Name: recognition of epidermal transglutaminase by iga and tissue transglutaminase 2 antibodies in a rare case of rhesus dermatitis
Uploaded: 2011-12-15T12:00:00
Description: 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

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, McMi...

<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>

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 ...

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