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Neurotransmitter serum levels in children with recurrent upper respiratory tract infections

Background. Schoolchildren are prone to frequent respiratory tract diseases. In the structure of general morbidity, this pathology ranks first. Its prevalence is more than 60 %, which leads to frequent school absenteeism, lack of active games and outdoor walks. So, there is an increase in the amount of time that a child spent on means of mass electronic communication and electronic games, resulting in high affection and addiction, forming hypodynamia, which in aggregate has a negative impact on the regulatory systems functioning. Recurrent upper respiratory tract infections (URTI) contribute to the strain of neuro-endocrine-immune regulation, causing an imbalance of neuropeptides — mediators of neurogenic inflammation. The purpose of this work is to study the neurogenic inflammation parameters in schoolchildren with functional disorders of the cardiovascular system and recurrent respiratory tract diseases. Materials and methods. We surveyed 130 children with URTI aged 6 to 9 years in the somatic well-being period. The analysis of the contents of vasoactive intestinal peptide (VIP), substance P and final stable metabolites of nitric oxide (FSM NO) in serum was carried out. An increase in substance P level in children with recurrent URTI was found, which had statistically significant differences with the healthy children indices (p < 0.05). Results. It has been found that high concentration of substance P increases the risk of recurrent URTI. It has been proved that in children with recurrent URTI due to increased FSM NO serum content that has a pro-inflammatory orientation, there is an increased neurogenic inflammation activity. It was found that the reliable reduction in the content of VIP as an anti-inflammatory transmitter in children with recurrent URTI can not suppress the activity of neurogenic inflammation, resulting in its excessive stimulation. Conclusions. Children with recurrent URTI in the somatic well-being period have increased serum levels of substance Р and FSM NO. In the somatic well-being period in schoolchildren with recurrent URTI, a decrease in the concentration of antiinflammatory neuropeptide VIP is observed.

studies have shown that VIP operates directly on the muco sal immune system, where increases IgA synthesis, indica ting the critical role of this peptide in inducing oral intole rance [5].
Substance P is a peptide consisting of 11 amino acids that regulates the mucosal immune balance and chronic inflammation activity [11]. It is produced by macrophages, Tcells, dendritic cells and eosinophils. The production of substance Р by macrophages induces bacterial lipopolysac charides [14]. Substance P is currently considered as the main neurogenic inflammatory mediator capable of caus ing pathophysiological reactions such as edema, mucus hypersecretion, bronchospasm, vascular tone reduction, in creased permeability of postcapillary venules, immune cell penetration into the tissues and secreting glands, enhancing monocyte/macrophags chemotaxis, control of T1 response, increasing the production of interferon γ [14], proinflam matory activity and secretion of tumor necrosis factor α, interleukins 1, 6, 8, 12 [9].
Another substance that has proinflammatory properties is nitric oxide (NO) -a biological messenger molecule syn thesized from the amino acid Larginine in endothelial cells, macrophages, and neurons. NO mediates a wide range of important biological processes such as vasodilation, platelet aggregation inhibition, inflammation, immunoregulation and information transmission between the central nervous system neurons and the peripheral nervous system [4,13,14]. NO stimulates perivascular neurogenic inflammation by facilitating the synthesis and release of immunoreactive neurotransmitters such as substance P from nociceptive af ferent fibers [4]. Increasing the NO concentration in chil dren is observed in conditions accompanied by a change in the cytokine activity -acute infection, bronchopulmonary chronic diseases, persistent intracellular infection. NO con centration in exhaled air in children correlates with neutro philia [11]. Indirect cytotoxic effect of NO is realized due to the action of free radical derivatives -peroxynitrite, nitrogen dioxide, hydroxyl radical, which initiate lipid per oxidation. NO metabolites -nitrates and nitrites -play an important role in the inflammation [14].
Given the role of neuropeptides in the immune response [11,14], it is important to determine the effect of these mechanisms on the initial vegetative tone status in school children with upper respiratory tract infections (URTI). The possible neuroimmune mechanism disorders, as well as the hypodynamia effects, due to frequent exemptions from physical education classes, the prohibition of sports sections attending, and functional disorders of the cardiovascular system are the risk factors of cardiac organic pathology [3].
The purpose was to study neurogenic inflammation parameters in schoolchildren with functional disorders of the cardiovascular system and recurrent respiratory di seases.

Materials and methods
One hundred and thirty children aged 6 to 9 years were examined. The analysis of the serum VIP, substance P and the biologically active substance -NO in the form of final stable metabolites (FSM NO) was carried out.
During the observation, children were divided into three groups: group 1 -those with functional disorders of the cardiovascular system and recurrent URTI (n = 50); group 2 -patients with functional disorders of the cardiovascular system and episodic URTI (n = 50); group 3 (controls)conditionally healthy children (n = 30).
The study of serum FSM NO (nitrates, nitrites) was carried out by restoring nitrates to nitrites with the deter mination of the latter in reaction with the Griess reagent. The optical length was measured on spectrophotometer at a wavelength of 540 nm. Calculation of nitrite amount was carried out according to the calibration graph built on nitrogen. A quantitative determination of serum sub stance P and VIP was carried out by the immunoassay on Sunrise photometry analyzer (TECAN, Austria) using the ELISA kits (Peninsula Laboratories, LLC, San Car los, USA).
The study uses statistical methods for information pro cessing obtained: Fischer's angle criterion, mean (M), mean error (m), Student's ttest, MannWhitney Utest. ROC analysis of diagnostic tests was performed based on calculating the predictive value of the area under the ROC curve (AUC) with a cutoff point calculation.

