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Immunity indexes at the system and local levels of pregnant women with mycoplasma infection.

Introduction

The problem of infectious diseases of female genital mutilation continues to be one of the most pressing and important, both in the medical--social and scientific and practical aspects. In recent years, there is a sharp increase in infections everywhere, including mycoplasma infection. Despite the considerable progress in diagnosis and treatment, these infections remain very significant in the structure of obstetrical pathology and perinatal morbidity and mortality (Kira, 2001; Prilepskaya, 2007).

Certain patterns were identified, showing significant changes in the immune system with mycoplasmosis, which have real significance in the pathogenesis of perinatal disorders. Nevertheless, peculiarities of the fundamental components of immunity, presence of cytokines in mycoplasmosis is poorly understood, while the available information is piecemeal and cannot be integrated and synthesized.

Material and methodology

The study included 90 pregnant women with mycoplasma infection and 30 pregnant women of the control group (healthy pregnant women) during the 2nd and 3rd trimesters of pregnancy.

Phenotypic profile of lymphocytes was investigated by indirect membrane immunofluorescence on flow cytometry using monoclonal antibodies to surface antigens of lymphocytes (TNF "Sorbent" by method of A. Filatov (1992): for CD3+--marker of mature T-lymphocytes; for CD4+--marker of T-helpers/inductors; for CD8+--marker of cytotoxic lymphocytes supressors; for CD16+--marker of natural killers; for CD25+ marker of L-receptor chain of interleukin 2; for CD95+--marker of Fas-receptor mediating apoptosis; for CD19+--marker of B-lymphocytes. Phagocytic activity of neutrophils was evaluated in the reaction of phagocytosis of latex particles by blood neutrophils; metabolic functional activity--by NST test (Frimmel, 1987); circulating immune complexes (CIC)--by precipitation with polyethylene glycol; cytokines (IL-1J3, IFN-[gamma], TNF-[alpha], IL-4)--by solid-phase immunoenzyme method using test kits IFA Best (Russia) on an automatic EIA analyzer PLAB "Adaltis" (Italy). Statistical processing included calculation of averages and their errors, variance, correlation analysis. The reliability of differences was assessed using Student's t test.

The material for study were peripheral blood, serum, plasma lymphocytes, cervical mucus.

Results of the study

Immunophenotyping of lymphocyte subpopulation composition of peripheral blood allowed revealing abnormalities and assessing the level of activated cells that are responsible for the production of proinflammatory cytokines and autoantibodies. Immunological profile of pregnant women with mycoplasma infection and control group is shown in Table 1.

As can be seen from the table, in pregnant women with mycoplasma infection the number of total population of T-lymphocytes (CD3+) in peripheral blood is significantly decreased compared to healthy pregnant women (68.12 [+ or -] 0.9 and 74.8 [+ or -] 1.88, D<0.05). Lack of stability in the numbers of the total population of T lymphocytes CD3+ in the blood during pregnancy is possibly due to the persistence of mycoplasma infection.

Studying the status of T-helper cells showed a significant (P<0.05) decrease in the number T-helpers/inductors (CD4+) compared to the control group (40.35 [+ or -] 0.98 and 54.5 [+ or -] 3.55, respectively) that is a manifestation of T-cell immunodeficiency. The number of T-suppressors/cytotoxic lymphocytes (CD8+) tended to increase, although the differences were not reliable (35.15 [+ or -] 1.28 and 33.6 [+ or -] 1.6). The marked increase of T-suppressors/cytotoxic lymphocytes (CD8+) in the literature is described as a phenomenon that is realized in the period of pregnancy at the "mother-fetus" frontier for the elimination of cytotoxic T-lymphocytes directed against fetal antigens. In addition, the increase in the number of T-suppressor/cytotoxic lymphocytes may be associated with an increase in antigen-specific cytotoxic cells in the immune response to an infectious agent.

Studying immunoregulatory index CD4+/CD8+(1.15 [+ or -] 0.003 and 1.62 [+ or -] 0.003) showed a significant decrease in this index, which is a negative sign and shows the predominance of the depressor component of immunity in patients with mycoplasma infection.

