Clinical and immunological characteristic of children with recurrent episodes of acute laryngotracheitis, acute respiratory infections, otorhi-nolaryngological deseases

Researching on pathogenesis and immunopathogenesis of acute respiratory infections in conjunction with recurrent episodes of acute laryngotracheitis (ALT) and otorhinolaringological diseases (tonsillopharyngitis, rhinosinusitis, otitis media)


Клинико-иммунологические особенности у детей с повторными эпизодами острого ларинготрахеита, респираторных инфекций и ЛОРзаболеваниями
Comorbidity with respiratory viral infections in frequently ill children, including obstructive laryngitis (OLT) (ICD 10 -J05.0) and acute laryngotracheitis (ALT) (ICD 10 -J04.2) are considered to be relevant interdisciplinary problems. The severity of clinical symptoms, laryngeal stenosis in OLT and other complications point to the need for urgent and emergency care [1,2]. The pathogenesis involves viruses of influenza, parainfluenza, adenoviruses, respiratory-syncytial virus, bocavirus, viral-viral associations, microbial-viral associations, contributing to the formation of inflammation in the subglottic part of the larynx [1,2,3]. The study of OLT immunopathogenesis indicates the presence of allergic component that is confirmed by an increase in total IgE, IL-4 [4,5]. OLT disappears in most children by age 7; acute laryngotracheitis (ALT) with no laryngeal stenosis is more common in older children [6]. It is important to study immunopathogenesis of repeated ALT using modern methods of allergy and immunodiagnostics, including indicators of innate immunity. We examined children with recurrent ALT and ARVI; bacterial and viral infections, acute ENT pathology (adenoiditis, otitis media, chronic rhinosinusitis, chronic tonsillopharyngitis, etc.) were reported in some of them. Bacterial lysates are known to be effective in chronic diseases of the ENT organs and ARVI [4,7]. We chose Broncho-Vaxom, there were no studies of its intended purpose for patients with recurrent ALT, ARVI and ENT-pathology in the available literature.

OBJECTIVE
Researching on clinical and immunological characteristics and efficiency of prescribing bacterial lysate (OM-85) to children with recurrent acute laryngotracheitis (ALT), ARVI and ENT diseases.

