β‐Glucan supplementation, allergy symptoms, and quality of life in self‐described ragweed allergy sufferers

This randomized, placebo-controlled, double-blind study compared the effects of daily supplementation for 4 week with 250 mg Wellmune WGP® β-1,3/1,6-Glucan (WGP) with placebo 250 mg/day (rice flour) on physical and psychological health attributes of self-described “moderate” ragweed allergy sufferers. Study participants (mean age = 36 ± 9 year; range 18–53 year) were recruited before the beginning of ragweed season (September) in Northeastern Ohio. Serum IgE concentration, allergy symptoms [via self-report, Visual Analog Scale (VAS), and Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ)], psychological well-being [Profile of Mood States (POMS)], and physical function (RAND SF-36 Medical Outcomes Study) were measured immediately prior to and after supplementation with WGP (n = 24) or placebo (n = 24) for 4 weeks. Data were analyzed using repeated measures analyses of variance (ANOVA). Compared with placebo, WGP reduced total allergy symptoms (28%), symptom severity (52%), and symptom rating on the VAS (37%) (P < 0.05), but had no effect on serum IgE levels. As measured by the POMS, WGP increased participants' rating of vigor (10%), but reduced tension (34%), depression (45%), anger (41%), fatigue (38%), and confusion (34%) (P < 0.05). Study participants given WGP reported increased physical health (11%), energy (19%), and emotional well-being (7%) compared with study participants given the placebo (RAND SF-36 Medical Outcomes Study). The WGP group also reported decreased sleep problems (53%), reduced nasal symptoms (59%), eye symptoms (57%), non-nasal symptoms (50%), activities (53%), emotions (57%), and improved quality of life (QOL) (56%), as well as improved global mood state (13%). Supplementation with WGP for 4 weeks improved allergy symptoms, overall physical health, and emotional well-being compared with placebo in self-described “moderate” ragweed allergy sufferers during ragweed allergy season.


