RU2613056C1 - Prognosis method of uveitis development in children with juvenile idiopathic arthritis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to the medicine, particularly, to the ophthalmology, and may be used for prognosis of the uveitis development in patients with the juvenile idiopathic arthritis (JIA). For this purpose, IL-10, INF-γ are determined, after that, the prognostic factor is calculated via the following formulation PF=(IL-10/INF-γ)100, and in the case the prognostic factor is more than 31,8±4,7, the development of the uveitis in patients with the juvenile idiopathic arthritis is prognosticated.

EFFECT: method allows increasing the accuracy of a prognosis of the development of the uveitis at the early stages of the disease course for the purpose of preventive measures against complication development leading to the ablepsia.

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Description

The invention relates to medicine, namely to ophthalmology, and can be used as a method for predicting the development of uveitis in children with juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) - arthritis of an unknown cause, lasting more than 6 weeks, developing in children under the age of 16 years with the exception of other joint pathology. JIA is one of the most frequent and most disabling rheumatic diseases found in children. The incidence of JIA is 2-16 per 100 thousand children under the age of 16 years. One of the extraarticular manifestations of JIA is an inflammatory disease of the vascular tract of the eye. It is known that uveitis is an autoimmune disease, which is based on a violation of the cytokine regulation of immune responses. The importance of assessing the systemic production of cytokines is determined by the ability of some of them to distant effects and penetration through the blood-brain barrier. Particular attention is paid to the search for immune mediators of inflammation responsible for the key links in the pathogenesis of uveitis. In biological fluids, a number of pro-inflammatory cytokines (IL-1, IL-2, IL-6, TNF-α, IL-5, IL-10, IFN-γ) and anti-inflammatory cytokines (IL-4, IL-10, TGF -β). Currently, the results of a study of cytokine network disorders in a number of infectious and non-infectious uveitis, as well as connective tissue diseases in children and adults, have been published. However, data on the results of studies of the immunopathogenesis of uveitis against JIA are presented only in a few works, while there is practically no information on comparing the immune status in children suffering from JIA without signs of uveitis and with clinical manifestations of uveitis. At the same time, this group of patients deserves special attention due to the severity of the underlying disease, aggravating the course of uveitis.

Much attention is paid to the method for predicting the development of uveitis in children associated with juvenile idiopathic arthritis.

Patent Search

Much attention is paid to the method for predicting the development of uveitis in children associated with juvenile idiopathic arthritis. When conducting a patent search, we studied the following patents.

1. A method for predicting the course of uveitis associated with joint damage in children. Authors: Katargina Lyudmila Anatolyevna (RU), Starikova Aleksandra Viktorovna (RU). RU 2002131179. 51, IPC G01N 33/569 (2000.01), application: 2002131179/15, 20.11.2002.

2. A method for predicting the course of endogenous uveitis in children. Author (s): Katargina L.A., Chesnokova N.B., Kuznetsova T.P. RU 2199123, IPC G01N 33/52, Moscow Research Institute of Eye Diseases named after Helmholtz, application: 2001116520/14.

3. A method for predicting the course of uveitis associated with rheumatic diseases in children under conditions of therapy with inhibitors of tumor necrosis factor alpha in relation to the underlying disease. Authors: Katargina Lyudmila Anatolyevna (RU), Slepova Olga Semenovna (RU), Denisova Ekaterina Valerievna (RU), Lyubimova Natalya Valerievna (RU), Petrovskaya Maria Stanislavovna (RU) RU 2479848, IPC G01N 33/68 (2006.01), Moscow Scientific Research Institute of Eye Diseases named after Helmholtz, application: 2011152764/15, 12/23/2011.

4. A method for predicting the adverse course of uveitis. Authors: Slepova Olga Semenovna, Bykovskaya Galina Nikolaevna, Katargina Lyudmila Anatolyevna, Denisova Ekaterina Valerievna, Velichko Tatyana Vladimirovna, RU 2298188, IPC G01N 33/68 (2006.01), application 2005128953/15, 09/19/2005.

