يدفع ترسب البلاك التهابات الجيوب الأنفية داخل الأبهر والشريان 10 اللامسماة 11. التهاب التدريجي داخل الشريان اللامسماة من P. -infected اللثوية يمكن رصد الفئران في الحيوانات الحية باستخدام التصوير بالرنين المغناطيسي في الجسم الحي. تشريحيا، والآفات الشريانية من P. -infected اللثوية الفئران تظهر زيادة تراكم الدهون accompaNIED بواسطة تفعيل بطانة الأوعية الدموية، وزيادة ارتشاح الخلايا المناعية، والتعبير مرتفع من وسطاء التهابات 12. استخدام هذا النموذج في الفئران خروج المغلوب وتوضيح دور مكونات الإشارات المضيفة وسطاء التهابات، وكذلك التفاعلات الخلية المحددة التي تدفع P. اللثوية يسببها الباثولوجيا المناعية 12-14. وبالإضافة إلى ذلك، حددت تجارب استخدام طفرات بكتيرية محددة P. حرجة اللثوية الفوعة العوامل المساهمة في التهاب مزمن في المواقع المحلية والنظامية 15. تفاصيل هذه المقالة منهجيات لتقييم P. اللثوية يسببها التهاب مزمن في المواقع المحلية والنظامية. نحن نقدم بروتوكول مفصلة لتحليل فقدان العظم السنخي بواسطة microCT باستخدام برنامج أميرة. بالإضافة إلى ذلك، نحدد فائدة المسلسل في الجسم الحي MRI الحيوانات الحية لتقييم تقدمية فيflammation داخل الشريان اللامسماة. ندرج منهجيات التصور النوعي والكمي لوحة التهابات الآفات في الشرايين، ووصف توصيف لهم النسيجي. استخدام الفئران المعدلة وراثيا والمسوخ البكتيرية المحددة يجعل هذا النموذج مناسبة خاصة لتحديد كل من المضيف والعوامل الميكروبية تشارك في بدء وتطور، ونتائج المرض. بالإضافة إلى ذلك، يمكن استخدام النموذج للكشف عن استراتيجيات علاجية جديدة، بما في ذلك التطعيم والتدخل الدوائي.

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			<td height="21" style="height:21px;width:508px;">Name of the Material/Eqiupment</td>
			<td style="width:196px;">Company</td>
			<td style="width:132px;">Catalogue Number</td>
			<td style="width:88px;">Comments&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Amira analysis software</td>
			<td>Visualization Sciences Group</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Anaerobic chamber DW Scientific Model MG500&#160;</td>
			<td>Microbiology International</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">BHI&#160;</td>
			<td>Becton-Dickinson&#160;</td>
			<td>211059</td>
			<td></td>
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			<td height="21" style="height:21px;">Hemin&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>51280-5G</td>
			<td></td>
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			<td height="21" style="height:21px;">Menadione (Vitamin K)</td>
			<td>Sigma-Aldrich&#160;&#160;</td>
			<td>M5625-25G</td>
			<td></td>
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			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Becton-Dickinson&#160;</td>
			<td>212750</td>
			<td></td>
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			<td height="21" style="height:21px;">Carboxymethyl cellulose (medium viscocity)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C-4888</td>
			<td></td>
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			<td height="21" style="height:21px;">Sulfamethoxazole and Trimethoprim Oral Suspension 200 mg/40 mg per 5ml</td>
			<td>Hi-Tech Pharmacal</td>
			<td>NDC 50383-823-16</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#956;CT 40&#160;</td>
			<td>Scanco</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">HistoChoice Tissue Fixative</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H2904</td>
			<td></td>
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			<td height="21" style="height:21px;">Sudan IV</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S4261-25G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">vertical-bore 11.7T Avance spectrometer&#160;</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Paravision</td>
			<td>Paravision</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">rat anti-mouse F4/80</td>
			<td>Serotec</td>
			<td>MCA497R</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">rat anti-mouse TLR2</td>
			<td>eBioscience</td>
			<td>13-9021-80</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Leica S4 Dissecting Scope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Microm HM 550 Cryostat</td>
			<td>Microm</td>
			<td></td>
			<td></td>
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</table>

a mouse model for pathogen-induced chronic inflammation at local and systemic sites

