PLACEBO-DRIVEN CLINICAL TRIALS OF TRANSFER POINT GLUCAN #300 IN CHILDREN WITH CHRONIC RESPIRATORY PROBLEMS: ANTIBODY PRODUCTION

The role of glucan in stimulation of immune reactio ns has been well-established. In this report, we fo cused on the antibody production in glucan-supplemented c hildren with chronic respiratory problems. We measured the levels of salivary IgA, IgM and IgG in 40 children aged 8-12 years and evaluated the effe cts of 100 mg d oral dose of glucan. We found a significant increa se in production of all tested antibodies in the glucan-stimulated group, but a decrease of anti body production in the control group. A thirty-day oral application of yeast-based natural immunomodulator β-glucan strongly stimulated the mucosal immunity of children with chronic respiratory problems.


INTRODUCTION
Upper respiratory infections are the highest incidence of acute illness in the developed world. According to the estimates, the average adult has 3 colds per year and the average schoolchild up to 10 (Spector, 1995;Vetvicka and Vetvickova, 2010). Although patients with complications may benefit from antibiotic or inhaler treatment, current medicine has very little to offer for uncomplicated infections. There is no doubt that a need for effective, safe and inexpensive treatment of chronic respiratory problemsexists. Our paper is focused on the natural immunomodulator β-glucan as a possible solution.
β-Glucans are well-known natural Biological Response Modifiers (BRM). Thus far, glucans are classified as "non-specific" because their biological effects are pleiotropic. On the other hand, for the past 30 years, Japan has sucessfully used several forms of mushroomderived β-glucan in cancer patients (Hamano et al., 1999).
Numerous rodent studies demonstrated that glucan supplements offset the increased risk of infection, usually via stimulation of immunological activities (Davis et al., 2004). In addition, recent findings also showed strong effects on humoral immunity, including antibody formation (Vetvicka and Vetvickova, 2008;. Up to now, the effects of glucan on mucosal immunity has not been studied. Recently, several preclinical and clinical studies showed that yeast-derived glucan reduced respiratory symptoms of women in stress (Talbott and Talbott, 2012) and helped to maintain the body's defense against pathogens (Auinger et al., 2013). For our clinical trial, we used the best characterized insoluble, yeast-derived glucan #300, which was repeatedly confirmed to be the most active commercially available glucan (Vetvicka and Vetvickova, 2007;.

AJI
In addition, our previous clinical trials showed a significant increase of salivary lysozyme in glucantreated children (Vetvicka and Vetvickova, 2009). In this report, we used the same model of children with chronic respiratory problems living in an area known for its extremely high level of pollution and focused on the effects of glucan administration on antibody response.

Protocol
The same protocol that was previously described (Vetvicka and Vetvickova, 2011) was been used throughout this study. Briefly, a randomized, doubleblind, placebo-controlled trial compared β-glucan #300 and placebo in children. Forty children (24 females, 16 males, age 8-12, average 10.7±2.3) from the sanatorium for respiratory diseases EDEL were enrolled in the 4week trial. 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 offor clinical testing for the Czech Republic. Parental consent was given in all cases.
Subjects were randomly assigned to groups which were blinded to intervention. During the intervention period, the subjects consumed 100 mg d −1 of β-glucan or placebo. Both glucan and placebo capsules looked identical. Subjects were routinely evaluated by the medical staff.

Tests
In all subjects we obtained saliva at the beginning of the study and at the conclusion of their stay in Sanatorium. We used identical times (between 8 and 9 AM) for sampling, so the possible influence of circadian rhythms could be eliminated.
Saliva was collected using a commercial Salivette device (Sarstead, Orsay, France). After 2 min of chewing,the cotton swab was added into a sterile container and centrifuged at 1,000 g for 15 min and stored at-15°C. We measured the levels of sIgA, sIgG and sIgM using nephelometer Siemens BM II as suggested by the manufacturer.

Statistical Analysis
Statistical significance was evaluated by a pair t-test using a GraphPad Prism 502 software (GraphPad Software, USA).

RESULTS
All children participating in this study are living at the same locality at Nothern Moravia, known for its extremely high level of pollution. Only children diagnosed with repeated upper airway infections, chronic bronchitidis, allergies and/or asthma were used in this study. All subjects were given an identical diet with optimal nutrition and were identically treated using climatotherapie and speleotherapie. In addition, a full medical examination was given at the beginning and end of the trial. The identification of glucan-treated and placebo-treated group was done only on the end of the study.
Our results of the specific antibody response are shown in Table 1. Our data suggest that at the beginning of the study, the levels of IgG were identical in both groups. However, the glucan-supplemented group showed a strong increase in IgG production, whereas placebo group expressed significant decrease of salivary IgG levels (Fig. 1).

