Esta labor fue apoyada por los Institutos Nacionales de Alergias y Enfermedades Infecciosas Grant P01 A1078894 a CAG

<ol>
	<li>Nathan, C., Ding, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonresolving+Inflammation.">Nonresolving Inflammation.</a> Cell. 140 (6), 871-882 (2010).</li><li>Karin, M., Lawrence, T., Nizet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+gone+awry:+linking+microbial+infections+to+chronic+inflammation+and.">Innate immunity gone awry: linking microbial infections to chronic inflammation and.</a> 124, 823-835 (2006).</li><li>Connor, S. M., Taylor, C. E., Hughes, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Pathogen-induced+inflammation+at+sites+distant+from+oral+infection:+bacterial+persistence+and+induction+of+cell-specific+innate+immune+inflammatory+pathways.">Review: Pathogen-induced inflammation at sites distant from oral infection: bacterial persistence and induction of cell-specific innate immune inflammatory pathways.</a> Molecular Oral Microbiology. 25 (5), 305-316 (2010).</li><li>Gibson, F. C., Ukai, T., 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=Engagement+of+specific+innate+immune+signaling+pathways+during+Porphyromonas+gingivalis+induced+chronic+inflammation+and+atherosclerosis.">Engagement of specific innate immune signaling pathways during Porphyromonas gingivalis induced chronic inflammation and atherosclerosis.</a> Frontiers in Bioscience: a Journal and Virtual Library. 13, 2041-2059 (2008).</li><li>Pihlstrom, B. L., Michalowicz, B. S., Johnson, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontal+diseases.">Periodontal diseases.</a> Lancet. 366 (9499), 1809-1820 (2005).</li><li>Baker, P. J., Evans, R. T., Roopenian, D. 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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. 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Los autores declaran que no tienen intereses financieros en competencia.

El P. gingivalis modelo infección oral proporciona una valiosa herramienta para el estudio de la inflamación crónica inducida por patógenos en los sitios locales y sistémicos. Este modelo único permite la caracterización de ambos mecanismos específicos de acogida y patógenos que contribuyen a la inflamación crónica y la inmunopatología. Además, el modelo puede ser utilizado para la detección de nuevas estrategias terapéuticas, incluyendo la inmunización y la intervención farmacológica. Los pas...