MATERIALS AND METHODS
50 children (22 boys and 28 girls) aged 3-9 years old in the initial period of monitoring (32 children aged 3-6 years and 18 children -7-9 years) with recurrent ALT (3-9 times a year), ARVI (6-12 times a year) and ENT-pathology (adenoids of I-III degree -in 16 (32 %)), chronic tonsillopharyngitis -in 40 (80 %), chronic rhinosinusitis -in 18 (36 %), recurrent acute moderate serous otitis media -in 8 (16 %)) were examined. The children were randomly divided into 2 groups Ia and Iб and received standard treatment; 25 children (subgroup Iб) additionally got bacterial lysate (Broncho-Vaxom) outside the acute period: 10 days a month, 3 months; 4 courses курса in 3 years; 1 courses in 1 st year, 1 course in year 2 and 3. The subgroups are similar in sex, age and clinical manifestations of the disease. Clinical and immunological screening was held within 6 months prior to treatment after the course of bacterial lysate (Broncho-Vaxom) and in 3 years (group Ia did not receive the drug). The children were examined outside ALT, ARVI and acute ENT-pathology, did not received antibacterial therapy while taking bacterial lysate, further-according to the indications. There was further examination of a comparison group of 30 children at age 3-9 years (21 children at age 3-6 years and 9 children -7-9 years) with ARI frequency 3-5 times a year without chronic respiratory tract infection and recurrent ALT. All children received standard treatment, follow-up period was 3 years. The study was conducted in "SRC Institute of Immunology" FMBA of Russia, all the protocols were approved by the Ethic Committee.
Before consulting an allergist-immunologist at the beginning of the study the children were diagnosed acute stenosing laryngotracheitis (OLT), they had been observed by an ENT doctor and pediatrician for 2 years. Thus, in the opening, the children aged 3-6 years old had: in 25 (50 %) -OLT of I degree, mild course (score of 3-5), according to the classification [1]; in 25 (50 %) -OLT of II degree, moderate course (score of 5-8) with inpatient treatment. Examination and monitoring of such patients are important and relevant. OLT diagnosis was made by an ENT doctor in accordance with the recommendations [8,9,10]. Further patient follow-up did not mark laryngeal stenosis symptoms, at an exacerbation accompanied by characteristic clinical manifestations; ENT diagnosed acute laryngotracheitis (ALT) [8][9][10][11]. Laryngitis and laryngotracheobronchitis are characterized by rough cough and hoarseness; there is no laryngeal stenosis phenomena and respiratory failure. ARVI can be accompanied by the following nosological forms: acute nasopharyngitis, acute pharyngitis, acute laryngi-tis, acute tracheitis, acute laryngotracheitis, acute laryngopharyngitis. ARVI symptoms can last on average 10-14 days [9,12,13]. In acute ARVI period the children were monitored and treated by a pediatrician at the place of residence. With recurrent ALT (3-8 episodes a year) the children were referred for an allergist-immunologist consultation outside the acute period. The children were examined by a pediatrician, allergist-immunologist, ENT doctor; pathology of the larynx and trachea is excluded. The diagnosis of ENT diseases (tonsillopharyngitis, rhinosinusitis, otitis media) was made by an ENT doctor [10,14]. All the children received rehabilitation of the foci of infection according to indications at the place of residence; antiseptic drugs were prescribed to irrigate the mucous of the tonsils and pharynx -benzyldimethyl [3-(myristoilamine)propyl]ammonium chloride monohydrate 0,01 % solution twice per day for 7 days. Considering the sensitivity, antibiotics were prescribed in the acute period (tonsillopharyngitis, rhinosinusitis) in the presence of complications, sowing from the pharynx, nose or tonsils mucosa Streptococcus haemoliticus β, Streptocоccus pneumonia, according to the recommendations [8,9,10,14].
The study included laboratory and instrumental methods: clinical blood test, urinalysis, biochemical blood test (according to indications), bacterial inoculation on flora and the sensitivity of microorganisms to antibiotics from the mucous of the pharynx and tonsils. Immunological indicators were determined by flow cytometry as instructed; monoclonal antibodies with mono-and double-label Beckman Coulter: CD3 − HLA-DR + -, CD3 + HLA-DR + -, CD3 − CD16 + -, CD3 + CD16 + -, TLR2 + CD14 + -, TLR4 + CD14 + -, CD14 + СD119 + -cells were used. TLR2 and TLR4 expression were determined using monoclonal antibodies by Hycult Biotech. The standard method of direct immunofluorescence was applied using monoclonal antibodies, labeled by a fluorescein isothiocyanate (FITC) or biotin, or phycoerythrin as instructed. The samples were analyzed using the flow cytometer Epics PROFILE-II (Cultronics) (FC 500 Beckman Coulter). Total IgE was defined by the ELISA method (Hema, Russia). Serum immunoglobulin concentration (А, М, G) was determined by the method of performing radioimmunoassay in Mancini gel [15]. Chemiluminescence study (spontaneous, induced, stimulation index) was conducted following the methology [16].
Statistical processing of the results was carried out using Standart program with Statistica 6,0 software package. Types of statistical analysis evaluating the reliability by Student's test, an interval estimate of standard deviation and dispersions of the normal distribution by χ 2 criterion, Mann-Whitney U-test were used.

REFINED INCLUSION CRITERIA FOR THE STUDY:
-children with ALT (3-8 episodes a year), with OLT I-II in anamnesis, aged 3-9 years old at the beginning of the strudy; -disease duration more than 1 year; -ARVI frequency 6-12 times a year; -ENT disorders (chronic tonsillopharyngitis, rhinosinusitis).

