https://orcid.org/0000-0001-8075-458X
Figures
Abstract
Considering the differences in molecular structure and function, the effects of β-1,3-glucans from Euglena gracilis and β-1,3/1,6-glucans from Saccharomyces cerevisiae on immune and inflammatory activities in dogs were compared. Four diets were compared: control without β-glucans (CON), 0.15 mg/kg BW/day of β-1,3/1,6-glucans (Β-Y15), 0.15 mg/kg BW/day of β-1,3-glucans (Β-S15), and 0.30 mg/kg BW/day of β-1,3-glucans (Β-S30). Thirty-two healthy dogs (eight per diet) were organized in a block design. All animals were fed CON for a 42-day washout period and then sorted into one of four diets for 42 days. Blood and faeces were collected at the beginning and end of the food intake period and analysed for serum and faecal cytokines, ex vivo production of hydrogen peroxide (H2O2) and nitric oxide (NO), phagocytic activity of neutrophils and monocytes, C-reactive protein (CRP), ex vivo production of IgG, and faecal concentrations of IgA and calprotectin. Data were evaluated using analysis of covariance and compared using Tukey’s test (P<0.05). Dogs fed Β-Y15 showed higher serum IL-2 than dogs fed Β-S30 (P<0.05). A higher phagocytic index of monocytes was observed in dogs fed the B-S15 diet than in those fed the other diets, and a higher neutrophil phagocytic index was observed for B-S15 and B-Y15 than in dogs fed the CON diet (P<0.05). Monocytes from dogs fed B-S15 and B-S30 produced more NO and less H2O2 than those from the CON and B-Y15 groups (P<0.05). Despite in the reference value, CRP levels were higher in dogs fed B-S15 and B-S30 diets (P<0.05). β-1,3/1,6-glucan showed cell-mediated activation of the immune system, with increased serum IL-2 and neutrophil phagocytic index, whereas β-1,3-glucan acted on the immune system by increasing the ex vivo production of NO by monocytes, neutrophil phagocytic index, and serum CRP. Calprotectin and CRP levels did not support inflammation or other health issues related to β-glucan intake. In conclusion, both β-glucan sources modulated some immune and inflammatory parameters in dogs, however, different pathways have been suggested for the recognition and action of these molecules, reinforcing the necessity for further mechanistic studies, especially for E. gracilis β-1,3-glucan.
Citation: de Souza Theodoro S, Gonçalves Tozato ME, Warde Luis L, Goloni C, Bassi Scarpim L, Bortolo M, et al. (2024) β-glucans from Euglena gracilis or Saccharomyces cerevisiae effects on immunity and inflammatory parameters in dogs. PLoS ONE 19(5): e0304833. https://doi.org/10.1371/journal.pone.0304833
Editor: Rui Tada, Tokyo University of Pharmacy and Life Sciences: Tokyo Yakka Daigaku, JAPAN
Received: December 15, 2023; Accepted: May 20, 2024; Published: May 31, 2024
Copyright: © 2024 de Souza Theodoro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The authors need to acknowledge the following institutions or companies, that made possible to conduct the study: This study was financed by Kemin Nutrisurance Nutrição Animal LTDA (Vargeão-SC- Brazil). The authors wish to acknowledge the financial support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; process 2020/04120–4) for a PhD's scholarship to S.S. THEODORO. We also acknowledge BRF Pet food, BRF Ingredints and Adimax Pet Food for the support to Laboratório de Pesquisa em Nutrição e Doenças Nutricionais de Cães e Gatos “Prof. Flávio Prada”, and Manzoni Industrial for the donation of the extruder used in the study. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
An increasingly adopted strategy in animal production is the use of food additives to improve the immune system, with the aim of reducing the incidence of diseases and overcoming the indiscriminate use of antibiotics and chemotherapy [1–3]. This strategy has also been explored in pet food with the aim of developing diets with health benefits in addition to an optimum nutrient supply [4–6]. Immunomodulatory compounds can beneficially modulate immune and inflammatory systems, thereby increasing host resistance [7,8]. β-glucans have been explored for this function, with interesting results for several species [9,10].
The β-glucans can be found in a variety of natural sources such as yeasts, mushrooms, bacteria, algae, barley, and oats, all of which have different structures and functions [11]. They have attracted attention because of their broad-spectrum biological activities, including anti-tumour, immunomodulatory, glycaemic, anti-aging, and anti-inflammatory properties [12–14]. However, only a few β-glucan structures, in particular β-1,3/1,6-glucans, have been studied for their immunomodulatory and inflammatory bioactivities, as they interact with membrane receptors on immune cells and induce specific biological responses [14–16].
In this context, β-1,3/1,6-glucans extracted from the cell wall of Saccharomyces cerevisiae have been widely investigated in veterinary medicine [17–19]. In dogs, β-1,3/1,6-glucans from S. cerevisiae stimulate interleukin (IL)-2 serum concentrations, phagocytic activity of peripheral cells, antibody production, and modulation of blood glucose concentrations [20]. In a subsequent study [21], evaluated the inclusion of two β-1,3/1,6-glucan molecules, with different purities incorporated into complete and balanced diets for dogs, piglets, mice, and chicks. Although differences in biological responses between the two molecules were observed, both modulated the immune system in a similar pattern in all tested species, inducing greater production of IL-2, a greater phagocytosis index of monocytes and neutrophils, and a greater production of antibodies, suggesting that vertebrates share similar mechanisms of β-1,3/1,6-glucan recognition and action.
In the last decade, seaweed polysaccharides have attracted interest because of their potential biological properties [22–24]. The unicellular alga Euglena gracilis is considered a potential source of β-1,3-glucan, particularly following the development of large-scale cultivation methods. When grown aerobically in the dark with a carbon source, high amounts of paramylon, an insoluble, non-digestible β-glucan composed of high molecular weight linear polymers of β-1,3-D-glucose, are deposited in the cytoplasm as highly crystalline discoid granules [25,26].
Studies on dried E. gracilis and purified paramylon (β-1,3-glucan) are available for some species. In mice, paramylon was tested in a model of atopic dermatitis skin lesions induced by 2,4,6-trinitrochlorobenzene, and the authors verified a reduction in the development of skin lesions, followed by inhibition of T-helper 1 and 2 cell responses [27]. Supplementation with fermented E. gracilis, containing more than 50% β-1,3-glucan was investigated through self-reported changes in upper respiratory tract infection symptoms in humans. This study provides evidence that supplementation can reduce or prevent symptoms of upper respiratory tract infection, provide immune support, and improve overall health [28].
To our knowledge, no studies on E. gracilis in dogs have been conducted. Considering the molecular differences between the β-1,3/1,6-glucans from S. cerevisiae, which present branched chains [15,29], and the high molecular weight linear polymers of β-1,3-glucans from E. gracilis [3,16], it is expected that these compounds differ in their interactions with immune cells and thus in their biological effects. Thus, we aimed to evaluate and compare the effects of β-glucans from E. gracilis and S. cerevisiae incorporated into complete and balanced diets on the immune and inflammatory activity of healthy adult dogs.
