RU2379050C2 - Method for prediction of clinical effectiveness in chronic purulent rhinosinusitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely otorhinolaryngology and can be used for prediction of clinical effectiveness in chronic purulent rhinosinusitis. The method involves prediction of clinical effectiveness in chronic purulent rhinosinusitis provided the recombinant cytokine immunotherapy based on genetic typing of polymorphism of cytokine gene. The therapy includes recombinant interleukine-1β, and the presence of low-producing allele cytokine gene IL-1β, increased phagocytic activity of neutrophils ensured by higher level IL-8 is predicted with high clinical effectiveness secured.

EFFECT: application of the invention allows predicting efficiency of the forthcoming therapy with recombinant interleukine-1β ensured by determination individual susceptibility of each patient considering genotype-based functional activity of cells.

2 cl, 4 dwg, 4 ex

Description

The invention relates to medicine, namely to methods for treating infectious and inflammatory diseases with recombinant cytokines, and can be used in immunotherapy of upper respiratory tract diseases.

In recent years, there has been a significant increase in diseases of the nose and paranasal sinuses. Despite the existence of many conservative and surgical methods for the treatment of chronic purulent rhinosinusitis (hereinafter HFS), it is rarely possible to achieve complete cure, but relapses of the disease have to be observed quite often.

Immunotherapy as a concept combines various ways of influencing the immune system in order to stop the pathological process in the body.

For specific treatment, antigens or antibodies are used that are specific for the pathogen or antigen. Nonspecific methods include exposure to the immune system of chemicals, physical factors and antigens that are nonspecific with respect to the arising pathological process. Non-specific immunotherapy is characterized by the fact that ready-made non-specific immunity factors and cells are administered to a patient who has their insufficiency.

Currently, there is a transition from mass therapy to individual therapy, which allows to cure a specific patient. However, the absence of significant prognostic factors of effectiveness makes such therapy often unpredictable, because depending on the conditions and doses of the acting agent, both stimulation and inhibition of a number of indicators of the immune system can be caused.

It is known to use for solving such problems recombinant cytokines, in particular cytokines that regulate inflammation, such as interleukin-1 (hereinafter IL-1), tumor necrosis factor (TNFα), "proliferative" - IL-2, or "allergy stimulants" - IL -4, IL-5 and others. They can compensate for the missing regulatory factors and thereby enhance the immune response.

At the body level, cytokines provide a link between the immune, nervous, endocrine, hematopoietic and other systems and serve to engage them in organizing and regulating a single defense reaction. Thus, cytokines serve as the organizing system that forms and regulates the whole complex of pathophysiological changes during the introduction of pathogens.

The pro-inflammatory cytokines currently include IL-1α, IL-1β, IL-2, IL-6, IL-8, TNFα, interferon gamma (IFNg), anti-inflammatory cytokines - the receptor antagonist IL-1 (IL-1RA), IL-4, IL-10. Currently, a large number of recombinant analogues of cytokines, including those involved in the regulation of inflammation, have been obtained, in addition, their number is constantly growing.

However, clinical experience with recombinant cytokine analogues has shown that they act selectively. Such therapy significantly helps many patients in a number of cases, allowing to cure after the first use, does not help some at all, and in a number of cases pronounced adverse reactions are observed.

Therefore, the prognosis of the effectiveness of cytokine therapy is relevant, and the determination of the patient’s personal sensitivity to cytokine therapy will make it possible to use it more reasonably and effectively.

A known method of treating chronic purulent rhinosinusitis by immunotherapy with Betaleikin, including its systemic and local application [1].

Recombinant IL-1b under the commercial name Betaleikin (hereinafter referred to as BL) is approved by the Ministry of Health of the Russian Federation for clinical use (No. 97/51/6). The biological properties of this cytokine, namely, stimulation of bone marrow hematopoiesis, restructuring of immunopoiesis, activation of connective tissue metabolism, increased proliferation of fibroblasts and epithelial cells (wound healing effect), are currently successfully used in clinical practice to treat a number of pathologies (oncological, infectious, surgical, ENT, etc.).

After BL therapy according to this method, the majority of patients (53%) sharply reduce the amount of pus in the washings, which disappears on the third day of treatment (excellent result), in some patients (33%) a small amount of pus in the washings is observed until the fourth day of treatment (good result), and in some patients (14%), against the background of a decrease in the number of secretions from the sinuses, pus streaks persist and there is swelling and hyperemia of the nasal mucosa (satisfactory result). Thus, the effect of therapy is different in different patients.

The disadvantage of this method is that there is no prognostic criterion that determines the individual susceptibility and side effect of BL therapy.

There is also a method for the diagnostic use of polymorphism of a gene encoding a receptor for TNF type II to identify patients who do not respond to anti-TNF therapy, in which, as a prognostic factor for the ineffectiveness of therapy with antibodies to TNFα, receptor gene polymorphism for this cytokine is detected [2].

This method allows you to identify respondents who do not respond to therapy with antibodies to TNFα, however, it does not predict the degree of its effectiveness.

The closest in technical essence to the claimed solution is a method for predicting the effectiveness of treatment with interleukin-1 [3].

This method includes the analysis of genotyping data on the basis of cytokine gene allelic polymorphism and allows predicting the effectiveness of treatment with IL-1 on the basis of the individual susceptibility of the patient to treatment with this drug to eliminate the side effect of the upcoming therapy. If a patient has a cytokine gene homozygous for a high-producing allele and changes in temperature upward, low treatment efficiency is predicted, and if a patient has a cytokine gene homozygous for a low-producing allele and no temperature increase, a high treatment efficiency is predicted.

