This work was funded by the Austrian Science Fund (FWF) grant P18820-B13.
Ethics statement:
The study was approved (PLUS_Ethik_090514) by the Ethics Committee for Experiments Involving Humans and/or Animals at the University of Salzburg complying with the Helsinki Declaraction as revised in 1983 and all patients participating gave their written informed consent.

1. Preparation of drug conjugates
<ol>
 <li>Dissolve 10 mg DF in 1 ml dH2O in a 1.5 ml reaction tube. In the further proceeding use 95 &mu;l of this DF solution. To conjugate PP to HSA dissolve 30.4 mg PP derivative (304 g/mol) in 500 &mu;l 0.3 M sodium hydroxide (NaOH).</li>
 <li>Add 430.6 &mu;l dH2O and 100 &mu;l 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 6.5 to 95 &mu;l of dissolved DF. Add 33.1 &mu;l dH2O and 140 &mu;l of 2 M MES buffer pH 2.9 to the PP sample.</li>
 <li>Add 57.5 &mu;l of a freshly prepared N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) stock solution dissolved in dH2O with a concentration of 100 mg/ml to the DF sample, or 10 &mu;l EDC solution to the PP sample. Vortex the samples for 1 minute.</li>
 <li>Add 16.9 &mu;l of HSA solution with a concentration of 118 mg/ml to each sample.</li>
 <li>Incubate the samples for 2 hours at room temperature while shaking at 300 rounds/min.</li>
 <li>Dialyze the DF and PP samples 3 times against 2 liters of phosphate-buffered saline (PBS) pH 7.4 for several hours each. Perform all dialyzing steps at 4&deg;C.</li>
 <li>Centrifuge the sample 10 minutes at 14,000 x g and collect the supernatant containing DF or PP conjugated to HSA.</li>
 <li>Check supernatant for proteins by performing a SDS-PAGE, using 12% gels. Load about 12 &mu;g of HSA conjugate into one gel slot.</li>
</ol>
2. Determine coupling rate of drug conjugates (Figure 2)
<ol>
 <li>For the analysis of the coupling rate of DF and PP conjugates by HPSEC use a 100 mM sodium phosphate buffer pH 6.5 containing 150 mM NaCl and 0.05% sodium azide. Use a 7.8 x 300 mm TSK-Gel-G2000SWXL column.</li>
 <li>Perform detection of signals by using an online-coupled RI and UV detector system.</li>
 <li>Prepare a HSA standard sample with a concentration of 1 mg/ml in dH2O. For detector calibration inject 50 &mu;l HSA standard solution.</li>
 <li>Calculate the RI constant kRI and the UV constant kUV for the detector. Use the formulas areaRI=kRI*[(dn/dc)HSA*c(HSA)] and areaUV=kUV*[(dA/dc)HSA*c(HSA)] with (dn/dc)HSA=0.185 and (dA/dc)HSA=0.5185.</li>
 <li>Prepare DF and PP derivative standards for HPSEC that 5, 10, and 30 nmol amounts of both standards can be easily injected.</li>
 <li>Calculate mean values for (dn/dc)drug and (dA/dc)drug for both standards. For DF, a (dn/dc) of 0.235 &plusmn; 0.010 and a (dA/dc) of 39.000 &plusmn; 0.493 was determined. For PP, a (dn/dc) of 0.328 &plusmn; 0.016 and a (dA/dc) of 53.155 &plusmn; 2.464 was determined.</li>
 <li>Inject drug conjugates on HPSEC and determine their RI and UV peak areas.</li>
 <li>Calculate the concentration of DF, PP derivative, and HSA by solving the two equations areaRI=kRI*[(dn/dc)drug*c(drug)+(dn/dc)HSA*c(HSA)] and areaUV=kUV*[(dA/dc)drug*c(drug)+(dA/dc)HSA*c(HSA)] for the unknowns.</li>
</ol>
3. BAT using drug conjugates
<ol>
 <li>Prepare seven flow cytometry vials and label them depending on their content: unstained control, negative control, positive control, and drug conjugates.</li>
 <li>Pipette 50 &mu;l of B-CCR-STB stimulation buffer (Flow2 CAST, B&uuml;hlmann Laboratories, Sch&ouml;nenbuch, CH) into the vials reserved for the unstained and negative control. As a positive control, use 50 &mu;l of an anti-Fc&#949;RI solution provided by the Flow2 CAST kit. Add the appropriate amounts of conjugate solution to the vials reserved for the drug conjugates by creating a 1:10 dilution series from 0.02 -20 &mu;g/ml DF conjugate and PP conjugate (final concentrations in an assay volume of 200 &mu;l). These titration experiments must be performed to determine the concentration of optimal basophil activation for each patient sample.</li>
 <li>Add 100 &mu;l stimulation buffer to each tube and 50 &mu;l patient's EDTA whole blood. Mix the samples carefully.</li>
 <li>Pipette 20 &mu;l Flow2 CAST staining reagent containing anti-CCR3 antibodies labeled with phycoerythrin (PE) and anti-CD63 antibodies labeled with fluorescein isothiocyanate (FITC) to each tube and mix again. Do not pipette staining reagent into the unstained sample!</li>
 <li>Incubate the samples for 45 minutes in a 37&deg;C water bath.</li>
 <li>Stop reaction on ice for 5 minutes.</li>
 <li>Lyse erythrocytes by adding 2.0 ml Flow2 CAST lysing reagent to each vial and vortex gently. Incubate samples in the dark for 10 minutes at room temperature.</li>
 <li>Centrifuge samples for 5 minutes at 500 x g and carefully decant supernatant.</li>