Materials and methods
This study was conducted at the Laboratory of Research in Nutrition and Nutritional Diseases of Dogs and Cats “Prof. Dr. Flávio Prada”, School of Agricultural and Veterinary Sciences, São Paulo State University (UNESP), Jaboticabal, SP, Brazil. All animal procedures followed the ethical principles of the Brazilian College of Animal Experimentation and were approved by the Ethics Committee on the Use of Animals (Protocol no. 001974/20).
Animals
Thirty-two adult Beagle dogs, 8 males and twenty-four females, with ages between 1 to 6 years old (2.8±2.3 years) and 10.7±1.4 kg were used. The mean body condition score was 5 on a scale of 1 to 9 [30]. Prior to the study, the dogs were subjected to physical, haematological, and serum biochemical evaluations by a veterinarian, and all were considered healthy. The animals were randomly distributed into treatments, but each group was balanced to present similar body weights and ages (P>0.05), and all was composed by 6 females and 2 males.
Experimental design
The study included four experimental diets and was conducted in a randomised block design with two blocks of 16 dogs each, with four dogs per diet in each block for a total of eight animals (repetitions) per diet (treatment). The blocking factor was time because of the structure available for research. Each block lasted 84 days, with a 42-day washout period (when all animals received the control diet) and a 42-day treatment period, during which dogs were sorted into one of the four experimental diets. Immediately before (day 42) and at the end of each period of experimental diets intake (day 84) the following analyses were conducted: serum and faecal cytokines (TNF-α, IL-2, IL-6, and IL-10), faecal calprotectin and immunoglobulin A (IgA), serum C-reactive protein (CRP), phagocytic activity of peripheral monocytes and neutrophils, in vitro production of hydrogen peroxide and nitric oxide in cell culture by peripheral neutrophils and monocytes, and IgG production in cell culture.
The amount of food offered was initially calculated by considering the food metabolizable energy content, estimated by its chemical composition, and the individual energy requirements of laboratory dogs [31]. Food was provided once a day (at 3 p.m.), and the amount offered, and leftovers were weighed to calculate intake. The dogs were weighed weekly, and the amount of food provided was adjusted to maintain a constant body weight throughout the study. Water was provided ad libitum. During the study dogs were housed in kennels measuring 1.5 m × 3.5 m with a solarium and released daily in a collective playground for exercise and socialization during 4 to 6 hours a day.
Ingredients evaluated
Two sources of β-glucan were studied. A purified source extracted from S. cerevisiae cell wall with 78.4% of β-1,3/1,6-glucans (MacroGard, Biorigin, Lençóis Paulista, Brazil), and a source constituted by dried E. gracilis with 58.5% of β-1,3-glucans (Pralisur, Kemin Industries, Iowa, USA). The β-glucan concentrations in the studied ingredients studied were analysed using an enzymatic assay (K-EBHLG, Megazyme© Ltd., Bray, Wicklow Country, Ireland) according to the manufacturer’s instructions.
Experimental diets
A single formulation based on corn grain, poultry by-product meal, poultry fat, and sugarcane fibre was used (Table 1), balanced for adult dogs according to the nutritional recommendations of the European Pet Food Industry Federation [32].
To obtain experimental diets, different amounts of β-glucan sources were included in the formulation, targeting to obtain specific intakes of the active compound per dog per day. The amount added was established based on a previously study [21], who suggested that 15 mg β-glucan per kg of body weight per day was an effective dosage of β-1,3/1,6-glucan for dogs. Considering the expected food intake by dogs during the study and the analysed β-glucan concentration in the products evaluated (Table 2), the following proportions was included (on an as-fed basis): control diet without inclusion of β-glucans source (CON), inclusion of 0.115% β-1,3/1,6-glucan source from S. cerevisiae cell wall of (Β-Y15), inclusion of 0.155% β-1,3-glucan source from E. gracilis (Β-S15), and inclusion of 0.310% β-1,3-glucan source from E. gracilis (Β-S30). The last treatment was designed to double the intake dosage seaweed β-glucan. The β-glucan sources addition was added replacing corn, not altering the remaining ingredients.
Diets were manufactured at the Extrusion Laboratory of the School of Agricultural and Veterinary Sciences, São Paulo State University (UNESP), Jaboticabal, SP, Brazil. A single lot of raw material was used for the four experimental diets. The ingredients were weighed and mixed before being ground in a hammer mill (Tigre, Moinhos Tigre, São Paulo, SP), fitted with a 0.8 mm sieve screen size, and extruded in a single-screw extruder (Model Mex-250, Manzoni Indústria Ltda, Campinas, SP, Brazil), with an average extrusion capacity of 250 kg/h. The temperature of the extruder preconditioner was maintained at >85°C using direct steam injection. After extrusion, the kibbles were dried in a forced air dryer at 105°C for approximately 20 min and coated with poultry fat and a liquid palatant.
Phagocytic activity
Phagocytic activity was measured at the beginning and end of each experimental period using a commercial kit (pHrodo Escherichia coli BioParticles; Molecular Probes Inc., Eugene, OR, USA). Blood samples were drawn via jugular venipuncture and placed in heparinised tubes. Then, 100 μL of each sample was incubated with 20 μL pHrodo E. coli BioParticles, a reagent provided by the commercial kit. For each blood sample, two sets of tubes were prepared: one was placed on ice, and the other was placed in a water bath at 37°C for 15 min. The samples were then lysed, centrifuged, and washed using reagents recommended by the manufacturer. On each collection day, two negative control samples were run: two tubes with no bioparticles, one placed on ice, and the other placed at 37°C. Samples were evaluated using a flow cytometer (FACSCanto; Becton Dickinson Immunocytometry System, Mountain View, CA, USA), and the results were reported as the percentage of fluorescence signal within the designated populations of monocytes and neutrophils. The target cell population was gated according to its volume and complexity [4,33].
Cytokines evaluation in serum and faeces
Blood samples (3 mL) were collected via jugular venipuncture and placed in tubes without anticoagulants. Subsequently, the samples were centrifuged at 3,500 × g for 10 min (5810R; Eppendorf, Hamburg, Germany). The resulting serum was frozen and stored at -80°C until further analysis.
Fresh faeces (immediately after elimination) were collected on three consecutive days. Faecal samples were pooled by dog and period, and faecal extraction was performed using saline solution [34]. Approximately one gram of faeces was weighed and diluted in 10 mL of extraction buffer composed of 0.01 M phosphate-buffered saline (PBS) (pH 7.4), 0.5% Tween (Sigma-Aldrich, St Louis, MO, USA), and 0.05% sodium azide. After homogenisation, faecal suspensions were centrifuged at 1,500 × g for 20 min at 5°C. Then, 1 mL of the supernatant was transferred to a sterile microtube containing 20 μL of protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). To remove the residues, the samples were centrifuged at 15,000 × g for 15 min at 5°C, and the supernatants were stored in microtubes at -20°C until further analysis.
The levels of interleukin (IL)-2, IL-6, IL-10, and tumour necrosis factor alpha (TNF-α) in faeces and serum were estimated using a Luminex kit specific to dogs, according to the manufacturer’s recommendations (MILLIPLEX MAP ELISA Canine Cytokine/Chemokine Magnetic Bead Panel—Immunology Multiplex Assay—Merck Millipore, St Charles, MO, USA).
