Este trabalho foi financiado pelo Instituto Nacional de Alergia e Doenças Infecciosas Grant P01 A1078894 para CAG

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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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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen-mediated+inflammatory+atherosclerosis+is+mediated+in+part+via+Toll-like+receptor+2-induced+inflammatory+responses.">Pathogen-mediated inflammatory atherosclerosis is mediated in part via Toll-like receptor 2-induced inflammatory responses.</a> Journal of Innate Immunity. 2 (4), 334-343 (2010).</li><li>Hayashi, C., Papadopoulos, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+for+TLR4+signaling+in+atherosclerosis+progression+as+revealed+by+infection+with+a+common+oral+pathogen.">Protective role for TLR4 signaling in atherosclerosis progression as revealed by infection with a common oral pathogen.</a> Journal of Immunology (Baltimore, Md.: 1950). 189 (7), 3681-3688 (2012).</li><li>Papadopoulos, G., Weinberg, E. 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Os autores declaram que não têm interesses financeiros concorrentes.

O P. gingivalis modelo infecção oral proporciona uma ferramenta valiosa para o estudo da inflamação crônica induzida por patógeno em sites locais e sistêmicos. Este modelo único permite a caracterização de ambos os mecanismos específicos de acolhimento e de patógenos que contribuem para a inflamação crônica e imunopatologia. Além disso, o modelo pode ser utilizado para o rastreio de novas estratégias terapêuticas, incluindo a imunização e a intervenção farmacológica. Os passos descritos n...

