RU2613718C1 - Pemphigus simulation method for mice by immunoglobulin g administration - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method comprises administering of immunoglobulin G (IgG) preparation, taken from patients with pemphigus vulgaris to newborn mice. Here IgG is taken from at least 5 patients. The resulting preparation is administered in a dose of 30 mg of IgG/mouse followed by animal euthanasia after 48 hours. The method can be reproduced in 100% of cases and provides clinical, histological and immunological features of pemphigus, which is important for the study of pathogenetic mechanisms of the disease and the development of new methods of severe autoimmune dermatosis diagnosis and treatment.

EFFECT: method is effective for establishment of an experimental pemphigus model in order to obtain the possibility of pathogenesis study, development of new methods of pemphigus diagnosis and treatment.

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Description

FIELD OF THE INVENTION

The invention relates to medicine, in particular to experimental biology, dermatovenerology, and in particular to a model in medicine, and relates to a method for modeling pemphigus in mice.

State of the art

Pemphigus (pemphigus) is a severe autoimmune bullous disease characterized by the formation of blisters and erosion on the skin and / or mucous membranes [Kubanov A.A., Abramova T.V. Pemphigus: textbook. allowance / A.A. Kubanov, T.V. Abramova; GBOU DPO "Russian Medical Academy of Postgraduate Education". - M .: GBOU DPO RMAPO, 2015 .-- 54 p .; Grando S.A. Pemphigus autoimmunity: hypotheses and realities // Autoimmunity. - 2012. - Vol. 45. - No. 1. - P. 7-35.].

The pathogenetic role in the formation of blisters in dermatosis - pemphigus is played by circulating autoantibodies with high tissue specificity, belonging to the class of immunoglobulins G (IgG), and causing destruction of desmosomes due to the release of proteolytic enzymes, which leads to acantholysis [Matushevskaya EV, Kubanova A. A., Samsonov V.A. Autoantibodies and autoantigens with pemphigus and pemphigoid // Bulletin of Dermatology and Venereology. - 1995. - No. 5. - S. 28-33; Kubanov A.A., Znamenskaya L.F., Abramova T.V., Svishchenko S.I. On the diagnosis of true acantholytic pemphigus // Herald of dermatology and venereology. - 2014. - No. 6. - S. 121-130 .; Joly R., Bernard P., Bedane C. et al. Pemphigus Guidelines for the diagnosis and treatment. Centers de reference des maladies huileuses auto-immunes. Societe Francaise de Dermatologie // Ann Dermatol Venereol. - 2011 .-- Vol. 138. - P. 252-258.]. It has been established that the main structures to which autoantibodies are produced during pemphigus vulgaris are the proteins of the desmosomal apparatus — type 3 desmogleins (Dsg 3)) [Stanley J.R., Yaar M., Hawley-Nelson P. et al. Pemphigus antibodies identify a cell surface glycoprotein synthesized by human and mouse keratinocytes // J. Clin. Invest. - 1982. - Vol. 70. - P. 281-288 .; Stanley J.R., Koulu L., and Thehletlet C. Distinction between epidermal antigensbinding pemphigus vulgaris and pemphigus foliaceus autoantibodies // J. Clin. Invest. - 1984. - Vol. 74. - P. 313-320; Ohyama M, Ota T., Aoki M. et al. Suppression of the immune response against exogenous desmoglein 3 in desmoglein 3 knockout mice: an implication for gene therapy // J Invest Dermatol. - 2003 Apr. - Vol. 120 (4). - P. 610-615.].

The first models for studying the pathogenicity of antibodies in pemphigus were human skin explants [Schiltz J.R., Michel B. Production of epidermal acantholysis in normal human skin in vitro by the IgG fraction from pemphigus serum // J. Invest Dermatol. - 1976 Aug. - Vol. 67, No. 2. - P. 254-260.] And mouse keratinocyte cell cultures [Farb R.M., Dykes R., Lazarus G.S. Anti-epidermal-cell-surface pemphigus antibody detaches viable epidermal cells from culture plates by activation of proteinase. Proc Natl AcadSci USA 1978 Jan; 75 (1): 459-463.].

J.R. Schiltz and B. Michel (1976) demonstrated the pathogenicity of antibodies of the blood serum fraction obtained from pemphigus patients in vitro on human skin explants [Schiltz JR, Michel B. Production of epidermal acantholysis in normal human skin in vitro by the IgG fraction from pemphigus serum . J. Invest Dermatol 1976 Aug; 67 (2): 254-60.]. Somewhat later R.M. Farb et al. Found that the IgG fraction of serum from pemphigus patients leads to the separation of epidermal murine BALB / c cells grown in a medium with a low complement content [Farb R.M., Dykes R., Lazarus G.S. Anti-epidermal-cell-surface pemphigus antibody detaches viable epidermal cells from culture plates by activation of proteinase. Proc Natl AcadSci USA 1978 Jan; 75 (1): 459-463.].

