Bu çalışma NIH hibe R01AI077610 tarafından finanse edildi.

Tüm işlemler Bakım ve Ulusal Sağlık Enstitüsü (NIH) Laboratuvar Hayvanları Kullanım Kılavuzu uyarınca yapılmıştır ve Johns Hopkins Üniversitesi Hayvan Bakım ve Kullanım Komitesi (JHUACUC) tarafından onaylanmıştır. Belirli uygulama usulleri ACUC protokolleri MO13M292 ve MO13M370 altında yapıldı. 1. Donör Cilt Hasat <ol><li> (- Fare konisi ile 2% 1 bakım,% 4 indüksiyon buharlaştırıcı) izofluran ile donör fare anestezisi. anestezi derinliği izlemek için ayak tutam çekilme refleksi kullanın. </li><li> Bir elektrikli tıraş makinesi kullanarak, hayvanın arka tıraş ve (örneğin, Betadine)% 10 povidon iyot ile dezenfekte edin. </li><li> areolar bağ dokusu düzeyinde künt diseksiyon ile steril makas, eldiven ve aseptik teknik, boyun kalça hasat donör arka cilt, kullanma. </li><li> hasat sonrası servikal dislokasyon ile hayvan Euthanizederi grefti. </li><li> Mikroskop altında, ince tenotomi makas kullanarak geri deriden bağ dokusu, yağ dokusu ve panniculus carnosus ayırın. panniculus carnosus cilt seğirmesi hareketleri sorumlu ince, şeffaf bir kastır. </li><li> Steril aletler tekniği kullanarak, 15 mm x 15 mm greft yatağına 10 mm x 10 mm geri deriden 15 mm x 15 mm greft kesip. </li><li> Buz üzerinde bir petri steril fosfat tamponlu salin (PBS) ile ıslatılmış etme yöntemleriyle ilgili greft saklayın. Not: 8-10 greft, bir verici bir fare elde edilebilir. </li></ol> 2. Alıcı Cilt Nakli <ol><li> (- Fare konisi ile 2% 1 bakım,% 4 indüksiyon buharlaştırıcı) izofluran ile alıcı fare anestezisi. anestezi derinliği izlemek için ayak tutam çekilme refleksi kullanın. </li><li> Ameliyat sonrası ağrı tedavisi için Buprenorfin 0.02 mg / kg BW yönetme. </li><li> Hayvanın arka tarafı Tıraş nerede greft will takılı olması ve% 10 povidon iyot ile dezenfekte edin. </li><li> makas kullanarak, derinin x 15 mm kare 15-mm 10 mm x 10 mm&#39;lik kesti. kusur boyutu greft daha (% 10) biraz daha büyük olması gerekir. kadar yüzeysel mümkün olduğunca kesin. panniculus carnosus ve damarları korumak için özen gösterin. panniculus carnosus onun hareketlilik ve buna yüzeysel koşmak kan damarları tarafından altta yatan fasya ayırt olabilir. </li><li> kenarları boyunca kıvrımlar kaçınarak, greft yatakta greft yerleştirin. </li><li> Köşelerde ve her kenarın ortasında 8 sütür yerleştirin. Her sütür için, çevredeki alıcı deri altına yatak greft panniculus carnosus yoluyla daha sonra greft içinden iğne geçmesi ve. </li><li> anestezi maskesi çıkarın ve hayvan yapışkan bandaj uygulamadan önce anestezi kısmen kurtarmak edelim. </li><li> greft üzerine katlanmış gazlı bez ile bir yapışkan bandaj alıcı fare sarın. kesme, iki bandaj birleştirerek bandaj yapmakyapışkan birinin parçası ve birlikte iki emici pedler yerleştirerek. </li><li> bandaj toraks gezi kısıtlayan ve nefes olmadığından emin olmak için kurtarma sırasında yakından fare izleyin. solunum sayısı azalır ya da hayvan sığ gasp ya da nefes başlarsa bandajı çıkarın. </li></ol> 3. Ameliyat Sonrası Bakım <ol><li> Enfeksiyon profilaksisi için ameliyattan sonra enrofloxacin 5 mg / kg yönetmek. </li><li> tamamen anestezi kurtarır kadar mikrodalga ısıtma yastığı üzerinde temiz bir kafes içinde nakledilen fare yerleştirin. </li><li> konut tesisine iade postoperatif önce 1 saat için fare gözlemleyin. </li><li> Yedi gün ameliyat sonrası Adım 2.1 gibi fare uyutmak. bandaj ventral tarafta boyunca sadece keserek bandaj çıkarın. </li><li> Gözlemlemek ve kabuklanma, kasılma veya sertlik belirtileri ertesi gün greft palpe. Varsa, greft uygun vasculariza elde olmayabiliryon ve teknik bir arıza düşünülmelidir. </li><li> ret belirtileri günlük izleyin. greft dokusunun ≥90% nekrotik hale geldiğinde reddedilen greft düşünün. </li><li> reddedilen hayvanları euthanize ve analiz için onları hasat. </li></ol>