Faecal calprotectin determination
To quantify faecal calprotectin concentration, the same extracts as previously described were used, and quantification was performed using a specific ELISA kit (Cloud-Clone Corp, Houston, TX, USA). Readings were performed using an ELISA microplate reader (Awareness Technology, Florida, USA).
Serum C-reactive protein
Serum C-reactive protein (CRP) was analysed using serum samples obtained as previously described using a specific ELISA kit (Cloud-Clone Corp, Houston, TX, USA) for dogs according to the manufacturer’s recommendations.
Determination of ex vivo hydrogen peroxide (H2O2) and nitric oxide (NO) production
This analysis was previously described [4], briefly, blood samples (30 mL) were drawn via jugular venipuncture and placed in heparin-containing tubes. These samples were then mixed with 4.5 mL of Histopaque 1119 and 3 mL and Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA) in 15-mL conical centrifuge tubes. The tubes were centrifuged at 700 × g for 30 min until two distinct opaque layers, representing mononuclear and granulocyte cells, were evident. Each layer was collected separately, transferred to a 50 mL conical centrifuge tube, and washed twice with isotonic phosphate-buffered saline by centrifugation at 360 × g for 10 min. If required, erythrocyte lysis was performed using 2 mL of ammonium-chloride-potassium solution (0.15 M ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM EDTA) for a maximum of 2 min.
The cells were suspended in complete medium (RPMI 1640; Merck KGaA, Darmstadt, Germany) supplemented with 40 mg/mL gentamicin and 10% foetal bovine serum. The cell concentration was adjusted to 3 × 105 neutrophils or monocytes/ml. Subsequently, the suspensions were placed in 96-well flat plates (100 μL/well). Mononuclear cells were maintained at 37°C in a humidified 5% CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA) for one hour to allow monocytes to adhere to the well surface. Afterwards, the supernatants were carefully removed using a pipette and each well was replenished with complete medium.
To produce H2O2, 100 μL of the sample was added to 36 wells. Among these, 18 wells contained non-stimulated cells suspensions, while the remaining 18 wells were stimulated with LPS (10 μg/well—E. coli lipopolysaccharide, Sigma Aldrich, St. Louis, MO, USA). The plates were then incubated at 37°C in a humidified incubator with 5% CO2 for 36 h. H2O2 production was quantified as previously described [35,36]. Buffer solution (100 μL/well) consisting of 7.8 mL distilled water (dH2O), 0.8 mL of solution A (800 mL dH2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4, 11.5 g Na2HPO4), 0.1 mL of solution B (100 mL dH2O, 1 g CaCl2), 0.1 mL of solution C (100 mL dH2O, 1 g MgCl2), 0.1 mL of phenol red (100 mL dH2O, 1 g phenol red), 0.1 mL of peroxidase (10 mg horseradish peroxidase, 2 mL PBS) and 1 mL of glucose (100 mL dH2O, 1 g glucose), was added to each well. In half of the non-stimulated wells and half of the LPS-stimulated wells, 10 μL/well phorbol myristate acetate (PMA) was added, followed by incubation at 37°C in a humidified incubator with 5% CO2 for 1 hour. For each dog, nine wells were used for non-stimulated cells, nine for non-stimulated cells and PMA, nine for LPS-stimulated cells, and nine for LPS-stimulated cells and PMA. After and incubation of one-hour, the reaction was stopped by the addition of 10 μL of 1 N NaOH. Absorbance was measured at 595 nm using a microplate reader (iMark Microplate Absorbance Reader 168–1135, Bio-Rad, Hercules, CA, USA). The results are reported in micromolar units of H2O2/3X105 cells. A hydrogen peroxide standard curve was constructed for each plate, with a range of 0.25 to 16.00 nM of H2O2.
NO concentration was determined using a colorimetric method (GRIESS reaction) [37]. This analysis was conducted with nine repetitions for LPS-stimulated and nine for non-stimulated cells (10 μg/well E. coli lipopolysaccharide, Sigma Aldrich, St. Louis, MO, USA), resulting in 18 wells per sample. GRIESS reagent (100 μL) was added and prepared by diluting a 1:1 solution containing n-(1-naftil)-ethylenediamine diluted to 0.1% in dH2O and 1% sulfonamide diluted in 5% H2PO4 (both from Sigma Aldrich, St Louis, MO, USA). Absorbance was measured at 540 nm using a microplate reader (iMark Microplate Absorbance Reader 168–1135, Bio-Rad, Hercules, California, USA). The results are expressed as μM of NO/3×105 cells. An NO standard curve was constructed for each plate with a range of 0.78 to 100 μM of NO.
IgG production in cell culture
The previously described cell separation process was used for IgG analysis. Lymphocytes were cultured (3×105) in the presence of LPS stimulus (5 μg/mL) for seven days and the supernatant was collected and frozen at -80°C until further analysis. The IgG concentration was analysed using a dog-specific IgG ELISA kit according to the manufacturer’s protocol (Cloud-Clone Corp, Houston, TX, USA).
Faecal IgA
Faecal collection and extraction were performed as described previously. Faecal IgA was analysed using an ELISA kit designed for dogs (Bethyl Laboratories, Montgomery, TX, USA) following the manufacturer’s recommendations. The optical density (OD) was measured at 450 nm using a Microplate Reader (MRX TC Plus; Dynex Technology, Chantilly, VA, USA). To establish IgA concentration, OD of the samples was compared with the OD of a standard with a known concentration of IgA. The kit included a standard canine IgA sample, and seven dilutions of the standard were prepared to develop a regression curve between the OD and IgA levels [4].
Statistical analysis
Each dog was considered an experimental unit. The sample size was established based on the results of IgA and interleukin 10 [4], considering a completely randomised block design. The test power was set at 0.8 (Opdoe package of R), α = 0.05, standard deviation = 149.2, and a standard error of approximately 31.7 for IgA. This analysis was performed using the sample size procedure of R, and a sample size of eight dogs was obtained. All the variables were tested for error normality and homoscedasticity. When necessary, logarithmic transformation (log10) was applied. Data were subjected to an analysis of covariance, considering the effects of the block and diet. Instead of examining the changes in the outcome variables, the differences between the final measurements were adjusted for baseline measures. This practice has been demonstrated to enhance the statistical power in intervention trials, as indicated in previous studies [4,38]. When the results of the F-test were significant, multiple comparisons of the means were performed using Tukey’s test (P<0.05). The analysis was conducted using the R software (version 4.2.0).
Results
All dogs ingested the food properly and maintained a constant body weight throughout the study. Considering the verified intake of each experimental diet, dogs fed Β-Y15 ingested 13.7±2.3 mg of β-1,3/1,6-glucans/kg/day, Β-S15 ingested 14.4±2.9 mg of β-1,3-glucans/kg/day, and dogs fed Β-S30 ingested 27.3±5.7 mg of β-1,3-glucans/kg/day. So, the verified intake was very close to the amount planned, with similar intakes of β-glucan in the treatments B-Y15 and B-S15, and a double intake for dogs fed the treatment B-S30. The β-glucan sources were well-tolerated during the 42 days of consumption, as there were no changes in the clinical condition of the animals.