Introduction
An estimated 40-50 million Americans are affected by allergic disease (National Institute of Allergy and Infectious Diseases 2003). Pollen from plants of the genus Ambrosia (which includes ragweed) is a primary cause of allergic rhinitis, also known as hay fever, during summer and fall (Frenz et al. 1995). In the U.S., ragweed season can extend from August through October (Pongdee 2011). The U.S. National Health and Nutrition Examination Survey III conducted from 1988 through 1994 reported a prevalence of 26.2% for ragweed allergy as determined by skin test reactivity (Arbes et al. 2005).
of life (QOL) (Leynaert et al. 2000;Meltzer 2001), and decreased work productivity, accounting for an estimated 3.5 million work days and 2 million school days lost per year (Nathan 2007). In addition to local inflammation, allergic rhinitis can induce systemic inflammation, which may result in inflammation of the upper and lower airways (Pawankar et al. 2011). Comorbid conditions for allergic rhinitis include asthma, rhinosinusitis, nasal polyposis, serous otitis media, and sleep disorders (Young 1998;Bousquet et al. 2001;Pawankar et al. 2011). Allergic conjunctivitis affects more than 20% of the U.S. population  and approximately 18% of children, aged 12-14 in the U.K. (Austin et al. 1999). Inadequate control of allergic rhinoconjunctivitis leads to decreased QOL, reduced productivity, and potentially, increased visits to doctor's offices.
Approaches to reduce the physical and psychological symptoms of allergic disease include allergen avoidance, pharmacotherapy, and immunotherapy (Bousquet et al. 1994). Standard allergen immunotherapy can prove problematic due to inconvenient dosing regimens and the potential for induction of systemic allergic reactions, such as anaphylaxis, because of the relatively large doses of allergen administered (Lockey et al. 1987;Du Buske et al. 1992;Bukantz and Lockey 2004). A safe, effective, and convenient therapy for allergic disease would provide relief of the physical and psychological effects of allergies and improve the QOL of allergy sufferers (Kim et al. 2011).
b-Glucans are a diverse class of long-chain glucose polymers that have a backbone of b-(1-3)-linked b-Dglucopyranosyl units with primarily b-(1,4)or (1,6)linked side chains. These naturally occurring substances have been shown to provide health benefits including wound healing (Delatte et al. 2001), decreasing blood lipid concentration Nicolosi et al. 1999), protection against infection (de Felippe 1993;Babineau et al. 1994a, b;Dellinger et al. 1999), inhibition of tumor development (Cheung et al. 2002;Hong et al. 2004) and metastasis (Yoon et al. 2008), and promotion of tumor regression (Driscoll et al. 2009). b-Glucans are known as biological response modifiers. Their effects have been investigated by administration through diverse routes; importantly, they exhibit biological activity when consumed orally (Hong et al. 2004;Rice et al. 2005) and may be combined with other immunotherapies to elicit a more powerful effect (Hong et al. 2004;Chen and Seviour 2007). They activate the entire immune system, which may be advantageous for their use as therapy for disease.
Identification of b-glucans as nonself molecules by mammalian cells induces innate and adaptive immune responses (Vetvicka and Yvin 2004;Brown and Gordon 2005). The initial response to b-glucans occurs via the innate immune system. This response is rapid and nonspecific, occurs without memory, and primarily involves phagocytic cells, such as macrophages and neutrophils. Following ingestion, b-glucans are bound to PRRs on macrophages and are internalized. As reviewed by Volman et al. (2008), b-glucans interact with the mucosal immune system including the intestinal intraepithelial lymphocytes and Peyer's patches, inducing cytokine production and increasing resistance to infection (Suzuki et al. 1990;Tsukada et al. 2003). Whole glucan particle (WGP), a purified yeast b-glucan sphere derived from standard food-grade Baker's yeast, Saccharomyces cerevisiae, enters the gut and is taken up by Microfold (M) cells in the Peyer's patches. Intestinal macrophages transport b-glucan to the organs of the immune system (e.g., the spleen, lymph nodes, and bone marrow) via the lymphatic system In the bone marrow, larger WGP are degraded into smaller fragments by macrophages (Hong et al. 2004). b-Glucan-induced signaling is not dependent on internalization of whole b-glucan particles (McCann et al. 2005). b-Glucans are believed to induce phagocytosis (Ladanyi et al. 1993;Kurashige et al. 1997;Brown et al. 2002), stimulate microbial killing by mechanisms such as respiratory burst (Tsoni and Brown 2008), and initiate production of innate immune system components including inflammatory mediators such as tumor necrosis factor alpha (TNF-a), interleukin-1 (IL-1) (Li et al. 2006), macrophage inflammatory protein 2-alpha (MIP-2), eicosanoids, reactive oxidants (Vassallo et al. 1999(Vassallo et al. , 2000Hahn et al. 2003), and local immunomodulators. Effects of b-glucans on nonimmune cells have also been reported (Hahn et al. 2003;Ramakers et al. 2007). Other cells are recruited to the infection site and activate the adaptive immune system.
The response of the adaptive immune system involves production of antibodies against specific substances by Blymphocytes. Assay of IgE can determine whether an individual has been sensitized to a substance such as ragweed. The adaptive immune system response also involves antigen presentation by leukocytes to T-lymphocytes. Cytotoxic and T-helper cells attack infected body cells (Volman et al. 2008) and depending on the antigen presented, results in the development of immature cells to type 1 helper T-lymphocytes (Th1) or type II helper T-lymphocytes (Th2) (Volman et al. 2008). Individuals experiencing an allergic response of asthma are thought to have an overactive Th2 response. b-1,3-glucan can stimulate macrophages, which secrete anti-inflammatory mediators, such as prostaglandin E2, tumor growth factor, and IL-10, and may inhibit the Th2 response (Sarinho et al. 2009).
In this study, we investigated whether supplementation with WGP would alleviate the physical and psychological effects of allergies and improve QOL in self-described "moderate" ragweed allergy sufferers during ragweed allergy season.