CRITERIA OF PATIENT EXCLUSION FROM THE STUDY:
-children with primary immunodeficiencies; -children with severe somatic diseases at the sub-and decompensation stage (hemorrhagic syndrome, diabetes, disease of liver, endocrine system, kidney and other internal organs; autoimmune diseases, active and latent tuberculosis); -children with severe allergic diseases (severe BA, diffuse AtD, severe AR, nasal polyposis II-IV); -children with ALV in the neonatal period, BPD; -children with diagnosed gastroesophageal reflux; -children, receiving immunotropic drugs for 6 previous months.

RESULTS AND DISCUSSION
When examining indicators of clinical and biochemical blood tests (total bilirubin, ALT and ACT), pathology of the general urine analysis was not found.
Infection persistence in children with recurrent ALT, ARVI and ENT pathology is presented in Table 1.
In children with recurrent ALT, ARVI and ENT diseases monoculture is identified in 42 %, 2 and more causative agents -in 54 %, Candida albicans -in 10 %, combined bacterial and fungal flora is marked in 10 % of examined children. Flora growth was not found in 16 % of children. Quan-titative indicators ranged from 4×10 4 CFU/ml to 6×10 6 CFU/ml. The results point to infection persistence on the pharynx mucous in children with recurrent ALT, ARVI and ENT diseases. When compared to the children with ARVI frequency 3-5 times a year with no OLT and foci of chronic respiratory tract infection there is significantly lower frequency of Staphylococcus aureus, Streptococcus pneumoniae, Candida albicans (p < 0,05; χ 2 > 3,8) sowing and less pronounced infection persistence. Quantitative indicators ranged from 3×10 3 CFU/ml to 4×10 5 CFU/ml. The groups are similar by sex and age. Table 2 presents the comparison of indicators in children with recurrent ALT, ARVI and ENT pathology and children with ARVI frequency 3-5 times a year, no ALT and ENT pathology. The children are presented without subdivision into age as dynamics of indicators were similar. There was a higher level of IgG, IgA, indicators of spontaneous and induced chemiluminescence, spontaneous production of IFN-γ cells, induced synthesis of IFN-α and IFN-γ in children without ALT and ARVI frequency 3-5 times a year with no ENT pathology (p < 0,05). Significant difference in the number of CD3 − CD16 + -, CD14 + -cells, stimulation index, IFN-α in serum and spontaneous production by IFN-α were not found. Children with ALT have a significant lower level of IgE, expression of TLR2, TLR4, CD119 receptors on CD14 + -cells. Most children with recurrent ALT experience increased expression of TLR2, TLR4, CD119 receptors on CD14 + -cells (p < 0,05). The results were obtained for the first time. Table 4 presents clinical efficacy data of prescribing bacterial lysate (BL) (broncho-vaxom) in children with recurrent ALT, ARVI and ENT diseases over 3 years of follow-up.
In the first follow-up year children received 2 courses of the drug, they experienced acute ENT-pathology, ARVI and recurrent ALT. In the 2 nd and 3 rd year there were fewer cases of acute ENT-pathology, ARVI and recurrent ALT. Thus, 1 course of BL (broncho-vaxom) a year was prescribed. In 3 years the control group had a significant decrease in the frequency of acute tonsillopharyngitis (р < 0,05), reduction in the need for antibiotics is significant on the Mann-Whitney test (р < 0,05), dynamics of other indicators were not reliable.
Group Iб, that received BL, showed a significant decrease in the frequency of tonsillopharyngitis and ARVI (р < 0,05) in the 3 rd year, all the indicators were lower, compared to Ia group (р < 0,05), ARVI frequency is 2,6 times as low.
tivation of innate immunity with chronic and recurrent infections, particularly TLR-receptors, is the starting point in the protection development.