A higher monocyte phagocytic index (Table 3) was observed in dogs fed the B-S15 diet (P<0.05) than that in the other groups, which did not differ significantly from each other. The neutrophil phagocytic index was higher in B-Y15 and B-S15 fed dogs, and lower in dogs fed the CON diet (P<0.05). Among the evaluated serum cytokines (Table 4), dogs fed the Β-Y15 diet exhibited higher IL-2 concentration than animals fed the Β-S30 diet (P<0.05), but similar values in comparison with the CON and Β-S15 treatment. No significant differences were detected for the other cytokines evaluated in the serum (P>0.05). The concentration of cytokines in the faeces was similar between dogs fed different treatments (P>0.05; Table 5).
No differences in faecal calprotectin levels were observed among treatments (P>0.05) (Table 6). Dogs fed both diets containing E. gracilis β-1,3-glucans presented higher CRP serum concentration than animals fed Β-Yeast (P<0.05), although all values observed was inside the reference interval for health dogs [39–41]. This may be related to the activation of the immune system, as can be observed by the higher ex vivo NO production by peripheral monocytes observed in dogs fed the same diet, in comparison with animals fed the CON and Β-Y15 diets (P<0.05; Table 7). The ex vivo production of NO by neutrophils did not differ significantly (P>0.05). For H2O2, however, the opposite was verified, with lower ex vivo production from monocytes of dogs fed diets containing β-1,3-glucans from E. gracilis than CON and Β-Y15 (P<0.05; Table 8). No differences in ex vivo H2O2 production by peripheral neutrophils were observed (P>0.05). The concentrations of IgG in the cell culture and IgA in the faeces did not differ (P>0.05) between dogs fed different experimental diets (Table 9).
Discussion
The current study confirmed that β-glucan intake had an impact on some inflammatory and immune parameters in dogs, but the induced responses varied depending on the sources used. It has been reported that differences in shape, molecular weight, degree of branching, solubility, molecular structure, and extraction process determine the biological activity of β-glucan, which can significantly differ between sources and manufacturers [16,42,43].
In the present study, S. cerevisiae β-1,3/1,6-glucan intake stimulated IL-2 secretion and ex vivo phagocytic index of neutrophils without increasing calprotectin in faeces, serum CRP, or NO and H2O2 ex vivo production. The increase in IL-2 (in line with the numerical, although not statistically significant, increase in serum IL-6) signals the activation of innate immunity, as previously reported in many studies [9,20,21,44–46]. The enhancement of innate immune function through the increased phagocytic activity of neutrophils was also evident in the present study [47,48]. Increased serum IL-2 levels regulate the growth, proliferation, and differentiation of T-cells [49,50]. In addition, IL-2 may be involved in the stimulation of natural killer cells and cytotoxicity of monocytes [51,52]. In clinical practice, there have been reports of treatments for autoimmune diseases, human immunodeficiency virus infection, and chemotherapy for metastatic neoplasms with direct administration of IL-2 [52–54], although no disease has been attributed to IL-2 deficiency or excess [52]. This may suggest a possible role of S cerevisiae β-1,3/1,6-glucan in these conditions, open opportunities of studies.
When included in the diet, β-1,3/1,6-glucans reach the small intestine intact and are absorbed by Peyer’s patches, where they are recognised by circulating antigen-presenting cells (dendritic cells or M cells) and present to other immune cells [16,55]. β-1,3/1,6-glucans are microbial structures recognised as pathogen-associated molecules that initiate an immune response. This recognition is dependent on identification and binding to different cell membrane receptors [9,16]. The main receptor for β-1,3/1,6-glucans is dectin-1, which recognises its triple-helix conformation [56]; however, lactosyl ceramide, elimination receptors, and complement receptor 3 may also be involved [9,57,58]. These receptors are found on various immune cells including monocytes, macrophages, dendritic cells, neutrophils, and natural killer cells [16,59,60]. The interaction between β-1,3/1,6-glucans and these receptors activates multiple and complex signalling cascades that induce pathogen phagocytosis, generation of a respiratory burst, microbial death, and release of chemical mediators that activate and recruit other immune cells [61,62].
The phagocytic responses of monocytes and neutrophils are significantly influenced by β-glucan consumption in different ways. In the present study E. gracilis β-1,3-glucans treatment stimulated monocyte phagocytosis, while neutrophils phagocytosis was stimulated by both E. gracilis β-1,3-glucan and S. cerevisiae β-1,3/1,6-glucan. Phagocytosis is first line of defence of the body when faced with an invading agent, and these results demonstrate a relevant effect of both β-glucans, corroborating previous findings in different species [21,63] and also in dogs [20,21].
The authors [64] measured the cytokines IL-6, IL-8, IL-10, and TNF- α in dog faeces by an ELISA method, considering it a potential technique for a non-invasive biomarker of inflammation in dogs. However, in the current study, no significant differences were detected between the treatment groups. This lack of an effect may be explained by the different stimuli, experimental conditions, and methods used (in this case, the MULTIPLEX Kit). Only healthy dogs were included in the present study, and the β-glucans intake did not induce an inflammatory condition, as calprotectin did not change and CRP levels, although higher in dogs fed β-1,3-glucans, were inside the reference value for healthy dogs [39,40,41], and possibly the effect on faecal cytokines was too low or absent in this situation.
The intake of E. gracilis β-1,3-glucans did not affect serum cytokine levels, highlighting the importance of properly characterising the actions of different β-glucan molecules. These results are in line with the findings of another study [65], which compared 13 different β-glucan sources using human whole blood cell cultures and found no stimulation of cytokine production by E. gracilis β-1,3-glucan.
However, a significant increase in ex vivo NO production was observed in dogs fed diets containing E. gracilis β-1,3-glucans, which was not observed for β-1,3/1,6-glucans. This effect of E. gracilis β-1,3-glucans was also noted [66] in Mollusca, suggesting the activation of a signalling cascade that may enhance the defence system of the organism. However, the activated system may be different because there is no stimulus for cytokine production, which is typically induced by β-1,3/1,6-glucans through dectin-1 recognition [46,65,67]. Therefore, other cell membrane receptors and mechanisms of action have been suggested for β-1,3-glucan recognition, whose molecular structure is linear and unbranched [68,69]. Interaction with cells expressing different receptors than dectin-1 in the intestinal lumen and Peyer’s patches was proposed [70]. One hypothesis is that the complement receptor-3 (CRP-3), found on neutrophils and monocytes, are activated facilitating phagocytosis and cytotoxic degradation [71]. Studies, however, that proposes potential molecular mechanisms of action of E. gracilis β-1,3-glucans are necessary to better understand it physiological function.
NO is an inflammatory mediator present in almost all cells in the body, and its action can modulate all immunological parameters [72]. Under physiological conditions, NO acts as a vasodilator [73], anti-tumour, immunosuppressant, pro-apoptotic factor [72], neuromodulator, and mediator of smooth muscle relaxation in the cardiovascular system and gastrointestinal tract [74]. Maintaining stable NO levels is essential for health because both deficit and excess of NO have been reported to reduce general health [73]. During an inflammatory state, pro-inflammatory cytokines stimulate NO production in monocytes, macrophages, and neutrophils, leading to a potential increase in NO levels of up to 1,000-fold [75, 76]. Since cytokines did not increase after E. gracilis β-1,3-glucans intake, it cannot be suggested that this compound induced an inflammatory status, what was reinforced by the very minor increase observed in serum CRP. E. gracilis β-1,3-glucans are much less studied than the S. cerevisiae β-1,3/1,6-glucan, with limited available publications. Although a biological effect was characterised in the present study, its extension, and implications for animal health require further investigation.
