この作品は、国立アレルギー·感染症研究所によってサポートされていましたCAGにP01 A1078894を付与します

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+antibody+response+to+oral+infection+precedes+but+does+not+prevent+Porphyromonas+gingivalis-induced+alveolar+bone+loss+in+mice.">Serum antibody response to oral infection precedes but does not prevent Porphyromonas gingivalis-induced alveolar bone loss in mice.</a> Oral Microbiology and Immunology. 14 (3), 194-196 (1999).</li><li>Gibson, F. C., Hong, C., 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=Innate+immune+recognition+of+invasive+bacteria+accelerates+atherosclerosis+in+apolipoprotein+E-deficient+mice.">Innate immune recognition of invasive bacteria accelerates atherosclerosis in apolipoprotein E-deficient mice.</a> Circulation. 109 (22), 2801-2806 (2004).</li><li>Hayashi, C., Viereck, J., 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=Porphyromonas+gingivalis+accelerates+inflammatory+atherosclerosis+in+the+innominate+artery+of+ApoE+deficient+mice.">Porphyromonas gingivalis accelerates inflammatory atherosclerosis in the innominate artery of ApoE deficient mice.</a> Atherosclerosis. 215 (1), 52-59 (2011).</li><li>Hayashi, C., Madrigal, A. G., 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=Pathogen-mediated+inflammatory+atherosclerosis+is+mediated+in+part+via+Toll-like+receptor+2-induced+inflammatory+responses.">Pathogen-mediated inflammatory atherosclerosis is mediated in part via Toll-like receptor 2-induced inflammatory responses.</a> Journal of Innate Immunity. 2 (4), 334-343 (2010).</li><li>Hayashi, C., Papadopoulos, G., 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=Protective+role+for+TLR4+signaling+in+atherosclerosis+progression+as+revealed+by+infection+with+a+common+oral+pathogen.">Protective role for TLR4 signaling in atherosclerosis progression as revealed by infection with a common oral pathogen.</a> Journal of Immunology (Baltimore, Md.: 1950). 189 (7), 3681-3688 (2012).</li><li>Papadopoulos, G., Weinberg, E. 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=Macrophage-Specific+TLR2+Signaling+Mediates+Pathogen-Induced+TNF-Dependent+Inflammatory+Oral+Bone+Loss.">Macrophage-Specific TLR2 Signaling Mediates Pathogen-Induced TNF-Dependent Inflammatory Oral Bone Loss.</a> The Journal of Immunology. , (2012).</li><li>Gibson, F. C., Hong , C., 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=Innate+immune+recognition+of+invasive+bacteria+accelerates+atherosclerosis+in+apolipoprotein+E-deficient+mice.">Innate immune recognition of invasive bacteria accelerates atherosclerosis in apolipoprotein E-deficient mice.</a> Circulation. 109 (22), 2801-2806 (2004).</li><li>Miyamoto, T., Yumoto, H., Takahashi, Y., Davey, M., Gibson, F. C., Genco, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen-accelerated+atherosclerosis+occurs+early+after+exposure+and+can+be+prevented+via+immunization.">Pathogen-accelerated atherosclerosis occurs early after exposure and can be prevented via immunization.</a> Infection and Immunity. 74 (2), 1376-1380 (2006).</li><li>Baker, P. J., Dixon, M., Roopenian, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+susceptibility+to+Porphyromonas+gingivalis-induced+alveolar+bone+loss+in+mice.">Genetic control of susceptibility to Porphyromonas gingivalis-induced alveolar bone loss in mice.</a> Infection and Immunity. 68 (10), 5864-5868 (2000).</li><li>Baker, P. J., Dixon, M., Evans, R. T., Roopenian, D. 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著者は、彼らが競合する金融利害がないことを宣言します。

P.歯周経口感染モデルは、局所および全身の部位で病原体により誘導される慢性炎症の研究のための貴重なツールを提供します。このユニークなモデルは、慢性炎症や免疫病理に寄与し、ホストと病原体の両方の特定のメカニズムの特徴付けを可能にする。さらに、モデルは、免疫化および薬理学的介入を含む新規治療戦略のためにスクリーニングすることができる。このプロトコル?...