The disadvantages of this method is that it does not take into account the ability of the functional activity of cells depending on the genotype.

The technical result of the invention is to predict the effectiveness of treatment of patients with chronic purulent rhinosinusitis with Betaleikin immunotherapy, carried out on the basis of the individual susceptibility of the drug to each patient and taking into account the ability of the functional activity of cells depending on the genotype.

To achieve the indicated technical result, in a method for predicting the effectiveness of treatment of chronic purulent rhinosinusitis, immunotherapy with recombinant cytokine is carried out, which is carried out on the basis of genotyping of allelic polymorphism of the cytokine gene.

According to the proposal, the therapy is carried out with recombinant interleukin-1β, and in the presence of a low-producing allele of the cytokine gene IL-1β, an increase in the phagocytic activity of neutrophils is predicted by increasing the level of IL-8 and guarantee high treatment efficiency.

The presence of distinctive features indicates the conformity of the claimed technical solution to the patentability criterion of "novelty."

The proposed method is based on the influence of functional polymorphism of cytokine genes on the nature of the immune response, while the effectiveness of therapy is characterized by an increase in the phagocytic activity of cells based on their recombinant analogues.

It is known that the activity of all cytokines involved in inflammation reactions is realized and regulated by a similar mechanism, therefore, we examined IL-1.

This cytokine is multifunctional and performs at least 50 different functions, the targets of which are cells of almost all organs and tissues. The main producers of IL-1 are monocytes and macrophages, and many other cells of the human body, in particular keratinocytes, can also produce IL-1.

IL-1 is an inducible protein, the synthesis of which begins in response to the introduction of microorganisms or tissue damage and which is necessary for the development of local inflammation and the implementation of the whole complex of protective reactions called acute phase response.

In the IL-1 family, it is customary to include IL-1α, IL-1β, IL-1 receptors (IL-1Re) and the receptor antagonist of this cytokine (IL-RA). The balance between production, expression and inhibition of protein synthesis of the IL-1 family plays one of the key roles in the development, regulation and outcome of the inflammatory process. The IL-1 gene cluster (430Kb) is located on the 2q13 chromosome and contains the IL-1α, IL-1β genes of the IL-1Re and IL-1RA receptors, the structure of which is quite conservative.

With a surge of IL-1β, the pro-inflammatory cytokine IL-8 is synthesized, which, as shown by experiments, stimulates the phagocytic activity of cells of the neutrophilic link.

IL-8 is synthesized by NK cells after activation of fibronectin receptors (β1-integrins) involving the intracellular signaling pathway Rac1 / p38 MAPK. IL-8 was found in the nasal secretions of patients infected with a synthic virus for sinusitis, as well as for bacterial infections.

It has been established that gram-positive and gram-negative bacteria can trigger IL-8 synthesis in different ways. The decrease in the level of production of IL-1 leads to a significant decrease in the synthesis of IL-8. The synthesis of IL-8, which began in response to endogenous stimuli, manifested in the focus of inflammation with the development of a local protective reaction to the introduction of pathogens, forms a cytokine network that regulates the local inflammatory response, including the activation of neutrophils.

Initially, IL-8 was characterized as a soluble factor with chemotactic activity for neutrophils. The participation of this cytokine in attracting neutrophils and activation of phagocytosis in the focus of inflammation is confirmed by in vivo experiments.

Subcutaneous administration of IL-8 to experimental animals caused an accumulation of neutrophils at the site of introduction. The introduction of specific neutralizing antibodies to IL-8 in similar simulated inflammatory reactions prevented neutrophil infiltration and neutrophil-mediated tissue damage. These observations led to the conclusion that IL-8 plays a key role in migration - activation of neutrophils in the focus of inflammation.

It is known that functional (responsible for the altered expression and production of the corresponding protein) polymorphism of genes encoding a number of known pro- and anti-inflammatory cytokines that carry small mutational changes (point nucleotide substitutions (SNP, single nucleotide polymorphism) or tandem repeats of gene parts (VNTR, variable number tandem repeat)), can lead to an imbalance of the inflammatory and anti-infectious immune response.

Allelic variants of the genes of a number of pro- and anti-inflammatory cytokines responsible for the increased production of the proteins encoded by them were identified: IL-1α - nucleotide substitution at (-889), IL-1β (+3953), IL-1RA (VNTR), TNFα (- 308)

IL-6 (-174), IL-4 (-590), IL-10 (-1082), IFNg (+874), IL-2 (-330), etc., the frequency of occurrence of which in a number of populations in a heterozygous form reaches 40-50%, and in homozygous 10-15%.

Thus, depending on the individual ensemble of high- or low-producing (normal) variants of these genes, the nature of the inflammatory response may vary between individuals with “polar” combinations: for example, the “pro-inflammatory genotype” - the majority of pro-inflammatory cytokine genes (IL-1α, IL-1β , TNFα, IFNgamma, etc.) are highly producing, and anti-inflammatory cytokines (IL-1RA, IL-4, IL-10, etc.) are low-producing; or "anti-inflammatory genotype" - the carriage of normal variants of pro-inflammatory cytokine genes in combination with highly producing variants of anti-inflammatory cytokine genes.

Some allelic associations of IL-1 family genes are responsible for the altered expression and production of the proteins encoded by them. A number of SNP markers of a highly producing variant of the IL-1β gene have been identified, usually inherited jointly.

