The authors have no acknowledgements.

<ol>
	<li>Douek, D. 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=Changes+in+thymic+function+with+age+and+during+the+treatment+of+HIV+infection.">Changes in thymic function with age and during the treatment of HIV infection.</a> Nature. 396 (6712), 690-695 (1998).</li><li>Zelm, M. C., Szczepanski, T., van der Burg, M., van Dongen, J. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+history+of+B+lymphocytes+reveals+homeostatic+proliferation+and+extensive+antigen-induced+B+cell+expansion.">Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion.</a> J. Exp. Med. 204 (3), 645-655 (2007).</li><li>Fronkova, E., 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=B-cell+reconstitution+after+allogeneic+SCT+impairs+minimal+residual+disease+monitoring+in+children+with+ALL.">B-cell reconstitution after allogeneic SCT impairs minimal residual disease monitoring in children with ALL.</a> Bone Marrow Transplant. 42 (3), 187-196 (2008).</li><li>Sottini, A., 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=Simultaneous+quantification+of+recent+thymic+T-cell+and+bone+marrow+B-cell+emigrants+in+patients+with+primary+immunodeficiency+undergone+to+stem+cell+transplantation.">Simultaneous quantification of recent thymic T-cell and bone marrow B-cell emigrants in patients with primary immunodeficiency undergone to stem cell transplantation.</a> Clin. 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Neuroimmunol. 240-241, 147-150 (2011).</li><li>Zanotti, 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=Peripheral+accumulation+of+newly+produced+T+and+B+lymphocytes+in+natalizumab-treated+multiple+sclerosis+patients.">Peripheral accumulation of newly produced T and B lymphocytes in natalizumab-treated multiple sclerosis patients.</a> Clin. Immunol. 145 (1), 19-26 (2012).</li><li>Sottini, A., 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=Pre-existing+T-+and+B-cell+defects+in+one+progressive+multifocal+leukoencephalopathy+patient.">Pre-existing T- and B-cell defects in one progressive multifocal leukoencephalopathy patient.</a> PLoS One. 7, e34493 (2012).</li><li>Quiros-Roldan, E., 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=Effects+of+combined+antiretroviral+therapy+on+B-+and+T-cell+release+from+production+sites+in+long-term+treated+HIV-1++patients.">Effects of combined antiretroviral therapy on B- and T-cell release from production sites in long-term treated HIV-1+ patients.</a> J. Transl. Med. 10, 94 (2012).</li><li>Mensen, A., 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=Utilization+of+TREC+and+KREC+quantification+for+the+monitoring+of+early+T-+and+B-cell+neogenesis+in+adult+patients+after+allogeneic+hematopoietic+stem+cell+transplantation.">Utilization of TREC and KREC quantification for the monitoring of early T- and B-cell neogenesis in adult patients after allogeneic hematopoietic stem cell transplantation.</a> J. Transl. Med. 11, 188 (2013).</li><li>Serana, F., 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=The+different+extent+of+B+and+T+cell+immune+reconstitution+after+hematopoietic+stem+cell+transplantation+and+enzyme+replacement+therapies+in+SCID+patients+with+adenosine+deaminase+deficiency.">The different extent of B and T cell immune reconstitution after hematopoietic stem cell transplantation and enzyme replacement therapies in SCID patients with adenosine deaminase deficiency.</a> J. Immunol. 185 (12), 7713-7722 (2010).</li><li>Borte, S., 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=Neonatal+screening+for+severe+primary+immunodeficiency+diseases+using+high-throughput+triplex+real-time+PCR.">Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR.</a> Blood. 119 (11), 2552-2555 (2012).</li><li>Baker, M. W., 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=Implementing+routine+testing+for+severe+combined+immunodeficiency+within+Wisconsin's+newborn+screening+program.">Implementing routine testing for severe combined immunodeficiency within Wisconsin's newborn screening program.</a> Public Health Rep. 125 (2), 88-95 (2010).</li><li>Lorenzi, A. 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=Determination+of+thymic+function+directly+from+peripheral+blood:+a+validated+modification+to+an+established+method.">Determination of thymic function directly from peripheral blood: a validated modification to an established method.</a> J. Immunol. Meth. 339 (2), 185-194 (2008).</li><li>De Boer, R. J., Perelson, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+T+lymphocyte+turnover.">Quantifying T lymphocyte turnover.</a> J. Theor. Biol. 327, 45-87 (2013).</li></ol>

The authors have nothing to disclose.

