Diese Arbeit wurde vom NIH DK080351 auf PC, und durch eine großzügige Schenkung des Terry und Kristine Gore Familie unterstützt.

Experimentelles Protokoll Sobald die Hypophyse Immunogen bereit ist (siehe begleitende Artikel), ist es subkutan in SJL Mäusen (The Jackson Laboratories, hat 000.686) injiziert. Eine zweite, identische Injektion ist 7 Tage später wiederholt. Mäuse werden dann verwendet, um in vivo und post-mortem Ergebnisse zu sammeln. Schritt 1. Die subkutane Injektion von Hypophysen-Immunogen in SJL Mäusen Mäuse werden zunächst betäubt durch die Injektion von 0,5 ml 20 mg / ml 2,2,2-Tribromethanol (Avertin, TCI America, T-1420) intraperitoneal. Mäuse werden dann subkutan mit 100 ul der Hypophyse Emulsion, die, wie in der Begleiter angegeben 2181 Artikel JoVE injiziert, enthält 1 mg Hypophysenextrakt und 0,25 mg von komplettem Freund-Adjuvans. Die Emulsion wird in der linken dorsalen Hinterbein Region ((50 ul) und der rechten Leistengegend (50 ul) injiziert. Die Hypophyse Emulsion wieder an Tag 7 in den gegenüberliegenden Seiten (50 ul in der rechten dorsalen Hinterbein Region und injiziert 50 pl in der linken Leistengegend). Mäuse sind täglich für die ersten 3 Tage überwacht und dann wöchentlich bis zum Tag der Opfer, die in der Regel 28 Tage nach der ersten Immunisierung. Schritt 2. Sammlung von in-vivo-Ergebnisse: Blood Assays Das Gewebe am häufigsten verwendet werden, um experimentelle Ergebnisse zu bewerten, während die Mäuse noch am Leben ist Blut. Blut wird routinemäßig 14 Tage nach der ersten Immunisierung, die Serumspiegel von Hypophyse Antikörpern oder anderen Markern wie Zytokine und Chemokine zu messen gezogen. Blut wird von lebenden Mäusen wie folgt gesammelt. Mäuse sind sicher durch die Genick gehalten und die Rückseite der Kieferknochen befindet. Sobald der Rückseite des Kieferknochens befindet, sind 4 mm Lanzetten zu durchbohren die submandibularis Leitbündel verwendet. Nach Blut gesammelt, mit Druck auf den auf dem Gelände einen Alkoholtupfer, um ein Hämatom zu vermeiden. Gesammelte Blut wird bei 2.000 g für 20 Minuten zentrifugiert und bei -80 ° C bis zur Verwendung. Schritt 3. Sammlung von Post-mortem-Ergebnisse: Pituitary Histopathologie Mäuse sind in der Regel 28 Tage nach der ersten Immunisierung geopfert. Der Kardinal-Funktion notwendig, um die Diagnose von EAH zu etablieren ist die Infiltration der Hypophyse durch hämatopoetische mononukleären Zellen. Mäuse sind, indem man sie in einem verschlossenen Plastikbeutel Kammer, die mit CO 2 durchströmt wird eingeschläfert. Mäuse sind in der Kammer für insgesamt 8 Minuten gehalten. Nach Euthanasie, ist die Hypophyse gesammelt wie in der Begleitung Artikel beschrieben. Eine erkrankte Hypophyse erscheint geschwollen und mehr fest mit dem umgebenden Dura mater. Sharp Pinzette werden die Hirnhäute rund um die Hypophyse zu lösen. Sobald die Hypophyse bewegt sich frei auf dem Keilbein, einem Ende der Hypophyse wird mit einer Pinzette gefasst und der Hypophyse wird vorsichtig angehoben. Die Drüse wird dann in ein Mikrozentrifugenröhrchen mit Beckstead das Fixiermittel 5 platziert. Die Hypophyse wird über Nacht, fest eingewickelt in Papier-Objektiv, und in einer Kassette. Die Drüsen werden anschließend mit dem folgenden Protokoll: <table border="1"><tbody><tr><td> Rufnummer </td><td> Reagens </td><td> Zeit in Minuten </td><td> Temperatur (Celsius) </td></tr><tr><td> 1 </td><td> 70% Alkohol </td><td> 5 </td><td> 25 </td></tr><tr><td> 2 </td><td> 70% Alkohol </td><td> 15 </td><td> 25 </td></tr><tr><td> 3 </td><td> 95% Alkohol </td><td> 15 </td><td> 25 </td></tr><tr><td> 4 </td><td> 95% Alkohol </td><td> 15 </td><td> 25 </td></tr><tr><td> 5 </td><td> 100% Alkohol </td><td> 15 </td><td> 25 </td></tr><tr><td> 6 </td><td> 100% Alkohol </td><td> 7,5 </td><td> 25 </td></tr><tr><td> 7 </td><td> 100% Alkohol </td><td> 7,5 </td><td> 25 </td></tr><tr><td> 8 </td><td> Xylol </td><td> 20 </td><td> 25 </td></tr><tr><td> 9 </td><td> Xylol </td><td> 20 </td><td> 25 </td></tr><tr><td> 10 </td><td> Xylol </td><td&gt; 20 </td><td> 25 </td></tr><tr><td> 11 </td><td> Paraffin </td><td> 30 </td><td> 58 </td></tr><tr><td> 12 </td><td> Paraffin </td><td> 5 </td><td> 58 </td></tr><tr><td> 13 </td><td> Paraffin </td><td> 5 </td><td> 58 </td></tr></tbody></table> Nach der Bearbeitung werden Hypophyse in Paraffin eingebettet und mindestens 5 nonconsecutive Abschnitte (5-mu m dick) werden von jeder Drüse abgeschnitten. Nach dem Anschneiden, sind die Drüsen mit Hämatoxylin und Eosin gefärbt und analysiert, um die mononukleäre Infiltration zu quantifizieren. Der Abschnitt mit den schwersten Infiltration ist für die Auswertung ausgewählt. Die Schwere wird auf der Grundlage einer subjektiven Einschätzung des Ausmaßes der Hypophysenvorderlappen Ersatz oder Zerstörung durch Immunzellen erzielt: Klasse 0, keine Krankheit, Grad 1, 2% -20% der Betroffenen; Grad 2, 20% -30%; Grad 3, 30% -50%; Grad 4, 50% -90%; Grad 5, mehr als 90%. Alle Abschnitte sind von zwei Personen geblendet, um die Quelle der Abschnitte erzielt.

