The authors wish to thank study participants and their parents. We would also like to thank Dr. R&#233;jean Lapointe (CHUM Notre-Dame, Montreal, Canada) for access to his ELISpot reader, Dr. Lubo Alexandrov for statistical analysis, and Denis Blais, Sandra Caron, Silvie Valois and Martine Caty for expert technical assistance. Supported by grants from le Fonds d&#8217;op&#233;ration pour les projets de recherche clinique et d&#8217;&#233;valution des technologies (CHU Sainte-Justine) to H.S. and P.O., by la Fondation Centre de canc&#233;rologie Charles-Bruneau, and by the Leukemia &#38; Lymphoma Society of Canada. I.S.F. was supported by scholarships from la Fondation CHU Sainte-Justine and le Fonds de la recherche du Qu&#233;bec-sant&#233; (FRQS). A.J.G. was the recipient of scholarships from the Department of Microbiology, Infectiology &#38; Immunology, Universit&#233; de Montr&#233;al (Gabriel-Marquis Scholarship), FRQS, and the Canadian Institutes of Health Research (CIHR). N.M. was supported by la Fondation CHU Sainte-Justine, the Cole Foundation, and FRQS.

This research protocol was approved by the Institutional Ethics Review Board of CHU Sainte-Justine, Montreal, Quebec, Canada, where the study was conducted. Informed consent was sought and obtained from all study participants, their parents or legal guardians. All procedures performed on days 1 and 2 must be carried out under sterile conditions (i.e. under a laminar flow hood). Standard safety procedures for handling human blood should be strictly observed.
1. Coating the Plates
<ol>
	<li>Permeabilize the polyvinylidene difluoride (PVDF) membranes at the bottom of 96 well MultiScreen IP white ELISpot plates by adding to each well 20 &#956;l of 35% ethanol for 1 min. Membranes should become slightly translucent.</li>
	<li>Immediately wash the wells 3 times with 200 &#956;l of 1x phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) using a multichannel pipette or similar device. This step is critical given that ethanol residues can affect cell viability and binding of capture antibody. ELISpot plates are fragile and should be handled carefully: the use of an automated plate washer is not recommended.</li>
	<li>Coat the wells with 10 &#956;l of purified mouse anti-human IFN-&#947; capture antibody diluted in 1x PBS to a final concentration of 10 &#956;g/ml. Cover the plates with regular plastic wrap and incubate overnight at 4 &#176;C.</li>
</ol>
2. Blocking Plates
<ol>
	<li>Empty the wells, tapping them dry, and wash plates 5 times with 200 &#956;l of 1x PBS.</li>
	<li>Fill the wells with 200 &#956;l of complete RPMI 1640 medium and incubate plates for 2 hr at 37 &#176;C (blocking step).</li>
	<li>Wash the plates 5x with 200 &#956;l of 1x PBS and keep the wells full of 1x PBS until cells are ready to be plated.</li>
</ol>
3. Cell Plating and Stimulation
<ol>
	<li>Prepare peripheral blood mononuclear cells (PBMC) from human venous blood samples by centrifugation on Ficoll-Paque gradients using standard procedures. Alternatively, using standard methods, thaw aliquots of PBMC frozen under liquid nitrogen. After the last centrifugation, resuspend cells at a final concentration of 2 x 106 cells/ml and incubate overnight at 37 &#176;C under a 5% CO2 atmosphere in a water jacket incubator.</li>
	<li>Remove PBMC from the incubator and resuspend them in a final volume of 5 ml of complete RPMI 1640 medium.</li>
	<li>Incubate PBMC in the presence of 10 &#956;l of genetically engineered endonuclease from Serratia marcescens for 5 min at room temperature.</li>
	<li>Remove a 50 &#956;l aliquot of PBMC and mix with 50 &#956;l of trypan blue dye. Count cells and estimate % viability using a haemocytometer and microscopic examination (dye exclusion method). Various automated methods for cell counting and assessment of cell viability can also be used. PBMC should be &#62;95% viable. Discard sample when viability is lower than 95%.</li>
	<li>Centrifuge PBMC at 700 x g for 5 min, discard supernatant, and resuspend cells in AIM-V medium supplemented with 2% (v/v) inactivated human serum (HIS) at a final concentration of 2 x 106 cells/ml. Keep at 37 &#176;C until stimulation mixtures are added to the plate.</li>
