Журнал «Здоровье ребенка» 1 (52) 2014

Epshtain-Barr virus (EBV) infection is an immune system infectious disease with chronic viral persistence. EBV possesses tropism to different cells, but its main target is B-lymphocytes and dendritic cells carrying receptor CD21 (or CR2 – receptor for C3d component of the complement system) [1–3]. EBV may also affect mucous membrane epithelium of the nasopharynx and laryngopharynx, ducts of salivary glands, gastrointestinal tract, blood vessel endothelium and smooth muscle cells [4, 5]. It was reported about possible viral infection of immunocompetent cells – Т-lymphocytes (CD3), natural killer cells (NK-cells CD16), macrophages, monocytes and neutrophils [6, 7]. At EBV infection, one may observe an immunological pattern, which is characteristic on the whole for persisting infections.

Various authors describe at EBV infection (frequently very contradictory) different changes of all components of immune system [8, 9, 10].  For example, Niedobitek G. et al. note absence of significant changes of B-lymphocytes with CD20+ phenotype in children with different severity of infectious mononucleosis (IM) as compared with healthy children or between themselves [11]. On the contrary, Kasahara Y., Yachie A., Ohga S. et al. observed increased CD21+ В-cells during the disease first 3 weeks in children with more severe IM [4, 12]. Other authors also note increase of B-lymphocytes (with CD72+ phenotype) in the same period [5, 13]. There are data about decrease of peripheral blood lymphocytes with phenotypes CD19+ (В- lymphocytes) and CD19+CD23+ (mature В-lymphocytes) during acute IM phase and in the period of early reconvalescence; such lymphocyte decrease was in direct correlation with severity of the disease [14]. At acute EBV infection, Т-cell immunity considerably changes, especially in content and functional activity of T-lymphocytes. According to different authors, such changes are also multidirectional [1, 8, 14]. Thus, at acute EBV infection, Zidovec L.S. et al., Novitsky V.V. et al. observed increase in relative and/or absolute level of T-lymphocytes (CD3+ cells) [15, 16].  Other researchers note decreased cells with a particular phenotype during acute IM phase [5, 13]. In accessible literature, changes of T-lymphocyte subpopulation are also described differently. During acute IM period or during early reconvalescence, Kasahara Y. et al., Niedobitek G. et al., Quintanilla-Martinez L. et al. detected increased content (or a tendency to increasing) of CD4+ cells [11, 12, 17]. However, according to Crawford D.H., Keltseva V.A. et al. and  Novitskiy V.V. et al., at acute IM, these cells do not change or their level lowers [10, 14, 16].  In the case of T-lymphocytes with CD8+ phenotype, the results of different authors are also controversial. Most authors point out at increased level of these cells in the acute IM phase [10, 13, 14, 16]. At that, they note the increase of activated T-lymphocytes with phenotype CD8+ CD38+ and CD8+HLA-DR+ [14, 16]. Besides, markers of CD8+ cells activity remain even in 4 months after the beginning of the acute EBV infection [18]. However, according to some data, at acute EBV infection, there is tendency towards lower content of CD8+ cells or this index dependence on the disease severity: CD8+ cells level is raised at slight IM form, but lowers at severe IM form [4, 12]. In acute IM phase, disbalance of the cell immunity reveals also in blood rise of cortical thymocyte predecessor cells (CD3+CD4+CD8+) and in different ratio between T- lymphocytes with phenotypes CD45RO+ (memory cells) and CD45RА+ (mature nonimmune or “naive” lymphocytes) [5, 10]. Immature large thymocyte population (phenotype CD3+CD4+CD8+) expresses CD21+ receptor. Obviously, this promotes these cells to be infected with the virus and explains EBV ability to affect T-lymphocytes in early stages of T-lymphopoiesis (still in thymus) [13]. Contradictory are also data concerning another effector cells – natural killer NK-cells (CD16+). Some studies point out to these cells growth (absolute and/or relative) during acute IM phase [5, 13, 17]. Other authors observed lowering of NK-cells for the same period, which were more expressed at severe IM course [4, 12].

EBV modulates functioning of both natural cytotoxicity system and immunogenesis, which may help the virus to escape protective factors of the human organism.