<ol>
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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=Orthotopic+Hind+Limb+Transplantation+in+the+Mouse.">Orthotopic Hind Limb Transplantation in the Mouse.</a> J Vis Exp. , (2016).</li><li>Oh, B., 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=A+Novel+Microsurgical+Model+for+Heterotopic,+En+Bloc+Chest+Wall,+Thymus,+and+Heart+Transplantation+in+Mice.">A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice.</a> J Vis Exp. , (2016).</li><li>Oberhuber, R., 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=Murine+cervical+heart+transplantation+model+using+a+modified+cuff+technique.">Murine cervical heart transplantation model using a modified cuff technique.</a> J Vis Exp. , e50753 (2014).</li><li>Jones, T. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+and+fate+of+skin+autografts+and+skin+homografts+in+rabbits:+A+report+to+the+War+Wounds+Committee+of+the+Medical+Research+Council.">The behaviour and fate of skin autografts and skin homografts in rabbits: A report to the War Wounds Committee of the Medical Research Council.</a> J Anat. 78, 176-199 (1944).</li><li>Billingham, R. E., Brent, L., Medawar, P. B., Sparrow, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+studies+on+tissue+transplantation+immunity.+I.+The+survival+times+of+skin+homografts+exchanged+between+members+of+different+inbred+strains+of+mice.">Quantitative studies on tissue transplantation immunity. I. 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Yazarlar ifşa hiçbir şey yok.

Tavşan ve farelerde daha sonra insan çalışmalarında ilk ve Medawar tarafından piyasaya sunulmasından bu yana, deri transplantasyonu allojenik bağışıklık tepkilerinin 6,7 çalışmaları için paha biçilmez bir model olmuştur. Bu yazıda, üst ve alt sırt derisi kullanılarak büyük ölçekli, non-vaskülarize, tam kalınlıkta deri nakli bir model sunuyoruz. Kuyruk deri veya Nakledilecek doku kaynağı olarak fare kulak derisi kullanılarak da dahil olmak üzere çeşitli alternatif yöntemler...

Organ nakli son dönem organ yetmezliği olan hastalarda tercih edilen tedavi olup, sonuçlar cerrahi işlemler ve immünsüpresyondaki gelişmeler ile son derece düzeldi. Ancak, uzun süreli immünsüpresyon önemli yan etkileri ile ilişkilidir ve hoşgörüyü teşvik yeni stratejilerin geliştirilmesi, modern nakli araştırmanın amacı kalır. Çeşitli hayvan modelleri allograft reddinin mekanizmaları incelemek ve immünosupresyon aşı reddinin engellenmesi için ve uzun vadeli dayanıklılık 1-3 teşvik etmek için yaklaşımlar test etmek için, transplantasyon temel araştırma için geliştirilmiştir. Fare modelleri nedeniyle tanı ve tedavi antikorlar ve iyi tanımlanmış kendilenmiş ve transgenik suşlar seçkin ve geniş müsaitlik durumuna immünolojik araştırmaların temelini haline gelmiştir. Cilt nakli özel mikrocerrahi beceri gerektiren ve can değil basit bir işlemdirKolayca postoperatif izlenecektir. Birlikte ele alındığında, fare deri nakli greft reddi 4,5 sırasında antijen sunumu, hücre kaçakçılığı ve doku yıkımı da dahil olmak üzere immün yanıt katılan birçok açıdan incelemek için olağanüstü bir araç olmuştur. Burada, fare modeli kullanılarak tam kalınlıkta deri transplantasyonu için adım-adım prosedürü göstermek ve biz nakledilen deri başarılı bütünleşme için gerekli önemli adımlar açıklanmaktadır.

