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"Child`s Health" 2 (61) 2015

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Leukocyte composition and immunophenotype of peripheral blood lymphocytes of DAAb-positive and DAAb-negative children with diabetes-associated autoantibodies (DAAbs) in preclinical and early clinical stages of type 1 diabetes mellitus.

Authors: Popova V.V. — SI “V.P. Komisarenko Institute of Endocrinology and Metabolism of Acad Med Sci of Ukraine”, Kyiv, Ukraine

Categories: Pediatrics/Neonatology

Sections: Clinical researches

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Type 1 diabetes mellitus (T1DM) is progressing rapidly in the world as well as in Ukraine. Current priority of this disease has led to an increase in basic and applied studies on pathogenesis of T1DM, a central role of which is in the study of natural and adaptive immunity indicators in preclinical latent period prior to clinical manifestation of the disease.
The purpose of the research. The study of immunity based on the determination of the blood leukocyte and lymphocyte immunophenotype in diabetes-associated autoantibodies positive and diabetes-associated autoantibodies negative children in preclinical and early clinical stages of type 1 diabetes mellitus. 
Materials and methods of the research.
There were examined 450 children and adolescents in the age range of 7 to 15 years (average age - 12,34 ± 0,82 years). Of these, 366 (81.3%) - the main group - were children and adolescents with normoglycemia who had close relatives with T1DM: parents or siblings.
This group of children on the results of the examination for radioimmunoassay simultaneously elevated titer diabetes-associated autoantibodies (DAAb), namely insulin autoantibodies (IAA), autoantibodies to protein tyrosine phosphatase (IA-2A) and autoantibodies to glutamic acid decarboxylase (GADA ) was divided into two subgroups - diabetes-associated autoantibodies positive (DAAb+) and diabetes-associated autoantibodies negative (DAAb-). DAAb + were 94 (25.7%) children required inclusion criterion which was necessary to have, by definition twofold, while elevated titer of at least two types of DAAb, mainly GADA and IA-2A. DAAb group were 272 (74.3%) patients with normal levels of blood glucose and the lack of high titer DAAb (DAAb-negative). 
Another group appeared as a logical chronological end autoimmune process in the group DAAb + children - a group of children with diabetes debut - 49 children (52.1%), which gradually over 6 months to 10 years (mean 30,9 ± 3 2 months) changed their "normoglycemic" DAAb-positive healthy children to clinical status of patients with T1DM debut. 84 healthy normoglycemic children (18.7%) composed the control group. Determination of the distribution and analysis of different lymphocyte immune phenotype was performed by using flow cytometry method and laser cytometry. 
The total number of leukocytes and leukocyte composition of blood in the DAAb-positive and DAAb-negative children in preclinical and early clinical stages of T1DM.
Researches which were conducted did not reveal any significant alteration in total number of leukocytes of the PB in DAAb+ and DAAb+ children with T1DM. Relative and absolute number of band and segmented neutrophils in DAAb-, DAAb+ before and after the manifestation of T1DM were close to those of a control group of healthy children. At the same time, the relative and absolute number of monocytes in DAAb+ children was significantly increased on the same indices in DAAb- children and children of the control group. Analyzing the content of monocytes in DAAb+ children with insulin-dependent diabetes debut  the monocytosis was also detected , but not so much as in DAAb+ group. With the development of clinical debut of T1DM, the lymphocytopenia was observed, but not as much as in DAAb+ children. Analyzing the relative and absolute content of large granular lymphocytes (LGL) which are morphological homologue of natural killer cells (NK) in DAAb+ group of children and DAAb+ group with T1DM debut, it was found significant probable decrease of relative and absolute number of lymphocytes compared with those in children of the control group which is confirmed by a simultaneous decrease in the number of lymphocytes expressing CD56 antigen. Thus, mild lymphocytopenia may be explained by a simultaneous decrease in the number of T-lymphocytes and NK/  LGL in the PB. 
Immunophenotype of lymphocytes in preclinical and clinical stages of insulin-dependent diabetes. Was found the lower relative and absolute number of the total of CD3+ cells in DAAb+ group comparing with DAAb- group, the control group, as well as DAAb+ children which T1DM debut. During the study of lymphocyte immunophenotype in DAAb+ children was shown a decreasing  of the number of CD3+ T-cells in PB which was  mostly due to a decrease in circulation of the absolute and relative number of CD4+ T-cells. Significant changes of relative number of CD8+ T-cells in DAAb- group, DAAb+ group, and in group of children with clinical debut of T1DM relatively to control was found. During calculating the absolute numbers of CD8+ T-cells, probable decrease of the number of these cells in DAAb+ children was observed. Index of СD4+/СD8+ was significantly reduced in both DAAb+ children and in children with insulin-dependent diabetes, comparing with control. In determining the number of B lymphocytes (CD20 cells) did any significant alteration in the relative number in PB not was not revealed. We obtained important data  in the study of the least studied subpopulations of lymphocytes - natural killer cells (NK-cells). In DAAb+ children and in children with T1DM debut, statistically significant decrease of relative and absolute number of lymphocytes expressing antigen CD56 in PB compared with a group of healthy children was observed. Similar changes were also identified by cytological analysis of NK-cells of PB smear based on counting LGL confirming the reliability of our data about the relative change in the number of this subpopulation of lymphocytes in preclinical and early clinical stages of T1DM.
Conclusions:
1. It was shown that the most significant changes in adaptive  and natural  immunity occur in the preclinical stage of the disease, which is caused by the most active phase of the autoimmune process..
2. In preclinical period of T1DM in DAAb+ children an increase in the absolute number of monocytes and decrease in the number of natural killer cells (CD56+ lymphocytes / LGL) in peripheral blood was observed, indicating a significant weakening of the pathogenesis of innate immunity at this stage of the disease progression. 
3. Latent, preclinical period of T1DM in children with elevated titers DAAb to islet antigens in comparison with marker-negative patients' availability of DAAb to islet antigens, and in healthy children of control group was  characterized by such traits as impaired adaptive immunity - a reduction in circulating total number of CD3+, CD4+, CD8+ T-cells which is a material substrate for determined autoimmune aggression in the preclinical stage of development of T1DM.

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