一般的な寄付基金、国立 - この作品は、国立多発性硬化症SocietyRG 4587-A-1、Civitan国際研究財団、マイク・L. Jezdimir横断性脊髄炎財団、アラバマ州保健財団の大学NINDS P30-NS069324によって賄われていました国立アレルギー感染症研究所、国立衛生研究所からの科学財団1355183、およびT32 AI007051。

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著者らは、開示することは何もありません。

MS患者は直接的にCNSを保護する治療選択肢の開発を正当化、CNSへのT細胞活性化および/または浸潤を減衰させる薬物を服用しながら、疾患の再発を経験し続けます。 EAEは、古典的MSの症状をモデル化するために使用され、 インビボで免疫系と中枢神経系との間の相互作用の性質を研究する際に強力なツールであることができます。 EAEにおいて治療上の考慮事項のタイミング、 例えば 、使用することは、疾患の開始前または後に、周囲にCNSにおける免疫細胞浸潤を調べ、増殖および活性化に関連して、免疫系の両方での処置の効果を描写することが可能であるとCNS。 C57BL / 6マウスにおけるEAEは、より広く利用されているが、これらのマウスは、実質における再発寛解型の表現型と免疫細胞の浸潤を持っているとして、SJLマウスにおけるEAEは、MSの症例の大部分をより表すことができます脳10の。 SJLマウスは、疾患が提示した後、それが可能な治療を開始することが、減少の炎症の時代に、ならびに寛解の間に明確な回復を持っています。 SJLマウスは、常に結果がプールされたときに、潜在的に大きなばらつきが生じ、同期して再発し、送金しないことを考慮することが重要です。そのため、一部の研究者は、疾患の進行に個別のポイントでFACS分析および組織学のためのマウスをしながら1動物から臨床スコアのための代表的な結果を示すことを選ぶことができます。 操作がEAEマウスに行われた場合を考慮すると、治療は免疫系や中枢神経系にどのように影響するかの決意を支援することができます。治療の開始時には多くのオプション、免疫細胞が中枢神経系に入っているとどのように彼らはCNSと対話することができるかどうかのために、独自的な意味合いを持つそれぞれがあります。症状の発症前に治療は、免疫細胞がまだ入力またはCNSへの損傷を引き起こしていないことを意味します。症状の発症後の治療は、免疫細胞が中枢神経系に入っているし、いくつかの被害をもたらしていることを意味します。 SJLマウスを使用して、治療はまた、免疫細胞が活発に浸潤し、炎症を引き起こしている再発、中、または免疫細胞が少ない炎症とCNSであまり普及しているかもしれ寛解、中に開始することができます。免疫細胞が治療中に病理学的過程のどこにいるか検討する際の治療はCNSおよび免疫系にどのような影響を与えるかについて初期仮説を行うことができます。 治療は、免疫細胞及びCNS、EAE症状の重症度を軽減するの最終結果にそれぞれ影響を与えることができるいくつかの方法があります。したがって、免疫細胞のCNS入力したか否かを、周囲及びCNSに影響を受けるかの免疫細胞を見てフローサイトメトリー分析および免疫組織化学を使用する必要があり、CNSは、治療に反応する方法。脊髄のフローサイトメトリー分析はどのように多くの細胞ヘクタールを判断することができますが与えられた時間にCNSを入力VEの免疫細胞の増殖は、脾臓で影響されない場合を除き、1は、この効果は減少した免疫細胞の輸送によるものであることを判断することはできません。末梢および中枢神経系の両方の組織を分析し、両方の組織を比較した場合の結果は、機構的に何を意味するかを決定することが必要です。免疫細胞活性プロファイルが調節性T細胞重プロファイルに病原性のヘルパーT細胞重いプロファイルのスイッチを有し、例えば、処理することにより変更することも可能です。