In view of the foregoing, the objective of our investigation was to study the state of T- and B-cell immunity in patients with acute EBV infection and attempt to correct the changes revealed. To this purpose, we were going to use the domestic multyprobiotic, which represents bacterial association of the genera Bifidobacterium, Lactobacillus, Propionibacterium and Lactococcus. The last contains 14 to 25 strains of probiotic bacteria with the living cell concentration 10⁹ to 10¹³ in one dose of the preparation [19].

Materials and methods.185 children aged 6–18 years were under our observation. They were sick with infectious mononucleosis of EBV etiology and were undergoing a course of treatment in the Kiev city children clinical infectious hospital during the last 7 years. The diagnosis was given on the basis of typical clinical aspect of disease. The examination of blood serum with immune-enzyme analysis (IEA) included determination of specific antibodies to EBV – immunoglobulins of М and G classes to core antigen (VCA), IgG to early antigen (EA) and IgG to nuclear antigen (EBNA). For detecting DNA of EBV in blood and saliva, polymerase chain reaction (PCR) was used (Vector-Best, Russia).

Expression of differential CD markers was studied in the immunology laboratory of the International center for astronomical and medical ecological investigation of National Academy of sciences of Ukraine (head of the immunology laboratory Taradiy N.N., candidate of medical science). There was studied expression of the following antigens: CD3+ (Т-lymphocytes), CD4+ (Т-helpers), CD8+ (suppressor-cytotoxic subpopulation, cytotoxic Т-lymphocytes (CTL)), CD7+ (Т-lymphocytes, FcμR-marker), CD16+ (natural killers or NK-cells), CD20+ (В-lymphocytes), CD22+ (В-lymphocytes), CD25 +IL-2R (Т-lymphocytes expressing on their surface a receptor to interleukine-2), CD45RA+ (mature nonimmune or “naive” lymphocytes) and CD95+ (Fas/Apo, apoptosis marker). State of Т- and В-cell component of immune system was determined in the acute phase of the disease. Reference values (n=15) were analyzed in the control group, which included 15 practically healthy children at the age of 6 to 18 years. Children were selected to this group taking account of anamnestic data: during last three months, there were no any diseases, vaccinations and injections of biologically active preparations. Children with signs of hypogammaglobulinemia or manifestations of allergic and chronic diseases were not included into the group.

Statistical treatment of the results were performed using methods of modern medical statistics. For data statistical processing, there was used MS Excel 2007 [20].

Results and discussion. Immunological examination of children with EBV infection showed changes in both Т- and В-cell components of immunity. In children with acute EBV infection, Т-cell component of immunity was characterized by considerable disbalance, signs of activation of antiviral protective mechanisms and signs of disturbance of the immune system regulation (figure 1).

Figure 1 – Expression of ICC differentiation markers in children with acute EBV infection; * – reliability of differences between indices in children with acute EBV infection and in children from the control group

In comparison with the control group, significantly increased was common level of lymphocytes due to relative and absolute increase of Т- и В-lymphocytes. Increased level of CD3+ cells (Т-lymphocytes) expression occurs primarily due to increased expression of differentiation markers CD95+ (Fas-ligand), CD45RA+ (mature nonimmune or “naive” lymphocytes), CD25+ (IL-2R), CD4+ cells (Т-helpers) and CD7+ Т-lymphocytes (FcμR-marker) (p < 0,05). There were estimated levels of differentiation markers of CD8+ (CTL) and CD16+ NK-cells that make a contribution into antiviral protection from EBV: CD16+ cells expression increased (p < 0,01), when the level of  CD8+ CTL cell was lowered (p < 0.05). At acute EBV infection, indices of В-cell immunity also changed; they were characterized by increased expression of differentiation markers of В-lymphocytes – CD20+ and CD22+ (p < 0,05). Thus, in examined children with acute EBV infection, we revealed changes in expression of differentiation markers, which characterize the state of Т- and В-cell immunity. We consider these changes as manifestations of immune disfunction of Т-cell type and development of clinically apparent activation of antiviral protection mainly by B-cell type. 

