RU2526812C1 - Diagnostic technique for autoimmune disorders of gastrointestinal vegetative structures - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: autoimmune disorder of gastrointestinal vegetative structures are diagnosed by examining patient's blood serum. The blood serum sample is examined for neuronal acetylcholine receptor ?3-subunit (ACR-?3) and gonadolibarin releasing hormone (GnRH) antibodies by enzyme-linked immunosorbent assay (ELISA) as shown by a reaction to an extracellular portion of neuronal ACR ?3-subunit and releasing hormon GnRH, while the ACR-?3 and GnRH levels are determined by straining using tetramethyl benzidine as a chromogene. An optical light absorption is determined by an ELISA analyser at wave length 450 nm in optical density (OD) units. If observing an increase of the ACR-?3 antibodies of more than 0.24 OD, and a decrease of the GnRH antibodies of less than 0.25 OD up to the full absence in relation to a reference, the autoimmune disorder of the peripheral vegetative nervous system at the level of gastrointestinal parasympathetic intramural ganglia and microganglia is diagnosed; if the GnRH antibody level is more than 0.25 OD, and the ACR-?3 antibody level is less than 0.24 up to the full absence, the autoimmune disorder of metasympathetic gastrointestinal intramural ganglia and microganglia is diagnosed.EFFECT: technique provides high-accuracy determination of the autoimmune involvement of the vegetative nervous structures controlling the gastrointestinal motility, and detecting the involved molecular target, particularly the parasympathetic and/or metasympathetic structures of the gastrointestinal vegetative nervous system.

Description

The invention relates to medicine, namely to neurology and gastroenterology, and can be used to diagnose autoimmune damage to various structures of the autonomic nervous system (ANS), regulating the motility of the gastrointestinal tract (GIT).

The nervous system is involved in the pathological process quite often, and its damage is both primary and secondary. Currently, autoimmune lesions are an urgent problem, since 5-7% of the world's population are prevalent and affect, mainly people of working age, which gives them socio-economic significance. The basis of their development is an unusual reaction of their own immune system, when antibodies produced in the norm, designed to protect the body, suddenly begin to interact with their own antigens, thereby destroying cells and tissues that carry these antigens. Proteins, nucleic acids, phospholipids and other cellular components can act as antigens. The resulting autoimmune process, as a rule, is chronic in nature and leads to long-term damage to tissues, since the autoimmune reaction is constantly supported by tissue antigens. The development of antibodies to a specific antigen determines the wide clinical polymorphism of this pathology, which usually involves almost all organs and systems of the human body.

Nervous regulation of gastrointestinal motility is multilevel in nature. Psychoemotional reactions, a general psychoemotional background, and organic lesions of the central nervous system exert an efferent effect on the state of the autonomic nervous system (sympathetic and parasympathetic), which regulates the functioning of smooth myocytes and glands through extraintestinal and intraintestinal ganglia, the intestinal nervous system.

Neurological diseases characterized by impaired motility of the gastrointestinal tract are very common. The greatest difficulties are presented by the diagnosis of the so-called "functional" gastrointestinal disorders, in which any modern examination methods (ultrasound, rectoscopy, gastroscopy, colonoscopy, irrigoscopy, biochemical and clinical tests of blood, urine, feces, cultures for dysbiosis, etc.) most often do not reveal deviations from the norm. However, for example, irritable bowel syndrome (IBS) is detected in 15-20% of the population and its incidence is 1% annually (Mayev I.V., Cheremushkin S.V. Irritable bowel syndrome: a guide for doctors. M .: Forte print, 2012, 52 p.). It is especially important to establish the cause of dyspeptic disorders in this group of patients due to the insufficient effectiveness of standard symptomatic therapy. Treatment, as a rule, is aimed at eliminating diarrhea or constipation, increased gas formation, and reducing pain. For this purpose, diet correction, various types of sorbents, enveloping agents, drugs to restore the normal intestinal flora, as well as antispasmodics (anticholinergics) and antidepressants are used. Treatment by gastroenterologists with the participation of psychotherapists and psychiatrists is not always effective and a number of patients with gastrointestinal motility disorders are examined according to an extended scheme repeatedly - up to 20-30 times. Moreover, some patients (often at their urgent request) undergo unreasonable surgical interventions.