However, when using the keratinocyte cell line, acantholysis is not observed, since the cells in the culture form only a monolayer.

Under natural conditions, mice do not develop pemphigus-like diseases. The administration of antibodies obtained from pemphigus patients to adult mice also does not cause acantholysis [van der Wier G., Pas H.H., Jonkman M.F. Experimental human cell and tissue models of pemphigus // Dermatol Res Pract. - 2010. - 143871.].

For the first time, a laboratory animal model (the closest prototype analogue) with passive transfer of antibodies to pemphigus patients was obtained by a group of Japanese scientists in 1982. Antibodies isolated from one pemphigus patient were introduced to newborn mice, and signs of pemphigus appeared in mice after 8-24 hours [ Anhalt GJ, Labib RS, Voorhees JJ et al. Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease // J. Med 1982. - Vol. 306, No. 20. - P. 1189-1196.]. However, the disadvantages of the described model is the lack of reproducibility (in less than 50% of animals), the problem with the selection of doses of immunoglobulins obtained from one patient. In some mice, pemphigus symptoms did not develop, which is associated with immune tolerance.

J.M. Jr.,

Α., Liu Ζ., Ding Χ., Swartz S.J., Fairley J.A., Diaz L.A. Mechanisms of acantholysis in pemphigus vulgaris: role of IgG valence // Clin Immunol Immunopathol. - 1997 Oct. - Vol. 85, №1. - P. 90-96.].Subsequently, it was shown that Fab fragments of IgG patients with pemphigus vulgaris cause the appearance of blisters in newborn mice [

JM Jr.,

Α., Liu Ζ., Ding Χ., Swartz SJ, Fairley JA, Diaz LA Mechanisms of acantholysis in pemphigus vulgaris: role of IgG valence // Clin Immunol Immunopathol. - 1997 Oct. - Vol. 85, No. 1. - P. 90-96.].

However, obtaining Fab fragments of IgG is laborious and financially expensive.

The inventive method differs from the prototype in that laboratory animals are injected with IgG preparations obtained from several patients with pemphigus, which increases the reproducibility of the experiment to 100%.

The objective of the invention is to provide a method for modeling pemphigus in laboratory animals - newborn BALB / c mice by the introduction of class G immunoglobulins obtained from several patients with pemphigus.

The aim of the invention is to create an experimental model (simulation) of pemphigus in newborn BALB / c mice by the method of administration of IgG isolated from several patients with pemphigus, i.e. accurate reproduction of clinical, pathomorphological, immunological signs of this disease.

SUMMARY OF THE INVENTION

A method for modeling pemphigus, which consists in the intraperitoneal administration of BALB / c IgG line to newborn mice, isolated from several pemphigus patients, at a dose of 30 mg / mouse, administered once, and the exposure time is 48 hours. This time is enough for the appearance of clinical, pathomorphological and immunological signs of pemphigus in animals.

Using the invention allows to obtain the following technical result.

The method allows to simulate pemphigus in newborn mice by administering an IgG preparation obtained from several pemphigus patients. In this regard, this method is reproducible in 100% of cases. Using the proposed method, clinical, histological and immunological signs of pemphigus are reproduced in laboratory animals, which is relevant for the search for new molecular biological targets, the study of the pathogenetic mechanisms of the development of the disease, the development of new methods for the diagnosis and treatment of severe autoimmune dermatosis.

The technical result is achieved due to the fact that for the first time after the administration of IgG preparations obtained from several (five) pemphigus patients, the authors created an experimental model of pemphigus in laboratory animals with 100% reproducibility.

The results can be explained by the fact that class G immunoglobulins (IgG) make up about 80% of serum immunoglobulins and are divided into four subclasses of IgG1, IgG2, IgG3 and IgG4, each with its own biological properties. IgG are formed at the height of the primary immune response and with repeated administration of the antigen (secondary response). The most common IgG1 (67%), less often IgG2 and IgG3 (22 and 7%, respectively), the rarest is a subclass of IgG4 (4%). In autoimmune bullous dermatoses, autoantibodies with high tissue specificity and IgG are produced [Schroeder H.W. Jr., Cavacini L. Structure and function of immunoglobulins // J Allergy Clin Immunol. - 2010 .-- Vol. 125 (2 Suppl 2). - P. S41-52.].

The presence or predominance of one or another subspecies of IgG antibodies in different people, including those with pemphigus, can probably vary, due to which the general combination of several subspecies (subclasses) gives as a result their always necessary concentration for the onset of the disease.