TREC and KREC quantification can be considered a good estimate of recent thymic and bone marrow output provided that some caveats are taken into account. Even though an absolute quantification method employing standard curve requires more reagents and more space on the real-time PCR reaction plate, it ensures highly accurate quantitative results because unknown sample quantities are interpolated from standard curves built upon known amounts of starting material. Moreover this method is better fitted to detect low amount ...

T-Zell-Rezeptor-Exzision Kreise (TRECs) und K-Löschen Rekombination Exzision Kreise (KRECs) sind kleine zirkularisiertes DNA-Elemente, die in einem Anteil von T- und B-Zellen jeweils während einer genomischen DNA-Rekombination herausgeschnitten werden, was zu dem Bildung einer stark diversifizierten Repertoire von T- und B-Zell-Rezeptoren. Sie haben keine Funktion, sondern weil sie stabil sind und nicht repliziert werden, werden sie nach jeder Zellteilung verdünnt, also nur in einer der beiden Tochter persistierenden Zellen. Daher kann ihre Ebenen im peripheren Blut als eine Schätzung des Thymus und des Knochenmarks Ausgangs angenommen werden. Während die TREC-Assay wurde weitgehend in den letzten 15 Jahren um das Ausmaß der Thymusfunktion auszuwerten, um 1 der KREC Assay, die ursprünglich entwickelt wurde B-Zell-Proliferation und ihren Beitrag zur B-Zell-Homöostase in Gesundheit und Krankheit, 2 messen wurde erst kürzlich als Marker der Knochen m vorgeschlagenenarrow Ausgang. 3,4 Hier beschreiben wir die Methode, die wir für die simultane Quantifizierung von beiden TRECs und KRECs entwickelt. 4 Mit diesem kombinierten Verfahren wird die Variabilität DNA Quantifizierung durch Echtzeit-PCR verbunden sind, durch die Verwendung eines einzigartigen Standardkurve durch Verdünnung einer Triple-Insert enthaltenden Plasmid Fragmente TRECs, KRECs und T-Zell-Rezeptor alpha konstant erhalten (TCRAC) beseitigt Gens in einer 1: 1: 1-Verhältnis. Dies ermöglicht eine genauere Bewertung der TREC und KREC Kopienzahl. Ferner ist die gleichzeitige Quantifizierung der zwei Targets in der gleichen Reaktion ermöglicht Reagenz Kostenreduzierung. Die vorgeschlagene TREC / KREC Assay kann nützlich sein, um das Ausmaß der T- und B-Zell-neo-Produktion bei Kindern oder Erwachsenen mit schwerer kombinierter Immundefizienz (SCID), 4 Common Variable Immunodeficiency, 5 Autoimmunerkrankungen, 6-8 und HIV-Infektionen zu messen Ferner. 9 zeigt, kann es verwendet werdenÜberwachung der Immun Erholung nach Transplantation hämatopoetischer Stammzellen, 10 Enzymersatz, 11 und antivirale 9 oder immunmodulierende Therapien. 6-8 Schließlich weil SCID-Patienten werden mit TREC-Test trotz der zugrunde liegenden genetischen Defekte erkannt und Agammaglobulinämie Patienten können mit KREC Quantifizierung identifiziert werden Die TREC / KREC Assay kann auch verwendet werden, um Immun in Neugeborenen-Screening-Programme erkennen. 12 In diesem Fall muss der Test an aus kleinen Flecken von Blut abgetupft extrahierten DNA durchgeführt, und auf Filterpapier getrocknet werden müssen hochempfindlich und spezifisch für sein die Zielkrankheiten, sowie in hohem Maße reproduzierbar und kostengünstig. Die Einführung KREC Quantifizierung im Test sollte Aufführungen von Neugeborenen-Screening für Immundefekte, die routinemäßig in den einigen Teilen der USA (WI, MA, CA) seit 2008 durchgeführt wurde, zu verbessern, wenn Wisconsin war der erste, um introduce die Analyse TRECs in seiner postnatale Screening-Programm. 13

