The comparative analysis of limfogramy’s indicators in adults with verified allegropathology on the background of replicative virus activity

Introduction. For the last few decades the prevalence of allergic, including atopic diseases, acutely grow up [15]. Atopic diseases are belonging to the multifactorial diseases  the development of which is determined by the influence of both genetic factors and environmental factors. It is crucial to talk that inherited not the specific allergic disease but only the predisposition to it [14]. As it is believed that genetic factors are not subject to the special dynamics in the population increase the frequency of allergic diseases due to the influence of epigenetic factors. First of all it is an increase external allergic load on immune system, which caused by negative environmental changes, features of modern urban food, by the extension of harmful habits, by uncontrolled using of drugs. Often it is epigenetic factors are the cause of the increase allergic pathology in developed countries. Among these factors group the main role belongs to the increasing prevalence of infectious load on the human body [1, 12]. Viruses and people have evolved together during the whole period of human existence, so immune system has formed certain protective antiviral mechanisms. However, some viruses, particular Herpes virus, have adapted to parasitism primarily due to a latent stage. In the scientific literature we can see expression that Herpes viruses are the part of the human “microbiome”, because they are adapted to the eternal infection their hosts [4]. It remains a high risk of viral reactivation, that can be a trigger of severe illness or complications of existing pathology. The opposite approach to this problem is to answer the question: what happens when the immune system will no longer coexist with viruses or meet them in limited quantities. According to many studies the results are the frequency prevalence of allergic diseases. First hygiene hypothesis, that infectious diseases in early childhood can have a preventive role in the formation of allergies was raised back in the 80-s of last century [9]. However, results of scientific research regarding to the association of viral infection as a trigger of allergic pathology today are controversial. In most cases they refer to the role of Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus type 6 (HHV 6), moreover, submitted different versions of the mechanisms of this effect [2, 3, 7]. However, most scientists motivated by the fact that viruses affect the differentiation of T cells, which leads to an imbalance between Th-1 and Th-2 (T-helper type1/type2) by immune answer [11].

Aim. The aim of our studies was to identify the phenotypic characteristics of lymphocytes and their active markers in patients with chronic persistent EBV and HHV 6 in stage of replicative virus activity with clinical and laboratory manifestations of allergy pathology.

Methods. It was examined 58 people who were in outpatient treatment and observation at Lviv Regional Medical Center of Clinical Immunology and allergy during 2014-2015 years. Patients age average value: 22.6±2.4 years, among whom were 33 (56.9%) women and 29 (43.1%) men.

Verification of the diagnosis carried out on the basis of clinical disease, anamnesis, including allergic. Patients made general laboratory and instrumental research, cytology research of pap-mark from the nasal mucosa, prick tests allergen extract (Diater, Spaine) determine the general and specific IgE (sIgE) by immune enzyme test using test system “Euroimmun”. Determination of DNA EBV, CMV and HHV 6 type in blood, saliva and scraping the mucous membrane of the posterior pharyngeal wall performed by PCR on the diagnostics “AmpliSens” (Russia) using “Rotor Geen 6000” (Corbett Recearch, Australia). Lymphocyte phenotyping and determination of basic expression of activation markers was performed with using monoclonal antibodies for flow cytoflyuorymetri “Bekton Diskenson” (USA).

Research results were analyzed with using the method of variation statistics using STATISTICA 6 (Statsoft, USA). The control group consisted of 20 healthy individuals of appropriate age and gender.

Results.  At the time of consultational examination, patients complained such: periodic nasal congestion, which accompanied by lacrimation, by itch, sneeze and some time by cough. Most of them (67.2%) pointed to deterioration in spring and summer (May and June), and 10.3% patients in that period had gasp. For 12 patients (20.7%) were complaint on dryness and irritation of the skin with periodic rash and itch on the face, exposed parts of the arms and legs, on shoulders and abdomen. These skin manifestations patients associated with meal, first of all raw vegetables/fruits (apple, pear, kiwi, celery, carrot, peach etc), forcing them to follow elimination diets. 12 patients pointed to improvement while outdoors and aggravation displays indoors. Three patients associated deterioration after contact with pets. Pathological manifestations listed above most of patients treated themselves with using antihistamines, feeling temporary improvement. Based on the anamnesis was detected that relatives of 11 patients has had the similar allergy but they did not apply to allergist. For four patients we can argue about a clearly aggravated atopic anamnesis because of verified asthma in relatives (three from the part of the mother and one from the father).