	<li>Add to the wells, one drop at a time, 100 &#956;l of the appropriate stimulation mixture. Three stimulation mixtures should be used as described below:
	<ol>
		<li>Use AIM-V medium supplemented with 2% (v/v) inactivated human serum (IHS) as negative control.</li>
		<li>Use anti-CD3 monoclonal antibody (clone OKT3) diluted in AIM-V medium supplemented with 2% (v/v) IHS to a final concentration of 0.5 &#956;g/ml as positive control).</li>
		<li>Use VZV antigen, in the form of either &#947;-irradiated (10,000 Gy; 30 min exposure time) live attenuated VZV vaccine (1,350 plaque forming units [PFU]/ml) diluted 1:200 in AIM-V medium supplemented with 2% (v/v) IHS) or 1 &#956;g of an equimolar mixture of 67 peptides (15 amino acid residues) synthesized based on the sequence of the VZV immediately early (IE63) phosphoprotein. Control irradiation efficacy by monitoring the absence of visible signs of cytopathic effects after 4 days in PBMC cultures.</li>
		<li>Prepare all wells in duplicate.</li>
	</ol>
	</li>
	<li>Add to the wells, one drop at a time, 100 &#956;l of cells prepared in step 5.4 to the appropriate wells. Two wells should be left without cells (negative control). Incubate for 20 hr at 37 &#176;C under a 5% CO2 atmosphere in a water jacket incubator. Do not shake, move, or stack the plates on top of one another during incubation.</li>
</ol>
4. Spot Development and Detection
<ol>
	<li>Discard the cells and wash the plates 10x with 1x PBS supplemented with 0.05% (v/v) Tween 20 (PBST). Tween 20 helps detach cells that have adhered to the PVDF membrane following overnight incubation.</li>
	<li>Using a multichannel pipette, add 100 &#956;l of 0.5 mg/ml biotin conjugated anti-IFN-&#947; monoclonal antibody (4S.B3 clone) diluted in 1x PBS containing 0.5% (w/v) bovine serum albumin (BSA). Incubate plates for 2 hr at room temperature.</li>
	<li>Wash the wells 6x with PBST and add each well 100 &#956;l of streptavidin conjugatd with alkaline phosphatase diluted 1:1,000 in 1x PBS containing 0.5% (w/v) BSA. Cover the plates and incubate for 1 hr at room temperature.</li>
	<li>Wash the plates 3x with PBST and 3x with 1x PBS. Add 100 &#956;l of a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium chloride (NBT) substrate solution and incubate 5 min at room temperature.</li>
	<li>Wash the plates under running distilled water. Remove the bottom section of the ELISpot plate and wash both sides of the membrane with distilled water. Air dry the plates.</li>
	<li>Examine the wells and enumerate the spots using a stereoscopic dissection microscope or scan the plates using an automated spot counter. Keep the plates away from light if they cannot be examined on the same day.
	<ol>
		<li>Average the numbers of spots counted in corresponding duplicate wells and subtract the number of spots counted in the negative control wells (no cells) from the number of spots counted in the test wells.</li>
		<li>Express IFN-&#947; production as the number of spot-forming units (SFU) per 106 PBMC.</li>
		<li>Consider that test samples are positive if the number of SFU is &#62;50 per 106 PBMC and 2 standard deviations (SD) above the negative control (cells + AIM-V medium supplemented with 2% (v/v) IHS).</li>
		<li>Use a value of 400 SFU per well when spots are too numerous to be counted.</li>
	</ol>
	</li>
	<li>Test whether ELISpot data are normally distributed or not using the Kolmogorov-Smirnov test. When data are normally distributed, perform statistical comparisons using Student&#8217;s t test (2 groups) or ANOVA and the Bonferroni post-test (multiple comparisons). If data are not normally distributed, use the Mann-Whitney U test (2 groups) or the Kruskal-Wallis test and Dunn&#8217;s post-test (multiple comparisons).</li>
</ol>

The authors declare that they have no competing financial interests.

Modifications and troubleshooting: IFN-&#947; ELISpot assays have been used to examine cell-mediated immune responses directed against a variety of microbial pathogens, including human immunodeficiency virus type 1 (HIV-1)24,25, hepatitis C virus (HCV)26,27, and Mycobacterium tuberculosis28,29, just to name a few. Here we described the development of an IFN-&#947; ELISpot assay to measure cellular immunity against, with the hope of defining correlates of VZV specific immune recon...