(+3953, -511, -3737, -1469, -999). Carrier of polymorphic variant 2 of the IL-1RA gene (VNTR) carrying a series of repeats of 86 bp (IL-1RA * 2) is associated with an increased level of circulating IL-1RA and the level of mRNA expression of this protein during inflammation.

In individuals homo- or heterozygous for a variant of the IL-1β gene (+3953), they produce, respectively, four times and twice as much of this cytokine in the inflammatory response than in individuals homozygous for the normal version of this gene (IL- 1β * 1).

The presence of combinations in the genome (the “+” sign - a variant of the gene is present, the “-” sign is absent) IL-1β (SNP) + / IL-1RA * 2-, IL-1β (SNP) - / IL-1RA * 2 + and IL-1β (SNP) + / IL-1RA * 2 + can have a significant effect on the ratio of expression and production of these proteins and is one of the main causes of dysregulation of the inflammatory response.

According to population studies, the IL-1RA * 2 variant is more often found in association with the normal (low-producing) variant of the IL-1β gene (IL-1β * 1) and is rarely present in conjunction with the high-producing variant, IL-1β * 2 (the marker is one of nucleotide substitutions listed above). The phenomenon of this inheritance is explained by the fact that these variants of genes located close to each other are usually inherited jointly.

In individuals heterozygous for these variants of genes, one of the chromosomes carries the haplotype IL-1β * 1 / IL-1RA * 2, the second - IL-1β * 2 / IL-1RA * 1, resulting in a combination of "1/2" IL -1β + "1/2" IL-1RA. The haplotype IL-1β * 2 / IL-1RA * 2 is found in 1-2% of the population and, most likely, is the result of meiotic recombination.

Thus, the presence of combinations of IL-1β (+3953) + / IL1RA * 2-, IL-1β (+3953) - / IL1RA * 2 + and IL-1β (+3953) + / IL1RA * 2 in the genome of patients with HGRS can significantly affect the production of these proteins and, accordingly, reduce the production of IL-8, which affects phagocytic cells.

The proposed method by identifying the cytokine gene homozygous for the normal version determines the high efficiency of therapy due to the activation of the phagocytic immunity and allows you to choose the treatment tactics for patients with chronic purulent-inflammatory diseases of the paranasal sinuses based on the individual genetically determined susceptibility of the patient to such therapy.

From the foregoing, it follows that the technical result of the invention is achieved by a new set of essential features both newly introduced and known, therefore, the claimed method meets the patentability criterion of "inventive step".

The method is illustrated in the drawing, where Fig. 1 shows a photograph of an agarose gel with variants of bands visually observed with alleles 1 and 2 of the IL-1RA gene (VNTR), 1/1 is the gene homozygous for variant IL-1RA * 1, 1 / 2 - heterozygous gene, 2/2 - gene homozygous for variant IL-1RA * 2; figure 2 shows table 1, which shows the carriage of combinations of variants of the genes IL-1β and IL-1RA in groups of patients with chronic hepatitis C and healthy donors (patients /%); figure 3 shows table 2, which presents the characteristics of the response of peripheral blood cells of patients with chronic rhinosinusitis to various inductors in the LHLC reaction (imp / s); figure 4 presents table 3, which shows the group of patients depending on the intensity of LHLC and combinations of genotypes.

The method is as follows.

Before immunotherapy, the patient takes material containing cells (blood, swabs from the maxillary sinuses), which is then examined.

First, molecular genetic determination of cytokine gene variants is carried out. From cells using one of the known methods, for example, using phenol / chloroform extraction according to Khomchinsky, samples of high molecular weight DNA are obtained.

Secondly, by the method of chemiluminescence (hereinafter CL), based on the fact that any biological process in a living organism is accompanied by the emission of a quantum of light, determine the functional activity of phagocytic cells of a neutrophilic unit. Since the light pulse is very small, you can catch it only by repeatedly amplifying it using special reagent applicators (luminol, luceginin). The amplified signal is recorded with luminometers.

Variants of gene alleles, normal and bearing point substitutions of SNP nucleotides, are determined by RFLP analysis of PCR amplification products of specific genome regions (RELF-PCR).

The PCR reaction is carried out using primers (Gene Bank library), limiting the portion of the IL-1β gene, which may contain the indicated nucleotide replacement. At the stage of restriction, the resulting amplification is incubated with individually selected restriction enzymes.

The restriction products are separated, for example, by electrophoretic agarose gel, stained with DNA dye and visualized. As a marker for the size of DNA fragments, for example, a set of molecular weight markers is used.

In the presence of a polymorphic mutation in the genome, the amplification product remains intact; in heterozygous individuals, both the whole and fragmented product are detected.

The presence of tandem repeats of parts of a gene (VNTR) is determined, for example, by PCR (RSC): the DNA region containing a different number of tandem repeats is amplified, the allelic type is determined by the length of the obtained amplification product during separation and visualization (Fig. 1).

The following abbreviations are used to briefly identify the alleles of the IL-1β and IL-1RA genes: the normal, non-detectable mutation allele is denoted by the number 1, polymorphic - 2, respectively, the homozygous normal gene - “1/1”, the homozygous highly producing gene - “2/2 ”, Heterozygous -“ 1/2 ”.

The chemiluminescence method determines the functional activity of phagocytic cells of a neutrophilic unit. The amplified signal is recorded using a multiscan luminometer, which allows the use of very small amounts of reagents.