The implementation of the invention

For the implementation of the invention, blood serum from at least 5 patients with a diagnosis of pemphigus containing antibodies to type 3 desmoglein (Dsg3) and 5 healthy individuals combined in pools of serum from patients and healthy individuals, respectively, are used. The presence of antibodies to Dsg3 in pemphigus patients is determined by ELISA using Anti-Desmoglein 3 ELISA kits (Euroimmun AG, Germany). To control the experiment, the blood serum of healthy individuals not containing antibodies to Dsg3 (Euroimmun AG, Germany) is used. Research is carried out according to the manufacturer's instructions.

The method of isolating IgG from human blood serum is based on the ability of bacterial protein G to specifically bind to Fc fragments of class G immunoglobulins. Protein G, immobilized on a carrier through which blood serum is passed, binds and retains serum IgG in patients with pemphigus.

Working process

A collapsible chromatographic column BioScale 10 ml (Bio-Rad, USA) is filled with a suspension of G-sepharose (Sepharose with immobilized protein G) (Bialexa, Russia), connected to a peristaltic pump (BioRad, USA) and equilibrated with washing buffer (0.1 M phosphate buffer, 0.15 M NaCl, pH 7.2-7.4) (5-10 column volumes). Next, the blood serum of pemphigus patients is added to the column at the rate of 2 ml of serum per 1 ml of G-sepharose. The column is then washed with a washing buffer with periodic measurement of the optical density of the buffer leaving the column in a BioPhotometer spectrophotometer with a wavelength of 280 nm (Eppendorf, Germany) until the optical density of the liquid is equal to the optical density of a clean washing buffer. When washing, the buffer exiting the column is collected in a separate container, forming the so-called "slip" - a mixture of proteins contained in blood serum.

The column is then washed with elution buffer (0.1 M glycine, pH 2.5). The eluate is collected in test tubes with 1 M Tris buffer (pH 8.0) in a ratio of 1 part Tris buffer to 4 parts of the eluate (the procedure is carried out to neutralize the pH of the eluate). The elution duration is controlled by measuring the optical density of the eluate solution flowing out of the column on a spectrophotometer with a wavelength of 280 nm. Elution is stopped when the optical density of the eluate solution is reduced to the optical density of the elution buffer.

The eluate and the slip are checked by the method of electrophoresis in polyacrylamide gel (PAG) according to Laemmli for the presence and degree of purification of antibodies. If IgG is detected in the slip, then the slip is re-applied to the column and cleaning is repeated. IgG-containing eluate fractions are combined and centrifuged in an Allegra X-12R refrigerated centrifuge (Beckman-Coulter, USA) at 3000 g in Amicon centrifuge filters (Millipore, France) with a filtration limit of 100 kDa for IgG concentration. During centrifugation, sterile isotonic buffer is added to the filters, resulting in a concentrated solution of IgG in the buffer. Next, from the filters (Amikon), the obtained IgG preparation is quantitatively transferred into 1.5 ml Eppendorf tubes.

Subsequently, 1 μl of the IgG preparation was dissolved in 1 ml of 0.9% NaCl. Protein concentration is measured in laboratory conditions by optical density using the UV method at a wavelength of 280 nm. The resulting absorbance value is multiplied by a factor of 1.4 to obtain an IgG concentration in mg / ml. After determining the concentration of IgG in the preparation, to prepare the necessary doses for administration to animals, the preparation is diluted with 0.9% NaCl. The resulting IgG preparation is stored at −4 ° C.

IgG preparations are injected with an insulin syringe with a 27G needle intraperitoneally to newborn BALB / c inbred mice weighing 2.5-3.5 g, at the age of less than 24 hours from birth, once at a dose of 30 mg / mouse, followed by euthanasia after 48 hours.

The practical applicability of the proposed method for modeling pemphigus in mice by the introduction of class G immunoglobulins obtained from 5 patients with pemphigus is confirmed to obtain 100% reproducibility, but is not limited to the following examples.

Example 1

The proposed method for modeling pemphigus was studied in a series of experiments on neonatal mice - males of the BALB / c inbred line with a mass of 2.5-3.5 g, less than 24 hours of age, provided by the Research Institute “Nursery of Laboratory Animals” Branch of the IBCh RAS Pushchino. The animal manufacturer provided the latest animal health control data confirming their SPF status (pathogen-free animals). Animals without signs of health deviations were selected for the experiment, so that the individual mass values did not deviate from the average value within the same sex by more than 20%. The basic rules for keeping and caring were in accordance with the guidelines in the Guide for Care and Use of Laboratory Animals. (ILAR publication, 1996, National Academy Press). Animals were kept under standard conditions and received food and water ad libitum.