La inflamación crónica es un importante motor de daño tisular patológica y una característica unificadora de muchas enfermedades crónicas en los seres humanos. Estas enfermedades incluyen neoplásicas, autoinmunes, y enfermedades inflamatorias crónicas 1. La etiología de muchas enfermedades crónicas no está clara, pero se entiende que es complejo y multifactorial, que involucra tanto la predisposición genética y la introducción de factores ambientales. Mientras que los perpetradores de la inflamación siendo difícil de alcanzar, los perfiles celulares y moleculares de la activación inmune se solapan considerablemente con los patrones observados en las respuestas del huésped a patógenos 2. La evidencia creciente implica la infección con patógenos microbianos en el desarrollo y progresión de la inflamación crónica y la diversidad de sus manifestaciones clínicas 2,3. Los patógenos pueden inducir y mantener la inflamación crónica directamente por subvertir el sistema inmune del huésped y el establecimiento de infecciones persistentes <sup&gt; 4. En la ausencia de persistencia microbiana, la infección puede precipitar inflamación crónica de las reacciones autoinmunes provocadas por mimetismo molecular a auto-antígenos, cambios en la auto-antígenos que las hacen inmunogénica, o el daño que libera antígenos del huésped previamente enmascarados. Rara vez, sin embargo tienen patógenos específicos sido identificados como la causa universal de una enfermedad crónica particular. Más bien, la mayoría de los datos disponibles sugieren que los patógenos usan distintos mecanismos para provocar la inflamación crónica con un amplio espectro de manifestaciones clínicas y de las enfermedades de la genéticamente susceptibles host 3. Por lo tanto, una comprensión detallada de los mecanismos por los que los agentes patógenos específicos inducen la inflamación crónica puede tener importantes implicaciones para la salud pública, así como el tratamiento y la prevención de muchas enfermedades crónicas. Aunque los mecanismos específicos de acogida y de patógenos que contribuyen a la inducción y mantenimiento de la inflamación crónica sonpoco conocidos, los avances en la modelización de la inflamación crónica inducida por patógenos han comenzado a avanzar en nuestra comprensión de estos procesos. El P. gingivalis modelo de infección oral es un modelo de ratón único, bien caracterizado de la inflamación crónica inducida por patógenos que permite el análisis de patógenos de acogida y mecanismos específicos que contribuyen a la inflamación crónica en el (la pérdida de hueso bucal) locales y sitios sistémicos (aterosclerosis) 5,6. P. gingivalis es un patógeno anaerobio oral de Gram-negativos implicados en la enfermedad periodontal humano, una enfermedad inflamatoria crónica infección impulsada caracterizado por la destrucción de los tejidos de soporte del diente 7. Además de la patología en el sitio inicial de la infección, acumulación de pruebas implica P. gingivalis inducida por la inflamación crónica en el desarrollo y progresión de enfermedades sistémicas, incluyendo la aterosclerosis 5, una enfermedad caracterizada por inflammatio crónican de la pared del vaso arterial. La infección oral de patógenos específicos ratones libres con P. gingivalis induce una respuesta inflamatoria local que resulta en la destrucción de hueso alveolar de soporte del diente 8. P. gingivalis puede recuperarse de las bocas de los ratones infectados hasta 42 días después de la infección 8 y ratones desarrollar altos niveles de títulos de anticuerpos séricos específicos de patógenos 9. En un modelo de ratón de la aterosclerosis establecida usando la apolipoproteína-E - / - ratones (ApoE - / -), la infección oral con P. gingivalis induce inflamación crónica que conduce a la deposición de placas inflamatoria dentro del seno aórtico 10 y la arteria innominada 11. Inflamación progresiva dentro de la arteria innominada de P. gingivalis infectados ratones pueden ser controlados en animales vivos que utilizan en la RM vivo. Histológicamente, las lesiones arteriales de P. gingivalis infectados ratones muestran una mayor acumulación de lípidos acomacompañada de la activación del endotelio vascular, un aumento del infiltrado de células inmunes, y elevada expresión de mediadores inflamatorios 12. El uso de este modelo en ratones knockout ha dilucidado el papel de los componentes de señalización de acogida y mediadores de la inflamación, así como la célula interacciones específicas que impulsan P. gingivalis inducida inmunopatología 12 - 14. Además, los experimentos que utilizan mutantes bacterianas definidas han identificado P. crítico gingivalis factores de virulencia que contribuyen a la inflamación crónica en los sitios locales y sistémicos 15. Este artículo detalla metodologías para la evaluación de P. gingivalis inducida por la inflamación crónica en los sitios locales y sistémicos. Proporcionamos un protocolo detallado para el análisis de la pérdida de hueso alveolar por microCT utilizando el software Amira. Además, se define la utilidad de la serie vivo MRI animal vivo en la evaluación de progresivo eninflamación dentro de la arteria innominada. Incluimos metodologías para la visualización y cuantificación de placa inflamatoria en las lesiones arteriales, y describimos su caracterización histológica. El uso de ratones transgénicos y mutantes bacterianas definidas hace que este modelo particularmente adecuados para identificar tanto huésped y factores microbianos implicados en la iniciación, progresión, y el resultado de la enfermedad. Además, el modelo se puede utilizar para la detección de nuevas estrategias terapéuticas, incluyendo la vacunación y la intervención farmacológica.

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			<td height="21" style="height:21px;">Anaerobic chamber DW Scientific Model MG500&#160;</td>
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			<td>M5625-25G</td>
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			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Becton-Dickinson&#160;</td>
			<td>212750</td>
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			<td height="21" style="height:21px;">Carboxymethyl cellulose (medium viscocity)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C-4888</td>
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			<td height="21" style="height:21px;">Sulfamethoxazole and Trimethoprim Oral Suspension 200 mg/40 mg per 5ml</td>
			<td>Hi-Tech Pharmacal</td>
			<td>NDC 50383-823-16</td>
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			<td height="21" style="height:21px;">&#956;CT 40&#160;</td>
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			<td>Sigma-Aldrich&#160;</td>
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			<td>Sigma-Aldrich&#160;</td>
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			<td>Bruker</td>
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			<td>Paravision</td>
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			<td>NIH</td>
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			<td>Microm</td>
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a mouse model for pathogen-induced chronic inflammation at local and systemic sites

