Pathophysiology of hepatitis С : case – control study of cell reactivity and adaptation tension level in patients with chronic HCV-infection at Bukovyna ( South-Western Ukraine region )

Introduction: Hepatitis C is a disease with a significant global impact. It had estimated that there are 2–5 million HCV-positive persons in Europe. It is very difficult to predict the individual course of hepatitis C due to the many factors influencing the disease progression. Сell reactivity and level of adaptive tension had been considered as additional parameters for the evaluation of individual immune response and may assist to predict an intensity of autoimmune liver inflammation caused by HCV.


Background
Hepatitis C is a disease with a significant global impact.According to the WHO, about 150 million people are chronically infected with hepatitis C virus, and more than 350 million people die every year from hepatitis C-related diseases (cirrhosis, hepatocellular carcinoma) [2,11].The cause of hepatitis C, HCV, is an enveloped, spherical, single-stranded RNA virus belonging to the Flaviviridae family.The natural targets of HCV are hepatocytes and, possibly, B-lymphocytes.Viral clearance is associated with the development and persistence of strong virus-specific responses by cytotoxic T lymphocytes and helper T cells [3].
Hepatitis C virus is a blood-borne virus and the most common modes of infection are through unsafe injection practices, inadequate sterilization of medical equipment, and the transfusion of unscreened blood and blood products [8].
The incubation period lasts 15-150 days for the acute phase, but only 15 % of patients will present the latter.Chronic symptoms appear 20-40 years after contamination.Symptoms of cirrhosis include weakness, anorexia, weight loss, and gynecomastia in men, a skin eruption on the palms, blood-clotting disorders, and telangiectasia [10].
There are considerable region differences.In some countries, e. g., Egypt, the prevalence is as high as 20 %.According to different estimates, up to 3 percent of people in Ukraine are ill with viral hepatitis C.There was no official statistics before 2009 and registration of people ill with chronic viral hepatitis С had not carried out in Ukraine until 1 July 2009.Detection rate of antibodies (anti-HCV) demonstrated that the percentage of HCV carriers varies in Eastern Europe from 1.5 to 4 %.The annual incidence rate of acute hepatitis C in Europe is 1-3 cases per 100 thousand populations [10].In Western Europe, the frequency of HCV 0.4-3 %.There are 6 genotypes of HCV, and genotypes 1a and 1b are more distributed [9].
In most infected people, viremia persists and is accompanied by variable degrees of hepatic inflammation and fibrosis.Findings from studies suggest that at least 50 % of hepatocytes may be infected with HCV in patients with chronic hepatitis C.
RNA-dependent RNA polymerase, an enzyme critical in HCV replication, lacks proofreading capabilities and generates a large number of mutant viruses well known as quasi species.These represent minor molecular variations with only 1-2 % nucleotide heterogeneity.HCV quasi species pose a great challenge to immune-mediated control of HCV and could explain the variable clinical course and the difficulties in vaccine development [3,9].
Hepatocytes, monocytes/macrophages, B-lymphocytes and polymorphonuclear leukocytes are targets of HCV.Direct infection of these immune cells determine immunological disorders of innate and adaptive immunity, which are registered in more than half of patients with chronic HCV-infection [7].In chronically infected patients, the T-cell response to the HCV is polyclonal and multispecific, although it is not as strong as the response in acutely infected patients who demonstrated a more vigorous T-cell response.Importantly, viral clearance in acutely infected patients is associated with strong CD4 (+) mediated T-cell response.Thus, the dominant cause of viral persistence during HCV infection may be due the development of a weak antiviral immune response to the viral antigens, with corresponding inability to eradicate infected cells.Liver cell damage may extend from virally infected to uninfected cells via soluble cytotoxic mediators and recruitment and activation of inflammatory cells forming the necrotic inflammatory response [8].
By literature data, up to 80 % of patients with acute hepatitis C develop chronic infection that manifests as persistent HCV RNA detected in the blood with / without abnormal liver function tests.Most patients with chronic hepatitis C have no or only mild symptoms, including fatigue, anorexia, and nausea [2,11].
Anti-HCV antibody is not protective, and its level does not necessarily remain elevated in patients with chronic hepatitis C. It is important to investigate the level of adaptation tension in patients with chronic hepatitis C as well as a cell reactivity status associated with indicators of innate and adaptive immunity [6].Current study aimed to recognize adaptation processes, and depending on it will be possible to support a prediction of the clinical course of disease and efficacy of holiatry.
Aim of the study was to investigate pathophysiologic mechanisms of cell reactivity and adaptation tension level of the patient's organism with chronic HCV-infection.