For the results of the general laboratory tests was found that in general and biochemical blood tests in most patients were not deviations from normal values, but for 14 people (24.1%) were found absolute mild eosinophilia, for ten (15.5%) — minor relative lymphocytosis, for 4 (6.9%) — increased the relative number of monocytes (from 12% to 14%,when normal is to 10%). In smears nasal mucosa for 28 (48.3%) people were found increased number of eosinophils (from 21% to 92% in sight) and isolated white blood cells that indicates on allergic rhinorhea because the eosinophils are indirect indicators of allergic reactions. Patients complaining of shortness of breath periodically evaluated the functional state of lungs under spirography. The results did not show pathological changes. All patients were screened for the presence of worms (IEA for the presence of immunoglobulins A, M, G to antigens for Giardia, roundworm, toxocara (“+”- ≥0,35) + stool for  helminth’s eggs and protozoa) and the results verified that 10 patients has had ascaridosis, 7 patients has had giardiasis, 3 — toxocarosis, 2 — ascaridosis+toxocarosis and one patient has had ascaridosis+giardiasis). All patients received appropriate treatment recommendations.

According to the consensus of molecular allergy diagnostics (A WAO-ARIA-GA2LEN consensus document on molecular-based allergy diagnostic, 2013), received medical history in the first phase of research, which indicating the presence of allergic disorders in all patients, we  moved to the second stage of diagnostic research [6].

For this purpose to all patients were made prick-tests with using allergens “Blend of herbs” (Phleum pratense, ryegrass, dactylis glomerata, Poa pratensis) and “Blend of pollen spring trees” (alder, birch, hazel, ash), extracts of household and epidermal allergens “Blend of acarus” (Dermatofagoides farina and Dermatofagoides pteronissinus)”.

For results of skin tests was found: 5 patients has had increased skin reaction on household allergens, in particular – “Blend of acarus of house dust”, 9 people has had reaction on “Blend of herbs”, 5 patients has had allergic reaction on “Blend of  pollen spring trees”, 22 patients has had reaction on “Blend of herbs” and “Blend of pollen spring trees”, 11 patients – on “Blend of herbs”, “Blend of pollen spring trees” and household allergens, 4 patients — “Blend of herbs”, “Blend of pollen spring trees”, cat and dog allergens. This skin reaction was different from “+” to “++++”, which indicated different degrees of sensitization to relevant allergens. For two persons the results of skin tests were negative.

In this manner, the result of the diagnosis of allergies indicated that for 19 people were found monosensitization, and for 32 people — polysensitization.

The next stage of research was to determine the general and specific  IgE-antibody with using ELISA. According to the research found that for 42 (72.4%) patients the general serum IgE was higher than normal levels and was in the range of 128-3012 IU/ml. For 11 patients (27.6%) the general indicators of IgE were within normal limits (to 100 IU/ml).

The research results of specific IgE (ELISA) had some differences with the skin prick-tests, namely: monosensitization (>0.35 kU/l) was found for 16 person, for 35-polysensitization. For patients with monosensitization to acarus of house dust, timophy, birch under the first stages of diagnostic (prick tests, ELISA) was not doubt of diagnostic. In accordance, we put the preliminary diagnosis and allergy immunotherapy was recommended. For patients with polysensitization, after selecting the correct treatment strategy they could pass the third stage of allergy diagnostic — carrying out components research.

To achieve our goal to all patients carried out the molecular genetic studies of saliva, blood and mucus scraping of posterior pharyngeal wall. The results of PCR established, that for 13 patients DNA-virus was not found. For another 40 persons (75.47%) identified: DNA EBV “+”- for 12 people, DNA HHV6 “+” — for 6 people, the remaining patients the association of DNA EBV and HSV6 “+” — 18 people, the association of DNA EBV, HHV6 an CMV for 4 patients.

So, in most of patients (45.0%) were found mixed infection of EBV and HHV 6,moreover the replication founded in difference biological environments, but most of all (55.6%) — while in scraping of the back wall of the throat and in saliva. Interesting is that in patients with allergic symptoms in anamnesis, which did not confirmed by following steps allergic diagnostic also was found  the chronic persistence EBV and HSV6 in the  the stage of replicative activity (PCR ”+” saliva). 85.0% of patients with virus activity identified elevated levels of general serum IgE, are often in mixed infection EBV, HHV 6 and CMV [16].