As it is known, the development of pregnancy is accompanied with a change in the number of blood NIC-cells, namely increased levels of lymphocytes with high cytotoxic potential. In studying the status of NIC-cells in pregnant women with mycoplasma infection significant increases in their numbers (25.16 [+ or -] 0.93 and 10.49 [+ or -] 0.76, respectively) were detected, which is 2.4 times more than in the control group. Increase of NI-cells indicates a significant activation of natural killer cells, resulting from antibody cytolysis during gestation and can occur in viral and bacterial infections. The number of B-cell antibody producers was significantly higher (25.61 [+ or -] 0.72 and 13.8 [+ or -] 0.13, P<0.05) than that of the control, which does not preclude the development of autoimmune processes.

The mycoplasma infection also results in reduction in the number of lymphocytes in the process of activation (CD 25), indicating that the expression of receptors for interleukin-2 was reduced compared to the similar data identified in healthy pregnant women. The reduced expression of the marker mediating apoptosis (CD 95) indicates a decrease in lymphocyte count circulating in blood, ready for death, as evidenced by lower apoptotic index CD 95/25 (0.52 [+ or -] 0.05 and 0.57 [+ or -] 0.08). The latter is regarded as a functional depression of the activation signals of apoptosis.

Mediator of cellular response to inflammation and stress is INF-y, which plays an important role in maintaining immune homeostasis, regulating the function of all the key immune system cells. In addition, it is known that INF-y is a proinflammatory cytokine and its variation characterizes the level of inflammation. In pregnant women with mycoplasma infection a significant increase in INF-y was detected, both in serum (136.7 [+ or -] 3.4 and 12.75 [+ or -] 0.05, P<0.001) and isolated peripheral blood lymphocytes (205.55 [+ or -] 1.97 and 28.4 [+ or -] 1.45, P<0.05) compared to those in the group of healthy pregnant women. Moreover, stimulation of lymphocyte mitogen PHA (30.6 [+ or -] 0.25 and 79.8 [+ or -] 1.3, respectively) showed the exhaustion of functional reserves of blood lymphocytes of infected pregnant women.

Comprehensive assessment of phagocytic immunity in pregnant women with mycoplasma infection in the NST test, and the reaction of phagocytosis showed generally the same changes. Found an increase in all indicators in 83% of women, including spontaneous and stimulated NBT test, which is a marker for activation of infection. At the same time spare capacity of neutrophils were retained and are consistent with indicators of healthy pregnant women at 39% and decreased in 61% of patients. Violation of the reserve capacity of neutrophils also shows the same type of index values induced activation of neutrophils (0.1 [+ or -] 0.003 and 0.1 [+ or -] 0.002). Probably, the neutrophil dysfunction contributed to the formation of circulating immune complexes, detected in 83% of women, whose number of infected pregnant women was significantly increased (0.046 [+ or -] 0.01 and 0.017 [+ or -] 0.003 conv. units, P<0.001). Assessing the level of lysozyme showed a significant decrease in its values (53.7 [+ or -] 4.24 and 44.8 [+ or -] 3.27) in pregnant women with mycoplasmosis. In the literature, the reduction in the activity of nonspecific factors of protection, namely the low value of lysozyme, is associated with risk of miscarriage, and intrauterine infection of fetus.

That means that in mycoplasma infection there is a significant violation of the immunity of women. At the level of the system immunity quantitative imbalance of T-cell component is generated, characterized by depression of circulating mature CD3, CD4 cells, increase of CD16 cells, stimulation of B-cell antibody, as well as decrease in the number of lymphocytes with activation markers CD 25 and CD 95. This is the evidence of violations in the universal system of regulation of homeostasis, responsible for activating the body's defenses.

We have investigated the content of pro-and anti-inflammatory cytokines IL-6, IL1B, INF[gamma], TNF-[alpha] and IL-4 in serum and locally in the cervical mucus from 30 pregnant women with mycoplasmosis and 30 pregnant women with physiological pregnancy.

Table 2 presents the results of a study of cytokines in peripheral blood of pregnant women with mycoplasma infection.

Analysis of cytokine profile in peripheral blood of pregnant women with mycoplasma infection revealed in all women a statistically significant increase in blood concentrations of proinflammatory cytokines (IL-1[beta]) compared to the control group (P<0.05), which supports the initiation of pro-inflammatory cytokine cascade.

Increased IL-6 in blood is the evidence of the presence of infectious process caused by mycoplasma. Also, it was found direct correlation between high levels of IL-6 in the cervical canal and preterm delivery. It was shown that the level of IL-6 in amniotic fluid is a sensitive test to detect the presence and severity of intrauterine infection of fetus.