Materials and Methods
Prospective case-control study was conducted within 2015 year at the Municipal Hepatology Center chaired at Chernivtsi Municipal Hospital.31 patients with chronic hepatitis C were enrolled.Patients with next gender allocation -17 male patients and 14 female, aged 18-65 years (average age 44.55 ± 1.14), were admitted to Municipal Hepatology Center and hospitalized for holiatry.The study included patients who had no other exacerbation of chronic diseases but hepatitis C. Patients with autoimmune, allergic diseases, tuberculosis and cancer of any localization, as well as patients who got three or more days of therapy with antibiotics were excluded.All persons involved into the study gave written informed consents.
The control group included 30 healthy volunteers (21 male and 9 female), aged 46.81 ± 2.41.All of them were enrolled for basic study group with preliminary diagnosis of HCV-infection.Presence of HCV and it viral load at the time of admission was atudied by PCR.
All patients and healthy individuals who were enrolled the current study underwent blood tests.Absolute and relative amount of basic populations of immunocompetent cells was determined by traditional method.We determined the following nonspecific adaptive response of the body of patients: stress, training reaction, calm response, and reaction of elevated activation [1,5,6].
We used adaptation index as an integrated test [1].Cell reactivity was studied with means of determination of leukocytic index of intoxication, endotoxemia nuclear index level, hematologic index of intoxication [4][5][6].
Cell reactivity and adaptation level in the organisms of patients were determined by the degree of immune disorders for each indicator [4].Sign «+» indicated hyperproduction of appropriate cell population, and «-» negative value considered the deficit accordingly.
The results of clinic and immunological studies were estimated with methods of variation statistics.Average error (M±m) was determined.Certain differences between the averages of samples performed by using t-test Student.

Оригинальные исследования
Differences of medium and relative frequencies considered as significant at the level of confidence (p) less than 0.05.The research results proceedings was performed by means of the software Statistica for Windows 5.0 (StatSoft Inc., USA).

Results
A leading role in providing of adaptation activity of a human organism belongs to immunocompetent cells in circulating blood.Simultaneously system of blood is one of a major transmitters of information about processes that being across the level of tissue structures.All immunocompetent blood cells as a part of peripheral immune system are very sensitive to the changes of environment as well as to internal state of organism.The parameters of absolute and relative amount of basic populations of immunocompetent cells changes can extend up to limit adaptation possibilities of organism.It had evidenced that power mechanism occupies the main place in the processes of adaptation.Use of adaptation reactions will give an opportunity to carry out the individual going near determination of adequacy of adaptation reactions with taking into account of the state of organism.The use of adaptation reactions will give an opportunity to carry out the individual determination of adequacy of adaptation reactions with taking into account the state of the organism.
Results assessment showed changes of absolute and relative amount of basic populations of immunocompetent cells of peripheral blood in patients with HСV-infection.Agranulocytes' relative amount was established on 8.19 % (p < 0.05).Consequently, a relative amount of monocytes decreased on 36.90 % and it was accompanied with tendency to decrease of relative amount of lymphocytes on 4.32 %.It was observed that absolute amount of monocytes fell down on 60.0 %.On this background the relative amount of granulocytic polymorph nuclear leucocytes increased on 8.06 % (p < 0.05) due to elevation of relative amount of neutrophils on 8.26 %.The absolute amount of thrombocytes diminished on 20.34 % in patients with chronic HCV-infection.All patients envolved in basic group demonstrated increase of erythrocytes sedimentation rate in average on 73.03 %.
Values of relative lymphocyte count and segmentic nuclear neutrocytes were assessed as well for determination of adaptation index for every patient.Results of absolute and relative quantities of the basic immuno-competent cells determination in enrolled patients described in Table 1 below.
Detailed comparison analysis of mentioned parameters allowed us to make conclusion about decrease of relative quantity of agranulocytes up to 8.19 % (p < 0.05).Moreover relative quantity of monocytes had drop down on 36.90 % and the tendency for decrease of relative lymphocytes amount had observed up to 4.32 %.Simultaneously the absolute figure of monocytes had declined up to 60.0 %.
It was determined that relative amount of polymorphic nuclear leucocytes was decreased on 8.06 % (p < 0.05) due to increase of relative amount of neutrophil granulocytes on 8.26 %.Absolute platelet count was reduced by 20.34 %.The obtained changes in the absolute and relative amount of major populations of immunocompetent cells of peripheral blood of enrolled patients attest to the systemic inflammatory process, evidence of which wass erythrocyte sedimentation rate significant increase in 73.03 %.
Based on the values of relative amount of lymphocytes and segmented neutrophils adaptation index for each patient was assessed [1].The prognostic value of adaptation index was well-proven.Reaction to the stress, training reaction, reaction of the «calm» activation and reaction of increasing activation of adaptation were assessed (Fig. 1).
Value of adaptation index in patients with HCV-infection and in volunteers being determined in the zone of «reaction on training», but this index for basic group had tendency to going down on 12.20 %.
Distribution by individual types of adaptation tension level in patients with chronic HCV-infection at Bukovyna was showed on Table 2.
Each range of values of adaptation index correspondingly answered for adaptation reactions.High values of adaptation index corresponded to the more favorable prognosis of the disease [1,5] (Table 3).
Stress response was diagnosed in 7 patients with HCV-infection, that testifies the unfavorable prognosis of clinical course.Favorable forecast was detected only in 9 (29.03%) patients, in majority -22 (70.97 %) it was predicted as unfavorable, that requires not only intensive facilities of treatment, but also an intensive psychological rehabilitation.