Based on our researches we have highlighted 4 groups of patients: 1st group-10 people with verified of preliminary investigations allergic diagnosis, DNA virus”-“, 2nd group — 10 people with allergic diagnosis and DNA EBV “+”; 3rd group — 12 people with allergic diagnosis, DNA “-“. Note that these groups are not included patients with atopic anamnesis and those persons with a history of infectious mononucleosis or sudden exanthema.

Patients of these groups held research of population and subpopulation of lymphocytes and their activation markers. Data listed in the table 1.

Table 1.

Comparative evaluation of lymfogramy and activation markers lymphocytes in patients of research group (М± m)

Indexes	Healthy	1st group, DNA (-) allerg.	2nd group, DNA ЕВV (+), allerg.	3rd group, DNA ЕВV, HHV6 (+), allerg.
	n=20	n=10	n=10	n=12
Leukocytes, G/l	6.53 ±0.46	5.12±0.95*	6.62±1.46	8.12±1.42
Lymphs (CD45+), %	34.40±1.87	34.08±1.43	31.31±1.52	30.14±1.50*
Lymphs (CD45+), G/l	2.22±0.15	1.71±0.07*	2.05±0.5#	2.44±0.7
CD3+, %	72.90±1.24	62.75±1.19*	68.0±1.20	64.8±0.20*
CD3+, G/l	1.61±0.10	1.06±0.03*	1.39±0.17#	1.56±0.5
CD4+, %	48.60±1.50	40.58±1.24*	46.0±1.12#	47.3±1.13^
CD4+ , G/l	0.78±0.04	0.68±0.02*	0.94± 0.22#*	1.15±0.5*
CD8+, %	24.30±1.01	20.17±1.12*	18.0±1.63*	17.09±1.61*
CD8+, G/l	0.37±0.03	0.34±0.22	0.35±0.06	0.42±0.5^
CD16+/56+, %	11.10±0.15	17.42±0.69*	8.08±1.19*#	21.06±1.21*^
CD16+/56+, G/l	0.24±0.02	0.29±0.01*	0.17±0.08*#	0.51±0.5*^
CD19+, %	9.60±0.61	10.00±0.83	12.10±1.15*	13.70±1.13*
CD19+, G/l	0.21±0.09	0.18±0.08	0.25±0.06#	0.33±0.09*^
IRI	2.04	2.05	3.83#*	2.77*
CD3+/CDHLA-DR+, %	10.7±0  1.9	7.10± 0.7*	11.24±0.92#	12.28±0.90
CD3+/CDHLA-DR+, G/l	0.16±0.02	0.07±0.01*	0.15±0.03#	0.19±0.5
CD4+/CD25+, %	10.50±0.40	11.00±0.58	17.21±1.39#*	19.03±1.41*
CD4+/CD25+, G/l	0.09±0.01	0.07±0.01	0.17±0.03#*	0.22±0.5*^

 Notes: 1) * reliability difference indices patients 1,2 and 3 groups with control  (p<0.05)

             2) # — reliability difference indices patients 1 and 2 groups  (р<0.05)

             3) ^ — reliability difference indices patients 2 і 3 groups (р<0.05);

              4) — reliability difference indices patients 1 і 3 groups (р<0.05)

As we can see in the table 1., patients with allergies pathology and DNA viruses (-) experienced a probable reduction of the absolute number of leukocytes (5.12±0.95 G/l, р<0.05) and lymphocytes (1.71±0.07 G/l, р<0.05) compared with the control group (respectively 6.53±0.46 G/l; 2.22±0.15 G/l). In general, studies limfogramy patients of group 1 indicated the formation of a kind of complex immunopathological features of T-cell deficiency parts of the immune system. This is corroborated likely decrease in absolute (1.06±0,03 G/l) and relative (62.75±1.19%) indices subpopulation of CD3+ -lymphocytes compared with healthy individuals (respectively 1.61±0.10 G/l; 72.90±1.24%, p<0.05). In persons of 1st group identified reducing the number of CD8+-lymphocytes that possessing suppressor-cytotoxic effect (20.17±1.12%, p<0.05), a significant reduction of absolute (0.68±0.02 G/l, p<0.05) and relative (40.58±1.24%, p<0.05) the number of CD4+ -lymphocytes compared with healthy individuals (respectively 48.60±1.50%; 0.78±0.04 G/l). The relative number of CD16+/56+ cells was significantly higher (17.42±0.69%, p<0.05) compared to the control group (11.10±0.15%), which probably indicates on the activation of antiviral parts of the immune system, that viruses in the latent stage. As to absolute indicators CD19+ -lymphocytes, their number was also lower (0.18±0.08 G/l) compared with the control group (0.21±0.09 G/l, p<0.05). The absolute number of activation markers of lymphocytes CD3+/CDHLA-DR+ was 2.3 times lower (p<0.05) than in the control group and approximately 2.7 lower (p<0.05) than in patients of 2 and 3rd groups which may indicate minimize inflammatory reactions at the site of persistent viruses. The absolute number of CD4+/CD25+ -regulatory lymphocytes has only a downward trend compared with healthy people and was lower (p<0.05) than in patients of 2 and 3rd groups.