Recruitment of study participants
The conduct of this study adhered to the Helsinki Declaration, which was revised in 1983, for clinical research involving humans. To recruit subjects, we posted flyers at a walk-in medical clinic where we have conducted previous studies, asking that "people with ragweed allergies" contact us. We screened 62 otherwise healthy selfdescribed "moderate" sufferers of ragweed allergy or individuals who considered themselves to have "hay fever" before peak ragweed allergy season in Northeastern Ohio in 2010 during September and October. Subjects were given a description of the study protocol. On the first visit, we conducted a physical examination of subjects. Individuals who had current URTI symptoms, were currently taking allergy or asthma medications, cited an inability to understand the study objective, were unable to complete all questionnaires and provide blood samples, were pregnant or lactating, or were currently using anti-biotics or other "immune" support products were excluded from the study. The Institutional Review Board at Aspire IRB, La Mesa, CA, approved the protocol for the study. All study participants provided written informed consent prior to the start of the study. The flow of the study participants is shown in Figure 1 and demographic information about the study population is shown in Table 1.

Study design and intervention
We conducted a randomized, placebo-controlled, doubleblind study. Study participants were randomly assigned using a random number generator to one of two treatment groups and were instructed to ingest a capsule containing 250 mg WGP (WGP group) or 250 mg of rice flour (placebo group) once per day, preferably in the  morning for 4 weeks. Fifty of the 62 individuals that we screened met the inclusion criteria for the study, and 25 study participants were assigned to each treatment group. Study participants reported to the test site 7 days prior to the start of the study. We supplied study participants with sufficient WGP and placebo capsules to last for the duration of the study. One subject from each group did not complete the study. The final subject population consisted of 17 men and 31 women (36 AE 9; range 18-53 years of age) (see Table 1).

Outcome measures
The outcomes of the study were ragweed-specific blood IgE concentrations and physical and psychological health attributes of the study participants. Measurements were made at baseline and after study participants consumed WGP or placebo for 4 weeks. IgE concentrations were measured using stored serum samples (N = 48). Measurements were made for three common ragweed allergens (Giant, Common, and False). IgE levels were measured using the IMMULITE 2000 3gAllergy test kit (Siemens AG, Eschborn, Germany; as described by Biagini et al. 2006). Subjects were asked to complete study surveys that provided information about their physical symptoms (allergy symptoms via self-report, the Visual Analog Scale [VAS; Hornblow and Kidson 1976;Price et al. 1983;Linder 1988;Hallen et al. 2001;Bousquet et al. 2007]; and Rhinoconjunctivitis Quality of Life Questionnaire [RQLQ; Juniper and Guyatt 1991;Juniper et al. 1996]); psychological well-being (Profile of Mood States [POMS; McNair et al. 1971McNair et al. , 2003); global mood state (POMS); and physical function (RAND SF-36 Medical Outcomes Survey [Ware 1992;Hays et al. 1993]).

Compliance and adverse events
Compliance with the study protocol was determined by completion and return of study surveys and verbal compliance to daily supplementation instruction. Pills were counted when subjects returned their bottles of capsules at the last visit. Except for two individuals, study participants were better than 90% compliant with the study protocol. The two subjects who did not comply with the study protocol were dropped from the study. Adverseevent information was collected by verbal disclosure; no adverse events (other than allergy symptoms, the primary outcome of the study design) were reported in this study.

Statistical analysis
Data were analyzed using repeated measures analyses of variance (ANOVA). Statistical analysis was conducted using JMP 8.0 (SAS Institute, Cary, NC) software and significance was set at P < 0.05. The study was set up as a pilot study to determine design criteria for future rhinoconjunctivitis studies; no power calculations were performed.