<ol>
	<li>Tzou, S. C., Lupi, I., Landek, M., Gutenberg, A., Tzou, Y. M., Kimura, H., Pinna, G., Rose, N. R., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+Hypophysitis+of+SJL+mice:+Clinical+Insights+from+a+New+Animal+Model.">Autoimmune Hypophysitis of SJL mice: Clinical Insights from a New Animal Model.</a> Endocrinology. , (2008).</li><li>Twarog, F. J., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+production+of+thyroid+autoantibodies+in+mice.">The production of thyroid autoantibodies in mice.</a> J Immunol. 101, 242-2450 (1968).</li><li>Patrick, J., Lindstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+response+to+acetylcholine+receptor.">Autoimmune response to acetylcholine receptor.</a> Science. 180, 871-872 (1973).</li><li>Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+hypophysitis:+an+underestimated+disease+in+search+of+its+autoantigen(s).">Autoimmune hypophysitis: an underestimated disease in search of its autoantigen(s).</a> J Clin Endocrinol Metab. 92, 2038-2040 (2007).</li><li>Beckstead, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+preservation+of+fixation-sensitive+antigens+in+paraffin-embedded+tissues:+addendum.">A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues: addendum.</a> J Histochem Cytochem. 43, 345-345 (1995).</li><li>Levine, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergic+adenophypophysitis:+new+experimental+disease+of+the+pituitary+gland.">Allergic adenophypophysitis: new experimental disease of the pituitary gland.</a> Science. 158, 1190-1191 (1967).</li><li>Yoon, J. W., Choi, D. S., Liang, H. C., Baek, H. S., Ko, I. Y., Jun, H. S., Gillam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+an+organ-specific+autoimmune+disease,+lymphocytic+hypophysitis,+in+hamsters+by+recombinant+rubella+virus+glycoprotein+and+prevention+of+disease+by+neonatal+thymectomy.">Induction of an organ-specific autoimmune disease, lymphocytic hypophysitis, in hamsters by recombinant rubella virus glycoprotein and prevention of disease by neonatal thymectomy.</a> Journal of Virology. 66, 1210-1214 (1992).</li><li>Davis, J. R., McMahon, R. F., Lowenstein, P. R., Castro, M. G., Lincoln, G. A., McNeilly, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+in+the+ovine+pituitary+gland+is+associated+with+hypophysitis.">Adenovirus-mediated gene transfer in the ovine pituitary gland is associated with hypophysitis.</a> J Endocrinol. 173, 265-271 (2002).</li><li>Rotondo, F., Quintanar-Stephano, A., Asa, S. L., Lombardero, M., Berczi, I., Scheithauer, B. W., Horvath, E., Kovacs, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenohypophysitis+in+rat+pituitary+allografts.">Adenohypophysitis in rat pituitary allografts.</a> Int J Exp Pathol. , (2010).</li></ol>

Vier Techniken berichtet worden, um Hypophysitis in Versuchstieren zu induzieren. Die Technik in diesem Artikel beschrieben wird, dass die Injektion von Hypophysen-Proteine ​​mit Freund Adjuvans vermischt wird, ist die älteste, seit 1967 6, und wurde in jüngerer Zeit 1 verbessert. Andere Techniken sind die Injektion von löslichen Rötelnvirus Glykoproteine ​​in 1992 7, die stereotaktische Injektion eines Adenovirus direkt in der Hypophyse in 2002 8 und die Transplantation von Hypophysen von einem Rattenstamm unter die Nierenkapsel einer anderen Rattenstamm in 2010 9 . Der wesentliche Vorteil der adjuvanten Ansatz der Freund s ist, dass es weniger invasive, konsequenter und vor allem genauere bei der Induktion einer Krankheit, die eng mit dem menschlichen Pendant. Die Technik in diesem Video Artikel beschrieben ergibt sich ein reproduzierbares Modell der Hypophysitis, die von Forschern genutzt werden können, um unser Verständnis dieses faszinierenden Krankheit zu verbessern.