 <li>Resuspend cells in 500 &mu;l Flow2 CAST wash buffer and store on ice until flow cytometry.</li>
 <li>Adjust the flow cytometer's voltage settings for forward and side scatter (FSC, SSC) while acquiring the unstained sample.</li>
 <li>Gate the cells predicted as lymphocytes from the SSC-FSC plot to the SSC-PE plot. Basophils are identified by a PE-labeled anti-CCR3 antibody as CCR3hi SSClo and gate them into the FITC-PE plot. The anti-CD63 antibody detecting activated basophils is labeled with FITC.</li>
 <li>Set the cut-off to maximum 2.5% of basophils as CD63hi in the negative control. A sample is considered positive for basophil activation if &gt;5% basophils are CD63hi and the mean fluorescence index (MFI) of the sample divided by the MFI of the negative control exceeds 2 (Figure 3).</li>
</ol>
4. Representative Results:
This experiment evaluates BAT as a beneficial tool to detect IgE-dependent drug hypersensitivity. Protein conjugates of NSAIDs are used for activation of basophils. By HPSEC with RI and UV detection aggregation state, coupling degree, and effective yield of the conjugates are determined. As Figure 4 shows, 43.5% of the DF conjugates remained monomeric with a coupling degree of 5.0 DF/HSA. For the aggregates a coupling degree of 9.5 was determined. The propyphenazone conjugate was shown to consist of 68.5% monomers with a coupling degree of 21.2. The coupling degree of the aggregates was 27.9. In total, the determined coupling degree of DF conjugates was 7.6 DF and that of PP conjugates was 23.2.
BAT performed with conjugates under optimized conditions (concentration of conjugates, stimulation time) allowed investigation of IgE-dependent reactions in NSAID hypersensitivity. As shown in Figure 5, only the PP conjugate was able to trigger basophil activation visualized by an upregulation in CD63 surface expression: 20.6% of basophils were activated characterized by a log-shift in fluorescence intensity. The MFI increased by a factor of 4.9 from 386 (negative control) to 1893. In contrast, using the DF conjugate only 3.1% of the basophils were activated which was below the 5% cut-off. Also the MFI increased only by a factor 1.1 (limit 2.0) from 197 (negative control) to 210. Assay validity is given by (i) &lt;5% basophil activation in the negative control, (ii) a log-shift in fluorescence intensity of a basophil subpopulation, and (iii) &gt;50% activated basophils (positive control).
<img alt="Figure 1" src="/files/ftp_upload/3263/3263fig1.jpg" /> 
Figure 1. Coupling of DF and PP derivative to HSA. After dissolving the drugs, water, MES buffer, and EDC (coupling reagent) are added. The samples are vortexed for 1 minute before HSA is pipetted to the drug solutions. Within 2 hours shaking at room temperature the drugs covalently bind to HSA. Monomers as well as aggregates may result.
<img alt="Figure 2" src="/files/ftp_upload/3263/3263fig2.jpg" /> 
Figure 2. Calculation of coupling degrees. First, the constants kRI and kUV are determined. Therefore, HSA standard with known concentration is injected into the HPSEC system. (dn/dc) and (dA/dc) of HSA are given values of 0.185 and 0.518, respectively 5. The peak areas are used for calculating the constants. Second, for each drug three standards are injected to determine drug- and concentration-specific RI and UV areas. Since the kRI and kUV constants are known from the steps above, the drug-specific (dn/dc) and (dA/dc) derivatives can be calculated. Third, drug-HSA conjugates are injected into HPSEC. The resulting peak areas are used to calculate the concentrations of drug and HSA per peak by solving the two equations with two unknown variables.
<img alt="Figure 3" src="/files/ftp_upload/3263/3263fig3.jpg" /> 
Figure 3. Basophil gating. Cells predicted as lymphocytes are gated side scatter versus forward scatter (SSC-FSC plot). The basophils are identified as CCR3hi SSClo from the gated lymphocytes (SSC-CCR3-PE plot). Basophils are analyzed for activation (CD63hi) in the CD63-FITC-CCR3-PE plot. The negative control is used to set the cut-off of maximum 2.5% basophils remaining CD63hi.
<img alt="Figure 4" src="/files/ftp_upload/3263/3263fig4.jpg" /> 
Figure 4. Determined coupling degrees of drug conjugates. RI and UV signals show two peaks representing aggregates (eluting at low retention volume) and monomers (eluting at high retention volume) of drug-HSA conjugates. Percentages of monomers and aggregates (determined from RI signals) and their coupling degrees are depicted (n=1).
<img alt="Figure 5" src="/files/ftp_upload/3263/3263fig5.jpg" /> 
Figure 5. Basophil activation test. Results of drug conjugate-activated samples, negative controls, and positive controls are shown in CD63-FITC-CCR3-PE plots (n=1).