In parallel with the increase in NO, a reduction in H2O2 ex vivo production was observed in dogs fed with E. gracilis β-1,3-glucans. H2O2 plays a crucial role in the immune response and cell signalling, working at very low physiological concentrations (nano-to micromolar). It modulates endothelial cell function through intricate mechanisms [77,78]. A reduction in H2O2 levels suggested a balanced immune response. An increase in NO may signal the preparation of the immune system, and a reduction in H2O2 does not expose the animal to an over-stimulated immune response or its negative effects on health [72,79].
Particularly when administered in lower doses, it is suggested that β-1,3-glucans may modulate innate immunity by exerting anti-inflammatory effects, as evidenced by reductions in IL-6 and TNF-α, alongside with increase in IL-10 [70,80]. These effects may occur without compromising the immune cells’ phagocytic capabilities, as indicated by enhanced phagocytosis rates observed in neutrophils and monocytes in the present study, and without impeding their ability to eliminate pathogens, as demonstrated here by elevated NO production.
Calprotectin, a protein constituting up to 50% of cytosolic proteins in neutrophils, is resistant to bacterial degradation in the colon [81]. It was used to assess possible undesirable body responses to β-glucan intake. Their low values, similar to those of CON and within the reference range for dogs, suggest that the products were well tolerated by dogs without inducing detectable intestinal alterations [82]. CRP is also among the group of proteins used to diagnose inflammation and is considered a sensitive test for detecting inflammation and disease in dogs in clinical practice [81,83,84]. Values of up to 10 or 20 mg/L are considered normal for healthy dogs [39–41]; thus, the results of the present study were well below the reference range, indicating no systemic inflammation. There is evidence that CRP is an important regulator of inflammatory processes and is not just a marker of inflammation and disease [85]. An increase in CRP is associated with higher NO production by monocytes [76,86,87], which may at least partially explain the concurrent increase in NO and CRP levels in dogs fed E. gracilis β-1,3-glucans in the present study. Therefore, considering both factors, preparation of the immune system without evidence of over-stimulation is suggested.
The effects of both β-glucans on adaptive immunity were assessed in the present study by ex vivo production of IgG by lymphocyte cell culture [88]. No significant effect was found, but the limitations of this ex vivo approach did not allow the exclusion of an in vivo effect of the β-glucan sources. Faecal IgA was also assessed because of its importance in intestinal immunity [89,90] and its possible dietary effects on faecal secretion [91]. However, no effects were observed on the tested products. Although not all studies with β-glucans found an effect on IgA secretion [92], the lack of an immune challenge and the fact that only healthy dogs were used in the present study does not allow us to exclude the effect of β-glucans on this parameter. Therefore, perhaps the main limitations of the present study that need to be considered in data interpretation include the use of only healthy Beagles, the absence of in vivo stimulation challenging the immune system of the animals, and all constraints related to the ex vivo methods adopted.
The E. gracilis β-1,3-glucan was tested in the present study in two dosages. Considering the body of information for S. cerevisiae β-1,3/1,6-glucan to dogs, the first dosage was similar to proposed by some authors to this compound [21]. Authors then selected another treatment with a double of this dosage, in order to explore it effects for the specie. No clear effect of intake amount was observed, since for monocytes phagocytosis dogs fed 30 mg/kg BW/day presented a lower effect than animals fed 15 mg β-1,3-glucan/kg BW/day, for IL-2 serum concentration the effect of the higher dosage was higher than that of the lower dosage, and for CRP, NO, and H2O2 the effects on dogs were similar between the two dosages. So, it is possible that the physiological effects of E. gracilis β-1,3-glucan may differ according to their dosage, but more studies are necessary to confirm this and to propose the better inclusion to influence dog’s health.
A limitation to consider in the present study is the lack of evaluation of the sex effect on the dog’s immune responses to the intake of the two β-glucan sources evaluated. In fact, very little is known about the effects of sex on prebiotic responses in dogs [93]. Most studies or used a variable number of male and females individuals but not explored the possible differences regarding sex on outcomes [94–97], as in the present study, or selected to only use females or males [92,98–100] not allowing the comparison between sex. Even in animal models’ studies, sex effects are little explored, and authors was able to localize only one study in rats, which described differences in prebiotic metabolization by male and female rat gut communities [101]. However, as evidenced in the present study, β-glucan have direct effects on immune system not necessarily related to alteration in the microbial community in the intestine, so any transference of these findings in rats might be premature not only due to the possible differences between species but also due to the particular mechanism of action on β-glucan, which directly interacts with the immune system. In any case, the present study adequately demonstrated the effects of β-glucan in dogs, regardless of sex, as 2 males and 6 females were used in each group, but the possible effects of sex on their organic actions in dogs remain unexplored.
Conclusion
Both β-glucan sources, S. cerevisiae and E. gracilis, have the ability to modulate immune and inflammatory parameters in dogs. The intake of S. cerevisiae β-1,3/1,6-glucan showed cell-mediated activation of the immune system, as evidenced by the increased serum levels of IL-2 and neutrophil phagocytic index, while E. gracilis β-1,3-glucan acted on the immune system by increasing the ex vivo production of NO by monocytes, monocyte and neutrophil phagocytic index, and serum CRP concentration. Different pathways have been suggested for the recognition and action of these molecules, reinforcing the necessity for further mechanistic studies, especially for E. gracilis β-1,3-glucan. Calprotectin and CRP values did not support inflammation or other health issues related to β-glucan source intake in the present study.
Acknowledgments
Authors acknowledge the Laboratório de Análises Especiais, Multi-user equipment network program, Faculdade de Medicina, Universidade de São Paulo (USP), for the cytokine analysis.
References
- 1. Pohlenz C, Gatlin DM III. Interrelationships between fish nutrition and health. Aquaculture. 2014; 431: 111–117.
- 2. Levine R, Horst G, Tonda R, Lumpkins R, Mathis G. Evaluation of the effects of feeding dried algae containing beta-1,3-glucan on broilers challenged with Eimeria. Poult Sci. 2018; 97(10): 3494–3500. pmid:30007294
- 3. Yamamoto FY, Sutili FJ, Hume M, Gatlin DM III. The effect of β‐1,3‐glucan derived from Euglena gracilis (AlgamuneTM) on the innate immunological responses of Nile tilapia (Oreochromis niloticus L.). J Fish Dis. 2018; 41(10): 1579–1588. pmid:30051484
- 4. Theodoro SdS Putarov TC, Tiemi C Volpe LM, de Oliveira CAF Glória MBdA, et al. (2019) Effects of the solubility of yeast cell wall preparations on their potential prebiotic properties in dogs. PLoS ONE 14(11): e0225659. pmid:31765439
- 5. Kroll FSA, Putrarov TC, Zaine L, Venturini KS, Aoki CG, Santos JPF, et al. Active fractions of mannoproteins derived from yeast cell wall stimulate inaate and acquire immunity of adult and elderly dogs. Anim. Feed Sci. Technol. 2020; 261(114392).