慢性炎症は、病理学的な組織の損傷およびヒトにおける多くの慢性疾患の統一特性の主要なドライバです。これらの疾患は、腫瘍性、自己免疫および慢性炎症性疾患1が含まれています。多くの慢性疾患の病因は依然として不明であるが、遺伝的素因および環境因子の導入の両方を含む、複雑で多因子性であると理解される。炎症のperpetuatorsはとらえどころのない残っていますが、免疫活性化の細胞および分子プロファイルは、病原体2に対する宿主応答において観察されるもののパターンとかなり重複する。 証拠が慢性炎症とその多様な臨床症状2,3の発症および進行中の微生物病原体の感染を暗示。病原体は、宿主の免疫系を破壊し、持続感染を確立することにより、直接、慢性炎症を誘導および維持することができます<sup&gt; 4。微生物の持続性の非存在下では、感染症、自己抗原分子擬態によりトリガ自己免疫反応は、免疫原性、それらをレンダリングする自己抗原、または以前にマスクされた宿主抗原を放出する損傷の変化による慢性炎症を沈殿させることができる。まれただし、特定の病原体は、特定の慢性疾患の普遍的な原因として同定されている。むしろ、利用可能なデータの大部分は、病原体は、遺伝的に感受性のホスト3における臨床症状および疾患転帰の広いスペクトルを有する慢性炎症を誘発するために異なるメカニズムを使用することを示唆している。したがって、特定の病原体が慢性炎症を誘導するメカニズムの詳細な理解は、主要な公衆衛生への影響だけでなく、多くの慢性疾患の治療および予防であってもよい。 慢性炎症の誘導および維持に貢献ホストと病原体の特定のメカニズムがありますがあまり理解されていない、病原体によって誘発される慢性炎症のモデル化における進歩は、これらのプロセスの理解を促進するために始めている。 P.歯周経口感染モデルは、ホストと病原ローカル（口頭骨の損失）で慢性炎症に貢献する具体的な仕組みや全身の部位（アテローム性動脈硬化症）5,6の分析、病原体によって誘発される慢性炎症のユニークな、十分に特徴付けられたマウスモデルである。 P.ジンジバリスは、人間の歯周病、組織7を支持歯の破壊を特徴とする感染主導の慢性炎症性疾患に関与するグラム陰性、嫌気性口腔病原体である。感染の初期部位における病理学に加えて、証拠が蓄積P.を暗示ジンジバリスは 、アテローム性動脈硬化症5を含む全身性疾患の発症および進行中の慢性inflammatioを特徴とする疾患を慢性炎症誘発動脈血管壁のnである。 Pと特定病原体フリーのマウスの経口感染ジンジバリスは、歯の破壊の結果は、歯槽骨8を支持することが局所的な炎症応答を誘導する。P.ジンジバリスは、病原体特異的血清抗体力価9の高レベルを開発するまでの42日感染後8およびマウス感染マウスの口から回収することができる。 - / -マウス（アポE - / - ）、P.経口感染アポリポタンパク質-Eを使用して、アテローム性動脈硬化症の確立されたマウスモデルにおいてジンジバリスは大動脈洞10と腕頭動脈11内炎症性プラーク沈着を駆動慢性炎症を誘導する。 Pの腕頭動脈内のプログレッシブ炎症歯周マウスは、生体内 MRI で使用して生きた動物でモニターすることができる感染した。組織学的には、Pから動脈病変歯周マウスは脂質accompa蓄積の増加を示し、感染した血管内皮の活性化、増加した免疫細胞浸潤、および炎症性メディエーター12の発現の上昇によって、防災科学技術研究所。ノックアウトマウスでのこのモデルの使用は、宿主シグナル伝達成分および炎症性メディエーターの役割、ならびに細胞P.を駆動特異的相互作用を解明している14 - 歯周は免疫病理12誘発 。また、定義された細菌の変異株を用いた実験では、重要なPを同定している歯周は、ローカルおよび全身の部位15で慢性炎症に貢献する要因を病原性。 この記事では、Pの評価のための方法論を詳述歯周は、ローカルおよび全身の部位での慢性炎症を誘発。私たちは、アミラソフトウェアを使用してマイクロCTによって歯槽骨喪失の分析のための詳細なプロトコルを提供する。さらに、当社は、プログレッシブでの評価のための生体内で生きている動物のMRI におけるシリアルの有用性を定義する腕頭動脈内のflammation。私たちは、動脈病変における炎症性プラークの可視化および定量化のための方法論が含まれ、それらの組織学的特徴づけを説明します。トランスジェニックマウスおよび定義された細菌の突然変異体の使用は、宿主および疾患の開始、進行および予後に関与する微生物因子の両方を識別するためのこのモデルは特に適している。さらに、モデルは、ワクチン接種および薬理学的介入を含む新規治療戦略のためにスクリーニングすることができる。