Thus, in patients with allegro pathology on the background DNA viruses «-» we observed a decrease in the number of T-lymphocytes by both T-helper and T-cytotoxic lymphocytes, which suggests the presence of these patients acquired / secondary immunodeficiency by lymphocytic type of infectious genesis in the first place — the presence of a viral infection in a latent stage, resulting in activation there is some innate antiviral cellular mechanisms. Reducing the number of activated CD3+/CDHLA-DR+ -lymphocytes indicates the absence of inflammation, as evidenced by the same number of regulatory lymphocytes (CD4+/CD25+) [17]. The patients need additional serological tests for detection of specific IgG EBV (VCA, EBNA) and HH 6.

As for 2nd group patients with allergies verified pathology on the background of chronic relapse EBV infection, the results of limfogramy had its own peculiarities. The first thing that attracted attention was that the absolute levels of white blood cells probably no different from those of healthy individuals and patients of 1 and 3rd groups. The absolute number of lymphocytes in the patients of 2nd  group  was significantly higher than in patients of 1st group (2.05±0.5 G/l and 1.71±0.07 G/L, p<0.05). The absolute number of CD3 + -lymphocytes in patients of 2nd group did not differ significantly from that of healthy individuals, however, was significantly higher than in patients of 1st group (respectively 1.39±0.17 G/l and 1.06±0.03 G/l, p<0.05).

If the absolute number of T-cytotoxic lymphocytes in patients of 2nd group significantly did not differ from that of healthy individuals and patients of group 1 was lower (0.35±0.06, p<0.05) only from the 3rd group (0.42±0,5), the number of T-helper cells for these patients was significantly higher and amounted to 46.0±1.12%, 0.94±0.22 G/l (p<0.05) compared with the control and with the 1st group. According to these indicators IRI increased to 3.83, which was significantly higher compared with patients of 1st group (2.05), 3rd group (2.77) and healthy individuals (2.04). Number of NK-cells in a test group of patients was significantly lower (8.08±1.19%) from that of healthy individuals and patients of 1st and 3rd groups (respectively 11.10±0.15% and 17.42±0.62%, 21.06±0.10% p<0.05). As for the B-lymphocytes, their absolute number was significantly higher for patients of 2nd group (0.25±0.06 G/l) compared with patients of 1st group (0.18±0.08 G/l, p<0.05) and lower than in the 3rd group (0.33±0.09 G/l, p<0.05). The number of activated T and B lymphocytes in patients of 2nd group remained stable and was not significantly different from these patients of 3rd group and healthy subjects and was higher than for patients of group 1 (p<0.05). Number of CD4+/CD25+ -lymphocytes for these patients was significantly higher than in the control group and patients of 2 group, both in absolute and in relative value(respectively 0.17±0.03 G/l and 17.21±1,39%, p<0.05).