At present, gastrointestinal motility disorders associated with impaired intestinal innervation are also poorly studied. Regulation of the motor and secretory function of the intestine is carried out by the sympathetic, parasympathetic and metasympathetic divisions of the ANS. The main mediators are adrenaline, norepinephrine, acetylcholine, but some peptides can also perform mediator functions: a vasoactive intestinal peptide, a pituitary adenylate cyclase-activating peptide, gonadoliberin releasing factor, and others. Hirschsprung’s disease is associated with congenital desimpat lack of sympathetic revertebral ganglia. Parasympathetic and metasympathetic structures of the ANS are represented by intramural ganglia and microganglia. Disruptions in the work of parasympathetic and metasympathetic structures can be caused by an autoimmune process both against the receptor apparatus and against peptides that perform mediator functions. In this regard, the development of methods for determining antibodies to the structures of the parasympathetic intramural ganglion (α3-AChR) and the peptide involved in the regulation of gastrointestinal motility through parasympathetic and metasympathetic structures of the ANS is relevant.

A known method of examining a patient with irritable bowel syndrome after treatment of her infertility with buserelin. Biopsy material was examined by histological analysis and immunohistochemistry. Plasma samples were examined using standard serological assays. The result of the study was the detection of antibodies against gonadotropin releasing hormone (GnRH), as well as the detection of a noticeable decrease in the number of intestinal GnRH-producing neurons. This suggests that this patient has developed irritable bowel syndrome due to buserelin-induced formation of anti-GnRH antibodies that destroy GnRH neurons originating from the muscular-intestinal plexus. (Ohlsson B, Veress B, Janciauskiene S, Montgomery A, Haglund M, Wallmark A: Chronic intestinal pseudo-obstruction due to buserelininduced formation of anti-GnRH antibodies., Gastroenterology, 2007, 132: 45-51). This study confirms the importance of identifying the causes of dysregulation of the motor and secretory function of the intestine, including the main lesion of the ANS divisions.

To date, a known method for diagnosing autoimmune lesions of the intramural ganglia of the gastrointestinal tract by determining in plasma and blood serum of patients suffering from impaired intestinal motility, irritable bowel syndrome (IBS) or paresis, C-reactive protein, soluble CD40 and antibodies to GnRH. Moreover, the analysis of antibodies to GnRH was performed by ELISA with optical light absorption of 405 nm. The measurement was carried out in a Titertek Multiscan photometer with a serum dilution of 1:40. The blood serum of 20 healthy donors served as a control. Moreover, antibodies against gonadotropin-releasing hormone and gastrointestinal motility disorders were found regardless of the treatment of the patient with GnRH analogues. The antibody titer also correlated with the level of CD40 (RS = 1,000, p <0,01), but not with the C-reactive protein. (Bodil Ohlsson, Eva Ekblad, Béla Veress, Agneta Montgomery and Sabina Janciauskiene RAesneatrcihb aorticdleies against gonadotropin-releasing hormone (GnRH) and destruction of enteric neurons in 3 patients suffering from gastrointestinal dysfunction, BMC, 2010, Gastroenter. This method is considered by us as the closest analogue. However, this method does not have high accuracy because of the small dilution, since it gives a large number of false positive results and reveals involvement in the autoimmune process and the defeat of only the intramural ganglia of the gastrointestinal tract.

The technical result is that our proposed method provides high accuracy in determining the involvement in the autoimmune process of the structures of the autonomic nervous system that regulate the motility of the gastrointestinal tract and the detection of an affected molecular target, in particular parasympathetic and / or metasympathetic structures of the ANS of the gastrointestinal tract.