- 6. Soares NMM, Bastos TS, Kaelle GCB, de Souza RBMdS, de Oliveira SG, Félix AP. Digestibility and Palatability of the Diet and Intestinal Functionality of Dogs Fed a Blend of Yeast Cell Wall and Oregano Essential Oil. Animals 2023,13,2527. pmid:37570335
- 7. Dawood MAO, Koshio S, Esteban MA. Beneficial roles of feed additives as immunostimulants in aquaculture: a review. Rev Aquac. 2018; 10(4): 950–974.
- 8. Koch JFA, Oliveira CAF, Zanuzzo FS. Dietary β-glucan (MacroGard®) improves innate immune responses and disease resistance in Nile tilapia regardless of the administration period. Fish Shellfish Immunol. 2021; 112: 56–63. pmid:33640538
- 9. Soltanian S, Stuyven E, Cox E, Sorgeloos P, Bossier P. Beta-glucans as immune stimulants in vertebrates and invertebrates. Crit Rev Microbiol. 2009; 35(2): 109–138.
- 10. Zielke C, Kosik O, Ainalem M-L, Lovegrove A, Stradner A, Nilsson L. Characterization of cereal β-glucan extracts from oat and barley and quantification of proteinaceous matter. PLoS ONE. 2017; 12(2): e0172034.
- 11. Zhu F, Du B, Bian Z, Xu B. Beta-glucans from edible and medicinal mushrooms: characteristics, physicochemical and biological activities. J Food Compost Anal. 2015; 41: 165–173.
- 12. Du B, Bian Z, Xu B. Skin Health Promotion Effects of Natural Beta-Glucan Derived from Cereals and Microorganisms: A Review. Phytother Res. 2014; 28: 159–166. pmid:23494974
- 13. Zhu F, Du Bin, Xu B. A critical review on production and industrial applications of beta-glucans. Food Hydrocoll. 2016; 52: 275–288.
- 14. Bai J, Ren Y, Li Y, Fan M, Qian H, Wang L, et al. Physiological functionalities and mechanisms of β-glucans. Trends Food Sci Technol. 2019; 88: 57–66.
- 15. Raa J. Immune modulation by non-digestible and non-absorbable beta-1,3/1,6-glucan. Microb Ecol Health Dis. 2015; 26: 27824 – pmid:26031679
- 16. Murphy EJ, Rezoagli E, Major I, Rowan N, Laffey JG. β-Glucans. Encyclopedia. 2021; 1(3): 831–847.
- 17. Li J, Li DF, Xing JJ, Cheng ZB, Lai CH. Effects of β-glucan extracted from Saccharomyces cerevisiae on growth performance, and immunological and somatotropic responses of pigs challenged with Escherichia coli lipopolysaccharide. J Anim Sci. 2006; 84(9): 2374–2381. pmid:16908640
- 18. Mo L, Chen Y, Li W, Guo S, Wang X, An H, et al. Anti-tumor effects of (1 → 3)-β-d-glucan from Saccharomyces cerevisiae in S180 tumor-bearing mice. Int J Biol Macromol. 2017; 95: 385–392. pmid:27838421
- 19. Wang M, Wang X, Zhang L, Yang R, Fei C, Zhang K, et al. Effect of sulfated yeast beta-glucan on cyclophosphamide-induced immunosuppression in chickens. Int Immunopharmacol. 2019; 74: 105690. pmid:31220696
- 20. Vetvicka V, Oliveira C. β (1–3) (1–6)-D-glucans Modulate Immune Status and Blood Glucose Levels in Dogs. J Pharm Res Int. 2014; 4(8): 981–991.
- 21. Oliveira CAF, Vetvicka V, Zanuzzo FS. β-Glucan successfully stimulated the immune system in different jawed vertebrate species. Comp Immunol, Microbiol Infect Dis. 2019; 62: 1–6. pmid:30711038
- 22. Pomin V, Mourão PAS. Structure, biology, evolution, and medical importance of sulfated fucans and galactans. Glycobiology. 2008; 18(12): 1016–1027. pmid:18796647
- 23. Wijesekara I, Pangestuti R, Kim SK. Biological activities and potential health benefits of sulfated polysaccharides derived from marine algae. Carbohydr Res. 2011; 84: 14–21.
- 24. Barahona T, Encinas MV, Imarai M, Mansilla A, Matsuhiro B, Torres R, et al. Bioactive polysaccharides from marine algae. Bioact Carbohydr Diet Fibre. 2014; 4(2): 125–138.
- 25. Barsanti L, Vismara R, Passarelli V, Gualtieri P. Paramylon (β-1,3-glucan) content in wild type and WZSL mutant of Euglena gracilis. Effects of growth conditions. J Appl Phycol. 2001; 13: 59–65.
- 26. Monfils AK, Triemer RE, Bellairs EF. Characterization of paramylon morphological diversity in photosynthetic euglenoids (Euglenales, Eugleno- phyta). Phycologia. 2011; 50: 156–169.
- 27. Sugiyama A, Hata S, Suzuki K, Yoshida E, Nakano R, Mitra S, et al. Oral Administration of Paramylon, a β-1,3-D-Glucan Isolated from Euglena gracilis Z Inhibits Development of Atopic Dermatitis-Like Skin Lesions in NC/Nga Mice. J Vet Med Sci. 2010; 72(6): 755–763. pmid:20160419
- 28. Evans M, Falcone PH, Crowley DC, Sulley AM, Campbell M, Zakaria N, et al. Effect of a Euglena gracilis Fermentate on Immune Function in Healthy, Active Adults: A Randomized, Double-Blind, Placebo-Controlled Trial. Nutrients. 2019; 11(12): 2926. pmid:31816842
- 29. Sivieri K, Oliveira SM, Marquez AS, Pérez-Jiménez J, Diniz SN. Insights on β-glucan as a prebiotic coadjuvant in the treatment of diabetes mellitus: A review. Food Hydrocoll Health. 2022; 2: 100056.
- 30. Laflamme DP. Development and validation of body condition score system for dogs. Canine Practices. 1997; 22: 10–15.
- 31.
NRC—National Research Council. Nutrient requirements of dogs and cats. Washington, DC: National Academy Press, 2006.
- 32.
FEDIAF—Fédération Européenne de L’industrie des Aliments pour Animaux Familiers. The European Pet Food Industry Federation. Nutritional Guidelines. 2019.
- 33. Neaga A, Lefor J, Lich KE, Liparoto SF, Xiao YQ. Development and validation of a flow cytometric method to evaluate phagocytosis of pHrodo™ BioParticles® by granulocytes in multiple species. J Immunol Methods. 2013; 390(1–2): 9–17. pmid:21767540
- 34. Peters IR, Calvert EL, Hall EJ, Day MJ. Measurement of immunoglobulin concentrations in the feces of healthy dogs. Clin Diagn Lab Immunol. 2004; 11(5): 841–848. pmid:15358641
- 35. Pick E, Keisari Y. A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J Immunol Methods. 1980; 38(1–2): 161–170. pmid:6778929
- 36. Pick E, Mizel D. Rapid microassays for measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J Immunol Methods. 1981; 46(2): 211–226. pmid:6273471
- 37. Grenn LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem. 1982; 126(1): 131–138. pmid:7181105
- 38. Vickers AJ. The use of percentage change from baseline as an outcome in a controlled trial is statistically inefficient: a simulation study. BMC Med Res Methodol. 2001(6); 1:6.