A inflamação crônica é um importante motor de danos no tecido patológico e uma característica unificadora de muitas doenças crônicas em seres humanos. Estas doenças neoplásicas incluem, auto-imunes, doenças inflamatórias crónicas e um. A etiologia de muitas doenças crônicas ainda não está claro, mas entende-se complexa e multifatorial, envolvendo tanto a predisposição genética ea introdução de fatores ambientais. Enquanto os perpetuadores da inflamação permanecem ainda desconhecidos, os perfis celulares e moleculares da ativação imune sobrepõem consideravelmente com esses padrões observados na resposta do hospedeiro a patógenos 2. Evidências crescentes implica a infecção por patógenos microbianos no desenvolvimento e na progressão da inflamação crônica e suas diversas manifestações clínicas 2,3. Patógenos podem induzir e manter a inflamação crônica diretamente por subverter o sistema imune do hospedeiro e infecções persistentes <sup&gt; 4. Na ausência de persistência microbiana, a infecção pode precipitar a inflamação crônica de reações auto-imunes desencadeadas por mimetismo molecular para auto-antígenos, mudanças na auto-antígenos que os tornam imunogênica, ou dano que libera antígenos do hospedeiro previamente mascarados. Raramente no entanto ter patogénios específicos foram identificados como a causa de uma doença universal crónica particular. Em vez disso, a maioria dos dados disponíveis sugere que os patógenos usar mecanismos distintos para provocar inflamação crônica com um amplo espectro de manifestações clínicas e evolução das doenças no hospedeiro geneticamente suscetível 3. Assim, uma compreensão detalhada dos mecanismos pelos quais os patógenos específicos induzem a inflamação crônica pode ter implicações importantes para a saúde pública, bem como tratamento e prevenção de muitas doenças crônicas. Embora os mecanismos específicos do hospedeiro e do patógeno que contribuem para a indução e manutenção da inflamação crónica estámal compreendida, os avanços na modelagem de inflamação crônica induzida por patógeno começaram a promover nossa compreensão desses processos. O P. gingivalis modelo de infecção oral é um rato modelo único, bem caracterizada de inflamação crônica induzida por patógeno que permite a análise de acolhimento e de patógenos mecanismos específicos que contribuem para a inflamação crônica no local (perda óssea bucal) e sítios sistêmicos (aterosclerose) 5,6. P. gingivalis é, um agente patogénico bucal anaeróbio Gram-negativas implicadas na doença periodontal humano, uma doença inflamatória crónica impulsionado-infecção caracterizada pela destruição de tecidos de suporte dos dentes 7. Além de patologia no local inicial da infecção, evidências acumuladas implica P. gingivalis induzida por inflamação crônica no desenvolvimento e progressão de doenças sistêmicas, incluindo a aterosclerose 5, uma doença caracterizada por inflammatio crônican da parede do vaso arterial. A infecção oral de-patógeno específico camundongos livre com P. gingivalis induz uma resposta inflamatória local, que resulta na destruição de osso alveolar de suporte do dente 8. p gingivalis pode ser recuperado das bocas de camundongos infectados até 42 dias após a infecção e 8 ratos desenvolver altos níveis de anticorpos séricos títulos específicos do micróbio 9. Em um modelo de camundongo estabelecido de aterosclerose utilizando-apolipoproteína E - / - murganhos (ApoE - / -), a infecção com P. bucal gingivalis induz inflamação crônica que leva a deposição de placa inflamatória dentro do seio da aorta 10 e 11 da artéria inominada. Inflamação progressiva dentro da artéria inominada P. gingivalis infectados camundongos pode ser monitorado em animais vivos, usando ressonância magnética in vivo. Histologicamente, as lesões arteriais de P. gingivalis infectados camundongos apresentam maior acúmulo de lipídios acomnhado por activação do endotélio vascular, um aumento infiltrado de células imunitárias, e a expressão elevada de mediadores inflamatórios 12. A utilização deste modelo em ratinhos knockout elucidou o papel de elementos de sinalização de acolhimento e de mediadores inflamatórios, assim como a célula de interacções específicas que dirigem P. gingivalis induzida imunopatologia 12-14. Além disso, as experiências utilizando mutantes bacterianas definidas identificaram P. crítico gingivalis fatores de virulência que contribuem para a inflamação crônica em sites locais e sistêmicos 15. Este artigo detalha metodologias para a avaliação de P. gingivalis induzida por inflamação crônica em sites locais e sistêmicos. Nós fornecer um protocolo detalhado para a análise de perda de osso alveolar por microCT usando software Amira. Além disso, definimos a utilidade da série in vivo MRI animal vivo para a avaliação da progressiva eminflamação dentro da artéria inominada. Nós incluem metodologias para a visualização e quantificação de placa inflamatória em lesões arteriais, e descrever sua caracterização histológica. O uso de camundongos transgênicos e bactérias mutantes definidos torna este modelo particularmente adequado para identificar tanto o hospedeiro e os fatores microbianos envolvidos na iniciação, progressão e evolução da doença. Além disso, o modelo pode ser utilizado para o rastreio de novas estratégias terapêuticas, incluindo a vacinação e a intervenção farmacológica.

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			<td style="width:88px;">Comments&#160;</td>
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			<td></td>
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			<td height="21" style="height:21px;">Anaerobic chamber DW Scientific Model MG500&#160;</td>
			<td>Microbiology International</td>
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			<td>Sigma-Aldrich&#160;&#160;</td>
			<td>M5625-25G</td>
			<td></td>
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			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Becton-Dickinson&#160;</td>
			<td>212750</td>
			<td></td>
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			<td height="21" style="height:21px;">Carboxymethyl cellulose (medium viscocity)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C-4888</td>
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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>Bruker</td>
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			<td height="21" style="height:21px;">Paravision</td>
			<td>Paravision</td>
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			<td height="21" style="height:21px;">ImageJ</td>
			<td>NIH</td>
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			<td>Leica</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