5. A method for predicting the course of traumatic uveitis. Tarasova L.N., Kiseleva T.N., Orlova N.S. RU 94035707, IPC A61B 5/00 (1995.01). Ural Institute for Advanced Medical Studies, Application 94035707/14 09/20/1994.

6. A method for predicting the course of traumatic uveitis. Aznabaev M.E., Gazdalieva L.M., Nikitin N.A., Dautova Z.A. RU 2003118152, IPC G01N 33/48 (2000.01). Ufa Research Institute of Eye Diseases. 2001118152/15 06/16/2003.

7. A method for predicting an unfavorable protracted course of rheumatoid uveitis. Shevchuk N.E., Gabdrakhmanova A.F., Malkhanov V.B., Zainutdinova G.X. RU 2325657, IPC G01N 33/68 (2006.01). State institution “Ufa Scientific Research Institute of Eye Diseases” of the Academy of Sciences of the Republic of Bashkortostan. Application 2007113135/15 03/30/2007.

8. A method for predicting the course of rheumatoid uveitis. Zainutdinova G.X., Malkhanov V.B., Shevchuk N.E., Marvanova Z.P. RU 2287164, IPC G01N 33/68 (2006.01). Ufa Research Institute of Eye Diseases. Application 2005121103/15 07/05/2005.

9. A method for predicting the course of post-traumatic uveitis with penetrating wound of the eyeball. Maracheva N.M. RU 2387417, IPC A61F 9/00 (2006.01), A61B 8/10 (2006.01). Application 2009110651/14 03/23/2009.

10. A method for predicting an adverse course of post-traumatic uveitis. Vinkova G.A., Vavilova G.I., Klimova Z.F. RU 2153283, IPC A61B 5/04 (2000.01), A61B 5/00 (2000.01). Ural State Medical Academy of Continuing Education. Application 99101993/14 02.02.1999.

11. A method for predicting the development of the chronic course of post-traumatic uveitis. Vinkova G.A., Kamilova F.X. RU 2149408, IPC G01N 33/68 (2000.01). Ural State Medical Academy of Continuing Education. Application 98121199/14 11/23/1998.

12. A method for predicting the complicated course of peripheral uveitis. Chudinova O.V., Hokkanyan V.M. RU 2149408, IPC A61B 8/06 (2006.01). Application 2009142010/14 11/13/2009.

The prototype, respectively, of the problem we solve, is considered the invention of EA Drozdova and L.N. Tarasova “A method for predicting the course of uveitis in systemic and syndromic diseases” (157541, IPC G01N 33/68 (2000.01) No. 2157541 dated 10.10.2000). In this invention, the prognosis of the course of uveitis in systemic and syndromic diseases is made by examining the lacrimal fluid (LF) in which the content of IL-4 and IL-6 in the tear is determined by calculating the prognostic coefficient according to the formula: PK = IL-6 / IL-4 * 100 . The magnitude of the prognostic coefficient predict the form of the course of RU. So, with PK = 0.59 ± 0.04, they indicate moderate severity with a favorable course. PK = 2.05 ± 0.1 makes it possible to predict a chronic, often recurring course. When PC = 5.39 ± 0.3, a complicated course of uveitis with a generalization of the inflammatory process is noted. In remission stage PC = 0.267 ± 0.04.

Our proposed method is more accurate in predicting the development of uveitis associated with JIA, since it allows the early stage of the disease, based on the determination of immunoregulatory cytokines at the systemic level (in blood serum), to identify a risk group for the development of uveitis among patients with JIA. The advantage of this method is the early follow-up of this group of patients by an ophthalmologist and the timely appointment of therapy.

The aim of the invention is to improve the accuracy of the prognosis of uveitis in juvenile idiopathic arthritis to prevent the development of complications (complicated cataracts, secondary glaucoma, recurrent cystic macular edema), leading to blindness and low vision of children.

