Quality of nutrition optimized to energetic needs of different age groups, living in different environmental conditions, is one of the basic requirements for maintaining quality health. Basic condition is adequate nutrition including all components necessary for optimal development of an organism. This also comprises elements for regeneration and resistance to environmental effects, conservation of physiological functions and strong immune responses. These conditions are particularly important during development of an organism (starting from pregnancy) as well as during aging and high physical or mental stress [1]. Adequate nutrition can support convalescence of an organism, which is under steady extreme stress from the high pollution of environmental conditions. It is therefore imperative to improve the quality of nutrition by augmenting it with food supplements corresponding by both the quality and quantity to the pressures of local exposition of well-defined harmful elements. Currently, nutritional supplements are gaining popularity. However, they do not always correspond to the requirements of individual health conditions, both in their quality and their purity. In addition, the marketing pressure of various companies often results in the situation where these supplements are often misused for reparation of physiological functions. On the current market, one can find an extensive spectrum of supplements without adequate animal and clinical trials, allowing us to define them as nothing better than placebo. This study is based on previous testing of a supplement with high levels of nucleotides (Imuregen). This particular material has already been tested for immunomodulation on several different types of patients, from children to patients with either cancer or cancer anamnesis. This data showed significant immune system improvements including NK cell reparation and higher production of sIgA, improved role of Fe in metabolism, modification of microbiome and of growth and maturing of gut mucosa [2,3]. This data supports the notion that Imuregen has immunoreparative effects. In addition to Imuregen supplementation, we also used supplementation with β-glucan. β-glucans represent the most studied immunomodulators with more than 30,000 published studies. They represent highly conserved structural components of cell walls in yeast, fungi, barley, and seaweed. They are effective biological response modifiers, able to both nonspecifically and specifically enhance the immune system. After several decades of intensive research on biological effects, β-glucan properties are well established. Various studies have definitively shown that β-glucans represent important substances exerting strong immunomodulatory properties [4]. These β-glucans may be included, among other substances, to act through an organism´s own biological response mechanisms as biological response modifiers [5].

Protocol

The same protocol that was previously described [6] has been used throughout this study. A randomized, double-blind, placebo-controlled trial compared β-glucan #300 and Imuregen in children. One hundred and five children (age 9 -14) were divided into individual groups and enrolled in the 4-week trial. A total of 71 children were from the Usti nad Labem region with the highest level of pollution (group UL). An additional 34 children were moved to the Sanatorium EDEL (group ZH) for the same supplementation. The UL group consisted of 45 girls and 26 boys, group ZH consisted of 14 girls and 20 boys. Ages of patients in individual groups and subgroups were identical with range between 10.5 and 12.5 years. The clinical trial was conducted at the Sanatorium EDEL (Zlate Hory, Czech Republic) and the study was approved by the Ethics Committees of the Public Health Institute and Sanatorium EDEL Czech Republic. This study was performed in agreement with Helsinki declaration (revised version 2000.09.01) and in full compliance with the rules of clinical testing for the Czech Republic. We explained the experimental protocol to every parent and obtained consent forms from all participating patients. Subjects were randomly assigned to groups, which were blinded to intervention. During the intervention period, the subjects consumed 0.25 mg of Imuregen, 100 mg/d of β-glucan or placebo. Both β-glucan and placebo capsules looked identical. Saliva was collected using a commercial Salivette device (Sarstead, Orsay, France). After two minutes of chewing, the cotton swab was added into a sterile container and centrifuged at 1,000 g for 15 minutes and stored at -15°C. We measured the levels of sIgA using nephelometer Siemens BM II as suggested by the manufacturer. Subjects were routinely evaluated by the medical staff.

Statistical analysis

Paired t-test statistical significance was evaluated (GraphPad Prism 5.04; GraphPad Software, USA). An average and standard deviation was evaluated after determining standard value composition (D’Agostino, Pearson). In case of nonstandard composition, values were converted into logarithms.

Comparison of the initial samples of children from the polluted environment with children in the Sanatorium EDEL found significant differences in levels of salivary IgA. These differences probably resulted in the bloodstream irritated by inflammation allowing higher penetration of IgA into the mouth cavity. When we compared these samples at the end of food supplementation, we did not find a significant decrease of sIgA levels. A small decrease is caused by supplementation, which clearly was not sufficient (either dose or duration) under persisting conditions. In patients from the Sanatorium EDEL, we found a statistically significant decrease of sIgA levels (p=0.0095); the decrease in the placebo group was not significant (p=0.075) (Figure 1). All children in group ZH got full medical treatment, which included not only food supplementation with tested material, but, most of all, a break from environmental pollution, optimal nutrition, climatotherapy and speleotherapy. Levels of sIgA in ZH group are elevated due to the fact that only children accepted to the Sanatorium EDEL are children with acute inflammation, particularly inflammation of respiratory tract. Children from control group had no clinical manifestation of any health problem.