<ol>
	<li>Himly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgE-mediated+immediate-type+hypersensitivity+to+the+pyrazolone+drug+propyphenazone.">IgE-mediated immediate-type hypersensitivity to the pyrazolone drug propyphenazone.</a> J Allergy Clin Immunol. 111, 882-888 (2003).</li><li>Demoly, P., Pichler, W., Pirmohamed, M., Romano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+questions+in+Allergy:+1--drug+allergy/hypersensitivity.">Important questions in Allergy: 1--drug allergy/hypersensitivity.</a> Allergy. 63, 616-619 (2008).</li><li>Ebo, D. G., Hagendorens, M. M., Bridts, C. H., Schuerwegh, A. J., Clerck, L. S., Stevens, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+in+vitro+activated+basophils,+specific+IgE+and+skin+tests+in+the+diagnosis+of+pollen-associated+food+allergy.">Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy.</a> Cytometry Part B (Clinical Cytometry). 64B, (2005).</li><li>Harrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diclofenac+hypersensitivity:+antibody+responses+to+the+parent+drug+and+relevant+metabolites.">Diclofenac hypersensitivity: antibody responses to the parent drug and relevant metabolites.</a> PLoS One. 5, e13707-e13707 (2010).</li><li>Wen, J., Arakawa, T., Philo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-exclusion+chromatography+with+on-line+light-scattering,+absorbance,+and+refractive+index+detectors+for+studying+proteins+and+their+interactions.">Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.</a> Anal Biochem. 240, 155-166 (1996).</li><li>De Weck, A. L., Sanz, M. L., Gamboa, P. M., Aberer, W., Bienvenu, J., Blanca, M., Demoly, P., Ebo, D. G., Mayorga, L., Monneret, G., Sainte Laudy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tests+based+on+human+basophils:+more+potentials+and+perspectives+than+pitfalls.+II.+Technical+issues.">Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. Technical issues.</a> J Investig Allergol Clin Immunol. 18, 143-155 (2008).</li><li>Ebo, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-assisted+allergy+diagnosis:+current+applications+and+future+perspectives.">Flow-assisted allergy diagnosis: current applications and future perspectives.</a> Allergy. 61, 1028-1039 (2006).</li><li>Poulsen, L. K., Quan, S., Kragh, C., Platzer, M. H., Skov, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basophil+granulocyte+in+allergic+reactions:+experimental+models+and+their+use+for+the+identification+of+drugs+with+effects+or+side+effects+on+basophils.">The basophil granulocyte in allergic reactions: experimental models and their use for the identification of drugs with effects or side effects on basophils.</a> Curr Med Chem - Anti-inflammatory &#38; Anti-Allergy Agents. 3, 167-180 (2004).</li><li>Sanz, M. L., Gamboa, P., de Weck, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+combined+test+with+flowcytometric+basophil+activation+and+determination+of+sulfidoleukotrienes+is+useful+for+in+vitro+diagnosis+of+hypersensitivity+to+aspirin+and+other+nonsteroidal+anti-inflammatory+drugs.">A new combined test with flowcytometric basophil activation and determination of sulfidoleukotrienes is useful for in vitro diagnosis of hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs.</a> Int Arch Allergy Immunol. 136, 58-72 (2005).</li></ol>

