The authors would like to thank Amanda Tardo, Dr. Mostafa Bouljihad, Stephanie Feely, Carol Coyne, Dr. Erin Ribka, Dr. Pete Didier and Dorothy Kuebler for their technical assistance. This work was supported by NIH grants R01-DK076653 and 3R01-DK076653-02S1 to K.S. and the base operating grant of the TNPRC RR000164.

1. Skin biopsy sample collection
<ol>
	<li>Prior to skin biopsy procedure, anesthetize animals intramuscularly with 2.5 mg/kg of tiletamine hydrochloride and zolazepam hydrochloride telazol mixture (Fort Dodge Animal Health, Fort Dodge, IA). Monitor the animals from administration of anesthetic until recumbency and then remove from their enclosure.</li>
	<li>Remove the hair from the skin area of interest utilizing an Oster Golden A5 Single Speed Veterinary Clipper with a size 40 blade (Oster Professional Products, McMinnville, TN) and aseptically prepare with alternating betadine scrub and alcohol. Secure a sterile fenestrated drape over the selected biopsy site.</li>
	<li>Using a sterile technique, place a 4.0 mm Miltex Punch Dermal Biopsy instrument (Miltex, York, PA) against the skin while rotating the instrument 180 degrees clockwise and counter clockwise with slight pressure until the biopsy punch transects through the dermal layers into the subcutaneous tissue. Remove the biopsy sample and grasp the transected portion of skin with forceps and free from the subcutaneous tissue.</li>
	<li>Close the skin defect with 3-0 nylon suture attached to a 3/8 circle cutting needle (Ethilon, Ethicon, Johnson &#38; Johnson Medical Limited, Berkshire, UK) in a cruciate pattern. Give all animals 0.01 mg/kg buprenorphine hydrochloride (Hospira, Lake Forest, IL) intramuscularly for post operative analgesia.</li>
</ol>
2. Sample processing
<ol>
	<li>Work with skin biopsy samples from chronic dermatitis and healthy control rhesus macaques. Obtain two to three (4 mm in diameter) biopsy samples from each animal.</li>
	<li>Fix first sample in zinc formalin (Z-fix, Anatech Ltd., Battle Creek, MI) for 24 hours, wash in water for 30 min, wash briefly in 70% ethanol, and place into ASP300 Leica tissue processor (Leica Microsystems Inc., Buffalo Grove, KS) where tissue is dehydrated with ascending grades of 70%, 80%, 95% and 100% ethanol 48 min each (Fisher Scientific, Pittsburgh, PA), followed by two changes of xylene (Fisher).</li>
	<li>Embed in paraffin media (Fisher) for long-term storage at room temperature. Place at -20oC for 20 min prior to sectioning. Prepare 6 &#956;m sections using a rotary microtome (HM325, Microm International, Waldorf, Germany). Place sections on charged slides (Fisher) and air dry at 60oC overnight. Stain with hematoxylin and eosin (H&#38;E) standard method (described below).</li>
	<li>Fix second sample in 2% paraformaldehyde (USB Corp, Cleveland, OH) for 30 min at room temperature, wash three times in phosphate buffered saline (PBS, Gibco-Invitrogen, Carlsbad, CA), place in 30% sucrose (Fisher) for 4 hours, and embed in OCT freezing medium (Sakura Finetek, Torrence, CA). Keep at -80oC for 20 min prior to sectioning. Prepare 15 &#956;m sections using the cryostat (HM560, Thermo Scientific, Kalamazoo, MI).</li>
</ol>
3. H&#38;E staining
<ol>
	<li>Deparaffinize embedded sections through three changes of xylenes (Fisher) and rehydrate through graded ethanols: three changes of 100%, two changes of 95%, one change of 80% and distilled water, 2 min each.</li>
	<li>Stain with hematoxylin (Richard-Allan Scientific, Kalamazoo, MI) for one min and follow by a brief wash in running tap water.</li>
	<li>Counterstain with eosin (Sigma) for 6 min.</li>
	<li>Dehydrate stained tissues through 95% and absolute ethanol, two changes of 2 min each and then clear in three changes of xylenes, 3 min each.</li>
	<li>Mount coverglass using Cytoseal Mounting Medium (Richard-Allan Scientific) over the H&#38;E-stained tissue sections.</li>
</ol>
4. Histopathological evaluation
Inspect H&#38;E-stained biopsy skin sections under a light microscope using the 4 - 40x magnification and Leica microscope with a SPOT Insight digital camera (Digital Instruments Inc., Michigan, USA).
5. Immunofluorescent staining
<ol>
	<li>Work with OCT-embedded skin sections for immunofluorescent staining. Prior to staining, soak for 20 min in PBS containing 0.2% fish skin gelatin (FSG, Sigma) with 0.1%Triton X-100 (Sigma) to remove the OCT and to permeabilize the tissue.</li>
	<li>After washing the slides, incubate with 10% normal goat serum (NGS, Sigma), diluted in PBS-FSG for 1 hour, at room temperature, in a humidified slide chamber.</li>
	<li>In order to visualize the deposition of IgA and tTG2 antibodies, incubate slides with anti-rhesus IgA and tTG2 primary antibodies, diluted in 10% NGS-PBS-FSG for 1 hour.</li>
	<li>Wash with PBS&#8211;FSG&#8211;Triton X-100 (Sigma) for 10 min, followed by PBS-FSG for 10 min.</li>
	<li>Incubate with secondary, isotype-specific antibodies tagged with Alexa Fluor fluorochrome 488 (green), 568 (red) or 633 (blue) (Molecular Probes-Invitrogen) at 1:1,000 dilution. Use the ToPro-3 dye to visualize the nuclear DNA. Use the 10% NGS-PBS-FSG as an antibody diluent.</li>