Discussion
It was proven that index of cellular reactivity of organism in patients with HCV decreased on 32.03 %.Haematological index of intoxication after V. Vasiliev increased on 32.25 %.Hypothesis about intoxication going outside interstitial liver space and it influence on toxins in circulatory peripheral blood increase was proved.Nevertheless, nuclear index increase on 81.25 % certified the presence of middle level of endotoxicosis.Parenchymal damage of cell membranes could lead to significant metabolic disorders, that plays a major role in liver fibrosis formation [7].

It was established an evident increase of intoxication
Patients with chronic HCV-infection abs.
Patients with chronic HCV-infection % Healthy volunteers abs.Original research index on 33.33 % and tendency to the elevation of leucocytic indexes of intoxication calculating by Ya.Kal-Kalif up to 5.73 % and by V. Rais on 8.63 %.At the same time the lymphocytic-granulocytic index increase on 4.40 % certifies level of endogenous intoxication and activating of processes of cells and tissues liver disintegration increase.Endogenic intoxication triggered hepatic cells cytolysis caused by viral reproduction with subsequent immune system reaction in HCV chronic phase.From pathophysiologic point of view, this syndrome was accompanied by lipid peroxidation processes increase, blood cells damage, disorders in antioxidant and immune system activity.

Conclusions
1. Level of adaptation processes decreased on 12.20 % in patients with chronic hepatitis С.The majority (70.97 %) of patients had determined in the zone of stress reaction (22.58 %) and reaction on training (48.39 %) by the adaptation index.
2. It have been established an evident increase of intoxication index on 33.33 % and tendency to the elevation of leucocytic indexes of intoxication calculating by Ya.Kal-Kalif up to 5.73 % and by V. Rais on 8.63 %.At the same time the lymphocytic-granulocytic index was increased on 4.40 % that certifies level of endogenous intoxication increase and activation of processes of cells and tissues liver disintegration.
3. Cellular reactivity of organism of patients with chronic hepatitis С decreased on 32.03 %, that was confirmed by an increase of nuclear index of endotoxicosis level on 81.25 %.In general, HCV-infected Caucasian persons was characterized by moderate endotoxicosis level accompanied by activating of processes of disintegration of cells and liver tissues.Оригинальные исследования

Table 1 .
Absolute and relative figures of main immune-competent cells of enrolled chronic HCV-infected persons at Bukovyna, M ± m

Table 2 .
Distribution by individual types of adaptation tension level in patients with chronic HCV-infection at Bukovyna, M ± m

Table 3 .
Cell reactivity of organisms of patients with chronic HCV-infection at Bukovyna, M ± m