This goal is achieved by determining the content of IL-10 and INF-γ in blood serum and calculating the coefficient by the formula:

the magnitude of which predict the risk of uveitis in children with JIA.

The cytokines secreted by the cells of the immune system are short-range mediators that determine local cell interactions in the foci of the development of processes in tissues. IL-10 is secreted by monocytes, macrophages activated by Th-2 cells. IL-10 has a powerful immunosuppressive effect, thereby inhibiting the production of INF-γ by lymphocytes and natural killers. INF-γ is released by CD4 + T cells (Th-0 and Th-1), CD8 + T cells, and activated natural killers. INF-γ is a pro-inflammatory cytokine. An imbalance in the ratio of cytokines at the systemic level can lead to depletion and dysregulation of the immune system, thereby creating favorable conditions for the onset of the disease.

The content of IL-10 in blood serum was determined in an enzyme-linked immunosorbent assay using reagents of the company "Vector-Best", Novosibirsk on the apparatus of Personal Lab. (Adaltis, Italy). 100 μl of sample dilution solution was added to all wells of the plate. In duplicates, starting from the upper wells of the first two strips, 100 μl of calibration samples were added; twenty; 40; 200; 500; and 1000 pg / ml. 100 μl of control sample was added to the next pair of wells. 100 μl of test samples were added to the remaining wells. Cut off the sticky film of the required size. The strips were sealed with a film and incubated at 37 ° C for 120 min in a thermostatically controlled shaker at a frequency of 700 rpm. At the end of the incubation, the adhesive film was removed and placed in a vessel with a disinfectant solution. Using washing devices, the wells of the plate were washed 5 times with solution 1, alternating aspiration and immediate filling of the holes of each strip. At least 350 μl of liquid was added to each well during each wash cycle. The time between filling and emptying the wells was at least 30 seconds. After washing, the moisture residues from the wells were carefully removed by tapping on the filter paper with the inverted plate. At the next stage, 100 μl of conjugate No. 1 was introduced into the wells of the tablet (a plastic bath and disposable tips included in the kit were used). The strips were covered with foil and placed in a shaker. Incubated for 60 min at 37 ° C, at 700 rpm. At the end of the incubation, the plate was washed 5 times according to the procedure described above. Next, 100 μl of conjugate No. 2 was added to the wells of the plate. The strips were covered with foil and placed in a shaker. Incubated for 30 minutes at 37 ° C, at 700 rpm. At the end of the incubation, the plate was washed again 5 times. Then, 100 μl of a solution of tetramethylbenzidine (TMB) plus was added to all wells. The tablet was kept in a dark place for 25 minutes at a temperature of 18-25 ° C. 100 μl of stop reagent were added to the wells at the same speed and in the same sequence as the tetramethylbenzidine solution. The optical density was measured using a spectrophotometer in two-wave mode: the main filter is 450 nm, the reference filter in the range of 620-655 nm. The measurement was carried out 2-3 minutes after stopping the reaction. The time between stopping the reaction and measuring the optical density did not exceed 10 minutes. A calibration graph was plotted against the optical density (ordinate axis) versus IL-10 concentration (abscissa axis) in the calibration samples. The results were expressed in pg / ml.