La inflamación crónica es un importante motor de daño tisular patológica y una característica unificadora de muchas enfermedades crónicas en los seres humanos incluyendo neoplásicas, autoinmunes y enfermedades inflamatorias crónicas. Nuevas pruebas implica la inflamación crónica inducida por patógenos en el desarrollo y la progresión de enfermedades crónicas con una amplia variedad de manifestaciones clínicas. Debido a la etiología compleja y multifactorial de la enfermedad crónica, el diseño de experimentos para la prueba de la causalidad y el establecimiento de vínculos mecanicistas es casi imposible en los seres humanos. Una ventaja de la utilización de modelos animales es que tanto los factores genéticos y ambientales que pueden influir en el curso de una enfermedad en particular pueden ser controlados. Por lo tanto, el diseño de modelos animales relevantes de la infección representa un paso clave en la identificación de patógenos de acogida y mecanismos específicos que contribuyen a la inflamación crónica. Aquí se describe un modelo de ratón de infla crónica inducida por patógenosmmation en sitios locales y sistémicas después de la infección con el patógeno Porphyromonas gingivalis vía oral, una bacteria estrechamente asociada con la enfermedad periodontal humano. La infección oral de ratones libres de patógenos específicos induce una respuesta inflamatoria local que resulta en la destrucción de hueso alveolar de soporte del diente, un sello distintivo de la enfermedad periodontal. En un modelo de ratón establecido de la aterosclerosis, la infección con P. gingivalis acelera deposición de la placa inflamatoria dentro del seno y innominada arteria aorta, acompañada por la activación del endotelio vascular, un aumento del infiltrado de células inmunes, y elevada expresión de mediadores de la inflamación dentro de las lesiones. Nos metodologías de detalle para la evaluación de la inflamación en los sitios locales y sistémicos. El uso de ratones transgénicos y mutantes bacterianas definidas hace que este modelo particularmente adecuados para identificar tanto huésped y factores microbianos implicados en la iniciación, progresión, y el resultado de la enfermedad. AdditionallY, el modelo puede ser utilizado para la detección de nuevas estrategias terapéuticas, incluyendo la vacunación y la intervención farmacológica.

Usando el modelo de ratón apropiada y el régimen de la infección oral, P. gingivalis induce la inflamación crónica y la inmunopatología al (cavidad oral) local y sitios sistémicos (arterias) (Figura 1). En ratones, la infección oral con P. gingivalis induce una respuesta inflamatoria local que conduce a la destrucción de hueso alveolar de soporte del diente 8. P. gingivalis infectados ratones desarrollan respuestas de anticuerpo...

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

Los modelos animales han demostrado ser herramientas muy valiosas en la definición de mecanismos de acogida y de patógenos específicos que contribuyen al desarrollo de la inflamación crónica. Aquí se describe un modelo de ratón de infección oral con el patógeno humano Porphyromonas gingivalis y metodologías detalle para evaluar la progresión de la inflamación en sitios locales y sistémicos.

Un modelo de ratón para Patógenos inducida inflamación crónica en los sitios locales y sistémicos

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

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Immunology and Infection

17.4K vistas.  Boston University School of Medicine.  Los modelos animales han demostrado ser herramientas muy valiosas en la definición de mecanismos de acogida y de patógenos específicos que contribuyen al desarrollo de la inflamación crónica. Aquí se describe un modelo de ratón de infección oral con el patógeno humano Porphyromonas gingivalis y metodologías detalle para evaluar la progresión de la inflamación en sitios locales y sistémicos. 

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

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

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

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

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

Trichuris muris infección: un modelo de tipo 2 inmunidad y la inflamación en el intestino

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

Investigación

Inmunología e Infección

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.

Uso del ratón humanizado BLT como un modelo de células madre del tumor Terapia génica basada en

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.

In vivo método de imagen para distinguir la inflamación aguda y crónica

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.

In vitro Ensayo de cocultivo para evaluar la migración transepitelial de neutrófilos inducida por patógenos

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.

Inyección sistémica de Neuronales células madre / progenitoras en ratones con EAE crónica

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.

La inducción de la inflamación intestinal murino por transferencia adoptiva de Efector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; T células en ratones inmunodeficientes

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.

Co-inmunoprecipitación de la proteína de ratón Mx1 con la Influenza A nucleoproteína del virus

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.

Un método optimizado para aislar y expandir Invariante Natural Killer células T de ratón Bazo

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.

Administración intra-traqueal de<em&gt; Haemophilus influenzae</em&gt; En modelos de ratón para estudiar las vías respiratorias inflamación

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.