RU2471195C1 - Method for determining indications for immune suppressive therapy following penetrating keratoplasty - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a method for determining indications for immune suppressive therapy following penetrating keratoplasty. Keratoplasty is preceded by determining a level of interleukin-2 (IL-2) in supernatants of recipient's lymphocyte cell cultures both stimulated and not stimulated by PHA; and the absence of a reliable difference in the IL-2 levels, the immune suppressive therapy is not considered to be indicated.

EFFECT: invention enables predicting a patient's preoperative immune response on a transplant and determining an adequacy of the immune suppressive therapy.

2 ex

Description

The present invention relates to ophthalmology and is intended to determine the indications for immunosuppressive therapy after end-to-end keratoplasty.

Corneal transplantation (cadaver) is one of the oldest operations in the history of transplantology. In the 21st century, thanks to the improvement of microsurgical techniques and techniques, the development of new drugs, keratoplasty has become an ordinary operation. Despite this, the long-term results of corneal transplantation remain unfavorable - keratograft rejection is noted in 5-70% (Gundorova R.A., Bordyugova G.G., Travkin A.G. Reconstructive operations on the eyeball. // M., Medicine, 1983. - P.224).

It is known that mandatory immunosuppression with the help of drugs such as cyclosporin A-CsA, mycophenolate-mofetil, tacrolimus FK-506 in combination with corticosteroids plays a decisive role in achieving success in transplanting allogeneic organs and tissues (including the cornea). However, even such an optimal approach does not guarantee a successful outcome of keratoplasty (Scheer S., Touzeau O., Borderie V., Laroche L. Immunosuppression in comeal transplantation // J.Fr.Ophtalmol. - 2003. - Vol. 26. - No. 6 . - P.637-47). This is due to the lack of clear indications for immunosuppressive therapy.

A known method for the prevention of transplant rejection after end-to-end keratoplasty in children at risk (patent 2297227, RU, 09.06.2005), including oral administration of cyclosporine and metipred. The combination of a certain administration regimen and doses of the drugs reduces the frequency of transplant rejection and clouding after SC in children in the high-risk group, which previously refused SC, and also reduces the toxicity of cyclosporine and metredred. However, this method does not reflect selective indications for the appointment of immunosuppression. Our observations have shown that some patients develop transplant disease also during immunosuppressive therapy.

A known method for predicting rejection of the corneal graft after keratoplasty for burn burns (patent 2210774, RU 22.10.2001). The method provides a high degree of reliability of predicting corneal transplant rejection in patients who have undergone keratoplasty for burn sores, and consists in determining the content of interleukin-1-beta (IL-1β) in blood serum and / or tear fluid. If it is detected in the pre- or early postoperative period, graft rejection is predicted. Moreover, IL-1β is determined using enzyme-linked immunosorbent assay. The method allows to predict the outcome of keratoplasty, but does not disclose possible approaches to treatment, namely, immunosuppressive therapy. A known method of the same purpose, including the determination of pro- and anti-inflammatory cytokines (IL1β, TNFα and IL4, IL10), as well as the levels of IL2 and / or IFNγ in the blood serum in the early postoperative period and hereinafter in dynamics, to detect immunologically “hyper- , normo-and hyporeactive "patients. Individual immunological reactivity determines the dosage and timing of immunosuppressive therapy. However, this method allows you to identify a possible response only after keratoplasty, since the release of cytokines occurs in response to surgery. It is practically impossible to identify “hyporeactivity” and, accordingly, a contraindication to the appointment of immunosuppression before surgery using in vitro tests. The effectiveness of this method of examining patients before keratoplasty is not high, since it is limited to the possibility of identifying only initially “hyperreactive” patients who are clearly shown to be immunosuppressed, while normal and hyporeactive patients by the presented method are not detected (Balayan TG “Differentiated tactics of immunosuppressive treatment in keratoplasty high risk "diss. candidate of medical sciences, M., 2008). Currently, immunosuppressive therapy is carried out for all patients with UPC, but graft engraftment is not observed in 100% of cases.

The task of the invention is to develop an adequate method for determining the indications for immunosuppressive therapy with end-to-end keratoplasty.

The technical result of the invention is the development of a differentiated approach in relation to immunosuppression for the management of patients with end-to-end keratoplasty in the pre and / or postoperative period.

