SU1727074A1 - Method for prognosis of uveitis relapses - Google Patents

Abstract

The invention relates to medicine and can be used to predict the development of uveitis in patients with somatic diseases. The purpose of the invention is to predict uveitis in a patient without symptoms of the underlying disease. Blood is taken from the patient, the level of lymphocyte blast-transformation on a non-specific mitogen is determined in it, lymphocytes containing la- and LFA-t-antigens are detected, the blood group is determined when the level of blast-transformation is reduced compared with healthy, the number of la-containing lymphocytes below 10%, the number of LFA-t containing lymphocytes below 5%, in a patient with blood group A (I) predict the development of uveitis. The method compared with the prototype allows predicting the development of uveitis in patients before the development of symptoms of the underlying somatic disease. WITH

Description

The invention relates to medicine, namely to ophthalmology, and is intended. to predict the course of endogenous uveitis and retinovasculitis.

The purpose of the invention is to predict the recurrence of uveitis in a patient before the development of symptoms of the underlying disease.

The method is carried out as follows.

Peripheral blood (15-20 ml) is taken from the cubital vein into a tube with 1: 100 diluted heparin, layered on a density gradient of ficollvereraphin 1077 (9 volumes of blood to 3 volumes of the gradient), centrifuged for 30 missions at 1000 rpm. Mononuclear cells are assembled with an interphase Pasteur pipette. The cells are washed with medium 199 (5-7 ml) by centrifuging for 20 min at 1000 rpm for 3 times.

The lymphocyte culture thus isolated was diluted with medium 199 to obtain a concentration of not more than 1.5-2 x 106 cells per ml.

Slides are prepared as follows.

Gas burners that are pre-degreased and slightly warmed up on the flame impose a piece of parafilm M with the cut out holes with a diameter of 3 to 5 mm and firmly press it against the glass in order to fully glue it. Heating should be carried out carefully so that the parafilm does not melt, otherwise the well will be shallow and its contents may be connected with the contents of the neighboring well, but also

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not weak, otherwise the contents of the hole will flow under the parafilm. Such glasses can be stored for a long time.

On the study day, poly-1-lysine (20-50 µg / ml) is applied to the wells and placed in a thermostat at 37 ° C for 1 hour for contact. Then, in medium 199, they are washed from an excess of Poly-Ischine, and a cell culture in the amount of 20-50 µl is placed in such prepared wells, depending on the diameter of the well, followed by incubation in a thermostat for 1 h. Then monoclonal antibodies are introduced into these wells and re-incubated for 1 h in a thermostat. Washed from excess MCA 5 times for 1 min in medium 199. For staining of cells, 20-50 µl of F (AB) 2-fragment of rabbit luminescent serum against mouse globulin is applied. The contact with the dye fragment is carried out for 1 hour at 4 ° C in a refrigerator / Then it is washed five times in glasses with medium 199 (30-50 ml of medium each) for 1 minute each. After that, 50% glycerin is added to the well, the wells are covered with a cover glass. The drug is ready for microscopic examination,

An ascetic liquid from hybridoma carrier mice, the BA B / C line at a dilution of 1: 100, served as the MCAT. ICA series ICO: ICO-1 against la-antigens and .ICO-11 against LFA-G antigen,

P (ab) 2 fractions were prepared from rabbit antiserum against immunoglobulins of a white mouse labeled with FITZ, and they were prepared according to standard methods, adsorbed with hepatic powder and human lymphocytes and used in the reaction at a dilution of 1:10.

Microscopy is carried out in phase-contrast and luminescent modes with the study of several visual fields alternately; then in one mode, then in another mode, with 200 cells of a specific type of immunofluorescence being counted with an increase in the lens x90-100, an eyepiece 2.5-10 under oil immersion.

According to literature data, la-antigens are involved in antigen presentation, cellular interaction, genetic restriction and the strength of the immune response are determined. The LFA-I-lymphocyte functionally associated antigen participates in the initial recognition-adhesion phase of the cytoxic T-lymphocyte reaction and plays the role of an adhesive glue. Failure. And the absence of Ga and LFA-1 antigens cause a defect in humoral and cellular immunity and lead to immunodeficiency.

In the control group of donors - practically healthy people (75 people) express

la-antigen ranged from 11% to 35% on average 29 ± 2.3%. The expression of the antigen in the control (20 people) ranged from 6–15% on average to 12.4 ± 1.4%. These data made it possible to regard the expression of expression from 10% and below for aa-yot 5% and below for LFA-T antigens as a manifestation of a deficiency of la- and LFA-L-positive cells.

Comparison of the expression of the expression of la- and LFA-1 antigens on blood mononuclear cells in patients with uveitis, depending on the nature of the course of uveitis, showed a significantly pronounced correlation between the deficiency of la- and LFA-1 positive cells and often

0 recurrent nature of uveitis (p 0.01;, 01) ..

