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Sadirova Shakhlo Sobirovna, The Research Institute of Virology, Tashkent, Uzbekistan E-mail: evovision@bk.ru
THE IMMUNE SYSTEM IN RARE AND FREQUENT RELAPSING LARYNGEAL PAPILLOMATOSIS IN CHILDREN
Abstract: Clinical and immunological examination of 54 children with rare (RRLP) and frequent (FRLP) relapsing laryngeal papillomatosis was carried out. As a control, 15 healthy children in comparable age were examined. A decrease in the number of CD16 +, CD3+ and CD4+ lymphocytes, IgA, IgG, and CD4+/CD8+ ratio, an increase in the number of CD95 + and CD19 + cells was revealed.
Keywords: laryngeal papillomatosis, lymphocytes, CD4 +/CD8 + ratio, humoral immunity, cellular immunity, immunoglobulins.
The problem of laryngeal papillomatosis (LP) in children remains one ofthe most urgent in modern otorhinolaryngology [1; 2; 3]. The significance of the problem is due to the fact that, papillomatous growth affecting the larynx leads to a narrowing of its lumen, disruption of vital functions - respiratory and voice formation. Etiological factor of PL is considered to be the human papilloma virus (HPV) is a member of the papovavirus family. Although the presence of a specific virus is considered to be the main cause of LP, the presence of the virus alone is not enough for the development of the disease [3; 7].
It is known that the supporting the homeostasis, in particular, the control of proliferating cells and virus infected cells maintained by system of immunobiological reactivity, by the factors of nonspecific and specific protection [4; 5; 6; 7]. Consequently, defects of the latter (primary and secondary nature) has effect on the life of viruses and the dynamics of proliferative growth. On the other hand, these processes, apparently, themselves can lead to the formation of immunodeficiency states.
Thus, the study of the immune status of the organism in LP seems appropriate and may have practical significance.
Based on the above, the aim of the study was to study the nature and significance of immune status disturbances in children with rare and frequent relapsing laryngeal papillomatosis.
Material and methods. We have studied and evaluated the pathogenetic significance of changes in the immune system in peripheral blood in 54 children patients in school-age with rare and frequent relapsing laryngeal papillomatosis, which were divided into 2 representative groups: 1st group - 35
patients with rare relapsing laryngeal papillomatosis (RRLG); 2nd group - 19 patients with frequent relapsing laryngeal papillomatosis (FRLP). The control group included 17 healthy children of comparable age.
Phenotyping of lymphocytes was performed using monoclonal antibodies CD3+, CD4+, CD8+, CD16+, CD19+, CD25+ and CD95+ (production of Sorbent, Russia, Moscow). The concentration of immunoglobulins of classes A, M and G were determined by radial immunodiffusion by Mancini (1964) using monospecific anti-serum made in the Institute of epidemiology and Microbiology named after N. F. Gamalea.
Mathematical data processing was carried out by methods of variation statistics using standard mathematical software packages with the definition of the average, its error, Student's t test.
Results and discussion. The results of the research showed that in preschool children in the preoperative period of RRLG revealed changes in some parameters of the immune system compared with the control group (healthy children). In the background of normal leukocyte and lymphocyte counts, it was found that in peripheral blood in children with RRLG the relative number of T-lymphocytes was - 50.8 ± ± 1.12%, which is significantly lower than the control values -58.9 ± 2.15% (P < 0.001). No significant deficiency of CD3 + + markers of T-lymphocytes in children with RRLG in comparison with the control data was found in the analysis of their absolute values - 1082.8 ± 55.52 cells/^l. against 1149.1 ± ± 107.32 cells /^l (P > 0.05) (table 1).