The technical result is achieved by comparing the levels of IL-2 in the supernatants of cell cultures of recipient lymphocytes stimulated by a non-specific mitogen phytogenagglutinin (PHA) compared with the level of IL-2 in the supernatants of cell cultures of recipient lymphocytes of a cell not stimulated by a non-specific mitogen of PHA.

In the absence of a difference in levels, we can talk about the recipient's reactivity, an unfavorable outcome and the choice of the optimal treatment regimen, immunosuppression is contraindicated in this case.

The proposed method allows to predict the immune response before surgery and determine the feasibility of using immunosuppressive therapy (CSA). The feasibility of identifying the type of patient reactivity before surgery is determined by the fact that in the postoperative period it is impossible to identify reliable areactivity, since the initial level of IL-2 in this particular patient is unknown. Immunosuppression is currently starting in the early postoperative period, which also makes it impossible to identify areactive patients to whom it is contraindicated.

The main point of influence of immunosuppression (cyclosporin A) is the suppression of the production of IL2 (T-lymphocyte growth factor) and autoimmune reactions, the development of which is promoted by its hypersecretion. The adverse course and outcome of the postoperative period is most often observed with "cytokine areactivity", namely a low level of IL2. To determine the type of reactivity before surgery, we proposed a method for determining the level of IL-2 in the supernatants of the cell culture of recipient lymphocytes, not stimulated and stimulated by phytohemagglutinin (before surgery). PHA is known to be a nonspecific activator of lymphocytes (major immunocompetent cells), whose in vitro exposure normally leads to stimulation of a number of immunomediators, in particular, the key regulatory protein IL-2. The theoretical basis of the proposed method is the assumption of a possible violation of the ability to stimulate in severe ophthalmopathology.

The method is as follows. Blood is taken from the ulnar vein into a tube treated with the anticoagulant EDTA or heparin. Lymphocytes are isolated from plasma, cultured in test tubes without the addition of preservatives and antibiotics. In parallel, the study is carried out in a culture stimulated by PHA and not stimulated by PHA; after 24 hours, cell supernatants (200 μl) were sampled from each tube and IL-2 production was examined by enzyme-linked immunosorbent assay (ELISA). The study is carried out using a commercial test system (Vector-Best) or an analogue. In the absence of a significant difference between the levels of IL-2 in PHA-stimulated and PHA-unstimulated cell supernatants, they are regarded as immunologically active patients who are contraindicated in immunosuppression.

Examples 1. Patient F., 47 years old, diagnosis: OD - alkaline burn of the cornea, extensive clouding of the cornea, neovascularization of the cornea. Visual acuity is the correct light projection. OS - without pathology. Somatically almost healthy. On the day of surgery, an immunological blood test to determine the level of interleukin-2 (IL-2) in the supernatants of cell cultures of recipient lymphocytes stimulated by PHA and not stimulated by PHA. No significant difference in the level of PHA of the stimulated and non-stimulated cultures was noted. The type of reactivity in patient F. is regarded as - reactive. In the postoperative period, the emphasis was on reparative and trophic therapy, without the use of immunosuppression. Complete epithelialization of the graft - on the 7th day after UPC. Sutures were removed 2 months after surgery. According to long-term results (after 12 months), the transplant is transparent, there is no neovascularization.

Example 2. Patient S., 35 years old, diagnosed with OS - post-inflammatory corneal opacity. OD - without pathology. Somatically almost healthy. A day before the operation, an immunological blood test was performed to determine the level of interleukin-2 (IL-2) in the supernatants of cell cultures of recipient lymphocytes stimulated by PHA and not stimulated by PHA. The level of PHA of the stimulated cell culture was significantly higher compared with the PHA of the non-stimulated culture. The patient is considered hyperreactive. After UPC, from the first day immunosuppressive therapy was prescribed cyclosporin 200 mg, once a day orally, and reparative therapy. Complete epithelialization of the transplant was noted on the 9th day after UPC, the sutures were removed after 3 months. The last examination 1 year 6 months after surgery - the transplant is transparent, there is no neovascularization.

Thus, the proposed method is an adequate method for predicting transplant disease and can be used to develop treatment tactics in relation to immunosuppressive therapy.

Claims (1)

A method for determining indications for immunosuppressive therapy after end-to-end keratoplasty, characterized in that prior to keratoplasty, the level of interleukin-2 (IL-2) in the supernatants of cell cultures of recipient lymphocytes stimulated by PHA and not stimulated by PHA is determined and in the absence of a significant difference in the level of IL- 2 between them consider immunosuppressive therapy not indicated.