Results
Fifty subjects were enrolled in the study; 48 individuals completed the study. Subject attrition, one from each treatment group, was due to failure to submit all completed surveys and questionnaires.
Physical Symptoms (Allergy Symptoms and RQLQ Survey) Figure 2 compares the serum IgE concentrations (reported in kU/L), total number of allergy symptoms, allergy symptom severity, and allergy symptom scores on the VAS (Hornblow and Kidson 1976;Price et al. 1983;Linder 1988;Hallen et al. 2001;Bousquet et al. 2007) before and 4 weeks after treatment with WGP or placebo. There were no differences in ragweed-specific serum IgE levels. Compared with the placebo post-treatment group, the total number of allergy symptoms (Sx) for the WGP group was reduced 28% (WGP group post-treatment = 4.2, placebo group post-treatment = 5.8, P < 0.001), and participants' ratings of symptom severity were reduced 52% (WGP group post-treatment = 6.9, placebo group post-treatment = 14.3, P < 0.001). The rating of symptoms on the VAS was reduced by 37% after study participants were treated with WGP compared with after treatment with placebo for 4 weeks (WGP group posttreatment = 15.2 vs. placebo group post-treatment = 24.2, P = 0.024). Figure 2. Allergy Symptoms. When compared with placebo treatment with Wellmune WGP ® for 4 weeks, there was a reduction observed in total number of allergy symptoms (28%), in symptom severity (52%), and in the rating of symptoms on the VAS (37%).*P < 0.05 compared with placebo. Figure 3 shows study participants' reports of level of tension, depression, anger, vigor, fatigue, and confusion as rated using the POMS before and after treatment with WGP and placebo. When compared with mean pretreatment values for Wellmune WGP, treatment with WGP for 4 weeks reduced tension by 34%: 7.3 versus 4.8, P < 0.05; depression by 45%: 5.1 versus 2.8, P < 0.05; anger by 41%: 6.6 versus 3.9, P < 0.05; fatigue by 38%: 6.8 versus 5.4, P < 0.05; and confusion by 40%: 5.3 versus 3.2, P < 0.05. Subjects reported increased vigor after WGP treatment compared with before treatment (+10%, WGP pretreatment = 18.3 vs. WGP posttreatment = 20.3). There were no significant differences between the placebo pre-and post-treatment groups or between the WGP post-treatment group and placebo post-treatment in tension, depression, fatigue, confusion, and vigor.

Profile of mood states
Global Mood State (cumulative POMS measurement) Figure 4 compares study participants' reporting of Global Mood State, a cumulative POMS measurement, pre-and post-4-week treatment with WGP or placebo. Global Mood State ratings were 110 and 108 pre-and post-treatment with placebo (n.s.), and 115 and 100 pre-and post-treatment with WGP (13%) for 4 weeks (P < 0.05). This demonstrates that subjects on Wellmune WGP had an improved Global Mood State over the 4-week period compared with WGP pretreatment levels.