For the study, heparin stabilized venous blood (20 IU / ml) and maxillary sinus rinse are used. The inflammatory cells contained in the flush are washed with an isotonic solution twice at 1500 rpm for 10 minutes, resuspended in Hanks solution at a concentration of 1 × 106 cells / ml and used in the following reaction.

The reaction is carried out as follows.

In the wells of a 96-well white, opaque plate, 20 μl of the test material (blood, rinse from the paranasal sinuses) is dug, the substances stimulating the functional activity of the inflammatory cells (phorbol myristate acetate, opsonized zymosan) contained in the test material are added, 40 μl of the solution luminol in a final concentration of 10-4 M, the total volume of the reactogenic medium is adjusted with Hanks solution to 200 μl. All studies are carried out in three parallels. The reaction is recorded for one hour at 37 ° C.

The result of the reaction is expressed by the light sum, that is, the number of pulses accumulated during the experiment.

Preparation of reagents is as follows.

Luminol - is a substance that accepts the entire pool of free radical forms of oxygen (superoxide radical, hydroxyl radical, singlet oxygen), as well as hydrogen peroxide and myelopyroxidase produced by inflammatory response cells (neutrophils, macrophages) upon their activation.

17.7 mg of luminol is dissolved in 10 ml of dimethyl sulfoxide (DMSO), a mother liquor is obtained, which is stored at the temperature of the refrigerator for one month. Before the study, a working solution is prepared by dissolving 1 volume of the mother liquor of luminol in 10 volumes of Hanks solution.

Phorbol myristate acetate (FMA) belongs to the group of phorbol esters, is a non-specific activator of the functional activity of inflammatory cells, acts directly through the protein kinase activation pathway, bypassing the receptor apparatus. PMA causes a series of sequential reactions in the cell, leading to a powerful oxidative explosion and the release of free radical forms of oxygen. Using this activator allows you to evaluate the oxidative potential of the cell. FMA is used in the reaction at a concentration of 10 ng / ml.

Zimosan - a fragment of the cell wall of baker's yeast - is also used in the reaction as an activator of the inflammatory response cells. The use of this activator allows one to evaluate the intensity of an oxidative explosion that occurs in neutrophilic cells as a result of the receptor interaction of the complement component zymosan, which is opsonized by components, with C3v receptors on the cell surface. Conducting a reaction with opsonized (ops) zymosan is a variant of the phagocytosis reaction.

Zimosan in an amount of 10 mg was diluted in 10 ml of 0.9% NaCl solution and boiled for 30 minutes to obtain a homogeneous suspension. The suspension is washed twice with an isotonic solution, resuspended in 2.5 ml of the same solution, an equivalent amount of human blood serum (at least 4 people) is added. Then placed for 40 minutes in a thermostat at a temperature of 37 ° C.

After washing off excess serum, zymosan is diluted with 10 ml of isotonic solution and 500 μl aliquoted in plastic tubes. Store at t-18 ° C for a month.

The levels of cytokines in blood serums, lavages and cell supernatants are determined by the method of solid-phase ELISA using polystyrene plates, test systems of Cytokine LLP (IL-1 and IL-8). The concentration of a particular cytokine in the sample is established by the calibration curve of the ratio of the optical density of the solution in the well and the known concentration of this cytokine as a standard, multiplying by the corresponding dilution of the sample.

The selection of patients for the study was carried out on the basis of the Federal State Institution "St. Petersburg Research Institute of Ear, Throat, Nose and Speech." We examined 98 patients with HGRS and 152 healthy donors aged 18 to 60 years. A total of 250 people, including 115 men and 135 women. Most of the patients selected for the study had a disease duration from 5 to 10 years.

Therefore, the disease had a fairly long course. Due to this, it is possible to objectively evaluate the results of examination of patients.

A detailed analysis of the distribution of combinations of polymorphic variants of the IL-1β and IL-1RA genes in patients with chronic hepatitis syndrome and healthy donors was carried out (table 1 in figure 2).

Polymorphism of the IL-1RA gene had an effect in group I of patients with low phagocytic activity. All patients of group I were carriers of the IL-1RA * 2 variant. The lowest values in comparison with the group of healthy donors were carriers of haplotypes “1/1” IL-1β + “2/2” IL-1RA, “1/2” IL-1β + “1/2” IL-1RA and “1 / 2 "IL-1β +" 2/2 "IL-1RA.

Patients of group II had the most mixed haplotypes. However, the most significant is the combination of the “1/2” IL-1β + “1/2” IL-1RA and “1/2” IL-1β + “2/2” IL-1RA genes.

When analyzing the IL-1β + IL-1RA haplotypes, it was found that individuals of homozygous normal variants of these genes had a high response to both inducers and entered group III; in these patients, the response to PMA of 30.6 imp / s was also higher compared to that the same group of healthy donors 20.7 - imp / s, (table 2 in figure 3).

However, patients of group III, except for carriers of “1/2” IL-1β + “1/2” IL-1RA and “1/2” IL-1β + “2/2” IL-1RA, had a rather low level of spontaneous LHCL, 5.1 imp / s in comparison with the response to zymosan and FMA. This caused a violation of the sequence of phases of phagocytosis, but a defect in the phagocytic system was not observed in these patients.

Thus, the carriage of the IL-1RA * 2 variant determined a decrease in the functional activity of venous blood cells in patients with chronic hepatitis C disease.

For diagnostic and therapeutic purposes, punctures of the maxillary sinuses were performed according to generally accepted standard methods under local anesthesia. A punctate LCHL study was conducted in 98 patients with chronic purulent rhinosinusitis.