異なる細胞型のマーカーを見て、処置および未処置動物の間のパーセント発現を比較するためにも重要な考慮事項です。 MS研究における新興の概念は、B細胞が自己免疫性脱髄において重要な役割を果たすことを示唆しています。これは、B細胞がT細胞20の再活性化に必要であることを示す研究に基づいています。この概念は、リツキシマブなどの治療の成功は、CD20元に対する抗体によってサポートされていますB細胞21,22の表面に押されました。臨床試験におけるモノクローナル抗体のオクレリズマブの成功によって示されるように、CD20の異なるエピトープを標的とする薬剤は、B細胞標的治療薬23の有効性を改善することができます。 ここに提示された技術の1つの制限は、免疫細胞がCNSに入るが、実質に移動することができなくすることが可能であることです。免疫組織化学、免疫細胞の血管周囲カフィングを検出し、処理および未処理動物の間で実質に移動した距離を評価するために使用することができます。別の潜在的な制限は、EAEの病因にmicrobiomeの影響を伴います。共生腸内細菌叢は重く疾患の病因24に影響を与えることができます。したがって、別のコロニーに、さらに別のケージで飼育したマウスは、疾患の重症度の広大な違いを持つことができます。以下のために、同じケージで飼育同腹子対照を使用することが可能な場合したがって、それは常に好ましいです。EAEを含む実験。最後の注意は、末梢における免疫細胞増殖の変化の影響を排除するために、実験が望ましい場合、受動的伝達の誘導ではなく、このプロトコルに記載の活性誘導を使用してそうすることが可能であるということです。 神経保護のためのさらなる確認は、細胞死または細胞型に選択的にタンパク質の欠失を可能にする条件付きノックアウトマウスの使用を介して特定の機構を試験する共培養系11を用いて達成することができます。さらに、神経保護されている薬理学的薬剤の探索を拡張するために、軸索切断および神経細胞死のマーカーが含まれるべきです。重要性の別の領域は、再ミエリン化です。負傷軸索は神経保護療法は、再ミエリン化療法の重要な一部である必要があり、さらにサポートを貸し再ミエリン化することはできません。さらに、無髄軸索がmyelinaよりも傷害に対してより脆弱ですテッドの軸索。これは、軸索は軸索損傷を防ぐことができますタイムリーな再ミエリン化を促進する脱髄治療的介入になるとことを示唆しています。これらの道を探索するために、脱髄および再ミエリン化のために他のin vivoモデル（ すなわち 、クプリゾン及びリゾレシチン）を用いることができます。本明細書に記載される方法は、ミエリンの損失を定量することによって神経保護を評価することに焦点を当てました。再ミエリン化の評価のための前駆細胞の数、ならびに増殖および成熟する能力も調査することが重要であろう。これらの代替モデルの言及で、1はまた、ウイルスに媒介される脳炎の異なるモデルを考慮しなければなりません。ミエリン損失を生成する2つのよく特徴付けRNAウイルスのモデルがあります：1は、タイラーマウス脳脊髄炎、非エンベロープピコルナウイルスであり、他方はマウス肝炎ウイルス、コロナウイルス科25,26のメンバーです。 EAEは、STのための貴重なツールです。操作または治療は、生体内で免疫系と中枢神経系にどのような影響を与えるかのudies。ここで説明するプロトコルは、それが周辺部で、血液脳関門に、またはCNSにあるかどうか、治療は病気のプロセスに影響を与えている場所を判断するのに役立ちます。 MSのための現在の治療は、時間の経過とともに、多くの場合、経験の減少疾患患者を治すません。同様に、急性散在性脳脊髄炎、横断性脊髄炎、および視神経脊髄炎、それは浸潤免疫細胞による攻撃の直下であるとしてCNSを保護欠如処理を含む免疫細胞のCNSへの浸潤およびミエリンの分解を伴う他の疾患。考慮治療のタイミングを取り、炎症および損傷を評価するためにCNSの免疫組織化学と関連して脾臓および脊髄のフローサイトメトリー分析を使用して治療についてなされるべきメカニズムの決定を可能にします。