All manipulations with animals were performed in accordance with the protocol approved by the bioethical commission of the FIBH RAS. The study is carried out in accordance with the National Standard of the Russian Federation "Principles of Good Laboratory Practice" (GOST 53434-2009); OECDTG407 Repeated Dose 28-Day Oral Toxicity Studyin Rodents. All procedures in the study are performed in accordance with an approved written Research Plan and Standard Operating Procedures. All animal procedures in the study were reviewed and approved by the Animal Care and Use Commission (IACUC) for regulatory compliance. A copy of the approved IACUC protocol was placed in the study data folder.

Isolation of IgG preparations from the pool of blood serum of 5 patients with pemphigus (performed according to the method described above) containing antibodies to Dsg3 (the presence of antibodies to Dsg3 was determined by ELISA using Anti-Desmoglein 3 ELISA, Euroimmun AG, Germany) and 5 healthy volunteers were performed using standard method - affinity chromatography on a column of protein G — sepharose (Sepharose with immobilized protein G) (Bialexa, Russia).

IgG preparations obtained from pemphigus patients and healthy individuals were administered to animals from one litter, which were divided into two groups: experimental and control. Mice from the experimental groups (n = 31) were injected with antibody preparations obtained from a pool of serum from pemphigus patients containing antibodies to Dsg3. The animals of the control groups (n = 15) were injected with IgG preparations obtained from the serum of healthy volunteers that did not contain IgG to Dsg3.

IgG preparations were injected with an insulin syringe with a 27G needle intraperitoneally according to the following schemes:

- once in doses of 10, 15, 20, 30 mg / mouse;

There were no deviations in the administration procedure.

The animals were euthanized by inhalation of CO ₂ after a certain period of time (exposure time) - 24-48 hours after the administration of IgG preparations, after which the skin was examined and autopsy skin material was obtained for pathomorphological examination (cystic element) and indirect immunofluorescence reaction ( nRIF) (apparently, a healthy area of the skin) (table. 1).

On visual examination, the severity of skin lesions in the form of blisters and / or erosion was assessed according to the following scale: 1+ — three or less morphological elements; 2+ - from 4 to 10; 3+ - more than 10 or the presence on the surface of areas of skin necrosis, comprising more than 20% of the epidermis of a laboratory animal.

Clinical symptoms of pemphigus were revealed in animals that were given 30 mg of IgG preparations once with an exposure time of 48 hours (n = 5). In 4 mice, erosion in the abdominal region was observed, on a 3-point scale assessed as 1+ (ie, three or less skin lesions), in 1 mouse, 2+ (Fig. 1. Erosion on the skin of newborn mice).

In a pathomorphological study of the autopsy material of the skin of mice, the pathognomonic sign of pemphigus - acantholysis - was detected in all histological preparations of animals from experimental groups that were injected with antibodies in an amount of 30 mg / mouse once and with an exposure time of 48 hours (Fig. 2. Histological picture. Epidermis of normal thickness , the layers are differentiated. A small loose keratosis. In the marginal zone of the biopsy specimen there is an intraepidermal bladder (at the level of the upper rows of the prickly layer of the epidermis), in the cavity of which acantholytic cells, lymphocytes and leukocytes live, and fragments of an isolated bladder cover are also present in the preparation. Follicle fragments are present in the dermis. A small histiolymphocytic infiltration is around the vessels, um, x200).

In the study of cryosections of the skin of mice from experimental groups by the nRIF method, IgG fixation was detected in the intercellular spaces of the epidermis in all mice injected with IgG from pemphigus patients at a dose of 30 mg / mouse (Fig. 3. nRIF. IgG preparation from a pemphigus patient. IgG fixation in intercellular spaces of the epidermis, x20).

In the control groups of animals that were injected with IgG preparations obtained from the blood serum of healthy individuals (n = 15), once at doses of 10, 20, 30 mg / mouse, clinical, pathomorphological signs of pemphigus, as well as IgG fixation in the epidermis were not observed.

The presence of acantholytic changes in the epidermis of mice of the experimental group during a pathomorphological study, as well as the fixation of human IgG in the epidermis, detected by the NRIF method only in animals that were injected with IgG preparations containing antibodies to Dsg3, indicate that the antibodies contained in the blood serum of pemphigus patients induce processes similar to pemphigus symptoms in the skin of animals.

Clinical, histological and histochemical signs of pemphigus were observed with the administration of IgG at a dose of 30 mg / mouse, administered once, and the exposure time of 48 hours.

Claims (1)

A method for simulating pemphigus vulgaris, including administering a class G immunoglobulin (IgG) preparation to newborn mice isolated from pemphigus vulgaris patients, characterized in that IgG is isolated from at least 5 patients and the resulting preparation is administered in a dose of 30 mg / mouse IgG followed by animal euthanasia after 48 hours.

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