The specified technical result is achieved by the fact that in order to diagnose autoimmune damage to the vegetative structures of the gastrointestinal tract, a patient’s blood serum is examined, and the antibodies to the α3 subunit of the neuronal acetylcholine receptor (α3-AChR) and gonadoliberin (GnRH) are determined by the solid-phase method. enzyme-linked immunosorbent assay (TIFA) for interaction with the extracellular region of the α3 subunit of neuronal AChR and the releasing hormone GnRH, and the level of antibodies to α3-AChR and GnRH determine by tearing using tetramethylbenzidine as a chromogen, the optical absorption of light is measured on an ELISA analyzer at a wavelength of 450 nm in units of optical density (OD) and with an increase in the level of antibodies to α3-AChR more than 0.24 OD, and to GnRH less than 0.25 OD and until they are completely absent with respect to control, they diagnose autoimmune damage to the peripheral autonomic nervous system at the level of parasympathetic intramural ganglia and microganglia of the gastrointestinal tract, and when the level of antibodies to GnRH is more than 0.25 OD and to α3-ACP less than 0.24 OD and to n Of their complete absence, an autoimmune lesion of the intramural ganglia and microganglia of the metasympathetic section of the gastrointestinal tract is diagnosed.

The method is as follows.

To verify the involvement of the parasympathetic and / or metasympathetic structures of the ANS in the autoimmune process, the patient is sampled from the cubital vein, then serum is separated from the blood clot by centrifugation for 15 minutes at 3000 rpm. Serum is tested for the presence of antibodies to the α3 subunit of the neuronal acetylcholine receptor (α3-AChR) and to releasing the hormone gonadoliberin (GnRH) in ELISA. Quantitative determination of the level of antibodies is carried out by the method of enzyme-linked immunosorbent assay (TIFA) using tetramethylbenzidine as a chromogen. As antigens, genetic engineering constructs of the α3-AChR extracellular domain and GnRH hormone releasing are used. The optical absorption of light at the final stage of ELISA is measured on an ELISA analyzer at a wavelength of 450 nm in units of optical density (OD). Elevated is the level of antibodies to α3-AChR more than 0.24 and to GnRH - more than 0.25 OD 450 nm.

To quantify the level of autoantibodies to α3-AChR and GnRH, the following study was performed using ELISA. On costar 96-well plates, the extracellular domain of the α3 subunit of neuronal AXP and the releasing hormone GnRH are sorbed at a concentration of 2 μg / well and incubated for 60 minutes at 37 ° C. The first row of wells is left free for conjugate control. To block non-specific binding, a 2% solution of BSA (Sigma) in PBS / Tween-20 at 200 μl / well was added to the wells and incubated for 60 minutes at 37 ° C. After incubation, serums of control donors are added to the second row, serums of patients with impaired regulation of motor and secretory function of the intestine are diluted 1: 100 in all subsequent rows and incubated for 60 min at 37 ° C followed by 3-fold washing (0.2% BSA in PB8 / Tween-20 at 200 μl / well) for 3 minutes. The presence of antibodies is determined by staining using a peroxidase conjugate with human anti-IgG (Sigma), which is added at 100 μl / well at a dilution of 1: 1000 and incubated for 60 minutes at 37 ° C, followed by 3-fold washing (0.2% BSA in PBS / TBHH-20 at 200 μl / well) for 3 minutes. Tetramethylenebenzidine (Sigma) is used as a chromogen: 10 mg is dissolved in 5 ml of nitrate buffer (pH 4.5) and 15 μl of hydrogen peroxide and 100 μl / well are added. Staining develops within 20 minutes. Inhibition of the reaction is carried out with a 0.25 M sulfuric acid solution - 100 μl / well. The measurement of optical light absorption at a wavelength of 450 nm is carried out on an ELISA analyzer. Results are expressed in units of optical density (OD).

The proposed method was examined 10 healthy donors and 14 patients with various disorders of the structures of the autonomic nervous system that regulate the motility of the gastrointestinal tract. In particular, patients suffering from impaired intestinal motility, irritable bowel syndrome (IBS) or paresis against a background of neurological pathology. In all cases, the level of antibodies corresponded to the condition of patients and healthy donors (control). In particular, in 5 patients with an increased level of antibodies to α3-AChR more than 0.24 OD, and to GnRH less than 0.25 OD and in some cases their complete absence to GnRH relative to control, an autoimmune lesion of the peripheral autonomic nervous system at the level of parasympathetic intramural ganglia and microganglia of the gastrointestinal tract. And in 9 patients with antibodies to GnRH more than 0.25 OD and to α3-AChR less than 0.24 OD or their complete absence, 4 out of 9 to α3-AChR showed autoimmune damage to the intramural ganglia and microganglia of the metasympathetic gastrointestinal tract. The average antibody content in the control group was: to α3 AChR - 0.2 ± 0.04 OD, to GnRH - 0.18 ± 0.07 OD.