The reaction of blast transformation of lymphocytes with a nonspecific mitogen is carried out according to the generally accepted method and

5, either phytohemagglutinin from Difko or Wellkome firms or domestic lymphocyte mitogen at a dose of 0.05 ml per 3 ml of culture mixture are used. After a three-day incubation in a thermostat by microscopy of a smear prepared from an incubated cell culture, the number of blast cells per 500 lymphocytes is determined and expressed as a percentage. Smears were stained with an aqueous solution of azureosin with the following composition: eosin 20 ml; Azur 26 ml; distilled water to 100 ml. The paint was applied to the smear in a volume of 3-4 ml for 10-15 minutes. Then the strokes were washed under running water. Paint applied

0 per smear in a volume of 3-4 ml for 10-15 minutes. Then the strokes were washed under running water. Paint was preparing ex-temporae. In the presence of less than 55% of the blasts, the proliferative response of lymphocytes is considered to be reduced and is regarded as an inhibition of the functional activity of T-lymphocytes.

According to our data, in 42.7% of patients often recurrent uveitis was observed

0 reduced lymphocyte proliferative response to a nonspecific mitogen.

The determination of the patient's group membership in the ABO system is carried out according to the standard method for determining erythrocyte antigens with the help of 5 commercial hemagglutinating sera of 4 blood groups: (O, #, / 3), U (A, $, III (B, a) , iV (A, B, 0),

Example 1. B. B., 23 years old, outpatient card No. 7870/88.

Diagnosis: generalized uveitis OI (both eyes), inactive. The disease duration is 2.5 years. The disease of the left eye (OS) occurred for no apparent reason in February 1987, the right eye (OD) fell ill a year later, in February 1987. Until August 1988, recurrences of uveitis OD were noted 3 times, the left eye - 10 times.

The primary immunological examination detected; low content of la and LFA-G1 cl. current (10 and 5%, respectively); decreased lymphocyte response to lymphocytic mitogen (42%); blood type A (I).

The data obtained suggested an unfavorable prognosis.

Clinical course: the occurrence of recurrent uveitis of the left eye after 6 months in the form of subretinal hemorrhage and retinal edema.

PRI mme R 2. B-A. Noah, 36 years old, outpatient card No. 6153/88. Diagnosis; angiitis od. The process is primary, the duration of the disease is 2 months. Arrived at the institute to clarify the diagnosis and treatment. Immunological examinations established low levels of la- and LFA-I containing cells (10 and 3%, respectively}, blood group A (II), decreased functional activity of lymphocytes (40% of blast forms). The prognosis is unfavorable by immunological parameters.

Clinical course: recurrence of hemophthalmus OD 2 months after the end of the course of treatment.

Froze B-th S., 38 years old, outpatient card No. 12388/88. Diagnosis: generalized assured OI inactive. The disease duration of the left eye is 25 years, the right eye is 1 year. Before May 1988, exacerbations on the left eye were noted 12 times, on the right eye - 2 times. At the time of the examination, blood group A (P), a low level of IA + and LFA-1 cells (8 and 1%, respectively), a reduced proliferative response of lymphocytes in the RBT to a nonspecific mitogen (45%) were determined.

The prognosis is unfavorable by immunological data. Clinical course; recurrence of the right eye - fresh polymorphic precipitates after 8 months.

The proposed method is simple. All studies are carried out simultaneously

The final result can be obtained in a fairly short time interval (4 days).

The proposed method for predicting the severity of uveitis is based on the latest achievements of fundamental immunology using the latest research methods, in particular monoclonal antibodies, which allow determining qualitative and quantitative defects in the immune system at the molecular level.

Using the proposed method based on the evaluation of the function of immunocompetent cells, the patient's group of patients according to the ABO system, makes it possible to predict the recurrence of uveitis, determine the groups of patients with an increased risk of recurrence who need regular follow-up, and the ability to prevent the development of severe outcomes through a timely preventive pathogenically reasonable course treatment with the use of immunomodulators of directional action and, as a result, the reduction and reduction of labor p, the increase in the duration of the working period of the patient.

The proposed method allows to predict the development of recurrence of uveitis without clinical manifestation of the underlying somatic disease, and according to the prototype it is possible to predict uveitis only in i. If a patient develops symptoms of a general disease.

Claims (1)

Formula from the range A method for predicting the recurrence of uveitis, including blood sampling, incubation with mitogen and determining the level of blast transformation, differs from the fact that, in order to early predict the development of symptoms of the underlying disease, lymphocytes containing la and LFA-I also develop antigens, establish blood group affiliation and with a decrease in blast transformation compared with healthy ones with an amount of la containing lymphocytes below 10% and a number of LFA-I containing lymphocytes below 5% in a patient with a blood group A (C), predict a recurrence of uveitis.