Normal pregnancy is associated with predominance in the decidual tissue of IL-4. Increasing concentrations of anti-inflammatory cytokine IL-4 in the blood of pregnant women with mycoplasmosis on our data indicates the activation of humoral immunity.

TNF is produced by cells extraembryonal fetal tissues (placenta and membranes), maternal tissues during gestation. Predisposition to hypersecretion of TNF may be one of the factors contributing to the development of complications such as habitual miscarriage, premature birth and infertility of infectious origin. Microbial products activate the macrophage-monocyte system and the cytokines being vacated at the same time; signal the onset of labor through the stimulation of the biosynthesis of prostaglandins by endometrial tissue. We identified an increase in the blood of the proinflammatory cytokine TNF in pregnant women infected with mycoplasmas, which can serve as criteria for the diagnosis or the presence of inflammation.

In pregnant women with mycoplasma infection a significant increase in the mediator of cellular immune responses of IFN-[gamma] was detected, both in serum (136.7 [+ or -] 3.4 and 12.75 [+ or -] 0.05, P<0.001) and isolated peripheral blood lymphocytes (205.55 [+ or -] 1.97 and 28.4 [+ or -] 1.45, P<0.05) compared to those in the group of healthy pregnant women. At this, stimulation of lymphocyte mitogen PHA (30.6 [+ or -] 0.25 and 79.8 [+ or -] 1.3, respectively) showed the depletion of the functional reserves of blood lymphocytes infected pregnant women. In addition, increase in IFN-[gamma] in the blood indicates the inflammatory process in pregnant women infected with mycoplasma. At the system level in pregnant women with mycoplasma infection in the blood we tested reinforcement of secretion of proinflammatory cytokines (IL-1[beta], IL-6, FNO[alpha], INF[gamma]), which is associated with activation of cells of TH-type 1, the accompanying pathology of pregnancy. Increase in the blood of pregnant women with mycoplasmosis of anti-inflammatory cytokine IL-4 in the initial state is likely to be associated with the development of specific humoral immune response and production of specific anti-mycoplasma antibodies.

Table 3 presents the results of a study of cytokines at the local level (cervical mucus).

Studies of the concentration of cytokines at the local level (cervical mucus) in pregnant women with mycoplasma infection showed a significant increase in concentrations of both proinflammatory (TNF-[alpha], IL-6) and anti-inflammatory (IL-4 in 4.5 times) cytokines compared with controls group (P<0.05 and P<0.01), which confirms the presence of an inflammatory process not only at the system level, but locally.

That means that pregnant women with mycoplasma infection, in comparison with healthy pregnant women, had violations of the regulation system of cytokines in peripheral blood and locally in the cervical mucus. It is characterized by increased TH-1 cell response to the increasing fusion of pro-inflammatory cytokines (IL-1, IL-6, IFN-[gamma], TNF-[alpha]), and pointed to the imbalance of their products, which may be a factor in this pregnancy complication.

Therefore, in accordance with modern concepts of major pathogenetic mechanisms of development of complications of pregnancy with mycoplasma infection of our study confirm the dysfunction of the immune system, therefore the study of immune responses is important to assess the orientation of the immune response, as well as course and outcome of pregnancy for both mother and fetus.

Conclusions

In women with persistent mycoplasma infection have been violations of immunity at the systemic and local levels, which are:

--the quantitative imbalance of populations and subpopulations of peripheral blood lymphocytes (reduced CD3+, CD4+, increasing the CD16+, CD19+, a decrease of lymphocytes in the process of activation of CD25+ and ready to apoptosis of CD95+ marker, P <0.05);

--reinforcing the Th-1 response with increased synthesis of proinflammatory cytokines (IL-[beta], IFN-[gamma], TNF-[alpha], IL-6, P<0.05) both at the systemic (serum, isolated lymphocytes) and local (cervical mucus) levels, as well as increased inducer of the humoral immune response (anti-inflammatory cytokine IL-4), 3.38 and 4.86 times, respectively;

--dysfunction of phagocytic immunity link, characterized by increased absorption and digestive function of peripheral blood neutrophils, as well as the activation of oxygen mechanisms in the NST test;

--reduction of factors of nonspecific immunity (lysozyme).

References

Filatov, A., 1992. "Differential antigens: Human lymphocyte subpopulations difference" [Differencialjnie antigerrbi: Subpopulyacionnaya raznica limfocitov cheloveka], in Russian, Synopsis of doctoral dissertation.