- 39. Eckersall PD, Conner JG, Parton H. An enzyme-linked immunosorbent assay for canine C-reactive protein. Vet Rec. 1989; 124(18): 490–491. pmid:2750033
- 40. Mylonakis ME, Ceron JJ, Leontides L, Siarkou VI, Martinez S, Tvarijonaviciute A, et al. Serum acute phase proteins as clinical phase indicators and outcome predictors in naturally occurring canine monocytic ehrlichiosis. J Vet Intern Med. 2011; 25(4): 811–817. pmid:21564293
- 41. Muñoz-Prieto A, Tvarijonaviciute A, Escribano D, Martinez-Subiela S, Ceron JJ. Use of heterologous immunoassays for quantification of serum proteins: The case of canine C-reactive protein. PLoS ONE. 2017; 12(2): e0172188. pmid:28222144
- 42. Sinthusamran S, Benjakul S. Physical, rheological and antioxidant properties of gelatin gel as affected by the incorporation of β-glucan. Food Hydrocoll. 2018; 79: 409–415.
- 43. van Steenwijk HP, Bast A, de Boer A. Immunomodulating Effects of Fungal Beta-Glucans: From Traditional Use to Medicine. Nutrients. 2021, 13(4): 1333. pmid:33920583
- 44. Li B, Cramer D, Wagner S, Hansen R, King C, Kakar S, et al. Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88- and Syk kinase-dependent pathways. Clin Immunol. 2007; 124(2): 170–181. pmid:17572156
- 45. Stier H, Ebbeskotte, Gruenwald J. Immune-modulatory effects of dietary Yeast Beta-1,3/1,6-D-glucan. Nutr J. 2014; 13: 38.
- 46. Rodrigues MV, Zanuzzo FS, Koch JFA, Oliveira CAF, Sima P, Vetvicka V. Development of Fish Immunity and the Role of β-Glucan in Immune Responses. Molecules. 2020; 25(22): 5378. pmid:33213001
- 47. Kobayashi SD, Voyich JM, Burlak C, DeLeo FR. Neutrophils in the innate immune response. Arch Immunol Ther Exp. 2005; 53: 505–517. pmid:16407783
- 48. Kumar V, Sharma A. Neutrophils: Cinderella of innate immune system. Int. Immunopharmacol. 2010; 10: 1325–1334. pmid:20828640
- 49. Nelson BH. IL-2, Regulatory T Cells, and Tolerance. J Immunol. 2004; 172: 3983–3988. pmid:15034008
- 50. Cornish GH, Sinclair LV, Cantrell DA. Differential regulation of T-cell growth by IL-2 and IL-15. Blood. 2006; 108(2): 600–608. pmid:16569767
- 51. Espinoza-Delgado I, Bosco MC, Musso T, Gusella GL, Longo DL, Varesio L. Interleukin-2 and human monocyte activation. J. Leukoc. Biol. 1995; 57: 13–19. pmid:7829965
- 52. Gaffen SL, Liu KD. Overview of interleukin-2 function, production and clinical applications. Cytokine. 2004; 28(3): 109–123. pmid:15473953
- 53. Glaspy JA. Therapeutic options in the management of renal cell carcinoma. Semin Oncol. 2002; 29(3): 41–46. pmid:12068388
- 54. Natarajan V, Lempicki RA, Sereti I, Badralmaa Y, Adelsberger JW, Metcalf JA, et al. Increased peripheral expansion of naive CD4C T cells in vivo after IL-2 treatment of patients with HIV infection. Proc Natl Acad Sci U S A 2002; 99(16): 10712–7. pmid:12149467
- 55. Reboldi A, Cyster JG. Peyer’s patches: Organizing Β-cell responses at the intestinal frontier. Immunol. Rev. 2016, 271(1): 230–245. pmid:27088918
- 56. Falch BH, Espevik T, Ryan L, Stokke BT. The cytokine stimulating activity of (1–3)-β-D glucans is dependent on the triple helix conformation. Carbohydr Res. 2000; 329(3): 587–96. pmid:11128587
- 57. Ross GD, Cain JA, Myones BL, Newman SL, Lachmann PJ. Specificity of membrane complement receptor type three (CR3) for beta-glucans. Complement. 1987; 4(2): 61–74. pmid:3040332
- 58. Brown GD, Gordon S. Fungal beta-glucans and mammalian immunity. Immunity. 2003; 19(3): 311–315. pmid:14499107
- 59. Li P, Wang F. Polysaccharides: Candidates of promising vaccine adjuvants. Drug Discov. Ther. 2015; 9(2): 88–93. pmid:25994059
- 60. Kardani K, Bolhassani A, Shahbazi S. Prime-boost vaccin estrategy against viral infections: Mechanisms and benefits. Vaccine. 2016; 34(4): 413–423. pmid:26691569
- 61. Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature. 2007; 449: 819–826. pmid:17943118
- 62. Barsanti L, Passarelli V, Evangelista V, Frassanito AM, Gualtieri P. Chemistry, physico‐chemistry and applications linked to biological activities of β‐glucans. Nat prod rep. 2011; 28(3): 457–466.
- 63. Vetvicka V, Vashishta A, Saraswat-Ohri S, Vetvickova J. Immunological Effects of Yeast- and Mushroom-Derived β‐Glucans. J Med Food. 2008; 11(4): 615–622.
- 64. Prakash N, Stumbles P, Mansfield C. Initial Validation of Cytokine Measurement by ELISA in Canine Feces. Open J Vet Med. 2013; 3(6): 282–288.
- 65. Noss I, Doekes G, Thorne PS, Heederik DJJ, Wouters IM. Comparison of the potency of a variety of β-glucans to induce cytokine production in human whole blood. Innate Immun. 2013; 19(1): 10–19. pmid:22653750
- 66. Bianchi VA, Castro JM, Rocchetta I, Nahabedian DE, Conforti V, Luquet CM. Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli. Fish Shellfish Immunol. 2015; 42(2): 367–378. pmid:25463294
- 67. Stier H, Ebbeskotte , Gruenwald J. Immune-modulatory effects of dietary Yeast Beta-1,3/1,6-D-glucan. Nutr J. 2014; 13: 38. pmid:24774968
- 68. Schley PD, Field CJ. The immune-enhancing effects of dietary fibres and prebiotics. Br J Nutr. 2002; 87(2): S221–S230. pmid:12088522
- 69. Ishibachi K, Nishioka M, Onaka N, Takahashi M, Yamanaka D, Adachi Y, et al. Effects of Euglena gracilis EOD-1 Ingestion on Salivary IgA Reactivity and Health-Related Quality of Life in Humans. Nutrients. 2019; 11(5), 1144. pmid:31121913
- 70. Kawano T, Miura A, Naito J, Nishida N, Ishibashi K, Yoshiyuki A, et al. High-parameter immune profiling and subjective health assessment of the immunomodulatory effects of paramylon-rich Euglena gracilis EOD-1: A randomized, double-blind, placebo-controlled, parallel-group study. Journal of Functional Foods. 2023; 105804.
- 71. Tsoni SV, Brown GD. Beta-glucans and dectin-1. Annals of the New York Academy of Sciences. 2008; 1143, 45–60. pmid:19076344
- 72.