<table border="0" cellpadding="0" cellspacing="0" width="923">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Straight micro forceps&#160;</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:136px;">F4017</td>
			<td style="width:415px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Curved micro forceps&#160;</td>
			<td style="width:125px;">Aesculap</td>
			<td style="width:136px;">BD333R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Curved Stevens tenotomy scissors&#160;</td>
			<td style="width:125px;">Aesculap</td>
			<td style="width:136px;">BC905R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mayo dissecting&#160; scissors</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:136px;">S3146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Micro needle holder</td>
			<td style="width:125px;">Aesculap</td>
			<td style="width:136px;">BM563R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sterile gauze</td>
			<td style="width:125px;">Covidien</td>
			<td style="width:136px;">441218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">6-0 Nylon suture&#160;</td>
			<td style="width:125px;">MWI</td>
			<td style="width:136px;">31849</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Plastic Strips Band-Aid</td>
			<td style="width:125px;">Johnson &#38; Johnson</td>
			<td style="width:136px;"></td>
			<td>Obtained from pharmacy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">10 cm Petri dish</td>
			<td style="width:125px;">Fisherbrand</td>
			<td style="width:136px;">FB0875712</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PBS</td>
			<td style="width:125px;">Quality Biological</td>
			<td style="width:136px;">119069131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Buprenorphine</td>
			<td style="width:125px;"></td>
			<td style="width:136px;"></td>
			<td>DEA Number required; Obtained from hospital pharmacy</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Enrofloxacin</td>
			<td style="width:125px;">Bayer Health Care</td>
			<td style="width:136px;">186599</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine full-thickness skin transplantation

Mürin tam kalınlıkta deri transplantasyonu alloimmun tepki ve greft reddini incelemek için iyi kurulmuş bir in vivo modeldir. insanlara sınırlı uygulanmasına rağmen, farelerde deri transplantasyon yaygın Nakil için kullanılmıştır. Prosedür öğrenmek ve kolay uygulanabilir olması ve hassas mikrocerrahi teknikler de kapsamlı bir eğitim gerektirmez. Ayrıca, bu modelde graft reddi çok tekrarlanabilir immünolojik reaksiyon meydana gelir ve kolayca doğrudan kontrol ve elle muayene ile izlenir. Buna ek olarak, donör uyumlu veya üçüncü taraf deri grefti ile ikincil deri nakli alternatif ve donör spesifik tolerans değerlendirmek için basit bir yöntem olarak daha karmaşık nakli modellerinde yapılabilir. komplikasyonlar düşük ve işlem sonrası anestezi doz aşımı ya da solunum sıkıntısı ile sınırlı genel olarak. Greft yetmezliği, diğer taraftan, gr yetersiz hazırlık sonucu sık görülürgreft yatakta kıç, yanlış konumlandırma, ya da bandaj uygunsuz yerleştirme. Bu yazıda, farelerde tam kalınlıkta deri transplantasyonu için bir protokol mevcut ve başarılı bir işlem için gerekli olan önemli adımları açıklar.