	<li>Use the anti-quenching solution (Sigma) to mount the stained tissue sections.</li>
</ol>
6. Confocal imaging
Perform imaging with a Leica TCS SP2 True Confocal Laser Scanning Microscope (Leica, Wetzlar, Germany) equipped with three lasers (six lines for excitation available): an argon-krypton laser at 488 nm (green), a krypton laser at 568 nm (red) and a helium-neon laser at 633 nm (blue), that span from the visible to the far-red side of the spectrum. Record differential interference contrast (DIC) images for evaluation of non-labeled tissue architecture.
7. Representative Results
An example of positive recognition of eTG by both tTG2 and IgA antibodies is shown in Fig. 3A. Consistent with situation in human patients and due to much lower prevalence of DH-like dermatitis than CD-like gluten sensitivity also in rhesus macaques, most of the skin biopsy samples are expected to show the positive reaction with tTG2 antibodies but without the presence of tTG2+IgA+ double positive cells (Fig. 4). These DH-unrelated cases, including dermatitis cases of microbial origin, will reveal some single-positive IgA+ cells scattered beneath the epithelium but not spectral overlap between the tTG2 and IgA-labeled markers (Figs. 3B and 4). Therefore, it is important to maintain the order of suggested testing procedures, starting with clinical examination (Fig. 1), followed by histopathological evaluation (Fig. 2) and only if no other, more common dermatitis-causing agents can be identified, confocal microscopy examination of skin biopsies stained for tTG2 and IgA is warranted. Clinical and/or histopathological evaluations alone are not sufficient for positive diagnosis of this condition. In those cases when double-positive (tTG2+IgA+) cells are identified around the area of dermal papillae, DH-like dermatitis diagnosis can be issued. In such cases, withdrawal of dietary gluten and administration of gluten-free diet is recommended in order to alleviate the dermatitis symptoms.
<img alt="Figure 1" src="/files/ftp_upload/3154/3154fig1.jpg" /> 
Figure 1. Inguinal region of EM96 macaque before (A) and after (B) withdrawal of dietary gluten for 5 weeks. Reintroduction of dietary gluten coincided with the reappearance of dermatitis (C).
<img alt="Figure 2" src="/files/ftp_upload/3154/3154fig2.jpg" /> 
Figure 2. HE staining of EM96 macaque skin biopsies collected before (A) and after (B) withdrawal of dietary gluten for 9 weeks. A: Epithelium is thickened (7-15 layers of epithelial cells) with a thick superficial crust of hyperkeratotic cells infiltrated with degenerating neutrophils. Neutrophils appear also inside the epithelium and are mixed with moderate numbers of lymphocytes, plasma cells, and histiocytes around the adnexa in the upper dermis. B: Some hyperkeratosis is still present, epithelial thickness is normalized (4-7 layers) with minimal lymphoplasmacytic infiltration in the upper dermis. C: Normal abdominal skin from FR56 macaque, with no lymphoplasmacytic infiltration, is included for comparison. Magnification 20x.
<img alt="Figure 3" src="/files/ftp_upload/3154/3154fig3.jpg" /> 
Figure 3. Confocal microscopy images of EM96 macaque skin biopsies collected before (A) and after (B) withdrawal of dietary gluten for 9 weeks. A: IgA staining (green), tTG2 (red) and ToPro-3 nuclear DNA marker (blue). Co-localization of IgA and tTG2 appears in yellow around the area of dermal papilae. B: Skin sample taken after 9 weeks of gluten-free diet shows no IgA within the epithelium while some subepithelial IgA is still present (green), tTG2 (red) and ToPro-3 nuclear DNA marker (blue).
<img alt="Figure 4" src="/files/ftp_upload/3154/3154fig4.jpg" /> 
Figure 4. Confocal microscopy images of GI24 rhesus macaque skin biopsies collected while animal was exhibiting the symptoms of chronic (DH-unrelated) dermatitis. The IgA staining is in green, tTG2 in red and ToPro-3 nuclear DNA marker is in blue. The tTG2+ cells are present inside the epithelium, few IgA+ cells beneath the epithelium, while no extensive spectral overlap between the red and green markers is seen.
<table border="1">
	<tbody>
		<tr>
			<td>Animal Tag</td>
			<td>Sex 
			[M/F]</td>
			<td>Age 
			[Years]</td>
			<td>Clinical Symptoms</td>
			<td>Plasma tTG2 
			antibodies</td>
			<td>Diet 
			[1,2]*</td>
		</tr>
		<tr>
			<td>HJ71</td>
			<td>F</td>
			<td>1.3</td>
			<td>Healthy</td>
			<td>-</td>
			<td>1</td>
		</tr>
		<tr>
			<td>HD13</td>
			<td>M</td>
			<td>1.4</td>
			<td>Healthy, occasional diarrhea</td>
			<td>+</td>
			<td>1</td>
		</tr>
		<tr>
			<td>FR56</td>
			<td>M</td>
			<td>5.4</td>
			<td>Chronic idiopathic diarrhea</td>
			<td>-</td>
			<td>1</td>
		</tr>
		<tr>
			<td>EM96</td>
			<td>F</td>
			<td>5.0</td>
			<td>Chronic dermatitis</td>
			<td>+/- (borderline)</td>
			<td>1 and 2</td>
		</tr>
		<tr>
			<td>GI24</td>
			<td>F</td>
			<td>4.6</td>
			<td>Chronic dermatitis</td>
			<td>-</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 1. TNPRC rhesus macaques used for collection of skin biopsies
* Diet 1 &#8211; regular monkey chow (gluten-containing); Diet 2 &#8211; gluten-free diet