A inflamação crônica é um importante motor de danos no tecido patológico e uma característica unificadora de muitas doenças crônicas em seres humanos, incluindo neoplásicas, auto-imunes e doenças inflamatórias crônicas. Emergentes evidência implica inflamação crônica induzida por patógeno no desenvolvimento e progressão de doenças crônicas com uma grande variedade de manifestações clínicas. Devido à etiologia complexa e multifatorial da doença crônica, projetar experimentos para a prova do nexo de causalidade e do estabelecimento de relações mecanicistas é quase impossível em humanos. Uma vantagem da utilização de modelos animais, é que ambos os factores genéticos e ambientais que podem influenciar o curso de uma doença particular pode ser controlada. Assim, projetar modelos animais relevantes de infecção representa um passo fundamental na identificação de acolhimento e de patógenos mecanismos específicos que contribuem para a inflamação crônica. Aqui nós descrevemos um modelo de mouse induzida por patógeno Infla crônicammation em sites locais e sistêmicos após a infecção com o patógeno Porphyromonas gingivalis via oral, uma bactéria estreitamente associada à doença periodontal humano. A infecção oral de agentes patogénicos específicos de ratos livre induz uma resposta inflamatória local, resultando na destruição de dente de apoio do osso alveolar, uma marca da doença periodontal. Em um modelo de camundongo estabelecido de aterosclerose, infecção com P. gingivalis acelera deposição de placas inflamatórias dentro da artéria inominada e sinus aórtico, acompanhado por activação do endotélio vascular, a um aumento da infiltração de células imunitárias, e a expressão elevada de mediadores inflamatórios em lesões. Nós metodologias detalhe para a avaliação da inflamação em sites locais e sistêmicos. O uso de camundongos transgênicos e bactérias mutantes definidos torna este modelo particularmente adequado para identificar tanto o hospedeiro e os fatores microbianos envolvidos na iniciação, progressão e evolução da doença. Adicionalmente podey, o modelo pode ser utilizado para o rastreio de novas estratégias terapêuticas, incluindo a vacinação e a intervenção farmacológica.

Utilizando o modelo de ratinho apropriado e o regime de infecção oral, P. gingivalis induz inflamação crônica e imunopatologia no local (cavidade oral) e sítios sistêmicos (artérias) (Figura 1). Em ratos, infecção oral com P. gingivalis induz uma resposta inflamatória local que conduz à destruição do osso alveolar dente de apoio 8. p gingivalis infectados camundongos desenvolver respostas de anticorpos séricos para este or...

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

Os modelos animais provaram ser ferramentas valiosas para a definição de acolhimento e de patógenos mecanismos específicos que contribuem para o desenvolvimento da inflamação crônica. Aqui nós descrevemos um modelo do rato de infecção oral com o patógeno humano Porphyromonas gingivalis e metodologias detalhe para avaliar a progressão da inflamação em sites locais e sistêmicos.

Um modelo de rato para induzida por patógeno inflamação crônica no local e sistêmica Sites

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 visualizações.  Boston University School of Medicine.  Os modelos animais provaram ser ferramentas valiosas para a definição de acolhimento e de patógenos mecanismos específicos que contribuem para o desenvolvimento da inflamação crônica. Aqui nós descrevemos um modelo do rato de infecção oral com o patógeno humano Porphyromonas gingivalis e metodologias detalhe para avaliar a progressão da inflamação em sites locais e sistêmicos. 

Vídeo: 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 Infecção: Um Modelo de imunidade do tipo 2 e inflamação no intestino

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

Imunologia e Infecção

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.

Utilizando o rato como um BLT humanizado baseado Stem Modelo Gene Therapy célula tumoral

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.

No método de imagem in vivo Distinguir inflamação aguda e crônica

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 Ensaio de cocultura para avaliar migração trans epitelial de neutrófilo induzida 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.

Injeção sistêmica de células-tronco neurais / progenitoras em ratos com 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.

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

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-imunoprecipitação da proteína do rato Mx1 com a nucleoproteína do vírus da gripe A

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.

Um método otimizado para o isolamento e expansão de células invariante Natural Killer T de Rato Spleen

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.

Administração intra-traqueal de<em&gt; Haemophilus influenzae</em&gt; No mouse modelos para o Estudo de inflamação das vias aéreas

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.