No conflicts of interest declared.

BAT is a well-established although not yet routinely used method for diagnosis of IgE-mediated allergic disease 6,7. For drug hypersensitivity, however, its applicability is compromised as low molecular weight compounds are not able to crosslink IgE receptors, a prerequisite for basophil activation 8. Therefore, drugs under investigation need to be covalently coupled to suitable carrier proteins (e.g. HSA). Importantly, the coupling degree (i.e. number of drug molecules per carrier protein) needs to be controlled to guarantee immunological activity (i.e. IgE receptor cross-linking) of conjugates. Theoretically, two haptens per carrier molecule should be sufficient for IgE receptor cross-linking and as few as five DF molecules per HSA have been shown to trigger mediator release in a cell-based assay4. Both conjugates, PP-HSA and DF-HSA, have been determined to display a sufficiently high coupling degree (HPSEC) and to be immunologically active making them suitable reagents for use in BAT. Another issue may be that drug metabolites may play a role, as a metabolite instead of the parent drug may cause the hypersensitivity reaction. In case of DF, this possibility has been evaluated in detail previously using five major Phase I metabolites and one linkage variant4. Important factors for mastering the described technique include quality of blood sample (&lt;12 hours since blood collection, number of detected basophils &gt;500) and optimization of stimulation conditions (time, dose). In addition, so-called non-responders who do not even react with the positive control (antibody directed against the IgE receptor) have to be identified. Therefore, validation criteria have to include an anti-Fc&#949;RI antibody as positive control. An important advantage of using drug conjugates is the fact that they appear non-toxic in contrast to pure drugs, as shown for DF-HSA conjugate by co-stimulation with anti-Fc&#949;RI antibody in BAT4. Pure DF, in contrast, displays cytotoxic effects at a concentration of 1.25 mg/ml causing problems with the interpretability of BAT9. Generally, testing for potential cytotoxicity is strongly recommended for every newly produced drug conjugate.
Here, we have shown the potential of PP-HSA conjugate used in BAT to reveal IgE-mediated hypersensitivity. In contrast, DF hypersensitivity is not associated with IgE demonstrated by the lack of basophil activation in response to DF-HSA conjugate. Importantly, in case of uncertainty concerning an IgE-mediated mechanism, conjugates have to be evaluated for immunological activity by in vitro cell-based assay systems as has been shown for DF4.
Using the described setup of conjugating low molecular weight drugs covalently to suitable protein carrier molecules a number of hypersensitivity reactions against drugs including antibiotics, other NSAIDs, radiocontrast media, muscle relaxants, anesthetics, etc. may be investigated for an involvement of an IgE-mediated mechanism. Hence, BAT may serve as an addition to the existing methods for diagnosis (skin test, oral provocation testing).