Based on the results obtained, immunoanalytic test systems have been developed that allow the quantitative determination of antibodies to these antigens in various forms of gastrointestinal motility disorders with the identification of an affected molecular target, in particular, intramural ganglia and parasympathetic and / or metasympathetic structures of the ANS of the gastrointestinal tract. In addition, these results also made it possible to recommend adequate differential therapy.

Thus, the proposed method has a high specificity for detecting the presence of antibodies to α3-AChR and to GnRH in TIFA and allows to accurately detect the absence or presence of antibodies and their quantitative level at which autoimmune lesions of the peripheral parasympathetic and metasympathetic part of the ANS of the gastrointestinal tract are diagnosed . This provides adequate timely treatment aimed at suppressing autoimmune damage to the parasympathetic and / or metasympathetic structures of the ANS of the gastrointestinal tract and improving the quality of life of patients with peripheral autonomic insufficiency (PVN).

Thus, the proposed method differs from the prior art in the following:

1) allows you to evaluate the structures of the ANS of the gastrointestinal tract by examining the blood of patients with the developed ELISA method in blood serum by interacting with the extracellular region of the α3 subunit of neuronal AChR and the releasing hormone GnRH;

2) allows you to assess the level of damage to the structures of the ANS of the gastrointestinal tract;

3) allows for differential diagnosis, to objectify the dynamics of the pathological process, and also facilitates the selection of adequate therapy in patients with signs of PVN and various forms of gastrointestinal motility;

4) is more economical and no less sensitive in comparison with other methods;

5) allows you to quantify the level of antibodies to α3-AChR and GnRH, which can characterize to some extent the severity of autoimmune damage at the level of parasympathetic and / or metasympathetic structures of the ANS of the gastrointestinal tract.

Method implementation examples

Example 1

Patient P., 54 years old, was admitted with complaints of constipation, replaced by frequent loose stools (up to 12-15 times per night), weakness in standing and sitting, inability to move around without pulling clothes, fatigue, dry skin and mucous membranes with proximal hyperhidrosis. Objectively, the patient has signs of peripheral autonomic failure progressing over 8 years: Horner's syndrome on the right, impaired accommodation with a distorted reaction of the pupils in the sample with adrenaline, an inverse reaction of blood pressure in the clinostatic and orthostatic sample (increase in the supine position and a significant decrease in the position standing), lack of dynamics of heart rate when changing posture. She received symptomatic therapy (enhanced water regimen, mineralocorticoids, skin-tightening clothes) with an insignificant temporary effect against the background of disease progression. To identify the causes of gastrointestinal motility, a study was conducted of the patient's blood serum by the proposed quantitative ELISA method. A significant increase in the level of autoantibodies to α3-AChR was detected: 1.78 OD 450 nm. Antibodies to GnRH were 0.01 OD 450 nm, which corresponds to an autoimmune lesion of the peripheral autonomic nervous system at the level of parasympathetic intramural ganglia and microganglia of the gastrointestinal tract. Based on these data, immunosuppressants (prednisone) were administered for 10 days. As a result of this treatment, the patient restored the motility of the gastrointestinal tract to normal.

