RU2501010C1 - Method for prediction of non-surgical treatment failure in ulcerative colitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is involved is the immunopathological examination using indirect immunofluorescence. At the first stage, patient's serum is incubated on monkey's renal tissue section; at the second stage; it is incubated with goat anti-species fluorescent serum. The reaction is recorded by fluorescence microscopy by specifying the number of renal tubules with a fluorescent basal membrane and expressing the measurement as a percentage. If the derived value exceeds 15%, a considerable increase of the probability of the non-surgical treatment failure is predicted.

EFFECT: method is easy to implement; it takes 2 hours to be implemented and requires no expensive chemicals to be used.

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Description

The invention relates to medicine, in particular to gastroenterology and immunology. The aim of the invention is to predict the ineffectiveness of drug therapy in severe cases of total form of ulcerative colitis (UC) in the stage of exacerbation of the disease.

Clinical, instrumental, and laboratory methods are known for determining the prevalence of colon damage, the severity of UC, and the activity of the inflammatory process (1, 7, 12). However, the data of these studies, necessary for establishing a diagnosis and determining treatment tactics, do not allow a high probability to predict the effectiveness of the upcoming conservative therapy. Minor changes in generally accepted metabolic parameters (weight deficiency, blood protein, albumin and hemoglobin) do not guarantee an adequate response to drug therapy, which is absent in 25% -30% of patients in this group. On the other hand, with a significant change in these indicators, a good response to treatment could be observed in 30% -40% of patients (4).

Known methods for determining the severity of UC by assessing insufficiency of exocrine pancreatic function (elastase-1 content in the feces) (2) and ultrasound examination of the diameter of the small intestine and reducing the flow rate of contents in it (3). The disadvantage of these methods is that they register changes secondary to the main pathological process, which can develop under the influence of a complex of mediated factors. The results of these studies do not contain data on predicting the effectiveness of treatment.

There is a method of evaluating the effectiveness of treatment of UC by changing the microscopic picture of a dried blood drop (line orientation, the presence of cracks, etc.) with a two-time (before treatment and after 7 days) staging (8). However, the authors do not provide data on the clinical characteristics of the examined group of patients, their distribution depending on the severity of the course, the prevalence of the lesion, the activity of the process in the colon and other factors that are crucial for choosing treatment tactics and evaluating its effectiveness.

The content of the proposed method is to determine the presence of antibodies to the epithelial basement membrane (EBM). EBM components are autoantigens and cause antibody production in a number of autoimmune inflammatory diseases of the kidneys, lungs, skin, and other organs (10, 14). In UC in the operated colon, immunopathological changes in EBM are observed, including deposition of IgG and components of the classical complement activation pathway with the presence of its terminal membrane-attacking complex (5, 9, 13). With a pronounced (destructive) inflammatory process, an increase in the expression of EBM antigens is detected (6), and antibodies to the basement membrane of the glomeruli of the kidneys are detected in the blood of 20% -40% of UC patients using various enzyme-linked immunosorbent test systems (11).

In accordance with the purpose of the invention, its task is to develop a simple, affordable quantitative method for recording the interaction of antibodies with a standardized material containing EBM antigens.

An immunohistological method was used, where slices of frozen monkey kidney tissue were used as a source of EBM for Euroimmun test systems (Germany), intended for the diagnosis of autoimmune glomerulonephritis, in which patient sera react mainly with renal glomerular BM (glomerulum).

Our method is characterized in that the morphological structure of the registration of the immunological reaction with EBM is the wall of the renal tubules. In contrast to the well-known antibody titration method, which requires additional reagent costs and time, the reaction is taken into account by calculating the percentage of fluorescent tubules.

The method is carried out using the aforementioned test system as follows. 25 μl of glycine buffer solution is applied to kidney sections located on the surface of standard biochips.

An incubation is carried out for 30 minutes, followed by washing the sections in saline buffer for 15 minutes. In the next step, the sections are incubated with the patient sera at a dilution of 1:10 in a volume of 25 μl for 30 minutes. Next, the serum is washed from the slices with a new portion of physiological buffer solution with washing in it for 5 minutes. 20 μl of goat immune serum to human IgG labeled with fluorescein is applied to the sections. Incubated for 30 minutes, rinse off the serum with physiological saline, wash the sections for 5 minutes, cover them with enclosing medium and coverslip. Calculation of the percentage of fluorescent tubules is performed under a luminescent microscope (JENALUMAR-a, Germany; eyepieces × 6.6, lens × 50).