Thus, in patients with allegro pathology and chronic EBV-infection with replicative virus activity on the background of the normal number of lymphocytes and T lymphocytes observed significant increase of T-helper cells and reduced cytotoxic T-lymphocytes, which greatly deepened the defect of specific antiviral defense. Strengthened the antiviral deficit reducing the number of NK-cells [8]. The increase in relative and absolute number of B lymphocytes probably contributed to the activation of these cells for products IgE, which correlates with its level in the blood of patients in 2nd group. As we can see from the results of limfogramy the most pronounced differences were indicators of patients with verified allergopathology and replicating activity of EBV and HHV 6. For these patients, on the background of a possible increase in the number of indicators compared to the healthy and the sick of the «DNA» virus «-» observed the likely increase in T-helper and T-cytotoxic lymphocytes, and especially — NK-cells, compared with patients with DNA EBV «+» (p<0.05),what is indicating a strong activation of specific antiviral protection and the formation of inflammation. The inflammatory process of patients with allergic pathology initiated not only IgE, but activated T and B lymphocytes, suggesting to increase the number of CD3+/CDHLA-DR -lymphocytes. In turn, the increase in CD4+/CD25+ -lymphocytes from one side to minimize the destruction of target tissues (primarily — local), on the other side slows the elimination of viruses from the body by reducing the inflammatory immune response to infectious process [17].

Thus, for patients with replicative activity of EBV and HHV 6 and verified allergic pathology defined the biggest disbalance of all parts of the immune system. From the one side the formation of inflammation correlated with severe clinical manifestations in these patients and increased production of general serum IgE, and on the other — indicating conditions for the formation of replicative virus activity in the host. Note that it is in (88.9%) these patients based on allergic diagnostic research, including the component of diagnostics, verified polysensitization.

Mixed infection of EBV and HHV 6 in the stage of replicative activity stimulates the allegro-pathological violations including the formation of polysensitization for  adult patients without atopic complicated history.

Conclusion.

1. For 75.47% of patients with verified allergic pathology were found the replication of viral activity.
2. Most of all (45.0%) in allergic pathology was found replication of EBV and HVV6, including — at 55.6% while in scraping from the back wall of the throat and saliva.
3. For 85.0% of patients with allergic pathology on the background of viral replication determined increased level of general serum IgE, most of all- with mixed infection EBV, HHV 6 and CMV.
4. For patients with allergic pathology on the background EBV-infection in stage of replicative viral activity observed a significant increase of T-helpers and decrease of T-citotoxic lymphocytes with activation of humoral link, an increased number of immunoregulatory lymphocytes, that enhances inflammation and deficit of antiviral mechanisms.
5. Mixed infection of EBV and HHV6 in the stage of replicative activity stimulates the allegro-pathological violations including the formation of polysensitization in adult patients without atopic complicated history.
6. In the scheme of treatment of such patients there is a need of a phased specific antiviral therapy and specific allergotherapy provided for in the relevant protocols of allergic diseases.

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17. Zubchenko S.O. «The prognostic significance of regulatory T-lymphocytes with Epstein-Barr virus infection», Natural and Technical Sciences. – 2014. – ІІ(3), Issue 21, Р. 38-41.[schema type=»book» name=»The comparative analysis of limfogramy’s indicators in adults with verified allegropathology on the background of replicative virus activity» description=»Objective. For the last few decades the prevalence of allergic, including atopic diseases, acutely grow up. In economically developed countries, this trend is associated with the effect of epigenetic factors, the major role influences the infectious strain on the human body. In this article are the results of examination of 53 patients who under general clinical laboratory and special research verified allergic pathology, and for 16 people was found monosensitization, for 35 — polysensitization. Method. Verification of the diagnosis carried out in the basis of objective and subjective data. General laboratory and instrumental studies/ citology smears of nasal mucosa researches/ prick-tests/ determination of total and specific IgE (sIgE), molecular genetic studies has been made. Result. .Based on the molecular genetic researches in 75.47% of persons defined replicating viral activity, most (45.0%) — replication of EBV and HVV6, including — at 55.6% while in scraping from the back wall of the throat and saliva. 85% of patients with allergic pathology on the background of viral replication determined increased level of general serum IgE, most of all- with mixed infection EBV,HHV6 and CMV. Patients with allergic pathology on the background EBV-infection in stage of replicative viral activity observed a significant increase of T-helpers and decrease of T-citotoxic lymphocytes with activation of humoral link, an increased number of immunoregulatory lymphocytes,that enhances inflammation and deficit of antiviral mechanisms. Conclusions. Mixed infection of EBV and HHV 6 in the stage of replicative activity stimulates the allegro-pathological violations including the formation of polysensitization in adult patients without atopic complicated history.» author=»Zubchenko S.O.» publisher=»БАСАРАНОВИЧ ЕКАТЕРИНА» pubdate=»2016-12-23″ edition=»euroasian-science.ru_25-26.03.2016_3(24)» ebook=»yes» ]