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	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amira analysis software</td>
			<td>Visualization Sciences Group</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anaerobic chamber DW Scientific Model MG500&#160;</td>
			<td>Microbiology International</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BHI&#160;</td>
			<td>Becton-Dickinson&#160;</td>
			<td>211059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemin&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>51280-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Menadione (Vitamin K)</td>
			<td>Sigma-Aldrich&#160;&#160;</td>
			<td>M5625-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Becton-Dickinson&#160;</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxymethyl cellulose (medium viscocity)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C-4888</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#956;CT 40&#160;</td>
			<td>Scanco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HistoChoice Tissue Fixative</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H2904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sudan IV</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S4261-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vertical-bore 11.7T Avance spectrometer&#160;</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paravision</td>
			<td>Paravision</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rat anti-mouse F4/80</td>
			<td>Serotec</td>
			<td>MCA497R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rat anti-mouse TLR2</td>
			<td>eBioscience</td>
			<td>13-9021-80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica S4 Dissecting Scope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microm HM 550 Cryostat</td>
			<td>Microm</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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

慢性炎症は、病理学的な組織の損傷及び腫瘍性、自己免疫および慢性炎症性疾患を含むヒトにおける多くの慢性疾患の統一特性の主要なドライバです。新たな証拠は、臨床症状の多種多様な慢性疾患の発症および進行中の病原体により誘導される慢性炎症を暗示。慢性疾患の複雑で多因子病因に、因果関係の証明および機構のリンクの確立のための実験を​​設計するヒトではほぼ不可能です。動物モデルを使用する利点は、特定の疾患の経過に影響を与える可能性の両方の遺伝的要因と環境要因を制御することができるということである。したがって、感染症の関連する動物モデルを設計する慢性炎症に寄与し、ホストおよび病原体特異的なメカニズムを特定するのに重要なステップである。 ここでは、病原体によって誘発される慢性inflaのマウスモデルを記述する口腔病原体ポルフィロモナス·ジンジバリス 、密接に人間の歯周病に関連する細菌による感染後の局所及び全身の部位でmmation。特定病原体フリーのマウスの経口感染が歯槽骨、歯周疾患の特徴を支える歯の破壊をもたらす局所的炎症応答を誘導する。アテローム性動脈硬化症の確立されたマウスモデル、P.による感染でジンジバリスは、血管内皮の活性化、増加した免疫細胞浸潤、および病変内の炎症性メディエータの発現の上昇を伴う大動脈洞および腕頭動脈内の炎症性プラーク沈着を加速させます。私たちは、局所および全身の部位での炎症の評価のための詳細な方法論。トランスジェニックマウスおよび定義された細菌の突然変異体の使用は、宿主および疾患の開始、進行および予後に関与する微生物因子の両方を識別するためのこのモデルは特に適している。 Additionallyは、モデルは、ワクチン接種および薬理学的介入を含む新規治療戦略のためにスクリーニングすることができる。

適切なマウスモデルおよび経口感染レジメン、P.を使用して歯周は （口腔）局所および全身の部位（動脈）（ 図1）で、慢性炎症や免疫病理を誘発する。 Pとマウスでは、経口感染ジンジバリスは、歯槽骨8を支持する歯の破壊を駆動する局所炎症応答を誘導する。P.歯周マウスは主にIgGアイソタイプ9の...

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

動物モデルは、慢性炎症の発展に貢献宿主および病原体特異的なメカニズムを定義する上で非常に貴重なツールであることが分かっている。ここでは、ローカルおよび全身の部位での炎症の進行を評価するために、ヒト病原体ポルフィロモナス·ジンジバリスとディテールの方法論で経口感染のマウスモデルを記述する。

局所および全身所における病原体により誘導される慢性炎症のためのマウスモデル

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 ビュー.  Boston University School of Medicine. 動物モデルは、慢性炎症の発展に貢献宿主および病原体特異的なメカニズムを定義する上で非常に貴重なツールであることが分かっている。ここでは、ローカルおよび全身の部位での炎症の進行を評価するために、ヒト病原体ポルフィロモナス·ジンジバリスとディテールの方法論で経口感染のマウスモデルを記述する。 

ビデオ: 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.

ネズミ鞭虫感染症：腸内のタイプ2免疫と炎症のモデル

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

免疫・感染学

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.

幹細胞ベースの遺伝子治療腫瘍モデルとしてBLTヒト化マウスを用いた

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.

急性及び慢性炎症を区別するために生体内イメージング法における

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.

インビトロ 病原体誘導性好中球トランス上皮移動を評価するコーカルチャーアッセイ

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.

慢性EAEを有するマウスにおける神経幹/前駆細胞の全身注射

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.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

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.

インフルエンザAウイルス核を持つマウスMx1のタンパク質の共免疫沈降

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.

マウスの脾臓から不変ナチュラルキラーT細胞を単離および拡張するための最適化方法

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.

の気管内の管理<em&gt;インフルエンザ菌</em&gt;マウスモデルにおいて気道炎症を研究します

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.