IFN status including circulating IFN; level of IFNα production by leukocytes infected with Newcastle disease virus; level of IFNγ production by lymphocytes induced by 10 µg/ml FGA («Difco»), level of spontaneous IFN production in vitro were assayed in whole heparinized blood. Units/ml of IFN were calculated as reverse dilutions protecting 50% of cellular monolayers from the virus-induced cytopathic effect [11].

ELISA

For the cultivation and mitogen stimulation of blood cells, a set of kit-reagents “Cytokin-Stimul-Best” was used and the concentration of IFN-α, IFN-γ, IL-1b, IL-6, IL-8, IL-17, IL-2, IL-4, IL-10, TNF-α was determined using the ELISA kits «Vector-Best» (Russia) and IL-13 (“Bender MedSystems”, Austria).

Statistical processing of the data was carried out using the BIOSTAT program (calculation of M±σ, reliability of differences in the mean values ​​from the paired Student test).

It was found that the majority of patients with exacerbation of AD in clinical analysis of the blood showed eosinophilia (95%) and increased concentration of IgE (70%) in the blood serum. All patients had a recurring course of the disease in the phase of exacerbation (3-5 exacerbations per year). The study of the IFN system by a biological method revealed differences in the rates between the examined and control groups. As can be seen from figure 1, in all the examined patients with AD, the IFN system was deficient in the production of α- and, especially, γ-IFN with blood leukocytes. The average IFN status was (in U/ml): the ability to produce α-IFN blood leukocytes 174.7±17.94; IFN-γ-12.53±0.77; serum IFN-7.87±2.32; spontaneous IFN-0.2±0.11. The titer of serum IFN was elevated in 6 patients (40%); the presence of spontaneous IFN-in 3 patients (20%).

Analyzing the data obtained to determine the levels of cytokines in the blood, we can note a number of features in patients with AD compared with healthy donors (control group). An increased content of proinflammatory cytokines, especially IL-8 (p<0.05), to a lesser extent, IL-6 (p>0.05), and at the same time a significant decrease in IL-2 (p<0.05) (Figure 2). Of the 15 patients, 5 values of serum IL-8 exceeded 100 pg/ml (97 to 575 pg/ml). After the treatment, the normalization of these indicators was noted. Serum TNF-α was detected in 3 (20%), IL-1β - in 5 patients out of 15 (33.3%).

It is known that Th2 cells are responsible for the development of the immune response of the humoral type and the hyperproduction of IL-4, IL-10, IL-13 in allergic diseases. However, IL-4 was not detected in the serum of the patients with AD, IL-10 was detected in 1 patient (6.7%), IL-13 in 3 patients (20%).

It should be noted that IFN-α and IFN-γ were not detected in the serum of patients with AD, although induced and spontaneous production of both types of IFN was observed, which was 4.5 times and 1.5 times (respectively) less pronounced in patients with AD, than in the samples of the control group (Figure 3). Between spontaneous and induced interferons production there is a statistically significant difference (p<0.05).

As can be seen from figure 4, significant (p<0.05) positive dynamics of interferon production with blood leukocytes with a tendency toward normalization, as well as normalization of serum IFN levels and the disappearance of spontaneous IFN in the in vitro reaction was noted after treatment with Cycloferon. In addition, the levels of proinflammatory cytokines, such as IL-8 and IL-6, in the blood serum after treatment, with a parallel increase in IL-2 and IFN-a, should be noted.

The combination of immune system disorders with the lack of ability to produce α- and γ-IFN by blood leukocytes in patients with revealed responsiveness of blood leukocytes to the IFN inducer Cycloferon gives grounds to consider it appropriate to use it in therapy of patients with AD exacerbations immediately after the basic therapy.

After complex therapy, there was a positive clinical dynamics in the form of: reducing erythema, reducing the intensity of itching, stopping the appearance of new elements, preservation of lichenification, as well as reducing the number of exacerbations and increasing the duration of remission.

