Granulocyte-dependent Autoantibody-induced Skin Blistering

I Am Cassian RO from the Department of Dermatology at the University of Fryberg Germany. The research of our laboratory is focused on the pathogenesis of autoimmune skin diseases. One of the autoimmune disease we're interested in is the Epidermolysis QUISTA or EBAA is an organ specific autoimmune disease with a well-defined antigen auto-antibody system and mutually complementary exvivo and animal models are available for this disease.

The blister formation of EBA can be reproduced by PE transferring antibodies against type seven collagen into mice. Therefore, in this video article, we will present the induction and analysis of skin blistering in mice by the passive transfer of collagen seven specific antibodies. Hi, I'm King OBA from CAS Group in the Department of Dermatology at the University Hospital of Freberg.

In this video, we are going to demonstrate you how the experimental mouse model of EBA is induced by the passive transfer of pathogenic auto antibodies. We use this procedure when examining the possible patho mechanisms in autoimmune skin disorders or when searching for pathogenic epitopes. When establishing the passive transfer model for EBA, a few additional steps must be performed prior to auto-antibody injection into wild type palp mice.

As a first step white rabbits areed subcutaneously with the purified recombinant antigen. Total IgG from rabbit serum is isolated by affinity column chromatography and subsequently injected into wild type mice. Injections are administrated subcutaneously every second day four times, and blood is drawn at two day intervals.

The mice are examined daily for their general condition and for evidence of cutaneous lesions. During the experiment, clinical scores reflecting the disease activity are calculated for each individual and when two weeks after the first injection, the animals are sacrificed serum samples as well as skin biopsies are taken and further analyzed. After the incubation Time, placed a column in a vertical support rack, open the bottom, tap and collect the flow through.

In 50 milli Falcon tubes, keep the flow through till you have the immune fluorescence analysis confirmation that it is specific antibody free concentrate diluted IgG by ultra filtration in special amicon tubes at 3, 220 times G for 30 minutes at four degrees. The concentration consists of letting the elate flow through a filter, discarding the flow through, refilling the tube and repeating till there is no elu left. As a last step wash with one times PBS collect the yellowish sticky concentrated IgG, measure its concentration and store it in properly labeled tubes.

Before starting with the actual injection of the autoantibodies, assess their specificity and their titer by indirect immunofluorescence. Incubate cryo sections of normal mild skin with serial dilution of the concentrated IGT preparation and visualize their deposition by an immune floor is simply labeled proper secondary antibody document by taking pictures Before going to The animal facility. Make sure that the animal protocol is written and all materials are prepared.

Data sheets for the clinical scoring of disease activity labeled tubes for blood and tissue samples. Antibody solution diluted so that the proper volume can be injected. Fresh anticoagulant and anesthetize sterilized surgical instruments as well as syringes.

A caliper and digital camera should also be included on the checklist. After cleaning the working surface and preparing the isof fluorine installation, heparinized the syringe for blood row and fill an insulin syringe with antibody solution. Take the mouse out Of the cage, check its identification number And weigh it.

Write all measured and observed data in the mice Personal data and clinical scoring sheet. When the mouse is already under ssis, measure the ear thickness, then check for the animal's skin and fur appearance. Pay special attention to the head area back and belly and limbs.

Take blood from the tail vein through a small incision applied on its upper one third disinfect the fur and by lifting the skin On the back carefully inject the IgG solution subcutaneously. When getting back to the laboratory, centrifuge the blood for 10 minutes at 1, 200 times G room temperature to obtain the serum. Transfer the sample to properly labeled Tubes and store them according to the plan.

Examine Mice daily for signs of disease when they start developing erythema alopecia and skin lesions. In addition to measuring their weight, evaluate the disease activity. Inject subcutaneously a ketamine cila in mixture to put the mouse under ssis.

Measure the ear thickness and subsequently start the general body check. Use curved fine point forceps to brush through the fur opposites to its growing direction to expose possible affected spots. Work your way around the face.

Eye snout moving towards the outer and inner part of the ear, and then check the Other side of the head as well. Continue with the mucosa of the oral cavity, The ventral part of the snout and abdominal area. The dors lateral, dorsal and neck area, as well as the front and hand limbs.

Quantify The extent of the lesions in record, the findings in the form based on the skin covered area, percentage of certain body parts and regions in comparison to the total skin surface of the mouse. A clinical disease score is calculated based on the total affected skin and mucus membrane area. Finally, take pictures of relevant body parts and diseased areas.

At the end of the experiment, sacrifice the animals under deep necrosis and collect blood as well as tissue samples. The ears, tail, and esophagus are the most frequently taken Ones in this experimental setting. Cut the lesional skin biopsies Into smaller stripes.

Place them into histology processing and embedding cassettes and keep them in 3.7%formaldehyde till embedding in paraffin. Subsequently, process the samples for h and e staining for immune fluorescence analysis. Embed the peral skin strips in Optimal cutting temperature medium and use a cryostat to cut six micrometer Sections.

Perform direct immune fluorescence analysis to visualize the tissue bound specific rabbit antibodies and mouse complement components as additional assay perform Eliza using recombinant antigen coated micro titration plates and serial dilution of the mouse serum in order to measure the levels of circulating auto antibodies. The passive transfer of collagen seven specific antibodies result in a full-blown disease in mice resembling at clinical histological and immuno pathological levels. The human EBA blisters, crusts, erosions and alopecia develop on the ear around the eyes, snout, paws, legs, and back of the mice.

The disease activity, which shows an increasing tendency throughout the experiment is calculated based on the affected skin and mucus membrane area. Representative results depicting deposition of specific rabbit IgG and mouse complement C3 detected by indirect immune fluorescence at the dermal epidermal junction. In cryo sections of peral skin are shown here.

No IgG or complement C3 was detected in the controls. Subsequent histological analysis of lesional skin reveals sub epidermal blisters and inflammatory infiltrate. However, sub epidermal blister formation is not seen in the control When performed.

As shown here, the model of autoantibody in use sub epidermal blistering will greatly the dissection of the ROY dependent inflammatory pathways triggered by autoantibodies resulting in tissue damage. It's robust design and a straightforward protocol make it suitable also for developing and testing new anti-inflammatory therapies in auto-antibody induced diseases.