الالتهاب المزمن هو المحرك الرئيسي للتلف الأنسجة المرضية وصفة توحيد العديد من الأمراض المزمنة في البشر بما في ذلك الأورام، أمراض المناعة الذاتية، والأمراض الالتهابية المزمنة. أدلة تورط الناشئة الناجمة عن مسببات المرض التهاب مزمن في تطور وتقدم من الأمراض المزمنة مع مجموعة واسعة من المظاهر السريرية. بسبب المسببات معقد ومتعدد العوامل من الأمراض المزمنة، وتصميم التجارب لإثبات العلاقة السببية وإنشاء الروابط الميكانيكية يكاد يكون من المستحيل في البشر. ميزة استخدام النماذج الحيوانية هي أن كل من العوامل الوراثية والبيئية التي قد تؤثر على مسار مرض معين يمكن التحكم. وهكذا، وتصميم النماذج الحيوانية ذات الصلة من العدوى يمثل خطوة رئيسية في تحديد آليات المضيف والممرض المحددة التي تساهم في الالتهاب المزمن. نحن هنا وصف نموذج الفأر التي يسببها الممرض infla المزمنmmation في المواقع المحلية والنظامية بعد العدوى مع الممرض عن طريق الفم Porphyromonas اللثوية، بكتيريا المرتبطة بشكل وثيق مع أمراض اللثة البشري. العدوى عن طريق الفم من معين الممرض الفئران الحرة يؤدي الى الاستجابة الالتهابية المحلية مما أدى إلى تدمير الأسنان العظم السنخي الداعم، وهو السمة المميزة لمرض اللثة. في نموذج الفأر أنشئت من تصلب الشرايين، والإصابة بفيروس P. اللثوية تسارع ترسب البلاك التهابات الجيوب الأنفية داخل الأبهر والشريان اللامسماة، يرافقه تفعيل بطانة الأوعية الدموية، وزيادة ارتشاح الخلايا المناعية، والتعبير مرتفع من وسطاء التهابات داخل الآفات. نحن منهجيات التفاصيل لتقييم التهاب في المواقع المحلية والنظامية. استخدام الفئران المعدلة وراثيا والمسوخ البكتيرية المحددة يجعل هذا النموذج مناسبة خاصة لتحديد كل من المضيف والعوامل الميكروبية تشارك في بدء وتطور، ونتائج المرض. Additionallذ، فإن النموذج يمكن أن تستخدم لكشف عن استراتيجيات علاجية جديدة، بما في ذلك التطعيم والتدخل الدوائي.

باستخدام نموذج الفأر المناسب عن طريق الفم ونظام العدوى، P. اللثوية يحرض الالتهاب المزمن والباثولوجيا المناعية في المحلي (تجويف الفم) ومواقع النظامية (الشرايين) (الشكل 1). في الفئران، والعدوى عن طريق الفم مع P. اللثو?...

Watch this Scientific Journal Video about A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites at JoVE.com

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

لقد أثبتت النماذج الحيوانية لتكون أدوات لا تقدر بثمن في تحديد آليات المضيف والممرض المحددة التي تسهم في تطوير الالتهاب المزمن. نحن هنا وصف نموذج الفأر العدوى عن طريق الفم مع الممرض البشري Porphyromonas اللثوية ومنهجيات التفاصيل لتقييم تطور التهاب في المواقع المحلية والنظامية.

نموذج الفأر لالتهاب الناجم عن مسببات الأمراض المزمنة في مواقع المحلية والجهازية

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

a-mouse-model-for-pathogen-induced-chronic-inflammation-at-local

Research

JoVE Journal

Immunology and Infection

17.4K Views.  Boston University School of Medicine.  لقد أثبتت النماذج الحيوانية لتكون أدوات لا تقدر بثمن في تحديد آليات المضيف والممرض المحددة التي تسهم في تطوير الالتهاب المزمن. نحن هنا وصف نموذج الفأر العدوى عن طريق الفم مع الممرض البشري Porphyromonas اللثوية ومنهجيات التفاصيل لتقييم تطور...

Video: A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

المسلكة الفأرية العدوى : نموذج من الحصانة من النوع 2 والتهاب في القناة الهضمية

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

باستخدام الماوس BLT إنساني وساق الموديل خلية مقرها جين ورم العلاج

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

في الجسم الحي أسلوب التصوير لتميز الالتهاب الحاد والمزمن

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

في المختبر المقايسة المشتركة لتقييم الهجرة العدلية المستحثة المسببة للأمراض عبر الظهارية

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

حقن النظامية من الخلايا الجذعية العصبية / السلف في الفئران مع EAE المزمن

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

تحريض الفئران المعوية التهاب عن طريق نقل بالتبني من المستجيب CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; عالية</sup&gt; خلايا T في الفئران العوز المناعي

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

شارك في مناعي من البروتين ماوس MX1 مع الأنفلونزا من الفيروسات البروتين النووي

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

والطريقة الأمثل لعزل وتوسيع قسما ثابتا الطبيعية الخلايا التائية القاتلة من الماوس الطحال

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

الإدارة داخل القصبة الهوائية لل<em&gt; المستدمية النزلية</em&gt; في نماذج الماوس لدراسة التهاب الشعب الهوائية

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.