The immune system of an individual has been burdened by environmental factors since early development. Long-term exposure of a population results in a burden to the entire organism. This vulnerability slowly, but steadily, results in damage of physiological mechanisms with subsequent clinical manifestation of numerous diseases. Out of these diseases, most are respiratory tract problems, higher frequency of allergic and cardiovascular diseases, and cancer [7,8]. An exposed population suffers from lifespan shortage and seniors, in particular, suffer from deteriorating health and productivity. Exposure to very small particles in utero leads not only to disruption of respiratory tract development and physiological functions, but also to risk of premature birth and subsequent disruption of physiological functions connected to the growth and development of the child [7]. Long-term subjection to these ultrafine particles results in high frequency of lung, cardiovascular and cancer diseases [8-10]. In addition, mechanisms of mucosal immunity, both humoral and cellular, are damaged. Some region of North Bohemia and North Moravia can serve as a model of these complications, with significant health problems already found at an early age (Figure 2). When we compared this situation with the WHO study, we found that the average age in these regions is approximately 3.5 years shorter than the entire country, and even 3 to 8 years shorter when compared to Scandinavia, Great Britain and Germany.

Children evaluated in our study are from the northwestern region of the Czech Republic, which is the region with approximately 800,000 inhabitants living in the 7,800 km² region with 4 large coalmines responsible for significant pollution and overall destruction of the entire area [11,12]. The situation is further aggravated by using the coal in six power stations and seven heating plants. In addition, current chemical industry contaminates the environment with formaldehyde, mercury, and organic contaminants. When we add the contamination caused by commercial transportation using an important commercial North-South axis, it is clear that this region can be characterized as a “black stain of Europe”. Sanatorium EDEL is a private medical institute focused on respiratory problems using ideal climatic conditions of Zlate Hory. This location is at the border between the Czech Republic and Poland with 410 meters of elevation. Presently, the sanatorium is used for children between 2 and 18 years of age. The average stay is five weeks and is dependent on diagnosis and overall health conditions. The children are under constant supervision of both pedagogical and medical staff. The nutritional diet is under direct supervision of trained nutritionists. The situation mentioned above supports the need for evaluation of novel compensatory treatments, similar to the ones which appeared in the Czech Republic during the 60^th and 80^th of the last century. The temporary transfer of children from a heavily polluted environment to a less polluted area, accompanied with food supplements and antioxidants, resulted in the reduction of respiratory diseases, allergic diseases and caused overall improvement of the immune system. Currently we are proposing to apply nutritional aspects using food supplements with well- characterized effects of immune reaction modulation and overall health. One possibility is the use of nucleotides, where positive effects were already established during the fetal period and during breast feeding [12,13]. These findings resulted in implementation of milk formulas with nucleotides [14] with clear manifestation of mucosal improvements, higher level of immune function [15-17], and healing of immune conditions [18]. In addition, reparation of the parenchyma in the liver [1,19] and expansion of secretory system [20-22] have been observed. Positive effects in modulation of the secretory immune response [23] resulted in application of nucleotides in patients suffering from bronchial asthma [24]. Availability of highly purified preparation resulted in application of nucleotides to individuals with high physical stress including athletes [2,3,25]. In our experimental group, we focused on supplementation with nucleotides, either alone or in combination with β-glucan, in patients treated for respiratory tract diseases [1,26,27]. Preparation of nucleotides used in our study is a mixture of nucleotides, polypeptides and amino acids and is currently intensively studied for its action on various diseases [28]. Levels of sIgA is heterogeneous until the age of five. This wide range is caused by various factors including length of birth, length of pregnancy, and type of pregnancy. In older children, the sIgA levels are more constant, influenced by health state and by possible contamination by blood during teeth growth. In general, around age 5 we find the sIgA levels in 50–100 mg/L range, which increased at age 10 to the 100–150 mg/L range. It might be important to note that in similar experiments it is necessary to observe the exact and identical time for collection of saline (between 7 and 9 AM in all our experiments), as the circadian rhythm can change the sIgA levels up to 5x. Our laboratory has long-term interests in evaluating effects of β-glucan concerning several types of diseases. The positive results obtained in adult population focused our attention on children in Sanatorium EDEL and in the North Bohemia region [19,26,29,30]. In this study, we focused our attention on a combination of preparation where we can expect reparative function (nucleotides) and activation (β-glucan). In addition, we evaluated potential effects on two groups of patients – one in a heavily polluted region, one in environmentally clear conditions at Sanatorium EDEL. In groups of children in the polluted region, no significant effects of food supplementation were found. We hypothesize that the reason is an underestimated dose and/or duration of supplementation. In addition, it is important to keep in mind that immune system repair and immune function development requires improved nutrition, balanced physical activity, rehabilitation, and additional processes. Future studies focused on higher doses of supplements are being developed.