多発性硬化症（MS）は、早期の疾患における脳の白質領域において主に炎症性病変によって特徴付けられます。長期進行した後、灰白質萎縮は、MRIイメージングによって検出され、疾患の神経変性相をマークします。反応性神経膠症、脱髄、および白質における軸索損傷は、CNS浸潤免疫細胞に起因しています。現在MSに使用される治療法のいずれも逆転しないか、直接CNSに神経変性を防止する - その代わりに、それらは、CNSへのT細胞活性化および/または浸潤を減衰させることによって炎症を低減します。そこにMSの治療法がなく、現在の治療法を使用している患者は、疾患の進行を経験し続けているため、脱髄およびニューロンの損失を防ぐ薬の発見は非常に重要です。 すなわち 、CNSへのダメージ低減を- -のように見えるが、免疫細胞への影響およびCNS上のものとを区別することは、結果として、実験的に困難な場合がありますかかわらず、それが発生し、それを通してメカニズムの雨。したがって、CNS保護の評価は、病気のメカニズムにどのように影響するかを薬理学的薬剤を決定するために周囲に免疫細胞と免疫細胞の増殖をCNSは、浸潤性の評価と提携する必要があります。 実験的自己免疫性脳脊髄炎（EAE）は、現在MS 1-4を治療するために使用される薬物の発見のための直接の原因であった自己免疫炎症性疾患の十分に確立された動物モデルです。マウスはしばしば、C57BL / 6マウスは、遺伝的変異体の利用可能性に基づいて、人気のある株であると、EAEのために使用されます。 EAEで誘導されたC57BL / 6マウスを、10日目、誘導後の周囲に発症する慢性疾患の進行を示します。脊髄実質および小脳の浸潤は、皮質実質5の浸潤の欠如と、これらの動物の組織病 ​​理学の特徴です。 Bでさらに、皮質病変および脱髄雨はC57BL / 6マウスでは比較的存在しない疾患6-9の顕著な特徴です。可能であればしたがって、MS 10と同様に表示され、脳および脊髄の両方に見られる再発寛解型疾患および病変を有するSJLマウスを、使用することが好ましいです。 免疫細胞が中枢神経系に到達しない場合の処置は、神経保護として分類することはできません。したがって、このプロトコルは、以前に11を示したように、脳、脊髄、およびEAEマウス由来の脾臓のフローサイトメトリー分析の使用は、免疫細胞のCNS ​​への浸潤および末梢における免疫細胞の増殖に対する処置の効果を決定することを可能にします。程度および神経保護の性質を決定するために、中枢神経系組織の免疫組織化学的分析も記載されています。これらの方法を組み合わせることにより、CNSがあったかどうかPR免疫細胞が活性化免疫細胞がCNSに進入するかどうかを、周辺で増殖されたかどうかの決意を可能にし、そして炎症や損傷からotected。神経保護効果は、免疫系への影響にもかかわらず、疑われる場合はCNSへの免疫細胞の浸潤が発生した後、実験者は、治療の開始時間を変更することができます。 ここでは、アクティブなEAE、MSのT細胞媒介動物モデルの2つの異なるモデルを使用してプロトコルを提示し、のさまざまな側面に実験的な治療法の有効性を決定するために、病気中の様々な時点での免疫組織化学と組み合わせたフローサイトメトリー分析MSの病因。この方法は、それが簡単に薬が疾患の病因に作用する方法を絞り込むことながら、中枢神経系の保護に対する免疫細胞の増殖と浸潤に対する効果を区別するの研究者を支援します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>22 x 22 x 20 mm embedding mold</td>
			<td>Fisher Scientific</td>
			<td>NC9719245</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 30 x 20 mm embedding mold</td>
			<td>Fisher Scientific</td>
			<td>NC9531194</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (55mM)</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Methylbutane</td>
			<td>Fisher Scientific</td>
			<td>O3551-4</td>
			<td></td>
		</tr>
		<tr>
			<td>30 x 22 x 20 mm embedding mold</td>
			<td>Fisher Scientific</td>
			<td>18-30</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD4 PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>552775</td>
			<td>0.2 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>anti-Foxp3-FITC</td>
			<td>eBioscience</td>
			<td>11-5773-82</td>
			<td>0.5 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>anti-GFAP (Cocktail)</td>
			<td>Biolegend</td>
			<td>835301</td>
			<td>1-3 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>anti-Iba-1 Polyclonal Antibody (50 ug)</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td>0.5 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>anti-IFN-&#947; APC</td>
			<td>eBioscience</td>
			<td>17-7311-82</td>
			<td>0.2 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>anti-IL-17A PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-7177-82</td>
			<td>0.2 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>anti-Ki-67 PE</td>
			<td>eBioscience</td>
			<td>12-5698-82</td>
			<td>0.2 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>anti-MBP (D-18)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-13912</td>
			<td>0.2 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>anti-TCR&#946; FITC</td>
			<td>eBioscience</td>
			<td>11-5961-85</td>
			<td>0.5 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>anti-TCR&#946; PE</td>
			<td>eBioscience</td>
			<td>12-5961-83</td>
			<td>0.2 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>Biotinylated Goat Anti-Rabbit IgG</td>
			<td>Vector Labs</td>
			<td>BA-1000</td>
			<td>1.5 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>Biotinylated Horse Anti-Mouse IgG</td>
			<td>Vector Labs</td>
			<td>BA-2000&#160;</td>
			<td>1.5 mg/mL stock concentration</td>
		</tr>
		<tr>
			<td>Citric Acid, Anhydrous, 99.5%</td>
			<td>Fisher Scientific</td>
			<td>AC42356-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA), tetrasodium salt dihydrate, 99%</td>
			<td>Fisher Scientific</td>
			<td>AC446085000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides, case of 10</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3/Transcription Factor Staining Buffer Set</td>
			<td>eBioscience</td>
			<td>00-5523-00</td>
			<td>&#8203;Foxp3 transcription factor staining reagents</td>
		</tr>
		<tr>
			<td>Golgi Plug</td>
			<td>BD Biosciences</td>
			<td>555029</td>
			<td>protein transport inhibitor</td>
		</tr>
		<tr>
			<td>Immedge Hydrophobic Barrier Pen</td>
			<td>Fisher Scientific</td>
			<td>NC9545623</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>EMD Millipore</td>
			<td>407952-5mg</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine, 100X</td>
			<td>Corning</td>
			<td>25-005-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential Amino Acids</td>
			<td>Corning</td>
			<td>25-025-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Near IR Live/Dead Staining Kit</td>
			<td>Life Technologies</td>
			<td>L10119</td>
			<td>viability dye</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Labs</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal horse serum</td>
			<td>Vector Labs</td>
			<td>S-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 96%</td>
			<td>Fisher Scientific</td>
			<td>AC416785000</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td>density gradient</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-500</td>
			<td>resinous mounting medium</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma</td>
			<td>P1585-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-Myelin Basic Protein Antibody</td>
			<td>BioLegend</td>
			<td>808401</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>10-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Corning</td>
			<td>25-000-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek CRYO-OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>nonionic detergent</td>
		</tr>
		<tr>
			<td>Vectastain Elite ABC Kit (Standard)</td>
			<td>Fisher Scientific</td>
			<td>NC9206402</td>
			<td>avidin-biotin-peroxidase&#160;complex (ABC) in immunoperoxidase&#160;</td>
		</tr>
		<tr>
			<td>Vector Laboratories Peroxidase Substrate Kit (DAB)</td>
			<td>Fisher Scientific</td>
			<td>NC9276270</td>
			<td>DAB solution</td>
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	</tbody>
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determining immune system suppression versus cns protection for pharmacological interventions in autoimmune demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