The content of INF-γ in blood serum was determined in an enzyme-linked immunosorbent assay using reagents of the company "Vector-Best", Novosibirsk on the apparatus of Personal Lab. (Adaltis, Italy). 100 μl of sample dilution solution was added to all wells of the plate. In duplicates, starting from the upper wells of the first two strips, 100 μl of calibration samples were added; twenty; 40; 200; 500; and 1000 pg / ml. 100 μl of control sample was added to the next pair of wells. 100 μl of test samples were added to the remaining wells. Cut off the sticky film of the required size. The strips were sealed with a film and incubated at 37 ° C for 120 min in a thermostatically controlled shaker at a frequency of 700 rpm. At the end of the incubation, the adhesive film was removed and placed in a vessel with a disinfectant solution. Using washing devices, the wells of the plate were washed 5 times with solution 1, alternating aspiration and immediate filling of the holes of each strip. At least 350 μl of liquid was added to each well during each wash cycle. The time between filling and emptying the wells was at least 30 seconds. After washing, the moisture residues from the wells were carefully removed by tapping on the filter paper with the inverted plate. At the next stage, 100 μl of conjugate No. 1 was introduced into the wells of the tablet (a plastic bath and disposable tips included in the kit were used). The strips were covered with foil and placed in a shaker. Incubated for 60 min at 37 ° C, at 700 rpm. At the end of the incubation, the plate was washed 5 times according to the procedure described above. Next, 100 μl of conjugate No. 2 was added to the wells of the plate. The strips were covered with foil and placed in a shaker. Incubated for 30 minutes at 37 ° C, at 700 rpm. At the end of the incubation, the plate was washed again 5 times. Then, 100 μl of a solution of tetramethylbenzidine (TMB) plus was added to all wells. The tablet was kept in a dark place for 25 minutes at a temperature of 18-25 ° C. 100 μl of stop reagent were added to the wells at the same speed and in the same sequence as the tetramethylbenzidine solution. The optical density was measured using a spectrophotometer in two-wave mode: the main filter is 450 nm, the reference filter in the range of 620-655 nm. The measurement was carried out 2-3 minutes after stopping the reaction. The time between stopping the reaction and measuring the optical density did not exceed 10 minutes. A calibration graph was plotted against the optical density (ordinate axis) versus INF-γ concentration (abscissa axis) in calibration samples. The results were expressed in pg / ml. The conducted studies revealed a risk group for the development of uveitis against the background of JIA. In each patient, PCs were determined that ranged from 60.3 ± 14.5 in patients with uveitis associated with JIA and 31.8 ± 4.7 in patients with JIA without uveitis (p≤0,000).

To confirm the submitted materials, we present clinical examples.

Example 1: Patient P., 10 years old, was observed at a pediatric rheumatologist with a diagnosis of JIA, oligoarticular variant, RF (-), activity 1, FC 2, from 7 years old. Received basic therapy methotrexate 15 mg / week. Remission for articular syndrome has been achieved. The child is sent for an examination to an ophthalmologist in a planned manner. On examination: anterior iridocyclitis of the right eye of moderate severity. OD - light pericorneal injection of the eyeball, a small amount of small and medium precipitates on the corneal endothelium, posterior synechia deforming the pupil along the pupil edge of the iris at 19.00; in the anterior layers of the vitreous body, a light suspension of inflammatory cells. The fundus is not changed. Visual acuity OD - 0.8, is not corrected. OS is healthy. IL-10 = 29.808 pg / ml, INF-γ = 28.058, PC = 106.24.

Enhanced anti-inflammatory treatment was carried out both systemic and local. The dose of methotrexate was increased to 20 mg / week. Within 1 month, uveitis was stopped, articular syndrome inactive, vision restored to 1.0. The child is observed during the year - there were no exacerbations of uveitis.

Example 2. Patient Z., 8 years old, is observed in a pediatric rheumatologist with a diagnosis of JIA, oligoarticular variant, RF (-), activity 1, FC 2 from 5 years. Receives basic therapy with methotrexate 15 mg / week, articular syndrome inactive. At the routine examination of the ophthalmologist, no pathology was revealed. IL-10 = 4.8 pg / ml, INF-γ = 18.667, PC = 25.7.

During the observation period of 2 years, the inflammatory process of the choroid was not diagnosed.

Claims (3)

A method for predicting the development of uveitis in children with juvenile idiopathic arthritis, including a study of the level of cytokines in the blood serum of children with juvenile idiopathic arthritis, characterized in that it is determined by IL-10 and INF-γ with the calculation of the prognostic coefficient by the formula PC = (IL-10 / INF-γ) 100 , with a prognostic coefficient of more than 31.8 ± 4.7, the development of uveitis in patients with juvenile idiopathic arthritis is predicted for the early diagnosis of diseases of the choroid.