Results
The examination of schoolchildren was carried out du ring the period of somatic wellbeing. The results of the study are presented in Table 1.
When determining serum levels of substance P, VIP, FSM NO in group 1 children in the period of somatic well being, there was a statistically significant (compared to group 3) increase in substance P concentration, FSM NO and a decrease in VIP content (p < 0.05). At the same time, a decrease in the serum VIP was noted compared to study group 2 (p < 0.05).
Analyzing the levels of substance P in children with re current and episodic URTI, one can conclude that no sig nificant difference was found (U = 1071.0; p > 0.05), this is confirmed by the data of ROC analysis (Fig. 1). ROC analysis of substance P serum concentration distri bution between the 1 and 2 observation groups showed that the cutoff point was 0.31 ng/ml. Higher levels of substance P are associated with an increased risk of recurrent URTI. The optimal cutoff point is 0.31 ng/ml (AUC 0.6) with sen sitivity of 26.0 % and specificity of 90.0 % (Fig. 1).
Among children with recurrent URTI and control group, there is a statistically significant difference in the concentration of substance P (U = 529.5; p < 0.05). Also, these differences in results are confirmed by data from ROC analysis (Fig. 2). ROC analysis of the distribution of sub stance P content in groups 1 and 3 showed that cutoff point is 0.3 ng/ml. Higher levels of substance R are associated with an increased risk of recurrent URTI. The optimal cut off point is 0.3 ng/ml (AUC 0.7) with sensitivity of 33.0 % and specificity of 100.0 % (Fig. 2).
Determination of serum VIP in children of groups 1 and 2 showed significant differences, as evidenced by data of ROC analysis (Fig. 3).
Cutoff point for the serum concentration of VIP was determined, which corresponds to the level of 0.34 ng/ml. Lower levels are associated with an increased risk of recur rent URTI (AUC 0.8) with sensitivity of 68.0 % and speci ficity of 100.0 %. Similar results are obtained when com paring groups 1 and 3. The differences are also confirmed by ROC analysis data (Fig. 4). The cutoff point for VIP serum concentration is 0.28 ng/ml. VIP indices below this index will be associated with the risk of developing recurrent URTI (AUC 0.8) with sensitivity of 100.0 % and specificity of 50.0 %.
The determination of FSM NO serum content showed a slight difference between groups 1 and 2 (U = 873.0; p > 0.05) and the reliable differences between groups 1 and 3 (U = 377.5; p < 0.05). The conducted ROC analysis de termined the cutoff point for FSM NO in the comparison groups 1 and 2 -18.11 ng/ml. Serum concentration of FSM NO above this indicator was associated with an increased risk of recurrent URTI (AUC 0.7) with sensitivity of 44.0 % and specificity of 90.0 % (Fig. 5).
When comparing the indicators in groups 1 and 3, the se rum level of FSM NO was 16.99 ng/ml by the cutoff point. Excess of this concentration is associated with a high risk of recurrent URTI (AUC 0.75) with sensitivity of 63.0 % and specificity of 100.0 % (Fig. 6).

Discussion
The inflammation pathogenesis goes beyond the scope of immune mechanisms -neuronal interactions are in volved in this process. This is due to the ability of immu nocompetent cells stimulated by inflammation to produce neuropeptides similar to those produced in the central ner vous system [5]. Some neuropeptides/neurotransmitters have antiinflammatory properties -one of them is VIP, and proinflammatory properties such as substance P and NO [8,10,14].
Based on the current theory of neuroendocrineim mune regulation, immune responses can be considered as components of neuroendocrine activity, and by themselves they are impossible without the cohesive participation of the nervous and endocrine systems. Humoral regulatory factors affecting both of these systems are involved in the imple mentation of neuroimmune interactions [12].
Recurrent URTI in schoolchildren has a negative ef fect on the functional status of respiratory, cardiovascular, central nervous systems, changing the mechanism of patho logical process, which is clinically manifested by the delayed recovery. The need to study the serum levels of neurotrans mitters in schoolchildren with recurrent URTI allowed us to determine the degree of participation of neuroimmune inflammation in the pathogenesis of the disease and to al locate a risk group for the development of recurrent respira tory tract diseases. A comparative analysis of the serum sub stance P in schoolchildren showed significant differences between group 1 and the control group (p < 0.05). Exceeded the established concentration of substance P increases the risk of recurrent diseases.
The proinflammatory orientation of FSM NO, espe cially in children of group 1, may increase the activity of neurogenic inflammation. Probably, this due to the fact that in children with recurrent URTI on the background of increased activity of immunocompetent cells as a result of prolonged viralbacterial stimulation, the production of central nervous system transmitters increased in response to a stressful situation, which is linked with recurrent di seases [12]. Exceeding established concentration of FSM NO in creases the risk of recurrent URTI.
A significant decrease in the content of antiinflamma tory transmitters, in particular VIP, in children with recur rent URTI can not suppress the activity of neurogenic in flammation.

Conclusions
1. In schoolchildren aged 6 to 9 years with recurrent URTI in the somatic wellbeing period, there is an increase in the serum concentration of neuroimmune proinflamma tory mediators -substance P and FSM NO.
2. The period of somatic wellbeing in schoolchildren aged 6 to 9 years with recurrent URTI is characterized by a decrease in the concentration of antiinflammatory neuro peptide VIP.
3. ROC analysis is a mechanism for predicting the risk of recurrent URTI.