Table 1.- Cellular immunity indicators
Immunity indicators Control group n = 17 RRLP n = 35 FRLP n = 19
1 2 3 4
Leucocytes, g/L 5.3 ± 0.30 5.6 ± 0.12 6.6 ± 0.38
1 2 3 4
Lymphocytes,% 35.9 ± 1.32 37.4 ± 1.05 39.4 ± 1.60
Lymphocytes, (abs) 1930.3 ± 159.90 2131.6 ± 98.35 2701.8 ± 252.87
CD3 +,% 58.9 ± 2.15 50.8 ± 1.12 42.8 ± 1.05
CD3 + (abs) 1149.1 ± 107.32 1082.8 ± 55.52 1191.7 ± 129.96
CD4 +,% 33.6 ± 1.75 25.4 ± 0.55 21.4 ± 0.80
CD4 + (abs) 663.3 ± 71.44 545.2 ± 31.44 608.7 ± 74.92
CD8 +,% 23.7 ± 0.93 25.1 ± 0.59 23.1 ± 0.81
CD8 + (abs) 463.9 ± 44.37 541.8 ± 32.05 655.4 ± 78.69
CD16 +,% 12.9 ± 0.65 10.6 ± 0.36 8.6 ± 0.54
CD16 + (abs) 250.3 ± 23.22 227.3 ± 14.16 249.8 ± 37.11
CD4+/CD8+ ratio 1.4 ± 0.08 1.0 ± 0.02 0.9 ± 0.02
Analysis of the results in FRLP showed the following ambiguous changes in cellular and humoral immunity. The relative number of leukocytes in children with FRLP (6.6 ± ± 0.38 thousand cells/^l versus 5.3 ± 0.30 thousand cells/^l in control, P < 0.02) was significantly increased.
In RRLP the tendency to increase (37.4 ± 0.71% and 2151.3 ± 128.26 cells/^l) of the relative and absolute total pool of lymphocytes (at the control of 35.9 ± 1.32% and 1930.3 ± 159.90 cells/^l) was observed. There was a significant decrease in patients with FRLP relative number of CD3 + + lymphocytes (42.8 ± 1.05%, P < 0.001, against the control values of 58.9 ± 2.15%).
During the study of the subpopulation composition of lymphoid cells in the examined children, peculiar changes in functional activity and redistribution of lymphocyte subpopulations were revealed.
Analysis of the results of the study of the relative and absolute content of T-helpers /inducers in RRLP showed a significant decrease in the number of CD4 + cells to an average of 25.4 ± 0.55%, which is 1.32 times lower than the control values (P < 0.001). The absolute number of CD4 + cells tended to decrease, but did not differ significantly from the control values. It is known that CD4 + lymphocytes play a key role in protection against viral infections.
We found that in the blood of healthy children the relative number of T-suppressors/cytotoxic lymphocytes was 23.7 ± ± 0.93% and the absolute number was 463.9 ± 44.37 cells/^l. In the preoperative period in children with RRLP the content of CD8 + cells, both relative and absolute, did not differ from the control group, was 25.1 ± 0.59% and 541.8 ± 32.05 cells/^l, respectively.
In the case of FRLP, there was a significant decrease in the relative (21.4 ± 0.80%, P < 0.001) number and a slight tendency to decrease in the absolute number (608.7 ± 74.92 cells/^l,
P > 0.05) of CD4 + cells in untreated patients with the corresponding control (33.6 ± 1.75% and 663.3 ± 71.44 cells/^l) parameters. There was a significant increase in the absolute number of t-suppressor cytotoxic (CD8 +) cells in FRLP (655.4 ± 78.69 cells/^l, P < 0.05). But the relative numbers of these cells remained within the control values.
Consequently, in RRLP and FRLP there is a decrease in the relative content of T-helpers, and the content of T-suppres-sors did not change, which explains the decrease (P < 0.001) of the immunoregulatory index.
The quantitative study of the relative content of natural killer cells (NKC) showed that in the preoperative period in children with RRLP and FRLP the content of CD16 +-lym-phocytes in peripheral blood was 10.6 ± 0.36% (P < 0.01) and 8.6 ± 0.54%, respectively (P < 0.001), which significantly differs from the control values - 12.9 ± 0.65%. The study of the absolute content of CD16 + - lymphocytes in RRLP revealed a tendency to their decrease to 227.3 ± 14.16 cells/^l. compared with the control data (250.3 ± 23.22 cells/^l.), then at FRLP their absolute content was within the control (P > 0.05).
During the immune response, B-lymphocytes (CD19+) differentiate into plasma cells which secretes antibodies. Our studies have shown that in the preoperative period in peripheral blood of children with RRLP the relative content of b-lymphocytes increased and differed significantly from the control group and averaged 27.1 ± 0.73% (table 2).
The absolute value of this indicator tended to increase, but did not differ from the control indicators and was determined within 582.8 ± 34.63 cells/^l. in the control 481,0 ± 49.27 cells/^l.
Relative and absolute numbers of CD19+ cells significantly increased in FRLP (31.1 ± 1.32% and 893.9 ± 113.93 cells/^l versus 24.6 ± 1.21% and 481.0 ± 49.27 cells/^l in the control).