We also studied the effect of polymorphism of the IL-1β and IL-1RA genes on the functional activity of neutrophils directly in the focus of inflammation - in the cells of the washing fluid of the maxillary sinuses.

The cellular composition of the swabs was mainly represented by neutrophilic cells. LZHL was also studied using 2 inductors. In order for the results of the study to be comparable, quantitative counting of washout cells was performed before the study.

The cell content in the test wash after washing 2 times with isotonic solution was adjusted to 1 million / ml. A study of LHCL swabs was performed prior to treatment.

An initial analysis of the results showed that the LHCL levels of rinses from the paranasal sinuses ranged in a wide range from 12, 2 imp / s during the study (30 minutes) to 2495.4 imp / s, in connection with which we identified 3 groups of patients in depending on the intensity of LHCF.

Response levels were defined as low, medium, high.

As can be seen from table 3 in figure 4, at a low level, the response to FMA did not exceed 35.8 ± 5.6 imp / s for 30 minutes of the study, and the opsonized zymosan was equal to 28.9 ± 4.8 imp / s. While the level of spontaneous LHCL was the smallest 11.2 ± 2.0 pulse / s.

A low level of response (table 3 in figure 4) was observed in patients with carriers of the “2/2” IL-1RA variant, and low response values for PMA, opsonized zymosan and spontaneous LHCL were also noted. The haplotypes “1/1” IL-1β + “2/2” IL-1RA and “1/2” IL-1β + “2/2” IL-1RA are noteworthy, the carriage of combinations of these genes influenced the clinical symptomatology. Thus, a clinical analysis of patients with a low level of LHCF response showed that (according to the anamnesis) the duration of the disease recurrence in this group of patients exceeded 15 days before going to the hospital. Patients noted the presence of a mucopurulent discharge in the inter-relapse period. This fact confirms the violation of phagocytosis and the impossibility of complete completion of the inflammatory process in the paranasal sinuses.

The average level of response to PMA was observed in patients with chronic hepatitis C and it was 290.8 ± 33.4 imp / s, for opsonized zymosan 253.8 ± 38.2 imp / s. Patients with an average level of response were carriers of various haplotypes, however, most of them were carriers of “1/2” IL-1β + “1/2” IL-1RA and “1/2” IL-1β + “2/2” IL-1RA. It should be noted that patients with an average level of response locally at the site of inflammation belonged to group II of patients in determining the chemiluminescent activity of peripheral blood (table 1 in figure 2).

In the group with high rates, the average response rates for FMA are -1012.1 ± 1472 imp / s, for the opsonized zymosan -1180.3 ± 144 imp / s.

The highest level was observed in the III group of patients, the highest medians of the values of spontaneous and induced LHLC cells were observed in patients with haplotypes of "1/1" IL-1β + "1/1" IL-1RA, "1/2" IL-1β + " 1/1 "IL-1RA and" 2/2 "IL-1β +" 1/1 "IL-1RA. The level of spontaneous LHC in patients of group III exceeded 12.8 times the level of spontaneous LHC in group II, the level of LHC in response to PMA was 28.9 times, and the level of LHC in response to opsonized zymosan was 42.1 times. This observation of combinations of the haplotypes “1/1” IL-1β + “1/1” IL-1RA, “1/2” IL-1β + “1/1” IL-1RA and “2/2” IL-1β + “ 1/1 "IL-1RA with high phagocytic activity characterizes the severity of inflammation in the paranasal sinuses. Patients with a high level of intensity had a history of the lowest number of relapses per year. However, arising periods of exacerbations were accompanied by a vivid clinical picture.

In all three groups of patients, a high spontaneous level of LHCL was observed. Even in the group with low LHCL activity, the spontaneous reaction level was two times higher than the background level (5-6 pulses / s). Consequently, the high spontaneous LHLC level of cells contained in nasal swabs is due to massive phagocytosis of microflora located in the paranasal sinus cavity, despite the fact that the “quality” of phagocytosis varies significantly.

Significant differences in the intensity of the chemiluminescent response to cell inducers from the focus of inflammation reflect the level of functional activity of these cells.

According to the screening, HFS associated with the IL-1RA * 2 (VNTR) gene polymorphism (73.4%) and the combinations “1/1” IL-1β + “2/2” IL-1RA, “1/2” IL -1β + “1/2” IL-1RA and “1/2” IL-1β + “2/2” IL-1RA, causes a decrease in the functional activity of neutrophils both in peripheral blood and in the focus of purulent inflammation. Consequently, the polymorphism of these genes may be one of the main reasons for the violation of phagocytosis. Thus, a decrease in the level of serum production of IL-1β (2.37 ± 1.2) in comparison with healthy donors of IL-1β (33.1 ± 20.1) is guaranteed to prolong the decrease in the synthesis of IL-8 (3.4 ± 2.1 ) relative to the same donors of IL-8 (36.1 ± 29.1), responsible for the stimulation of phagocytosis.

In healthy donors, the combination of “1/2” IL-1β + “2/2” IL-1RA genes was found in 1.9% of cases, and in the examined patients with hGH, this genotype was found in 25.5% of cases. The analysis of indicators of the functional activity of phagocytic cells of the neutrophilic link in patients with HGH showed that the cells of swabs from the maxillary sinuses had two types of response to the used inflammatory inducers.