<table border="0" cellpadding="0" cellspacing="0" width="861">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Name of Reagent/ Equipment</td>
			<td style="width:211px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Histopaque-1077</td>
			<td style="width:211px;">Sigma Aldrich SRL</td>
			<td style="width:119px;">10771-500 ML</td>
			<td style="width:235px;">density gradient separation method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp DNA Blood Mini Kit (250)</td>
			<td style="width:211px;">QIAGEN</td>
			<td style="width:119px;">51106</td>
			<td style="width:235px;">DNA extraction</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Unmodified DNA Oligonucleotides HPSF 0.01 mmol</td>
			<td style="width:211px;">Eurofins MWG Operon/Carlo Erba Reagents S.r.l&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">Resuspend the lyophilized product to 100 picmol/&#181;l</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:296px;">AmpliTaq DNA Polymerase: including 10x Buffer II and 25mM MgCl2</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;">N8080156</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">GeneAmp dNTP Blend (100 mM)</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;">N8080261</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TOPO TA Cloning Kit for Subcloning</td>
			<td style="width:211px;">Invitrogen/Life-Technologies</td>
			<td>K4500-01</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XL1-Blue Subcloning Grade Competent Cells</td>
			<td style="width:211px;">Stratagene</td>
			<td>200130</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureYield Plasmid Miniprep System</td>
			<td style="width:211px;">Promega</td>
			<td>A1223</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SpeI 500U</td>
			<td style="width:211px;">New England Biolabs</td>
			<td>R0133S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HindIII-HF 10,000 U</td>
			<td style="width:211px;">New England Biolabs</td>
			<td>R3104S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureYield Plasmid Midiprep System</td>
			<td style="width:211px;">Promega</td>
			<td>A2492</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">XhoI 5,000 U</td>
			<td style="width:211px;">New England Biolabs</td>
			<td>R0146S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIS Utrapure</td>
			<td style="width:211px;">Sigma Aldrich SRL</td>
			<td>T1503</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td style="width:211px;">Sigma Aldrich SRL</td>
			<td>E5134</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer (1 mM TRIS and 0.1 mM EDTA)</td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TaqMan Universal PCR Master Mix</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;">4364338</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dual labeled probes HPLC 0.01 mmol</td>
			<td style="width:211px;">Eurofins MWG Operon/Carlo Erba Reagents S.r.l&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">Resuspend the lyophilized product to 100 picmol/&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop 2000c spectrophotometer</td>
			<td style="width:211px;">ThermoFisher</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Applied Biosystems 2720 Thermal Cycler</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td>4359659</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fast 7500 Real-Time PCR system</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">SDS Sequence Detection Software 1.4</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:235px;"></td>
		</tr>
	</tbody>
</table>

simultaneous quantification of t-cell receptor excision circles (trecs) and k-deleting recombination excision circles (krecs) by real-time pcr

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Der Test wurde in einer repräsentativen Stichprobe von 87 gesunden Kontrollpersonen durchgeführt: 42 Kinder im Alter von 0 bis 17 (männlich / weiblich: 25/17) und 45 Erwachsene im Alter von 24 bis 60 (Männer / Frauen: 29/16). Die Ergebnisse wurden als TRECs und KRECs pro 10 6 PBMC und dann den TRECs und KRECs pro ml Blut wurden berechnet erhalten. Die Anzahl der TRECs nimmt mit dem Alter aufgrund Thymusinvolution, 4 insbesondere in einem sehr scharfen Mode 0-3 - 4 J...

Watch this Scientific Journal Video about Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR at JoVE.com

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

Simultane Quantifizierung von T-Zell-Rezeptor-Excision Kreis (TRECs) und K-Löschen Rekombination Excision Kreis (KRECs) durch Echtzeit-PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

simultaneous-quantification-t-cell-receptor-excision-circles-trecs-k

Research

JoVE Journal

Immunology and Infection

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

16.3K Aufrufe.  Spedali Civili di Brescia. Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

Serie: Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Transnuclear Mäuse mit vordefinierten T-Zell-Rezeptor Spezifitäten gegen Toxoplasma gondii Via SCNT Erhalten

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Forschung

Immunologie und Infektion

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Ein TIRF Mikroskopie-Technik für Echtzeit-, Einzigartige Bilder aus dem TCR und der damit verbundenen Signalproteine

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

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

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

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

Nachweis von microRNAs in Mikroglia durch Real-time PCR in Normal-und ZNS Während Neuroinflammation

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

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Echtzeit-Live-Imaging von T-Zell-Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Hochdurchsatz-quantitative Echtzeit-RT-PCR-Assay zur Bestimmung Expressionsprofile der Typen I und III Interferon-Formationsglieder

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Retrovirale Transduktion von Knochenmark-Progenitorzellen zu generieren T-Zell-Rezeptor Retrogenic Mäuse

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

Genotypisierung<em&gt; Staphylococcus aureus</em&gt; Durch ribosomalen Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Echtzeit-Bildgebung und Quantifizierung der Pilz Biofilm-Entwicklung mit einem Zweiphasensystem rezirkulierenden Flow

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

Identifikation von Mediatoren der T-Zell-Rezeptor Signalgebung über das Screening von chemischen Inhibitors Bibliotheken

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.