CLINICAL FEATURES OF THE COURSE OF CHRONIC EPSTEIN-BARR VIRAL INFECTION DEPENDING ON THE TYPE OF IMMUNE REACTION OF ORGANISM

The results of the study of the main regulatory cytokines levels in patients with chronic forms of EBV-infection (CEBV) are presented the work. Aim of research. To establish the types of immune reaction for prognostication of the clinical course of disease in patients with chronic forms of EBV-infection on the base of the analysis of multidirectional synthesis dynamics of anti-inflammatory cytokines (IL-1β, TNF-α, IL-6), regulatory IL-2 and anti-inflammatory cytokines (IL-4 and IL-10). Methods. The research oт the study of the main cytokines levels by IEA method in the disease dynamics included patients with EBV-infection (n=80). The serum concentrations of studied cytokines: IL-lβ, TNF-α, IL-6, IL-2, IL-4, IL-10 were determined using test-systems by LTD “Protein outline” (Saint Petersburg, Russia), using the producer’s instruction, by sIEA. Statistical analysis of the results of research was carried out using the program Statistika 6.0 for Windows (Stat Soft Inc, USA). Results. The analysis of cytokine profile dynamics in patients with EBV-infection allowed reveal the multidirectional changes of synthesis of studied anti-inflammatory and proinflammatory cytokines that became a base for establishing of two types of immune reaction: dissociative and hyporeactive. Dissociative type was characterized with the low parameters of anti-inflammatory cytokines and regulatory IL-2 on the background of moderately increased values of anti-inflammatory cytokines, whereas the hyporeactive type was characterized with the low concentrations of all studied parameters. Conclusions. The received results of researches prove the existence of cytokine imbalance at EBV-infection. The established types of immune reaction testify to the inadequate cellular-humoral reactivity of organism under conditions of EBV prolonged persistency that is manifested by inclination to suppression of cellular-mediated and intensification of humoral mechanisms of immune response and is reflected in clinical-biochemical manifestations of disease that results in prolonged wavy course. Just the functional imbalance of immunity links to the priority of Тх2 type, which biological effects are directed on the limitation of intensity and prevalence of inflammatory process in different organs, favors the prolongation of viral persistency.


Introduction
In last years it was proved, that the initiators of immune response in human organism are cytokines that not only actively participate in formation of regulatory defense reactions but also provide homeostasis of organism in whole [1,2].Imbalance in the system of cytokine regulatory chain is a node link of immune disorders at EBV-infection.
It was established, that EBV-infection and HIV-infection is a disease of immune system, characterized with prolonged persistency of virus.This virus is a lymphotropic agent, causing the development of the lymphoproliferation and immune deficiency syndromes.Only in the case of infectious mononucleosis (IM) the one of main persistency places are B-lymphocytes that essentially influence the state of humoral immunity link, causing immune suppressive states.In connection with these properties of EBV immune system loses the ability to control the infectious process, induced by this virus or caused by the present outside microflora [2][3][4].
Prognostication of the results of EBV-infection depends on the presence and intensity degree of immune dysfunction, genetic predisposition to the one or another EBV-associated diseases and also on the series of external factors (stresses, infections, surgical interventions, unfavorable influence of environment), that injury the immune system.The scientists established that EBV has the large number of genes that give it a possibility to avoid the human immune system to certain extent.Particularly, EBV produces proteins -analogues of human interleukins and their receptors

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that change the immune response [5].In the period of active reproduction the virus produces IL-10-like protein that suppresses T-cellular immunity, function of cytotoxic lymphocytes, macrophages, disturbs the all stages of the natural killers functioning.The other viral protein can block the activity of cells-killers by inhibition of interleukin-12.At the same time EBV has a high mutable ability that allows it avoid for some time the influence of specific immunoglobulins (produced to the virus before its mutation) and the cells of the host's immune system.Thus, the EBV reproduction in human organism can be the cause of appearance of secondary immunodeficiency [6][7][8][9][10][11][12][13][14][15][16][17].
The disturbance of balance between the main regulatory cytokines obviously can lead to prolonged EBV persistency, often relapses and progressing of disease.From these positions EBV-infection can be considered as "unbalanced cytokinemia" [3][4][5].
The researchers think that predominant participation of cytokines, produced by Тh-2-lymphocytes is associated with prolonged viral persistency and chronization of process, whereas the activation of Тh-1 type -with spontaneous recovery at the acute EBV forms [18][19][20][21].That is why the study of data about the production of main regulatory cytokines with establishing of the type of immunologic reaction at EBV-infection is topical.

Aim of research
To establish the types of immune reaction for prognostication of the clinical course of disease in patients with chronic forms of EBV-infection on the base of the analysis of multidirectional synthesis dynamics of anti-inflammatory cytokines (IL-1β, TNF-α, IL-6), regulatory IL-2 and anti-inflammatory cytokines (IL-4 and IL-10).