Frimmel, G., 1987. Immunological methods [Immunologicheskie metodi], in Russian, Moscow: Medicine.

Kira, E., 2001. Bacterial vaginosis [Bakterialjniy vaginoz], in Russian, St-Petersburg.

Prilepskaya, V., Fofanova I., 2007. "Mycoplasma infection and pregnancy," Obstetrics and Gynecology [Akusherstvo i ginekologiya], in Russian, No. 4, pp. 5-8.

Nagima Mamedaliyeva, Saule Issenova, Ludmila Dzoz

Scientific Centre of Obstetrics, Genecology and Perinatology

Kazakhstan
TABLE 1. INDICATORS OF CELLULAR IMMUNITY IN PERIPHERAL
BLOOD IN THE STUDY GROUPS (UNITS)

Populations and subpopulations         Differentiated
of lymphocytes (%)                         antigen

Mature T-cells                              CD3+

T-helpers/inductors                         CD4+

T-suppressors                               CD8+

Natural killers (NK)                        CD16+

B-cells                                     CD19+

Marker of [pounds sterling]-link            CD25+
if IL-2 receptor

Marker of Fas-receptor mediating            CD95+
apoptosis

Apoptosis index                           CD95/CD25

Immunoregulatory index                     CD4/CD8

                                        Studied group
Populations and subpopulations
of lymphocytes (%)                       main group

Mature T-cells                      68.12 [+ or -] 0.9 *

T-helpers/inductors                 40.35 [+ or -] 0.98 *

T-suppressors                        35.15 [+ or -] 1.28

Natural killers (NK)                25.16 [+ or -] 0.93 *

B-cells                             25.61 [+ or -] 0.72 *

Marker of [pounds sterling]-link     5.0 [+ or -] 0.5 *
if IL-2 receptor

Marker of Fas-receptor mediating     2.6 [+ or -] 0.6 *
apoptosis

Apoptosis index                     0.52 [+ or -] 0.05 *

Immunoregulatory index              1.15 [+ or -] 0.003 *

                                        Studied group
Populations and subpopulations
of lymphocytes (%)                      control group

Mature T-cells                       74.8 [+ or -] 1.88

T-helpers/inductors                  54.5 [+ or -] 3.55

T-suppressors                         33.6 [+ or -] 1.6

Natural killers (NK)                 10.49 [+ or -] 0.76

B-cells                              13.8 [+ or -] 0.13

Marker of [pounds sterling]-link      7.0 [+ or -] 0.7
if IL-2 receptor

Marker of Fas-receptor mediating      4.0 [+ or -] 0.2
apoptosis

Apoptosis index                      0.57 [+ or -] 0.08

Immunoregulatory index               1.62 [+ or -] 0.003

Note: *  D<0,005 differences are reliable between
the main and control groups.

TABLE 2. CONCENTRATION OF CYTOKINES IN PERIPHERAL BLOOD
OF PREGNANT WOMEN WITH MYCOPLASMA INFECTION

Index (pg/ml)        Main group           Control group         D

IL-1[beta]      104.5 [+ or -] 8.9 *    25.0 [+ or -] 1.3    <0.05
IL-6             78.5 [+ or -] 1.2 *    30.0 [+ or -] 1.9    <0.05
INF-y           136.7 [+ or -] 3.4 **   25.0 [+ or -] 2.3    <0.001
TNK-a            4.25 [+ or -] 0.59 *    2.5 [+ or -] 0.35   <0.05
IL-4             79.7 [+ or -] 2.1 *    20.0 [+ or -] 1.7    <0.05

TABLE 3. CONCENTRATION OF CYTOKINES IN CERVICAL MUCUS
OF PREGNANT WOMEN WITH MYCOPLASMA INFECTION

Index (pg/ml)                 Main group           Control group

[TEXT NOT REPRODUCIBLE    85.7 [+ or -] 1.2 *    25.0 [+ or -] 1.1
IN ASCII]

[TEXT NOT REPRODUCIBLE    10.1 [+ or -] 0.59 *    2.5 [+ or -] 0.3
IN ASCII]

[TEXT NOT REPRODUCIBLE   121.5 [+ or -] 1.5 **   25.0 [+ or -] 1.1
IN ASCII]

Note: * D<0,05; D<0,01 between the main and the control group.
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Author:Mamedaliyeva, Nagima; Issenova, Saule; Dzoz, Ludmila
Publication:Medical and Health Science Journal
Article Type:Report
Date:Jan 1, 2011
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