As follows from the data obtained, with AD develops a pronounced IFN deficiency, especially the production of γ-IFN of blood leukocytes. There arises a completely natural assumption: are the defects of innate immunity one of the causes of AD? It is known that a prolonged persistence of viruses leads to secondary immunodeficiency, while in turn secondary immunodeficiency promotes the activation of the viruses themselves [12,13]. In any case, in the pathogenesis of the disease, the immune competence of the patient’s body is of great importance. Against the background of IFN-immune deficiency may often occur exacerbations of AD.

In this connection, the inclusion of IFN inducers in therapy seems reasonable. Its immune regulatory properties are mediated through the activation of IFN-γ. An increase in its production contributes to the restoration of the T-cell link of immunity: it normalizes the levels of subpopulations of CD3+, CD4+, CD8+, as well as the number of CD16+, CD72+. Earlier it was shown that the use of Cycloferon is quite effective in patients with chronic and recurrent viral and bacterial infections and in patients with a secondary immunodeficient status (herpetic and cytomegalovirus infections) [13]. Cycloferon therapy is also relevant for patients with relapsing AD. The treatment with Cycloferon reduced the number of exacerbations and increased the duration of remission in patients with AD.

The ability of blood leukocytes to produce cytokines under the influence of stimuli is clearly pronounced, and the ability of leukocytes of patients with AD to IFN-γ synthesis is significantly higher than the synthesis of IL-4, i.e. there is clearly a classical picture of the production of cytokines in the long-term inflammatory process. In our case, this is a chronic relapsing form of AD, which is very difficult to treat. The vast majority (85%) of the examined patients with AD have a persistent herpesvirus infection, which, as is known, can lead the patient’s body to the state of IFN-immune deficiency. It is known that these viruses affect more than 1/3 of the population worldwide and are responsible for a wide range of human diseases, ranging from mild herpesvirus infection to disseminated forms such as Eczema herpeticatum in patients with atopic eczema or in those with severe immunodeficiency. These viruses have an intracellular location, displaying a clinically latent persistent form of the disease. In addition, viruses of the Herpesviridae are able to influence the normal functioning of regulatory cells of the Treg, weighting the course of the underlying disease.

IL6, IL8 and IFN-γ serve as mediators of subacute and chronic inflammation in atopic dermatitis. The decrease in the level of proinflammatory cytokines, especially IL8, after complex treatment, as well as an increase in IL2 level, indicates a decrease in the inflammatory process.

The absence of IFN-a in the serum can lead to recurrent infections [14]. IFN-γ, secreted by activated viral antigen Th1 lymphocytes, can suppress the reproduction of the virus, disrupting the intracellular processes of viral replication. However, this mechanism does not work well or does not work at all if there is a deficiency/defect in the IFN system (for example, in herpetic infection). Induced (by various stimuls, mitogens) synthesis of cytokines reflects the potential, reserve capacity of cells to respond to an antigenic stimulus (in particular, the action of drugs). Reduced induced production of cytokines in vitro can serve as one of the signs of an immunodeficiency state.

With the action of various viral agents and Cycloferon, a whole cascade of signals is induced, which involve the “cytokine network” of the organism. As is known, the functioning of the immune system is built on the balance of lymphocytes (Th1 and Th2) and on equivalent production of the appropriate regulatory cytokines [15]. One of the important directions in the formation of a full-fledged immune response is the regulation of the synthesis of IFNs. The production of IFN-γ Th1 by cells and NC is carried out by stimulation with so-called IFN-γ-inducing cytokines, such as IL-12, IL-2 and TNF-a. In addition, IL-18, being a potential inducer of IFN-γ synthesis, acts as a synergist of IL-12, and both these cytokines lead in the rapid activation of the monocyte / macrophage system by stimulating the production of IFN-γ, which is actively stimulated by the action of Cycloferon.

Thus, the new knowledge about the imbalance of cytokines in AD makes it possible to add an additional criterion for the severity of this uneasy allergic disease (decreased ability to produce IFN-a and especially IFN-γ by blood leukocytes by a biological method, an increased level of IL-8 and IL-6 in serum, especially significant in patients with burden of the disease, a reduced level of IL-2) for further exposure to therapeutic agents.

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