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<td>SJL mice (The Jackson Laboratories)</td>
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<td>stock 000686</td>
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<td>2,2,2-Tribromoethanol (Avertin)</td>
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<td>TCI America</td>
<td>T-1420</td>
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<td>Beckstead's fixative</td>
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induction of experimental autoimmune hypophysitis in sjl mice

Autoimmune Hypophysitis kann experimentell durch die Injektion von Hypophysen-Proteine ​​mit einem Adjuvans in anfälligen Mäusen 1 gemischt wiedergegeben werden. Mausmodelle erlauben uns zu untersuchen, wie Krankheiten zu entfalten, bieten häufig eine gute Nachbildung der gleichen Prozesse beim Menschen auftreten. Für einige Autoimmunerkrankungen wie Typ 1A Diabetes, gibt es Modelle (der NOD-Maus), die spontan ein Krankheitsbild ähnlich wie das menschliche Gegenstück. Für viele andere Autoimmunerkrankungen, muss jedoch das Modell experimentell induziert werden. Ein gemeinsamer Ansatz in dieser Hinsicht ist die Maus mit einer dominanten Antigen aus dem Organ untersucht abgeleitet injizieren. Zum Beispiel, injizieren Forscher im Bereich der Autoimmun-Thyreoiditis interessiert Mäuse mit Thyreoglobulin 2, und derjenigen, die unter Myasthenia gravis interessiert spritzen sie mit der Acetylcholin-Rezeptor 3. Wenn das Autoantigen für einen bestimmten Autoimmunerkrankungen nicht bekannt ist, injizieren Forscher eine Proteinrohextrakt von der Orgel durch die Autoimmunreaktion ausgerichtet. Für autoimmune Hypophysitis, bleiben die pathogenen Autoantigen (s) zu identifizieren 4, und damit eine grobe Hypophyse Protein-Präparation verwendet. In diesem Video-Artikel werden wir zeigen, wie experimentelle autoimmune Hypophysitis in SJL Mäusen zu induzieren.

Watch this Scientific Journal Video about Induction of Experimental Autoimmune Hypophysitis in SJL Mice at JoVE.com

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Dieses Video zeigt, wie autoimmune Hypophysitis in SJL Mäusen und wie sie ihre Strenge durch Histopathologie beurteilen zu induzieren.

Induktion der experimentellen autoimmunen Hypophysitis in SJL Mäuse

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

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Immunology and Infection

11.4K Aufrufe.  The Johns Hopkins University. Dieses Video zeigt, wie autoimmune Hypophysitis in SJL Mäusen und wie sie ihre Strenge durch Histopathologie beurteilen zu induzieren.

Serie: Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Vorbereitung der Maus Pituitary Immunogen für die Induktion der experimentellen autoimmunen Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Forschung

Immunologie und Infektion

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Myelin Oligodendrozyten Glykoprotein (MOG<sub&gt; 35-55</sub&gt;) Induzierten experimentellen autoimmunen Enzephalomyelitis (EAE) in C57BL / 6 Mäuse

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

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.

Die Induktion von Murine Darmentzündungen durch adoptiven Transfer von Effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Hoch</sup&gt; T-Zellen in immundefizienten Mäusen

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

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.

Die Induktion der experimentellen autoimmunen Enzephalomyelitis bei Mäusen und die Auswertung des Krankheitsabhängige Verteilung von Immunzellen in verschiedenen Geweben

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

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Ein Nicht-invasive und in technischer Hinsicht nicht intensive Verfahren zur Induktion und Phänotypisierung für experimentelle bakterielle Pneumonie bei Mäusen

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

Die Induktion einer entzündlichen Reaktion in der Primär Hepatozyten-Kulturen von Mäusen

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Induktion der medikamenteninduzierten Autoimmunhepatitis bei BALB/c-Mäusen zur Untersuchung ihrer pathogenen Mechanismen

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

Beeinträchtigung der Endothelzellen und glialen Barrieren des Referats neurovaskuläre während experimentelle Autoimmune Enzephalomyelitis In Vivo Visualisierung

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

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.

Adoptive Immuntherapie von iNKT-Zellen in Glucose-6-Phosphat-Isomerase (G6PI)-Induzierte RA-Mäuse

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

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Flow Cytometric Analyse der Lymphozyteninfiltration im Zentralnervensystem während der experimentellen Autoimmunenzephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...