<ol>
	<li>Diosdado, B., Wijmenga, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+the+adaptive,+innate+and+regulatory+immune+responses+in+the+intestinal+mucosa+of+celiac+disease+patients.">Molecular mechanisms of the adaptive, innate and regulatory immune responses in the intestinal mucosa of celiac disease patients.</a> Expert. Rev. Mol. Diagn. 5 (5), 681-700 (2005).</li><li>Schuppan, D., Dennis, M. D., Kelly, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celiac+disease:+epidemiology,+pathogenesis,+diagnosis,+and+nutritional+management.">Celiac disease: epidemiology, pathogenesis, diagnosis, and nutritional management.</a> Nutr. Clin. Care. 8 (2), 54-69 (2005).</li><li>Sardy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+transglutaminase+(TGase+3)+is+the+autoantigen+of+dermatitis+herpetiformis.">Epidermal transglutaminase (TGase 3) is the autoantigen of dermatitis herpetiformis.</a> J. Exp. Med. 195 (6), 747-757 (2002).</li><li>Rose, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+against+epidermal+transglutaminase+are+a+sensitive+diagnostic+marker+in+patients+with+dermatitis+herpetiformis+on+a+normal+or+gluten-free+diet.">Autoantibodies against epidermal transglutaminase are a sensitive diagnostic marker in patients with dermatitis herpetiformis on a normal or gluten-free diet.</a> J. Am. Acad. Dermatol. 61 (1), 39-43 (2009).</li><li>Bethune, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+transport+and+enzymatic+detoxification+of+gluten+in+gluten-sensitive+rhesus+macaques.">Transepithelial transport and enzymatic detoxification of gluten in gluten-sensitive rhesus macaques.</a> PLoS ONE. 3 (3), e1857-e1857 (2008).</li><li>Mazumdar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+transepithelial+passage+of+the+immunogenic+33-residue+peptide+from+&#x03B1;-2+gliadin+in+gluten-sensitive+macaques.">Visualization of transepithelial passage of the immunogenic 33-residue peptide from &#x03B1;-2 gliadin in gluten-sensitive macaques.</a> PLoS ONE. 5 (4), e10228-e10228 (2010).</li><li>Schuppan, D., Junker, Y., Barisani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celiac+disease:+from+pathogenesis+to+novel+therapies.">Celiac disease: from pathogenesis to novel therapies.</a> Gastroenterol. 137 (6), 1912-1933 (2009).</li><li>Hunt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+identified+genetic+risk+variants+for+celiac+disease+related+to+the+immune+response.">Newly identified genetic risk variants for celiac disease related to the immune response.</a> Nature Genet. 40 (4), 395-402 (2008).</li><li>Abadie, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+genetic+and+immunological+insights+into+a+model+of+celiac+disease+pathogenesis.">Integration of genetic and immunological insights into a model of celiac disease pathogenesis.</a> Annu. Rev. Immunol. 29, 493-525 (2011).</li></ol>