Discussion
In this study, we showed that administration of WGP during allergy season reduced the total number of allergy symptoms, rating of symptom severity, and symptom rating on the VAS, but had no effect on IgE concentration compared with placebo treatment in self-described ragweed allergy sufferers. Compared with pretreatment, WGP decreased study participants' ratings of tension, depression, anger, fatigue, and confusion and increased vigor as measured by the POMS (P < 0.05 for all). WGP improved global mood states (POMS) and had an improved effect on physical health, energy, and emotional well-being according to the SF-36 questionnaire, which has been validated for internal consistency, test-retest and interobserver reliability, discriminative properties, and evaluative properties (McHorney and Ware 1994;Leplege et al. 1995). In addition, study participants reported a decrease in sleep problems, nasal and eye symptoms, and QOL in the placebo and WGP treatment groups as reported using the Rhinoconjunctivitis QOL questionnaire.
The potential usefulness of b-glucan as a food additive is supported by work showing that yeast b-glucan exhibits immune-modulating function (Ikewaki et al. 2007); however, our study is one of few that have investigated whether b-glucan can alleviate the symptoms of allergic disease. Yamada et al. (2007) demonstrated a beneficial effect of oral consumption of b-1,3-glucan, derived from shiitake mushroom (Lentinus edoses Berk [Sing]), on symptoms of rhinoconjunctivitis and observed that the size of b-glucan particle influences efficacy. Their randomized, placebo-controlled, double-blind study showed that oral administration of superfine dispersed b-1,3-glucan (SDG) for 8 weeks improved ongoing symptoms of rhinoconjunctivitis and rhinitis in Japanese individuals with seasonal allergy to cedar pollen and perennial allergy symptoms (P < 0.0002), compared with an identical amount of nondispersed b-1,3-glucan (NDG) that was administered during the same period to a different group of study participants (Yamada et al. 2007). SDG, with a particle size of 0.08 lm, is thought to be absorbed easily by the intestinal mucosa, in contrast to NDG, which has a particle size of 288 lm. SDG reduced symptoms during the 8-week follow-up period that occurred immediately after the intervention (P < 0.0001) and prevented symptom development when taken prior to symptom onset (P < 0.05). The study also showed that consumption of SDG decreased allergen-specific and total IgE titers (initial Figure 6. Rhinoconjunctivitis Quality of Life. After 4 weeks of treatment with Wellmune WGP ® , there was a reduction in sleep problems (53%), nasal symptoms (59%), and eye symptoms (57%) and an elevation in QOL (56%); *P < 0.05 compared with placebo. high-IgE group, P < 0.043; initial low-IgE group, P < 0.03) and exhibited a good correlation with allergenspecific and total IgE titers (Yamada et al. 2007). Caveats of this study include a short observation period and a small study population.
Similarly, Szabo et al. (2000) showed that b-glucan alleviated some symptoms of allergic conjunctivitis, Sarinho et al. (2009) showed the effect of b-glucan on elevating IL-10 production and relieving asthma symptoms, and Damiani et al. (2011) showed the combination of colloidal silver and b-glucan on viral rhinitis and upper respiratory symptoms in children.
Other studies have also reported no increase or a small increase in Ig concentrations after b-glucan consumption. Lehne et al. (2005) reported no increase in IgA or IgG in serum or saliva after healthy subjects, age 20-30 years, orally consumed 100 mg/day yeast b-glucan for four consecutive days compared to concentrations at baseline. Consumption of 400 mg/day b-glucan for the same amount of time elicited an increase only in salivary IgA (mean AE SD = day 1: 39.6 AE 21.1 vs. 105.4 AE 73.9, P < 0.05). Browder et al. (1990) observed an increase in serum IL-1 in trauma patients, ages 18-65, 3 days after they had been treated with b-glucan (143.4 AE 193% vs. 78.6 AE 11.7%; P < 0.05), but there was no difference after that time point. b-Glucan had no effect on TNFa concentrations.
Allergic rhinitis is an IgE-mediated disease that presents with elevated allergen-specific IgE titers, IgE-dependent activations of mast cells, recruitment of activated eosinophils and T cells to mucosal surfaces, inflammation, and disease (Holgate 1999;Kay 2001a, b) T helper (Th) 2 cells contribute to the induction of IgE-mediated disease by overproducing Th2 cytokines  at the inflammation site (Romagnani et al. 1991;Huang et al. 1995). However, Th-1 cytokines  inhibit the Th2 immune response. In a randomized, placebo-controlled, double-blind study of 24 subjects with seasonal allergic rhinitis, Kirmaz et al. (2005) investigated whether b-glucan from S. cerevisiae reversed the Th2mediated immune response in individuals who were sensitized to Olea europea. Compared with pretreatment values, 20 mg b-glucan administered for 12 weeks reduced IL-4 (mean AE SEM = 5.48 AE 0.92 vs. 3.66 AE 0.64 pg/mL, P = 0.027) and IL-5 concentrations (mean AE SEM = 8.58 AE 1.58 vs. 5.81 AE 0.83 pg/mL, P = 0.04). Although there was no difference between the placebo group (n = 12) and the b-glucan group (n = 12) in the percentage of eosinophils in nasal lavage fluid after nasal provocation with Olea europea, the percentage of eosinophils in nasal lavage fluid was significantly reduced after a nasal provocation test with Olea europea compared with concentrations measured before the nasal provocation test (P = 0.01). In contrast, IL-12 levels increased after b-glucan treatment compared with pretreatment values (mean AE SEM = 11.08 AE 2.43 vs. 17.31 AE 2.75 pg/mL, P = 0.008). There was no change in IFN-c (mean AE SEM = 6.19 AE 1.18 vs. 7.83 AE 1.22 pg/mL, P = 0.1) in the bglucan group, and no changes in any cytokines for the placebo group. Furthermore, the percentage of eosinophils was unchanged for both treatment groups. The authors proposed that b-glucan is potentially an adjunct to standard therapy in individuals with allergic rhinitis.