In the first type of response, the response to PMA was lower than that of the opsonized zymosan. In the second type of response, we observed a decrease in the response to opsonized zymosan compared with the response of cells to PMA. This type of response indicates a decrease in the number of C3b receptors and, accordingly, a defect in the phagocytosis of cells from the focus of inflammation.

Interestingly, in the group with a high response to inducers (table 3 in figure 4), only four out of 32 patients had a second type of response. At the same time, in groups with a low and medium level of response to inflammatory inducers in most patients (62%), a decrease in the phagocytic activity of inflammatory cells obtained from the focus of inflammation was noted. Therefore, a decrease in the response to inflammatory stimuli reflects the degree of inhibition of the phagocytic activity of inflammatory cells.

An analysis of the results obtained by us in determining the functional activity of phagocytic cells of a neutrophilic link in peripheral blood and in rinses from the paranasal sinuses of patients with chronic hepatitis C syndrome indicates a correlation between the carriage of the “2/2” IL-1RA variant and the level of cell chemiluminescence.

According to this conclusion, overproduction of IL-1RA genetically determines a decrease in IL-1β → IL-8 production and, accordingly, reduces the activity of phagocytic cells in response to the introduction of a pathogen.

Genetically prolonged dysregulation of IL-1β / IL-IRA production in patients with chronic hepatitis C disease leads to a prolonged inflammatory response and, accordingly, the inability to eliminate invading foreign agents (bacteria, viruses, fungi, etc.).

Studies have also shown that patients with HGRS have different activity of phagocytic cells of the neutrophilic link, depending on the patient’s genotype.

The effect of functional polymorphism of the IL-1β and IL-1RA genes on the phagocytic link of the immune system can be described in the form of the following patterns: carriage of normal gene variants determines adequate phagocytic activity of phagocytic cells; in carriers of genetic preponderance towards IL-1β, phagocytosis proceeds more intensively; in carriers of a genetically determined preponderance towards the production of IL-1RA, phagocytosis is slowed down or reduced, which may cause inhibition of pathogen elimination from the focus of inflammation, and thereby contribute to the chronicity of the purulent process.

After the treatment, the following data were obtained.

Carriers of the haplotypes “1/1” IL-1β + “1/1” IL-1RA, “1/2” IL-1β + “1/1” IL-1RA and “2/2” IL-1β + “1 / 1 "IL-1RA had a response rate to opsonized zymosan higher than carriers" 1/1 "IL-1β +" 2/2 "IL-1RA," 1/2 "IL-1β +" 1/2 "IL-1RA and “1/2” IL-1β + “2/2” IL-1RA.

In groups with a higher level of response to opsonized zymosan, seven days later, the neutrophil functional activity indices quickly returned to normal values under the action of recombinant IL-1β (BETALEYKIN).

If a cytokine (agonist) has an antagonist mediator that suppresses the functions of an agonist, for a more accurate prognosis, the nature of the carriage of combinations of agonist and antagonist genes was determined.

When a patient identifies combinations of “1/1” agonist gene (IL-1β) and “1/1”, “1/2” or “2/2” antagonist gene (IL-1RA), high therapy efficacy is predicted due to phagocytic activation neutrophil activity.

If a patient identifies combinations of “2/2” of the agonist gene (IL-1β) and “1/1” or “1/2” of the antagonist (IL-1RA), low efficacy of recombinant IL-1β therapy is predicted.

The above data were obtained by genotyping 250 people.

The control group consisted of 152 donors. As a marker of a highly producing variant of the IL-1β gene, the replacement of C / T nucleotides at position (+3953) was determined by RT-PCR (RELF-PCR), as a marker of IL-1RA, the presence of 86 bp tandem repeats (VNTR) by PCR method.

Statistical processing of the results was carried out using the exact Fisher method, Mann-Whitney and Kruskal-Wallace criteria, Cox regression.

It should be noted that the joint carriage of homozygous “2/2” variants of the IL-1β and IL-1RA genes in patients and donors was not found in our studies.

Thus, the identification of the alleles of IL-1β (+3953) +, IL-1RA * 2 and functionally polar genotypes in relation to the production of the corresponding proteins allows us to predict an increase in the functional activity of phagocytic cells of the neutrophilic immunity unit upon administration of the BL preparation.

Analysis of the presented data shows that the introduction of exogenous IL-1β regulates the described imbalance of the inflammatory response in carriers of combinations of the IL-1β and IL-1RA genes.

The use of BL in the treatment of patients with HGRS showed that this drug is highly effective for carriers of the normal and heterozygous variant of the IL-1β gene and the IL-1RA * 2 variant. Within 2-5 days, therapy of patients carrying such genotypes led to the complete disappearance of purulent discharge and the cessation of the inflammatory process in the maxillary sinuses, as confirmed by the chemoluminescent method.

The introduction of exogenous IL-1β had a stimulating effect on the functions of the immune system of such patients, regulated the ratio of IL-1RA and IL-1β in the blood serum and the inflammatory focus by enhancing phagocytosis and thereby eliminating the pathogen from the body, activating the functional state of the immune response directly in the inflammatory focus and accelerating the completion of the purulent-inflammatory process in the paranasal sinuses.

After BL therapy, in most patients, a return of the disease within 1.5 years was not observed.

Thus, for patients with HGRS, who, according to the genotyping carried by the polymorphic variant of the IL-1RA * 2 gene, BL therapy was a highly effective method of immunocorrection.

In addition, for people sensitive to BL therapy after the first use, this drug is one of the most effective means for relapsing the disease.