DISCUSSION
Salivary anti-microbial systems are usually divided into immune (secretory antibodies) and non-immunoglobulin (lysozyme, agglutinins, lactoferrin). Most children with respiratory problems show a normal immunological status (Hosking et al., 1978), probably with exception of IgA (Ostergaard, 1980). Additional reports showed serum IgG and IgM levels to be significantly elevated, with sIgA remarkably low, suggesting the important role of salivary IgA in fighting respiratory infections (Lehtonen et al., 1987). Children living permanently in a region with a high level of pollution were found to have increased levels of sIgA (particularly in children with high frequency of respiratory diseases), which is usually increased after several weeks in nonpolluted areas (Richter and Pelech, 1996).
After the early research on mucosal immunity, recent decades show a revival of interest in salivary proteins in clinical immunology (Ambatipudi et al., 2009;Castagnola et al., 2011;Fabian et al., 2012). Some of the reasons might be the non-invasiveness of saliva collection, the lack of need of trained personnel and the lack of a risk of infection. Saliva is often considered to be a "body mirror" with little used abilities in diagnostics (Zhang et al., 2009). Proteomic studies lately helped to overcome most of the criticism suggesting that the saliva content is influenced by either lifestyle, genetic markup, age or sex (Pfaffe et al., 2011;Pink et al., 2009). Today we are able to find in saliva over 2,400 components, whereas only 20% of them can be found in plasma (Ambatipudi et al., 2009;Castagnola et al., 2011;Pfaffe et al., 2011;Lehne et al., 2006;Topkas et al., 2012). Our laboratory has been specializing in salivary immunity for quite some time for both clinical evaluation and for studies of the effects of environmental pollution on large populations. The current use of new Salivette devices significantly increased the quality of results (Topkas et al., 2012).
For our study we choose glucan #300, which is currently not only highly studies glucan, but also a glucan with the highest and most pleiotropic activities (Vetvicka and Vetvickova, 2010; regardless the rout of administration (Vetvicka and Vetvickova, 2010). This study is a follow-up of our original finding of the effects of this particular glucan on secretory proteins in saliva, including CRP and lysozyme (Vetvicka and Vetvickova, 2011). Again, we tested the effects of glucan supplementation on children living in region with extreme environment pollution and suffering from chronic respiratory problems. Properly functioning immune system is crucial to defend against all invading pathogens, including common cold viruses. Currently, yeast-derived glucans were confirmed to have beneficial effects against common cold and respiratory symptoms (Talbott and Talbott, 2012;Auinger et al., 2013) and against allergy symptoms via decreasing proinflammatory cytokines such as IL-6 and TNF-α and increasing of secretion of IL-10 and accession of cellular antioxidants (Senoglu et al., 2008). These studies, however, were restricted to adults. In addition, glucan was found to significantly improve the immunosuppression caused by various toxins (Vetvicka and Vetvickova, 2012), making it an optimal molecule for children from environmentally poluted areas.
Whereas immunomodulatory effects of glucan in both animal models and clinical studies are well established (Novak and Vetvicka, 1987;Ramberg et al., 2010), very little is known about the effects on salivary immunity (Lehne et al., 2006). No clinical study evaluating glucan affecting antibody response of children with respiratory problems exists in the literature.
sIgA represents the most studied salivary component. Studies on dogs (Stuyven et al., 2010) and humans (Lehne et al., 2006) demonstrated fast and significant increase of sIgA levels. Different situation was found in case of sIgG: an increase in dogs (Stuyven et al., 2010), but no changes in humans (Lehne et al., 2006). Our data showing strong parallel increases in the levels of both sIgA and sIgG suggest possible effects of dose and the quality of glucan. Some suggest that the correlation of levels of salivary IgA and IgG are in inflammation influenced by permeability of capillaries (Gleeson et al., 1995), others hypothesize the effects of stress factors (Ambatipudi et al., 2009;Brown et al., 1977;Francis et al., 2002), which cannot be overlooked in our model of children during their early days at the sanatorium. Low IgG level in control group (C2) correlates with albumin levels (Vetvicka and Vetvickova, 2011). Decrease in albumin level (as indicator of inflammation) is in compromised children caused by reduction of mucosal irritation in respiratory tract, which is correlated by glucan-induced stimulation of IgG production.
The findings of increased levels of sIgM just add another stone to the mosaic of glucan-mediated immune response in disease-prone children. The increase of sIgM after glucan supplementation correspond to (Stuyven et al., 2010), the higher levels in control group (C1) were most probably caused by sublinical infection in some children, which in a relatively low number of samples could significantly influence the average amount. Our data also showed significant improvement of the overall health of tested children.

CONCLUSION
Taken together, our results strongly suggest that natural immunomodulator glucan can significantly improve the health of children with chronic respiratory problems, probably via stimulation of their mucosal immunity.

ACKNOWLEDGEMENT
This study was supported by Technology Agency of the Czech Republic TACR TA 0202094.