Alıcı fare üzerindeki bandajın yerleştirme işleminin önemli bir adımdır. Deri grefti omuz, kalça, omurga ve (Şekil 1) arasında, alıcı gövde üzerinde konumlandırılmış. Bandaj katlanmış gazlı bez ve iki plastik yapışkanlı bandaj kombinasyonu ile yapılır. Alıcı fare bandajın ortasına gazlı bez üzerinde aşağı greft ile yerleştirilir. iki kavisli mikro forseps kullanılarak, bandajın alt ucu, ilk çekilir ve daha sonra bir bandaj üst ka...

Watch this Scientific Journal Video about Murine Full-thickness Skin Transplantation at JoVE.com

Murine Full-thickness Skin Transplantation

Mürin tam kalınlıkta deri transplantasyonu alloimmun ortamda reddini incelemek için iyi kurulmuş bir modeldir. &gt; C57BL / 6 tam kalınlıkta deri nakli - Burada bir BALB / c performans katılan her adımda bir öğretici sağlar.

Mürin Tam kat Cilt Nakli

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

murine-full-thickness-skin-transplantation

Research

JoVE Journal

Immunology and Infection

19.9K Görüntüleme.  Johns Hopkins University School of Medicine.  Mürin tam kalınlıkta deri transplantasyonu alloimmun ortamda reddini incelemek için iyi kurulmuş bir modeldir. > C57BL / 6 tam kalınlıkta deri nakli - Burada bir BALB / c performans katılan her adımda bir öğretici sağlar. 

Video: Murine Full-thickness Skin Transplantation

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Murin Model B hücreleri CD40 aktivasyonu

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

İmmünoloji ve Enfeksiyon

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Sıçanlarda Ortotopik Hind-Ekstremite Nakli

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Granulocyte-dependent Autoantibody-induced Skin Blistering

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to Witebsky's postulates, one of the criteria in diagnosing a disease as autoimmune is the reproduction of the disease in experimental animals by the passive transfer of autoantibodies. For epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune disease of skin and mucous membranes, several experimental models were recently established. In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human EBA 1. Full-blown widespread disease is usually seen 5-6 days after the first injection and the extent of the disease correlates with the dose of the administered collagen VII-specific IgG. The tissue damage (blister formation) in the experimental EBA is depending on the recruitment and activation of granulocytes by tissue-bound autoantibodies 2,-4. Therefore, this model allows for the dissection of the granulocyte-dependent inflammatory pathway involved in the autoimmune tissue damage, as the model reproduces only the T cell-independent phase of the efferent autoimmune response. Furthermore, its value is underlined by a number of studies demonstrating the blister-inducing potential of autoantibodies in vivo and investigating the mechanism of the blister formation in EBA 1,3,-6. Finally, this model will greatly facilitate the development of new anti-inflammatory therapies in autoantibody-induced diseases. Overall, the passive transfer animal model of EBA is an accessible and instructive disease model and will help researchers to analyze not only EBA pathogenesis but to answer fundamental biologically and clinically essential autoimmunity questions.

In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.

Granülosit-bağımlı otoantikor indüklenen Cilt Kabarcıklanma

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Farelerde allojenik CD4 + T hücre Cevapları Eğitim için Kuyruk Cilt Nakli

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

İzolasyon ve Farklı Yaşlı Fare Timik Greft Nakli.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

Vitro ve In Vivo değerlendirme T, B ve Myeloid hücrelerin süpresif etkinlik ve nakli alıcılardan gelen Humoral yanıt

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

Robot sistemleri kullanarak işlemek ve histolojik analizler için kolon fare örnekleri katıştır

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2&#8217;-deoxyguanosine (to deplete donor&#8217;s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient&#8217;s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

We provide a simple and efficient method to transplant 2&#8217;-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

Çıplak farelerde 2 '-Deoxyguanosine tedavi Murine embriyonik Thymus renal subkapsüler nakli

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Lamina Propria Mononükleer Hücrelerin Murine Kolondan Kollajenaz E Kullanarak İzolasyon

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Murin Kalp Nakli için Manşet Tekniğinin Optimizasyonu

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...