And the presence in the genotype of a highly producing variant of the pro- or anti-inflammatory cytokine gene is a factor determining the inefficiency of additional administration of a recombinant analog of this cytokine into the body for immunotherapy, and can cause a pronounced side effect.

The method is illustrated by the following examples.

Example 1

Patient Selkina T.G. appealed to the Federal State Institution "St. Petersburg Research Institute of Ear, Throat, Nose and Speech" with complaints of mucopurulent discharge from the nose, a feeling of running down the back of the throat, weakness, and fatigue. The patient considers himself since childhood. The number of relapses of the disease is 5-6 times a year.

During the examination, computed tomography revealed a picture of bilateral sinusitis with the presence of fluid contents in the maxillary sinuses and ethmoid labyrinth cells. Objectively:

mucous congestion with a bluish tinge, edema is not pronounced. In the common nasal passages - purulent discharge. There are purulent strips under the middle turbinate. A puncture of the maxillary sinuses was performed, and a thick viscous pus of green color was obtained in an amount of approximately 4 ml. Diagnosed with bilateral purulent sinusitis.

Prescribed treatment: BL at a dose of 10 ng in 1 ml of isotonic solution once daily after washing the sinuses with isotonic solution.

According to the genotyping data, the patient is a carrier of the heterozygous IL-1β gene and the IL-1RA * 2 gene ("1/2" IL-1β + "2/2" IL-1RA) that is homozygous for the highly producing variant.

According to the study of CL, the patient has an average response level - so the level of spontaneous LHCL was 5.9 imp / s; and the level of LHCL in response to ops. Zimozan 14.3 imp / s; the LHCL level in response to the FMA was 40.5 cps. This fact indicated an insignificant phagocytic activity of neutrophils, the degree of local activation in the focus of inflammation in a patient carrying the genotype “1/1” IL-1β + “2/2” IL-1RA.

After the third injection of the drug, the patient noted improvement, decreased purulent discharge. After the fourth (and last) use of BL, the wash liquid does not contain pathological impurities. She feels well. Five days after the end of BL therapy, a control puncture was performed - the wash fluid is clean, the fistula function.

Relapse was reported one year after recombinant IL-1β therapy. The treatment was carried out according to a similar scheme.

Example 2. Patient Uspensky G.Yu. appealed to the Federal State Institution "St. Petersburg Research Institute of Ear, Throat, Nose and Speech" after exacerbation for 2.5 weeks with complaints of purulent nasal discharge, weakness. He considers himself sick since childhood. The number of relapses of the disease is 5-6 times a year.

During the examination, computed tomography revealed a picture of bilateral sinusitis with the presence of fluid contents in the maxillary sinuses and ethmoid labyrinth cells. Objectively: mucous congestion with a bluish tinge. In the common nasal passages - mucopurulent discharge. There are purulent strips under the middle turbinate. A puncture was made, a viscous pus was obtained in an amount of approximately 4 ml. Diagnosed with bilateral purulent sinusitis.

Prescribed treatment: BL at a dose of 10 ng in 1 ml of isotonic solution once daily after washing the sinuses with isotonic solution.

According to the genotyping data, the patient is a carrier of the heterozygous IL-1β gene and homozygous for the highly producing variant of the IL-1RA * 2 gene ("1/2" IL-1β + "2/2" IL-1RA).

According to the study of CL, the patient had an average response level - so the level of spontaneous LHCL was equal to 131.9 imp / s; and the level of LHCL in response to ops. Zimozan 253.1 imp / s; the LHCL level in response to the FMA was 411.9 cps. This fact indicated an insignificant phagocytic activity of neutrophils, the degree of local activation in the focus of inflammation in a patient who is a carrier of the “1/2” IL-1β + “2/2” IL-1RA genotype.

After the fourth injection of the drug, the patient noted a sharp improvement, decreased purulent discharge. After the fifth (and last) application of BL, the wash liquid does not contain pathological impurities. She feels well. Five days after the end of BL therapy, a control puncture was performed - the wash fluid is clean, the fistula function.

He relapsed 3 years after the treatment with recombinant IL-1β.

Example 3. Patient Kovba N.V. appealed to the Federal State Institution "St. Petersburg Research Institute of Ear, Throat, Nose and Speech" with complaints of purulent nasal discharge, weakness. He considers himself sick since his youth. The number of relapses of the disease is 5-6 times a year. Prior to treatment, the exacerbation period is within 2 weeks. He was treated in the clinic at the place of residence, where he was treated with broad-spectrum antibiotics by intramuscular injection and washing the maxillary sinuses with a solution of dioxidine. However, no improvement was observed.

According to genotyping, it is a carrier of the IL-1β gene homozygous for the high-producing variant and the IL-1RA * 1 gene (2/2 "IL-1β +" 1/1 "IL-1RA) homozygous for the low-producing variant.

According to the study of CL, the patient had a high level of response: so the level of spontaneous LHCL was 112.6 imp / s; and the level of LHCL in response to ops. Zimozan 302.0 imp / s; while the LHCL level in response to the FMA was 2495.4 imp / s. This fact indicated a high phagocytic activity locally in the focus of inflammation in a patient carrying the 2/2 IL-1β + “1/1” IL-1RA genotype.

During the examination, computed tomography revealed a picture of bilateral sinusitis with the presence of fluid contents in the maxillary sinuses and ethmoid labyrinth cells. Objectively: the mucous membrane is stagnant, edematous pale pink. In the common nasal passages - purulent discharge. There are purulent strips under the middle turbinate.