<table border="1">
<tbody>
<tr>
<th>Name of the reagent/device</th>
<th>Company</th>
<th>Catalogue number</th>
<th>Comments</th>
</tr>	
<tr>
<td>2-(N-Morpholino)ethanesulfonic acid</td>
<td>Sigma</td>
<td>M3671</td>
<td>-</td>
</tr>	
<tr>
<td>7.8 x 300 mm TSK Gel -G2000SWXL column</td>
<td>Tosoh Bioscience</td>
<td>08540</td>
<td>-</td>
</tr>	
<tr>
<td>BD FACSCanto II</td>
<td>Becton Dickinson</td>
<td>338962</td>
<td>-</td>
</tr>	
<tr>
<td>Bench top centrifuge</td>
<td>Sigma</td>
<td>-</td>
<td>-</td>
</tr>	
<tr>
<td>Diclofenac sodium salt</td>
<td>Sigma</td>
<td>D6899</td>
<td>-</td>
</tr>	
<tr>
<td>EDTA-Vacutainer</td>
<td>Becton Dickinson</td>
<td>368589</td>
<td>-</td>
</tr>	
<tr>
<td>Flow2 CAST Kit</td>
<td>B&uuml;hlmann Laboratories</td>
<td>FK-CCR</td>
<td>Effective June 14, 2011 Flow2 CAST kit is now Flow CAST</td>
</tr>	
<tr>
<td>HP1100 HPLC system</td>
<td>Hewlett Packard</td>
<td>-</td>
<td>-</td>
</tr>	
<tr>
<td>Human Serum Albumin</td>
<td>Sigma</td>
<td>A9511</td>
<td>-</td>
</tr>	
<tr>
<td>Laboratory centrifuge</td>
<td>Eppendorf</td>
<td>-</td>
<td>-</td>
</tr>	
<tr>
<td>N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)</td>
<td>Sigma</td>
<td>E6383</td>
<td>-</td>
</tr>	
<tr>
<td>PBS tablets</td>
<td>AppliChem</td>
<td>A9199</td>
<td>-</td>
</tr>	
<tr>
<td>Propyphenazone derivative</td>
<td>Department of Molecular Biology, University of Salzburg, Austria </td>
<td>-</td>
<td>-</td>
</tr>	
<tr>
<td>Shaker</td>
<td>Eppendorf</td>
<td>-</td>
<td>-</td>
</tr>	
<tr>
<td>Sodium azide</td>
<td>Sigma</td>
<td>S8032</td>
<td>-</td>
</tr>	
<tr>
<td>Sodium chloride (NaCl)</td>
<td>Sigma</td>
<td>S1679</td>
<td>-</td>
</tr>	
<tr>
<td>Sodium hydrogen carbonate</td>
<td>Sigma</td>
<td>90421C</td>
<td>-</td>
</tr>	
<tr>
<td>Sodium hydroxid</td>
<td>Sigma</td>
<td>S5881</td>
<td>-</td>
</tr>	
<tr>
<td>Sodium phosphate</td>
<td>Sigma</td>
<td>342483</td>
<td>-</td>
</tr>	
<tr>
<td>Triple detector array</td>
<td>Viscotek</td>
<td>TDA302</td>
<td>-</td>
</tr>	
<tr>
<td>Personal BioVortex V-1 plus</td>
<td>Peqlab</td>
<td>90-V-1</td>
<td>-</td>
</tr>	
<tr>
<td>Water bath 1012</td>
<td>GFL</td>
<td>1012</td>
<td>-</td>
</tr>	
</tbody>
</table>

basophil activation test for investigation of ige-mediated mechanisms in drug hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Watch this Scientific Journal Video about Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity at JoVE.com

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

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33.4K Views.  University of Salzburg. Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Video: Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

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.

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

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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.

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 ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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

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

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

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

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.