Materials and methods
The work was carried out at the department of general and clinical immunology and allergology of the medical faculty of Kharkov national university, named after V. N. Karazin and clinical bases of the department of Regional clinic infectious hospital of city Kharkov and MMI "City polyclinic № 6", Kharkov in 2009-2015.
The group of comparison included 20 clinically healthy young people without the signs of acute or any chronic pathology, among which 16 were examined as to the EBV markers, mean age 24,1±3,2 years.In anamnesis of their life were absent the data about IM and "serologic profile" was characterized with the presence of ЕВNА -Ig G only and absence of EBV DNA in the blood plasma and saliva.All patients with IM and lymphadenopathy necessarily underwent bacteriological study of smear from nasopharynx as to pathogenic flora and diphtheria.
The complex of examination of patients included clinical analysis of blood, revelation of atypical mononuclears, determination of specific Ig to EBV by the method of solid-phase immune-enzyme analysis (sIEA), revelation of EBV DNA by the method of polymerase chain reaction (PCR) in the blood and saliva, activity of asparaginic and alanine transaminases (ASAT, ALAT), lactate dehydrogenase (LDG), and creatine phosphokinase (CPG), content of C-reactive protein (CRP), fibrinogen in the dynamics of disease.
For verification of diagnosis the complex of serologic and molecular-genetic studies was carried out besides the general blood analysis.Heterogenetic test in Hoff-Bauer (HB) modification was applied as a screen express-analysis of blood as to the presence of EBV-infection (Chireshkina N. M., 1973).
Molecular-genetic studies included the determination of EBV replicative activity on the base of revelation of EBV DNA in the blood serum by the qualitative method of PCR using test-systems, made by SPF "Litech" (Russia).
The serum concentrations of studied cytokines: IL-lβ, TNF-α, IL-6, IL-2, IL-4, IL-10 were determined using test-systems by LTD "Protein outline" (Saint Petersburg, Russia), using the producer's instruction, by sIEA.The material of the study was the blood serum of patients with CEBV, taken in the period of aggravation of disease.The study included 80 patients with CEBV.
Technical realization of the analyses was carried out in the clinical-diagnostic laboratory of region clinic infectious hospital of city Kharkov (RCIH), medical laboratory "SINEVO".
Statistical processing of the results was carried out using the program Statistika 6.0 for Windows (Stat Soft Inc, USA) on personal computer with processor Pentium II Celeron 850 PPGA.The mean arithmetical (М), mean square deviation (σ), mean error of mean arithmetical (m) were calculated for each variational series.The assessment of reliability of the mean values (р) was carried out using Student-Fisher criterion (t).The divergences were considered as reliable ones at the value р<0,05.The data, received at examination of this category of patients, were presented in the article as absolute values.

Results of research
Having analyzed the cytokine profile dynamics in patients with EBV-infection we revealed the multidirectional changes of synthesis of studied anti-inflammatory and proinflammatory cytokines that became a base for establishing of two types of immune reaction: dissociative and hyporeactive.
The data about the reveled types of immune reaction are presented on the

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The results of analysis of the influence of established types of immune reaction on CEBV clinical course and of the study of the parameters of mean duration of the main clinical symptoms in patients with the different types of immune reaction are presented in the Тable 1.As it can be seen from the presented data, in patients with CEBV with established hyporeactive type of immune reaction (1 group) the parameters of main clinical symptoms were characterized with the less duration comparing with analogous parameters in patients with dissociative type (2 group) on the background of antiviral therapy.

Discussion of results of research
The studies of last years established that cytokine spectrum at EBV-infection depends on the balance of the links of organism immune response and the disturbance of balance of Th1/Th2 cytokines production by cells plays an important role in immunopathogenesis.The most scientists are agreed that the predominant participation of cytokines, produced by Тh-2-lymphocytes, is associated with the viral persistency and chronization of process and Тh-1 -with spontaneous recovery and elimination of causative agent.
Despite the significant attainments in this field, many questions as to the character of cytokine producing ability of immunocompetent cells and their immunopathogenetic features at CEBV-infection remain unexplained for today, and the literary data are not unambiguous and sufficiently grounded.
Thus, in patients with dissociative type of immune reaction was observed the low production of anti-inflammatory cytokines and regulatory IL-2, whereas the levels of proinflammatory IL-4 and IL-10 reliably increased in correspondence with the process intensity.IL-4 exceeded the parameters of control group in 5,7 times and IL-10 exceeded in 6 times the mean values of control group (р<0,05).

Table 1
Duration of the main clinical symptoms depending on type of immune reaction in patients with CEBV (n=80)