After activation of TLR-receptors there is a signal transmission to the nucleus, activation of the synthesis of proinflammatory and anti-inflammatory cytokines, acute phase proteins [17,18,19]. TLRs family includes 5 subfamilies (TLR2, TLR3,  TLR4, TLR5, TLR9). Sensitivity to a pathogen for tuberculous meningitis depends on TLR2 polymorphism [20]. The interaction of lipopolysaccharides and CD14-receptors through TLR4 leads to activation of cytokine synthesis [21]. We studied the expression of TLR2-and TLR4-receptors on CD14 + -cells (TLR4 + CD14 + , TLR2 + CD14 + ), receptors to IFN-γ (CD14 + CD119 + ) with recurrent ALT, ARVI and ENT diseases for the first time. Children with no ALT and with ARVI frequency 3-5 times a year had significantly lower IgE, lower expression of TLR2 + , TLR4 + , CD119 + receptors on CD14 + -cells. The results were obtained for the first time. It is known that combined activation of IFN-γ synthesis and TLR (TLR4) expression results in activation of STAT1, IRF-1 transcription factors and cytokine genes (TNF, IL-6, IL-12) that triggers the immune system activation [22]. According to our data, IFN-γ decrease combined with a rise in the expression of TLR4 and TLR2 receptors on CD14 + -cells in most patients points to dysregulation of immunological interactions. At the same time, there is reduction in IFN-α synthesis in 30-40 % of children of differ-ent ages that decreases antiviral protection. Children with no ALT and ENT-pathology with ARVI frequency 3-5 times a year have less pronounced changes in the immune system. These results are not found in the available literature.
TLR-receptors are known to recognize conservative patterns on pathogens and to launch protection mechanisms, activating signaling pathways and cytokine genes. Imbalance of TLR-mediated IFN-α production is found in patients with CVID. According to M.V. Horeva, the number of TLR2 + CD14 + -cells in children aged 10-15 years was 29,6 ± 9,5 %, in adults aged 20-40 years old -39,7 ± 11 %; TLR4 + CD14 + -cells in children aged 10-15 was 7,5 ± 2,4 %, in adults aged 20-40 -6,8 ± 1,3 %б respectively. The expression of TLR2and TLR4-receptors increased in acute myocardial infarction with inflammation [23]. According to our data, the number of TLR2 + CD14 + -cells and TLR4 + CD14 + -cells in children with recurrent ALT, ARVI and ENT disorders was higher with the diagnosed persistence of bacterial flora and infectious inflammation. Reduction in ARVI frequency and a decrease in the persistence of bacterial flora lower TLR2 and TLR4 expression that coincides with the data of other researchers [24]. Viral-bacterial association, specifics of microbiome [25] and local immunity [7,26] often occur with respiratory infection. Our study shows that recurrent course with the drug (broncho-vaxom) over 3 years of follow-up help to reduce the frequency of acute tonsillopharyngitis, rhinosinusitis and the * р < 0,05 by comparing the data in a year and 3 years in the control group.
** р < 0,05 by comparing the data in a year and 3 years in the group that received the bacterial lysate.
*** р < 0,05 by comparing the data in the control group and the group that received the bacterial lysate in 3 years.
need for antibiotics that confirms the possibility and feasibility of its administration in the complex treatment of recurrent ALT, ARVI and ENT-pathology. The administration of the bacterial lysate can activate innate immunity through TLR2/6-and TLR9-receptorр, signaling pathways and IFN-γ, β, α synthesis and lead to the decrease of ARVI frequency, obstruction episodes in children and adults with COPD [27,28] that correlates with our results on the administration of the drug (broncho-vaxom) with recurrent ALT, ARVI and ENT-pathology.
We have shown previously that patients with recurrent ALT and ARVI may experience allergic diseases that requires observation and treatment by an allergist-immunologist [29]. Thus, patients with an allergic pathology and the development of ENT diseases may be identified among children with recurrent ALT and ARVI that allows to choose a differentiated treatment approach. CONCLUSION 1. In children with recurrent ALT, ARVI and ENT-pathology the expression of TLR2 and TLR4 on CD14 + -cells (TLR4 + CD14 + , TLR2 + CD14 + ) and receptors to IFN-γ (CD14 + CD119 + ) are significantly higher than in children with no ALT and ARVI frequency 3-5 times a year, accompanied by a decrease in IFN-γ and IFN-α synthesis that is associated with the development of the immune system dysregulation.