The concentrations of immunoglobulins in the blood serum in healthy children of preschool age showed that the level of IgG was 1168.8 ± of 38.87 mg/%, and in the preop-
Table 2.- Indicators
The children in the control group, the levels of IgA and IgM in the serum are contained within - 131.0 ± 4.52 mg/% 132.9 ± 7.06 mg/%, and in children with RRLP before the operation, their contents were, respectively,- to 124.7 ± 4.37 mg/% 131.0 ± 5.13 mg/%, which did not differ from indicators of control. The concentration of IgA decreased significantly and amounted to 114.7 ± 2.83 mg% (P < 0.01) in FRLP.
Any immunological reaction, regardless of the predominance of humoral or cellular response, begins with proliferation. One of the criteria for evaluating the proliferative process may be an increase or decrease in the number of lymphocytes with a receptor for IL-2 (CD25+).
In RRLP, there was a tendency to increase the relative content of lymphocytes with a receptor to apoptosis (CD95 +) to an average of 28.2 ± 0.56% in control of 25.1 ± 1.73%, and in the absolute number of these cells had a significant increase -up to 606.8 ± 32.98 cells/^l. (P < 0.05), at control of 484.6 ± ± 46.69 cells/^l.
In patients with FRLP relative and absolute values of CD95 + lymphocytes significantly increased and amounted to 31.2 ± 0.85% (P < 0.01) and 863.5 ± 99.87 cells/^l (P < < 0.001), respectively.
So, in children with RRLP in the preoperative period, significant changes in the immune system were found in comparison with similar indicators of healthy children. Significant decrease in the total number of CD3, CD4 and CD16 lymphocytes and IgG levels was found. There is a tendency
erative period in children with RRLP and FRLP there was a significant decrease in IgG, respectively - 1011.3 ± 23.01 mg/% (P < 0.001) and 906.6 ± 33.73 mg% (P < 0.001).
of humoral immunity
The study of the number of lymphocytes with markers of early activation of CD25 + showed that in the preoperative period in children with RRLP, the number of these cells tended to decrease compared to the control group, but did not differ statistically. Thus, the number of CD25 + lymphocytes in the control group averaged 28.1 ± 1.37% and in the main group - 25.7 ± 0.73% (P > 0.05). In patients with FRLP, the relative values of CD25+ significantly decreased to 23.7 ± 0.85% (P < 0.01). The absolute values of these cells also did not differ in comparison with the control values (Table 3).
to decrease the number of CD25 cells and a tendency to increase the number of CD8, CD19 and CD95 cells. In FRLP significant increase in the number of CD19 and CD95 cells, a decrease in the number of CD3, CD4, CD16, CD25 lymphocytes and the level of immunoglobulins A and G were found.
Conclusion
The state of the immune system in patients with RRLP and FRLP is characterized by a lack of nonspecific immune defence and T-cell immunity, which is manifested by a decrease in the expression of marker receptors of CD16+, CD3+ and CD4+ lymphocytes; a decrease in the immu-noregulatory index (CD3+/CD4+ ratio); activation of CD95+; as well as in the background of increase of contents of B-lymphocytes the concentrations of immunoglobulins IgA and IgG are reduced.
Immunity indicators Control group n = 17 RRLP n = 35 FRLP n = 19
CD19+,% 24.6 ± 1.21 27.1 ± 0.73 31.1 ± 1.32
CD19+ (abs. number) 481.0 ± 49.27 582.8 ± 34.63 893.9 ± 113.98
IgA, mg% 131.0 ± 4.52 124.7 ± 4.37 114.7 ± 2.83
IgM, mg% 132.9 ± 7.06 131.0 ± 5.13 136.1 ± 3.02
IgG, mg% 1168.8 ± 38.87 1011.3 ± 23.01 906.6 ± 33.73
Table 3.- The performance of some activation markers of the immune system
Immunity indicators Control group n = 17 RRLP n = 35 FRLP n = 19
CD25+, % 28.1 ± 1.37 25.7 ± 0.73 23.7 ± 0.85
CD25+ (abs) 526.0 ± 38.84 554.7 ± 33.15 657.1 ± 74.55
CD95+, % 25.1 ± 1.73 28.2 ± 0.56 31.2 ± 0.85
CD95+ (abs) 484.6 ± 46.69 606.8 ± 32.98 863.5 ± 99.87
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