Watch this Scientific Journal Video about Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination at JoVE.com

自己免疫脱髄における薬理学的介入における免疫系抑制とCNS保護の決定 |免疫学と感染症 |ビデオ

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

自己免疫性脱髄に薬理学的介入のためのCNS保護に対する免疫系の抑制を決定します

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

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12.1K ビュー.  University of Alabama at Birmingham. This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

ビデオ: Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

免疫・感染学

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

移入、抗原チャレンジにより実験的自己免疫性脳脊髄炎（EAE）抵抗性マウス系統の応答がないことを克服する

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

ノーマルCNSのリアルタイムPCRによると神経炎症時のミクログリアにおけるマイクロRNAの検出

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

新生児と大人の中枢神経系から培養ミクログリア

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

酵素消化および勾配分離を使用したマウス皮膚から浸潤白血球の単離

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

マウスおよび種々の組織における免疫細胞の疾患に依存する分布の評価における実験的自己免疫性脳脊髄炎の誘導

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

炎症反応に関与する新規血管内皮レギュレータを特徴付けるための試金をスクリーニング

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

マウス小腸管免疫系からリンパ球を分離すること

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

グルコース-6-リン酸イソメラーゼ(G6PI)誘発RAマウスにおけるiNKT細胞の導入免疫療法

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...