Human studies have reported that b-glucan can improve QOL for individuals with cancer or upper respiratory influenza infections. In a randomized, placebocontrolled, double-blind, parallel group intervention study, Feldman et al. (2009) reported an improvement from baseline indicated by scores on two QOL measures of the SF36v-2 questionnaire after study participants, aged 18-65 years, consumed 500 mg/day WGP for 90 days as compared with consuming a rice flour placebo for the same amount of time. Study participants who consumed WGP reported an improved Physical Component Summary score (57.5 AE 4.5 vs. 55.5 AE 3.5; P = 0.029) and a greater General Health summary score (58.7 AE 7.0 vs. 52.0 AE 14.6; P < 0.038) compared with study participants who consumed the placebo. The b-glucan, lentinan, improved QOL in individuals with advanced cancer when administered intravenously (Yoshino et al. 2010) or orally for gastric cancer (Nakano et al. 1999;Kataoka et al. 2009), and orally for pancreatic (Shimizu et al. 2009) and colorectal cancer (Hazama et al. 2009). Nakano et al. (1999) reported that the total QOL score for gastric cancer subjects, in particular appetite and sleep quality, improved with lentinan consumption. In another study of 20 individuals between the ages of 38 and 84 years with advanced malignancies who were undergoing chemotherapy, b-1,3-glucan consumption resulted in a sense of well-being for 60% of study participants, and less fatigue during chemotherapy was reported by 40% of subjects who reported being fatigued (Weitberg 2008).
Immunotherapy has also been used to decrease the effects of ragweed allergy. A randomized, double-blind, placebo-controlled study, investigated the effectiveness of immunotherapy with a Ragweed-Toll-like receptor 9 agonist vaccine for allergic rhinitis (Creticos et al. 2006). Vaccination with the Ragweed-Toll-like receptor 9 agonist did not change the vascular permeability response to nasal provocation in the first season, as reflected in the albumin level in nasal lavage fluid, but improved peakseason rhinitis scores (P = 0.006) on the visual analog score, peak-season daily nasal symptom diary scores (P = 0.02), and mid-season overall quality-of-life scores (P = 0.05). The vaccine also inhibited the seasonal elevation of Amb a 1-specific IgE antibody. In a later ragweed season, compared with placebo, there were improvements in the peak-season rhinitis visual analog score (P = 0.02) and the peak-season daily nasal symptom diary score (P = 0.02). As with our study, the authors noted no change in IgE antibody titer during ragweed season (P = 0.19).
Some studies have reported that airway exposure to b-(1,3)-D-glucan, found in house dust, indoor molds, and some bacteria, can increase the airway allergic response as well as induce other health effects (Rylander et al. 1998). As summarized in Douwes (Douwes 2005), these data are inconsistent and further investigation is necessary to determine whether inhalation of b-(1,3)-D-glucan affects respiratory health. Infants who are exposed to a high concentration of b-(1,3)-D-glucan (60 lg/g) have a reduced risk of recurrent wheezing (adjusted odds ratio = 0.39, 95% CI = 0.16-1.93) and recurrent wheezing combined with allergen sensitization (adjusted odds ratio = 0.57, 95% CI = 0.30-1.10) (Iossifova et al. 2007).
One limitation of our study is the small study population. Additional studies including more study participants should be conducted. Furthermore, ragweed pollen levels were not measured during the study. In addition, there was inadequate characterization of the ragweed allergy status of study participants. Our study involved participants who were self-described allergy sufferers and had not received a clinical diagnosis of ragweed allergy. In contrast, Juniper et al. (1992) characterized subjects' ragweed allergy by requesting a 2-year history of symptoms for which treatment was necessary during the ragweed pollen season and required a positive skin-test result to ragweed pollen extract as a criterion for inclusion in the study.
Our findings suggest that the b-1,3/1,6-glucan, WGP, can be used to decrease the symptoms of allergy in individuals with self-described ragweed allergy. b-Glucans can be given orally, are typically less costly than immunotherapies, and have fewer side effects than immunotherapies (Murphy et al. 2010). Furthermore, they occur naturally, and are effective. Additional studies investigating the effect of b-1,3/1,6-glucan on allergies should be conducted.

Acknowledgments
S. T and J. T participated in the design of the study S. T, J. T, T. T, and E. D carried out the recruitment and coordination of the study, S. T performed the statistical analysis of the study. S. T drafted the manuscript. All authors read and approved the final manuscript. The authors are employees of GLH Nutrition, LLC. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This manuscript has not been or is currently not under consideration for publication elsewhere, and no portion of the manuscript has been previously published or posted on the Internet. This research was funded by Biothera, Inc. No public funds were used in the funding of this study.