No conflicts of interest declared.

Our newly established non-human primate model of gluten sensitivity was recently used to study the intestinal permeability changes triggered by dietary gluten 5,6. Plasma tTG2 antibodies as well as intestinal tTG2+IgA+ deposits were detected in some gluten-sensitive macaques. Since we recently identified rare incidents of idiopathic dermatitis in colony rhesus macaques, where no usual (infectious/metabolic) etiology could be established, objective of this study was to evaluate if tTG2+IgA+ antibody deposits co...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent or equipment</td>
 <td>Company</td>
 <td>Catalogue Number / Product Code</td>
 <td>Note</td>
 </tr>
 <tr>
 <td>Anti-Human IgA-FITC *</td>
 <td>Sigma, St. Louis, MO</td>
 <td>F-5259</td>
 <td>1:100 working dilution</td>
 </tr>
 <tr>
 <td>Anti-Human tTG2</td>
 <td>Thermo Scientific, Kalamazoo, MI</td>
 <td>MS-300-P1ABX</td>
 <td>1:100 working dilution</td>
 </tr>
 <tr>
 <td>Anti-Mouse IgG1 **</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>A-21124</td>
 <td>1:1,000 working dilution</td>
 </tr>
 <tr>
 <td>ToPro-3 DNA probe ***</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>T-3605</td>
 <td>1:1,000 working dilution</td>
 </tr>
 <tr>
 <td>Zinc Formalin 
 (Z-fix)</td>
 <td>Anatech Ltd., Battle Creek, MI</td>
 <td>171</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>OCT Freezing 
 Medium</td>
 <td>Sakura Finetek, Torrence, CA</td>
 <td>4583</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cytoseal Mounting 
 Medium</td>
 <td>Richard-Allan Scientific, Kalamazoo, MI</td>
 <td>8312-4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-Quenching 
 Solution</td>
 <td>Sigma, St. Louis, MO</td>
 <td>P 3130</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethilon 
 (Nylon Suture)</td>
 <td>Ethicon, Johnson &amp; Johnson, Berkshire, UK</td>
 <td>663G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Miltex Punch Dermal Biopsy Instrument</td>
 <td>Miltex, York, PA</td>
 <td>NA</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Oster Golden A5 Single Speed Vet Clipper with a size 40 blade</td>
 <td>Oster Products, McMinnville, TN</td>
 <td>78005-050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Leica Tissue Processor</td>
 <td>Leica Microsystems Inc., Buffalo Grove, KS</td>
 <td>ASP300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rotary Microtome</td>
 <td>Microm International, 
 Waldorf, Germany</td>
 <td>HM325</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cryostat</td>
 <td>Thermo Scientific, 
 Kalamazoo, MI</td>
 <td>HM560</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Confocal Microscope</td>
 <td>Leica, Wetzlar, 
 Germany</td>
 <td>TCS SP2</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
* Fluorescein isothiocyanate 
 ** Secondary (Alexa Fluor 568-conjugated) antibody used after tTG2 primary antibody 
 ** Dye

recognition of epidermal transglutaminase by iga and tissue transglutaminase 2 antibodies in a rare case of rhesus dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Watch this Scientific Journal Video about Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis at JoVE.com

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

recognition-epidermal-transglutaminase-iga-tissue-transglutaminase-2

Research

JoVE Journal

Immunology and Infection

24.4K Views.  Tulane National Primate Research Center. Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

Video: Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...