 At acute EBV infection, increased lymphocytes quantity in peripheral blood occurs due to expansion of marrowy origin cells (СD16 NK-cells, СD20 and СD22 В-lymphocytes) as well as owing to increased level of differentiation markers of Т-lymphocytes (СD 3 cells). Via СD7 (FcμR)-marker, there may be realized the following events: increase of intracellular calcium concentration, crossed reactions with СD25 (IL-2R), СD71, СD54 and HLA-DR, stimulation of interferon secretion (IFN-γ), increase of NK-cell cytotoxic activity, initiation of NK-cell proliferation and mediation of adhesion to fibronectin. At acute EBV infection, significant expression of Fas-ligand (apoptosis marker СD95/Fas) occurs. This marker binds to Fas-receptor on activated Т-lymphocytes and initiates thereby apoptosis of the last. Expression level of differentiating markers of stimulated lymphocytes (СD45RA and the high-affinity receptor IL-2 СD25 (IL-2R)) reflects intensity of the immune reaction: increasing quantity of these cells corresponds to high activity of immune response, which is associated with acute phase of the infection process and pronounced infectious inflammation [1].

Due to pronounced antigen load, acute EBV infection results in activation of antigen specific immunity mechanisms and antigen nonspecific immunity mechanisms (associated with natural killers and В-cells). Antigen nonspecific immunity mechanism is, on the one hand, insufficiently effective for elimination EBV viral antigens, but, on the other hand, it is the basis for generating autoimmune reactions and lymphoproliferative diseases in the remote catamnesis after acute EBV infection [2, 8]. Significance of expression of natural killer (СD16 cells) differentiating markers may be explained by the fact that these cells take a prime place in anti-infectious and antitumoral protection and are closely associated with autoimmune cytotoxic and lymphoproliferative processes [4, 12]. СD16 cells are the ones that circulate only in blood (without recirculation between blood and lymph) and may complete differentiation into mature forms in bloodstream. Besides receptors for cytokines, membrane of natural killers contains receptors for МНС-1 antigens and receptor for Fc-ends (IgG-FcyRІІІА (CD16)); they are the only inhibitory receptors that interact with soluble ligands. Natural killers CD16 take part in recognition of non-peptide antigens in complex with СD1-molecules, produce interleukin 4 (IL 4) and activate humoral immune response stimulating differentiation of Th0 into Th2; they also express a lot of Fas-ligand (CD95), which binds to Fas-receptor on activated Т-lymphocytes and triggers these cells apoptosis [4, 12]. 

At acute EBV infection, increased expression of differentiating markers of B-lymphocytes may perform, on the one hand, a compensatory role. But, on the other hand, such expression may testify that active elimination of EBV infected (EBV+) B-lymphocytes does not take place (may be due to lowered expression of the CTL differentiating marker (for СD8-cells)).

From our point of view, among studied indices of  Т- и В-cell immunity, predictors of transition from acute EBV infection into the chronic condition are expression levels of: CD8+ Т-lymphocytes, CD16+ natural killers, CD95+ (Fas-ligand, apoptosis marker), CD 20+ and CD22+ В-lymphocytes. Exactly, expression levels of differentiating markers of these cells in acute period and during course of the disease indicate the transition from the acute infection into the chronic state.

Thus, patients with acute EBV infection had variations of expression of all studied differentiating markers of Т- и В-cell immunity. Accordingly, in order to correct revealed disorders, our children received the multyprobiotic – one packet once a day during a month [21]. All children received a basic therapy, including antibacterial and antihistaminic preparations, enterosorbents, symptomatic and local treatment and, when needed, disintoxication infusion therapy with inclusion of glucose-salt solutions. Immunity parameters of children were studied in 1 month after the beginning of therapy.

We estimated influence of the developed therapy scheme with addition of the probiotic on immunologic indices in children using single-factor analysis of variance; the data obtained are shown in figure 2.

Figure 2 – Expression of ICC discriminative markers in children with acute EBV infection at admission to hospital and in 1 month after the beginning of treatment; * – significance of differences for studied indices  in children with acute EBV infection in 1 month after the beginning of treatment as compared with the control group; ^# – significance of differences for studied indices  in children with acute EBV infection in 1 month after the beginning of treatment in comparison with the indices at admission

In children with acute EBV infection, in a month after the beginning of treatment, we observed that levels of expression for CD4+Т-lymphocytes-helpers, CD8+Т-lymphocytes (CTL), CD7+Т-lymphocytes (FcμR-markers), differentiating markers of activated lymphocytes  CD25+ (IL-2R), CD45RA+ (mature non-immune lymphocytes) and CD95+ (Fas-ligand, apoptosis marker) were approaching to the indices for children from the control group (р > 0,05). The expression levels of differentiating markers for other immunocompetent cells (ICC) – CD3+lymphocytes (common lymphocyte pool due to both Т- and В-lymphocytes), CD16+cells (NK-cells, natural killers), CD20+В-lymphocytes and CD22+В-lymphocytes –  maintained increased in comparison with the control group (р < 0,05) (fig. 2).