A puncture was made, a liquid of pale green odorless pus was obtained in an amount of approximately 7 ml. Diagnosed with bilateral purulent sinusitis.

Prescribed treatment: BL at a dose of 10 ng in 1 ml of isotonic solution once daily after washing the sinuses with physiological solution. After the first injection of the drug, the patient noted an increase in nasal congestion, but purulent discharge decreased. During the repeated puncture, an abundance of mucous discharge is observed without an admixture of pus. The state of health is satisfactory.

It was decided to conduct an additional examination of the patient in order to identify antibodies to allergens in the blood of the patient. The result was positive - the patient was allergic to household and food allergens and the final diagnosis was made: year-round allergic rhinitis. Acute recurrent rhinosinusitis.

Seven days after the anti-allergic treatment, in combination with antibacterial and vasoconstrictive therapy, the patient was discharged from the hospital. A control puncture was performed on the seventh day of treatment - the wash fluid is clean, the anastomoses function in full. Computed tomography data 14 days after treatment: no pathology from the paranasal sinuses was detected.

Thus, there was a positive trend in a short time, due to the correct and timely diagnosis and the ability to determine the treatment tactics of the patient.

The patient with relapse within 2.5 years did not contact.

Example 4. Patient Morozova N., 43 years old, went to the clinic “Pathology of the upper respiratory tract” of the Federal State Institution “SPb Research Institute LOR Rosmedtehnologii” with complaints of purulent discharge from the nose for one month, moderate nasal congestion, heaviness in the area of projection of the paranasal sinuses, weakness, drowsiness. The duration of the exacerbation is about one and a half months. The number of exacerbations reaches five times a year. In the inter-relapse period, the discharge from the nose decreases in volume, but does not disappear completely. The patient suffers from sinusitis from a youth period.

Objectively: nasal breathing is moderately difficult. Nasal septum in the midline. The nasal passages are filled with mucopurulent discharge.

According to CT scan of the paranasal sinuses: a picture of bilateral sinusitis with the presence of fluid in the maxillary sinuses and cells of the ethmoid labyrinth.

A puncture of the maxillary sinuses was performed, aspirate - liquid green pus, odorless, in a volume of ≈ 7 ml.

Diagnosed with exacerbation of chronic purulent bilateral maxillary sinusitis, bilateral ethmoiditis.

Treatment: Betaleukin at a dose of 10 ng, diluted in 1 ml of isotonic solution (0.9% NaCl), in each sinus daily after preliminary washing of the sinuses with isotonic solution in order to remove pathological contents.

After the first injection of Betaleikin, the patient noted an improvement. Separated from the sinuses acquired a mucous character and purification of the sinuses by the third day.

According to the genotyping data, the patient is a carrier of the homozygous IL-1β gene for the low-producing variant of the gene and the homozygous for the high-producing variant of the IL-1RA * 2 gene ("1/1" IL-1β + "2/2" IL-1RA).

According to CL, the patient had low levels of response - so the level of LHCL is 5.1 imp / s, the level of LHCL in response to ips. Zimozan is equal to 12.3 imp / s.

The LHCL level in response to the FMA is 11.7 imp / s. This assay spectrum indicates a decrease in the functional activity of neutrophils due to the carriage of the genotype “1/1” IL-1β + “2/2” IL-1RA.

Five days after triple administration of the BL preparation, a control puncture of the maxillary sinuses was performed. The washing liquid does not contain pathological inclusions, the anastomoses function in full. Nasal breathing is free.

The patient turned to the ENT Research Institute a year after immunotherapy with a relapse of the disease. The treatment was carried out according to a similar scheme.

Thus, the presence of a highly producing IL-1RA * 2 gene in the combinations “1/1” IL-1β + “2/2” IL-1RA, “1/2” IL-1β + “2/2” IL-1RA can be one of the main reasons for the violation of the phagocytic function of neutrophils in chronic heart disease. According to our observations, in healthy donors, the variant of combining the genes “1/2” IL-1β + “2/2” IL-1RA was found in 1.9% of cases, and in the examined individuals with HGRS this genotype was detected in 25.5% of patients . An increase in the level of the receptor blocker can have a significant decrease in the level of IL-1β both in blood serum and locally in the focus of inflammation, which in turn does not provide the level of IL-8, a factor that stimulates the phagocytic activity of neutrophils.

It follows from the foregoing that the proposed method provides a technical result, does not cause difficulties, involves the use of developed materials and standard equipment, which indicates the compliance of the claimed technical solution with the patentability criterion of "industrial applicability".

Information sources

1. Patent KP№2161983, A61K 38/20, A61B 17/24, 2001.

2. EP patent No. 1172444, C12Q 1/68, 2002.

3. Patent RU No. 2301012, АВВ 5/01, 2007.

Claims (2)

1. A method for predicting the effectiveness of the treatment of chronic purulent rhinosinusitis with immunotherapy with recombinant cytokine, based on the genotyping of allelic polymorphism of the cytokine gene, characterized in that the therapy is carried out with recombinant interleukin-1β and in the presence of a low-producing allele of the cytokine gene IL-1β, an increase in phagocytic activity is predicted increased levels of IL-8, and guarantee high treatment effectiveness. 2. A method for predicting the effectiveness of treatment of chronic purulent rhinosinusitis according to claim 1, characterized in that when the patient identifies combinations of "1/1" of the agonist gene of IL-1β and "1/1", "1/2" or "2/2 »IL-1RA antagonist gene predict high therapy efficacy due to activation of phagocytic activity of neutrophils.

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