Zamora R; Billiar TR. Nitric Oxide: A True Inflammatory Mediator. In: Mayer B. eds. Nitric Oxide. Handbook of Experimental Pharmacology. Springer; 2000. Chap 19. Vol 143. https://doi.org/10.1007/978-3-642-57077-3_20
- 73. Wang C, Deen WM. Nitric oxide delivery system for cell culture studies. Ann Biomed Eng. 2003. 31: 65–79. pmid:12572657
- 74. Wiley JW. The many faces of nitric oxide: cytotoxic, cytoprotective or both. Neurogastroenterol Motil. 2007; 19(7): 541–544. pmid:17593134
- 75. Sharma JN, Al-Omran A, Parvathy SS. Role of nitric oxide in inflammatory diseases. Inflammopharmacology. 2007; 15(6): 252–9. pmid:18236016
- 76. Sproston NR, Mohamed EM, Slevin M, Gilmore W, Ashworth JJ. The effect of C-reactive Protein isoforms on nitric Oxide Production by U937 Monocytes/Macrophages. Front Immunol. 2018; 9: 1500. pmid:30013561
- 77. Halliwell B, Clement MV, Long LH. Hydrogen peroxide in the human body. FEBS Letters. 2000; 486:10–13. pmid:11108833
- 78. Cai H. Hydrogen peroxide regulation of endothelial function: Origins, mechanisms, and consequences. Cardiovascular Research. 2005; 68: 26–36. pmid:16009356
- 79. Forstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012; 33(7): 829–837. pmid:21890489
- 80. Russo R, Barsanti L, Evangelista V, Frassanito AM, Longo V, Pucci L, et al. (2017), Euglena gracilis paramylon activates human lymphocytes by upregulating pro-inflammatory factors. Food Sci Nutr, 5: 205–214.
- 81. Angriman I, Scarpa M, D’Incà R, Basso D, Ruffolo C, Polese L, et al. Enzymes in feces: Useful markers of chronic inflammatory bowel disease. Clin Chim Acta. 2007; 381(1): 63–68. pmid:17368600
- 82. Heilmann RM, Suchodolski JS, Steiner JM. Development and analytic validation of a radioimmunoassay for the quantification of canine calprotectin in serum and feces from dogs. AJVR. 2008; 69(7): 845–853. pmid:18593232
- 83. Matijatko V, Mrljak V, Kis I, Kucer N, Forsek J, Zivicnjak T, et al. Evidence of an acute phase response in dogs naturally infected with Babesia canis. Vet Parasitol. 2007; 144(3–4): 242–250. pmid:17116368
- 84. Bathen-Noethen A, Carlson R, Menzel D, Mischke R, Tipold A. Concentrations of acute-phase proteins in dogs with steroid responsive meningitis-arteritis. J Vet Intern Med. 2008; 22(5): 1149–1156. pmid:18691368
- 85. Sproston N, Ashworth JJ. Role of C-Reactive Protein at Sites of inflammation and infection. Front Immunol. 2018; 9: 754. pmid:29706967
- 86. Ikeda U, Yoshikazu M, Yamamoto K, Shimada K. C-reactive protein augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes. Cardiovasc Res. 2002; 56(1): 86–92. pmid:12237169
- 87. Hattori Y, Matsumura K, Kasai K. Vascular smooth muscle cell activation by C-reactive protein. Cardiovasc Res. 2003; 58(1): 186–95. pmid:12667961
- 88. Lebrec HO´Lone R, Freebern W Komocsar W, Moore P. Survey: Immune function and immunotoxicity assessment in dogs. J Immunotoxicol. 2012; 9(1): 1–14. pmid:22059464
- 89. Zaine L, Ferreira C, Gomes MOS, Monte M, Tortola L, Vasconcellos RS, et al. Faecal IgA concentrations is influenced by age in dogs. Br J Nutr. 2011; 106: S183–S186.
- 90. Pabst O, Slack E. IgA and the intestinal microbiota: the importance of being specific. Mucosal Immunology. 2020; 13:12–21. pmid:31740744
- 91. Maria APJ, Ayane L, Putarov TC, Loureiro BA, Neto BP, Casagrande MF, et al. The effect of age and carbohydrate and protein sources on digestibility, fecal microbiota, fermentation products, fecal IgA, and immunological blood parameters in dogs. J Anim Sci. 2017; 95(6): 2452–2466. pmid:28727033
- 92. Stuyven E, Verdonck F, Van Hoek I, Daminet S, Duchateau L, Remon JP, et al. Oral administration of β-1,3/1,6-glucan to dogs temporally changes total and antigen specific IgA and IgM. Clin Vaccine Immunol. 2010; 172(2): 281–285.
- 93. Perini MP, Pedrinelli V, Marchi PH, Henríquez LBF, Zafalon RVA, Vendramini THA, et al. Potential effects of prebiotics on gastrointestinal and immunological modulation in the feeding of healthy dogs: a review. Fermentation. 2023; 9, 693.
- 94. Rychlik A, Nieradka R, Kander M, Nowicki M, Wdowiak M, Kolodziejka-Sawerska A. The effectiveness of natural and synthetic immunomodulators in the treatment of inflammatory bowel disease in dogs. Acta Veterinaria Hungatica. 2013; 61(3): 297–308. pmid:23921342
- 95. Ferreira LG, Endrighi M, Lisenko KG, de Oliveira MRD, Damasceno MR, Claudino JA, et al. 2018 Oat beta-glucan as a dietary supplement for dogs. PLoS ONE 13(7): e0201133. pmid:30063762
- 96. Paris S, Chapat L, Pasin M, Lambiel M, Sharrock TE, Shukla R, et al. β-Glucan-Induced Trained Immunity in Dogs. Front. Immunol. 2020; 11:566893. pmid:33162983
- 97. Ferreira CS, Vendramini THA, Amaral AR, Rentas MF, Ernandes MC, Silva FL, et al. Metabolic variables of obese dogs with insulin resistance supplemented with yeast beta-glucan. BMC Veterinary Research. 2022; 18:14. pmid:34980115
- 98. Alexander C, Cross TL, Devendran S, Neumer F, Theis S, Ridlon JM, et al. Effects of prebiotic inulin-tyoe fructans on blood metabolite and hormone concentrations ond faecal microbiota and metabolites in overweight dogs. British Journal of Nutrution. 2018; 120: 711–720.
- 99. Nogueira JPDS, He F, Mangian HF, Oba PM, De Godoy MR. Dietary supplementation of a fiber-prebiotic and saccharin-eugenol blend in extruded diets fed to dogs. Journal of Animal Science. 2019; 97(11): 4519–4531. pmid:31634399
- 100. Lin C-Y, Carroll MQ, Miller MJ, Rabot R, Swanson KS. Supplementation of Yeast Cell Wall Fraction Tends to Improve Intestinal Health in Adult Dogs Undergoing an Abrupt Diet Transition. Front. Vet. Sci. 2020; 7:597939. pmid:33263019
- 101. Shastri P, McCarville J, Kalmokoff M, Brooks SPJ, Green-Johson JM. Sex differences in gut fermentation and immune parameters in rats fed an oligofructose-supplemented diet. Biology of Sex Differences. 2015; 6:13. pmid:26251695