On the background of ongoing therapy with inclusion of the multyprobiotic, we registered the following changes in the disease dynamic of our patients. The expression level of differentiating marker for CD3+ lymphocytes, representing general quantity of Т- and В-lymphocytes, maintained raised and was equal 1,58 ± 0,08 g/l, which is close to the index at admission (1,53 ± 0,05 g/l) (р > 0,05). The expression level of differentiating markers for major cells, CD4+Т-lymphocytes-helpers and CD7+ Т-lymphocytes (FcμR- marker), which take part in antiviral protection and in relevant reaction of the immune system and of all organism, was twofold lower in comparison with the index at admission to hospital (p < 0,05). The expression level of cytotoxic Т-lymphocytes (CTL) (CD8+Т- lymphocytes) in dynamics on the background of the therapy increased up to 0,68 ± 0,06 g/l as compared with the level at admission (0,43 ± 0,03 g/l) (р < 0,05) (fig. 2). The expression level of differentiating marker for B-lymphocytes (CD20+ and CD22+) on the background of the applied therapy lowered twofold as compared with the level at hospitalization (р<0,05). The expression level of natural killers, which takes active part in antiviral protection and are a predictor for transferring from acute EBV infection into the chronic phase, also decreased twofold in examined children (p < 0,05) (fig. 2). The expression level of differentiating markers for activated ICC (CD25+ lymphocytes (IL-2R)) decreased threefold, for CD45RA+ lymphocytes (mature non-immune lymphocytes) – fourfold and for CD95+ lymphocytes (Fas-ligand, apoptosis marker) – fivefold as compared with the index at hospitalization (p < 0,05).

Thus, in children with acute EBV infection on the background of prescribe therapy with addition of the probiotic, changes of indices of Т- and В-cell immunity had marked positive dynamics, which testifies about liquidation of pronounced inflammatory reaction from the side of all organism. This associates with adequate clinical recovery, the absence of prolonged infectious process and absence of transfer into chronic EBV infection.

Lower we will go into detail on the dynamics of expression of ICC differentiating markers that play the role of predictors for transfer from acute EBV infection into the chronic state; these are CD8+Т-lymphocytes, CD16+natural killers, CD95+Fas-ligand, CD 20+ and CD22+В-lymphocytes. Level of CTL expression (CD8+Т-lymphocytes) at hospitalization was lowered (0,53 ± 0,03) versus the index for children from the control group (p<0,05). In the dynamics of disease at the background of prescribed therapy, it increased (0,68±0,06) and reached the level characteristic for children from the control group (p > 0,05). At hospitalization, expression level of СD16+natural killers in children with acute EBV infection was increased threefold (1,32 ± 0,07) versus the index in the control group (0,45 ± 0,02) (р < 0,01). During treatment this index lowered twofold (1,02 ± 0,13) (р < 0,05), but still was insignificantly raised versus the control group (р < 0,05). Expression levels of B-lymphocytes differentiating markers at admission to hospital were raised for CD20+В-lymphocytes fourfold (2,12 ± 0,30) and for CD22+В-lymphocytes – sevenfold (2,38 ± 0,40) versus the index for children from the control group (p < 0,05). At the therapy background these levels lowered twofold (1,02 ± 0,13; 1,02 ± 0,02) (р < 0,05), but still remained insignificantly increased versus the index for the control group (0,54 ± 0,06;  0,34 ± 0,04) (р < 0,05). Expression level of CD95+Т-lymphocytes Fas-ligand in patients with acute EBV infection at hospitalization was sharply increased (5,29 ± 0,50) and surpassed tenfold analogous index in the control group (0,54 ± 0,09) (р < 0,001). In course of the disease, this level lowered fivefold (1,0 ± 0,2)  (р < 0,001) but still did not reach the index in the control group (р < 0,05).

Therefore, inclusion of the multyprobiotic into the treatment of acute EBV infection improves efficiency of the treatment, has marked clinical effectiveness, promotes clinical recovery, prevents from prolonged course of the disease and opposes to transfer into the chronic state. The multyprobiotic has an effect upon the following components of the immune system: Т- и В-cells via normalization of expression levels of differentiating markers of basic ICC, exerts an immunoregulatory influence (especially at activation of cell immune response), has an immunomodulating effect, which is specially pronounced at insufficient cell immunity.