Example 2

Patient X., 29 years old, was admitted with a diagnosis of vegetative-vascular dystonia, irritable bowel syndrome, multiple surgical interventions on the pelvic organs and appendectomy (performed at the urgent request of the patient). The patient revealed symptoms of autonomic dysfunction in the form of attacks of tachycardia, sweating, abdominal pain and gastrointestinal motility disorders. To identify the causes of gastrointestinal motility and abdominal pain, a study of blood serum was conducted by the proposed quantitative ELISA method. An increase in the level of antibodies to GnRH was revealed: 0.78 OD 450 nm. Antibodies to α3-AChR were absent. Based on the analysis, a diagnosis was made of an autoimmune lesion of the intramural ganglia and microganglia of the metasympathetic division of the autonomic nervous system of the gastrointestinal tract. Based on these data, the patient was prescribed neuromidin - a drug that facilitates the release of acetylcholine and thus improves cholinergic transmission. After 7 days, the patient's condition improved markedly, the above symptoms disappeared.

Example 3

Patient I., 47 years old, observed in the Neurology Scientific Center of the Russian Academy of Medical Sciences with a diagnosis of myasthenia gravis, generalized form. The patient complains of periodic diarrhea, followed by short-term constipation, not associated with errors in the diet. Objectively, there are symptoms of autonomic dysfunction in the form of attacks of tachycardia, sweating with dry mucous membranes, visual impairment (in addition to diplopia associated with myasthenic external ophthalmoparesis, periodically - "defocusing" of the contours of objects at an average distance), in addition to weakness and fatigue of voluntary muscles, general weakness, fatigue. The treatment with AChE acetylcholinesterase inhibitors did not restore the gastrointestinal motility. To identify the causes of gastrointestinal motility and abdominal pain, a study of blood serum was conducted by the proposed quantitative ELISA method. The absence of antibodies to both α3-AChR and GnRH was detected. This allowed these disorders to be considered as not related to autoimmune damage, neither at the level of the parasympathetic, nor at the level of the metasympathetic departments of the autonomic nervous system of the gastrointestinal tract. Further studies showed that gastrointestinal motility disorders and abdominal pain in the patient arose as a result of dishormonal disorders (operated on for thyroid cancer 4 years ago, there are irregularities in the menstrual cycle with alternating uterine bleeding and long periods of amenorrhea). The drug therapy adjusted for these data allowed already on the 8th day to improve the patient's condition in relation to abdominal pain, diarrhea and constipation.

Thus, the determination of antibodies to α3-AChR and to GnRH by ELISA in blood serum by interaction with the extracellular region of the α3-subunit of neuronal AChR and to GnRH reflects the ganglion level of lesion of the parasympathetic part of the ANS and autoimmune damage to gastrointestinal motility. The inventive TIFA method allows to improve the diagnostic process in patients with signs of peripheral autonomic insufficiency and gastrointestinal motility in a short time, to trace the dynamics of the pathological process and choose the right treatment tactics. With high specificity, the method is simpler and more economical and makes it possible to diagnose autoimmune damage to the vegetative structures of the gastrointestinal tract.

The method also allows differential diagnosis of the so-called "functional" dyspeptic phenomena associated with disorders of the autonomic digestive tract innervation of the autoimmune genesis.

Claims (1)

A method for the diagnosis of autoimmune damage to the vegetative structures of the gastrointestinal tract, including a study of the patient’s blood serum, characterized in that antibodies to the α3 subunit of the neuronal acetylcholine receptor (α3-AChR) and the releasing hormone gonadoliberin (GnRH) are determined by the solid-phase method Enzyme-linked immunosorbent assay (TIFA) by interaction with the extracellular region of the α3 subunit of neuronal AChR and the releasing hormone GnRH, and the level of antibodies to α3-AChR and GnRH determines staining m, using tetramethylbenzidine as a chromogen, the optical absorption of light is measured on an ELISA analyzer at a wavelength of 450 nm in units of optical density (OD) and with an increase in the level of antibodies to α3-ACP more than 0.24 OD, and to GnRH less than 0.25 OD and until they are completely absent with respect to control, they diagnose autoimmune damage to the peripheral autonomic nervous system at the level of parasympathetic intramural ganglia and microganglia of the gastrointestinal tract, and when the level of antibodies to GnRH is more than 0.25 OD and to α3-ACh less than 0.24 and complete them from in the absence, an autoimmune lesion of the intramural ganglia and microganglia of the metasympathetic section of the gastrointestinal tract is diagnosed.