Under observation were 43 patients with UC. All patients underwent the necessary clinical and instrumental (rectoscopy, colonoscopy, gastroduodenoscopy, ultrasound examination of the abdominal organs, x-ray studies, etc.) and laboratory studies. As a control group, the blood serum of 15 donors was examined. The age of patients with UC was 35.5 ± 1.7 (17-67) years; there were 23 men, 20 women. A severe form of the disease was observed in 30 patients, a moderate form in 13. A total colon lesion was diagnosed in 38 patients, a left-sided lesion - in 5. An acute course of UC was observed in 7 patients, a chronic recurrent course - in 11 and chronic continuous course - in 25 patients. UC patients were divided into 2 subgroups: 1-19 patients with a positive effect of drug therapy, and 2-24 patients in whom there was no response to treatment. A hormone-resistant form of UC was observed in 12 patients and a hormone-dependent form in 8. Patients with UC in the form of toxic dilatation of the colon developed in 4 patients; intestinal bleeding was observed in 1 patient. Surgical treatment to remove the affected colon due to the inefficiency of conservative therapy and developed complications was performed in 23 patients.

The number of positively reacting (fluorescent) renal tubules in the donor group was 5.0 ± 1.4% (0-15.5%); in patients with UC in group 1 - 27.5 ± 5.0% (0-94%) (p <0.001 compared with the control group), in patients with UC in group 2 - 4.7 ± 1.4% (0- 21.8%) (p> 0.05 compared with the control group and p <0.001 compared with group 1).

Thus, the level of antibodies to EBM, mediated by the number of responsive renal tubules, is significantly increased in patients with UC resistant to drug therapy, compared with patients with an adequate response to therapy, in which the level of antibodies is not increased compared to the control group.

An increase in the level of antibodies in comparison with the control group taken as normal (M ± 2SD, 15%) was observed in 15 of 24 (62.5%) patients with UC in group 1 and in 1 of 19 (5.2%) patients in group 2. Of the 24 patients with UC of group 1, 21 patients underwent surgical treatment during ongoing hospitalization (at the time of the examination) and 2 patients under next hospitalization after 6 months and 1 year. In patients of group 2, surgical treatment was not used. In total, among patients of these groups, an increase in antibody level was observed in 16 patients, of which 14 (87.5%) patients underwent surgical treatment due to the ineffectiveness of drug therapy; of 27 patients whose antibody level was not increased, operations were performed in 9 (33.3%) (p <0.001). Thus, an increase in the level of antibodies to renal tubule EBM in patients with total UC significantly increases the likelihood of conservative therapy failure.

Example 1. Patient K., 21, was undergoing treatment at the State Center for Coloproctology from 02.20 to 03.29.2012 with a diagnosis of ulcerative colitis, total lesion, chronic continuous course, severe hormone-dependent form. The result of immunohistological examination is 35.8%. Due to the ineffectiveness of conservative treatment and taking into account long-term (for 1 year) hormone therapy, the patient as a first stage of surgical treatment underwent colectomy with resection of the rectum according to the Hartmann type of surgery, Brook ileostomy.

Example 2. Patient B., 67 years old. I was treated at the State Center for Coloproctology from April 26 to May 28, 2012 with a diagnosis of ulcerative colitis, total lesion, chronic recurrent course, severe form. Anemia. The result of immunohistological examination is 29.5%.

During a planned computed tomography of the abdominal cavity, signs of covered perforation of the ascending colon were revealed. In this regard, a surgical operation was performed in the amount of colectomy with resection of the terminal ileum; abdominal-anal resection of the rectum, Brook ileostomy.

Example 3. Patient I., 31, was undergoing treatment at the State Center for Coloproctology from June 25 to August 3, 2007 with a diagnosis of ulcerative colitis, total lesion, chronic continuous course, severe hormone-resistant form. The result of immunohistological examination is 2%. On the background of treatment with prednisone at doses of 180 mg - 80 mg per day intravenously, methotrexate 30 mg intravenously 1 time per week, there was a persistent clinical improvement with positive endoscopic dynamics.

Example 4. Patient G., 42 years old. I was at the SSC of Coloproctology from February 26 to March 7, 2007. I was admitted for the purpose of examination and correction of treatment while taking prednisolone at a dose of 60 mg per day. Diagnosis: ulcerative colitis, total damage to the colon, clinical and endoscopic remission. The result of an immunohistological study - no antibodies were detected.

Literature

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Claims (1)

A method for predicting the ineffectiveness of conservative treatment of ulcerative colitis with total colon damage by immunohistological examination, characterized in that the patient’s blood serum using indirect immunofluorescence on sections of a monkey’s kidney determines the presence of antibodies to the epithelial basement membrane of the renal tubules, expressing the result in percentage of fluorescent and fluorescent with an indicator of more than 15%, a high probability of failure of conservative therapy is predicted.