First performed in 1989, UCBT is increasingly used as part of the treatment of various neoplastic and nonneoplastic blood disorders in children1. VZV is a cytopathic human alphaherpesvirus which causes two different diseases, varicella (after primary infection) and herpes zoster (after reactivation). Following primary infection, VZV persists throughout the life of the host sheltered within sensory nerves of dorsal root ganglia. One of the most threatening infectious complications following UCBT is associated with VZV2-4. In our clinical center, in absence of VZV prophylaxis, the cumulative incidence of VZV disease VZV disease at 3 years postUCBT was 46%2. In these patients, de novo infection with or reactivation of VZV is often associated with visceral dissemination to the central nervous system, lungs and liver5-7. As a result, acyclovir, valacyclovir or famciclovir prophylaxis is commonly administrated to UBCT recipients8,9. However, this treatment strategy does not take into account the protective potential of VZV specific T lymphocytes or the kinetics of reconstitution of VZV specific T cell responses. Potential problems associated with the expanding use of long term antiherpetic prophylaxis include a) patient overtreatment; b) the development of antiviral drug resistance10,11; and c) impairment of VZV specific immune reconstitution12,13. Because detection of functional VZV specific T lymphocytes correlates with the presence of long term protection from VZV infection and improved clinical outcome4,14,15, monitoring cell mediated immune responses directed against VZV during the posttransplant period might result in a more rational use of antiviral treatment by enabling medical practitioners to distinguish patients who would benefit from VZV prophylaxis from those whose immune system is capable of controlling VZV replication4,13.

The IFN-&#947; ELISpot assay is widely used for the monitoring cell mediated immune responses in a variety of experimental systems and clinical conditions. Spots are generated following the cleavage of a chromogenic substrate, generating a visible and stable precipitate at the site of the reaction. Each individual spot thereby represents the footprint of an individual cytokine-producing cell. IFN-&#947; ELISpot not only measures the ability of individual cells ex vivo to produce IFN-&#947; in response to in vitro stimulation with cognate antigen, but it also provides an estimate of the frequency of responding cells in a given cell population16,17. In addition to its high sensitivity, IFN-&#947; ELISpot is straightforward to perform, making its use possible in the context of personalized clinical protocols aimed at guiding initiation or cessation of antiviral treatment. The procedure detailed below describes an ELISpot assay that is specifically designed to detect and measure the production of IFN-&#947; by peripheral blood mononuclear cells following in vitro stimulation with VZV derived antigens.

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leucocep tube</td>
			<td style="width:101px;">VWR</td>
			<td style="width:143px;">89048-936/89048-932</td>
			<td style="width:397px;">12 ml or 50 ml tubes may be used depending on the volume of blood.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ficoll-Paque</td>
			<td style="width:101px;">GE Healthcare</td>
			<td style="width:143px;">17-1440-02</td>
			<td>Protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzonase nuclease</td>
			<td style="width:101px;">Novagen</td>
			<td style="width:143px;">70746-3</td>
			<td>Keep at -20 C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MultiScreenHTS-IP Filter Plate</td>
			<td style="width:101px;">Millipore</td>
			<td style="width:143px;">MSIPS4W10</td>
			<td>Sterile with pore size of 0.45 &#181;m.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse anti-human IFN-&#947; capture antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">551221</td>
			<td>NIB42 clone.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pepmix VZV IE63&#160;</td>
			<td style="width:101px;">JPT Peptide Technologies</td>
			<td style="width:143px;">PM-VZV-IE63</td>
			<td>Dissolve contents of one vial in 40 &#956;L of DMSO. Use within 6 months.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotin-conjugated anti-IFN-&#947; monoclonal antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">554550</td>
			<td>4SB3 clone.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Streptavidin conjugated with alkaline phosphatase&#160;</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-3554</td>
			<td>Dilute for use on the same day.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BCIP/NBT</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-6432</td>
			<td>Protect from light.</td>
		</tr>
	</tbody>
</table>

development of an ifn-&#947; elispot assay to assess varicella-zoster virus-specific cell-mediated immunity following umbilical cord blood transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

The IFN-&#947; ELISpot protocol detailed above was developed and optimized in our laboratory to measure the magnitude and the quality of cell mediated immune responses directed against VZV4. Diverse sources of VZV antigen can be used for the stimulation step. These include: a) commercially available detergent inactivated extracts from VZV infected Vero cells18; b) pools of overlapping synthetic peptides from specific VZV encoded proteins, including IE6315 and ORF419; c) live at...

Watch this Scientific Journal Video about Development of an IFN-Î³ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation at Jo

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

development-an-ifn-elispot-assay-to-assess-varicella-zoster-virus

Research

JoVE Journal

Immunology and Infection

15.6K visualizações.  Université de Montréal.  Novel gerações de ensaios funcionais, tais como o interferão gama (IFN-γ) ELISpot, que detectam a produção de citoquinas ao nível de células individuais e fornecer tanto a caracterização quantitativa e qualitativa das respostas das células T pode ser utilizado para avaliar as respostas imunes mediadas por células contra a varicela zoster vírus (VZV). 

Vídeo: Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...