Alberto Estrada

Immunomodulatory Activities of Oat β-Glucan In Vitro and In Vivo

Previous studies have shown that β‐glucans extracted from yeast or fungi potentiate immune responses. In the present study, the immunomodulatory activities of β‐(1→3, 1→4)‐glucan, derived from oats, were investigated. The ability of oat β‐glucan (OβG) to stimulate IL‐1 and TNF‐α release from murine peritoneal macrophages and the murine macrophage cell line P338D1, was assessed. In vitro stimulation of macrophages with OβG resulted in the production of IL‐1 in a dose and time‐dependent manner, whereas only small amounts of TNF‐α could be detected in the culture supernatants. OβG also induced the production of IL‐2, IFN‐γ and IL‐4 secretion in a dose‐dependent manner in cultured spleen cells. The intraperitoneal administration of OβG in mice resulted in the accumulation of leucocytes, predominantly macrophages, in the peritoneal cavity. Furthermore, OβG was tested for its ability to enhance non‐specific resistance to a bacterial challenge in mice. Survival of mice challenged with Staphylococcus aureus was enhanced by a single intraperitoneal administration of 500 μg of OβG 3 days prior to bacterial challenge. In conclusion, these studies demonstrated that OβG possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.

Isolation and evaluation of immunological adjuvant activities of saponins from Polygala senega L.

We have identified saponins in the root of Polygala senega L., a plant indigenous to the Canadian prairies, which display immunopotentiation activity to protein and viral antigens. By two-step extraction and hemolytic activity-guided fractionation by silica flush chromatography six saponin fractions were generated and their HPLC profiles determined. Two dominant fractions, designated as PS-1 and PS-2, were tested for adjuvant activity in mice immunized with ovalbumin, and hens immunized with rotavirus. The resulting adjuvant activity was compared with that of Quil A saponin. The P. senega saponins increased specific antibody levels to the antigens, in both mice and hens. In mice, there was a preferential increase of the IgG2a subclass, and upon in vitro secondary antigen stimulation, high IL-2 and IFN-gamma levels were observed in spleen cell cultures from P. senega saponins-immunized animals. The saponins were tested for their toxicity by lethality in mice and were found to be less toxic at the same dose than their counterpart Quil A. The results of this study indicated the potential of P. senega saponins as vaccine adjuvants to increase specific immune responses.

β-Glucan, extracted from oat, enhances disease resistance against bacterial and parasitic infections

The effect of beta-glucan, extracted from oats, on the enhancement of resistance to infections caused by Staphylococcus aureus and Eimeria vermiformis was studied in mice. In vitro study using macrophages isolated from the peritoneal cavity showed that beta-glucan treatment significantly enhanced phagocytic activity. In vivo study further demonstrated that beta-glucan treatment induced a significant (P<0.05) protection against the challenge with 5 x 10(8) of S. aureus in mice. Fecal oocyst shedding in the C57BL/6 mice infected with E. vermiformis was diminished by beta-glucan treatment by 39.6% in intraperitoneal and 28.5% in intragastric group compared to non-treated control. Patency period was shorter and antigen (sporozoites and merozoites) specific antibodies were significantly (P<0.05-0.01) higher in beta-glucan-treated group compared to non-treated control group. There were an increasing number of splenic IFN-gamma-secreting cells in glucan-treated group via intraperitoneal route, which might be responsible for the enhancement of the disease resistance. Glucan treatment was able to effectively change the lymphocytes population (Thy 1.2(+), CD4(+) and CD8(+) cells) in the mesenteric lymph nodes and Peyers patches in mice infected with E. vermiformis. In conclusion, the oral or parenteral oat beta-glucan treatment enhanced the resistance to S. aureus or E. vermiformis infection in the mice.

Effect of the dietary supplementation of fructooligosaccharides and Bifidobacterium longum to early-weaned pigs on performance and fecal bacterial populations

Two experiments were conducted to evaluate the effect of administration of fructooligosaccharides (FOS) and Bifidobacterium longumon growth performance and fecal bacterial populations in pigs weaned at 18 d of age. In exp. 1, two groups of 20 pigs each were fed diets containing 0 or 0.5% FOS for 21 d. On days 1 and 3, pigs receiving FOS were administered an oral dose of 1010 B. longumcells. During week 1, average daily gain was higher and feed efficiency was improved (P < 0.05) in pigs fed FOS and bifidobacteria relative to the control. On day 7, supplemented pigs had reduced numbers of total anaerobes and clostridia, and increased numbers of bifidobacteria in faeces (P < 0.05). In exp. 2, 80 pigs were divided into four groups of 20 animals each in a 2 x 2 factorial design. Main effects included FOS supplementation (0 or 0.5% of diet) and B. longum supplementation (0 or 107 cells per gram feed). FOS supplementation reduced growth while B. longum supplementation increased growth (P< 0.05). On days 12 and 1...

Immunomodulatory Effects of Oat β-Glucan Administered Intragastrically or Parenterally on Mice Infected with Eimeria vermiformis

The immunostimulatory effect of intragastrically or parenterally administered β‐(1→3; 1→4) glucan, extracted from oats (OβG), on disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Multiple administrations of OβG by intragastric or subcutaneous routes reduced fecal oocyst shedding compared to the non‐treated control group. The administration of OβG by subcutaneous route resulted in higher levels of total serum immunoglobulins and antigen (sporozoite and merozoite)‐specific immunoglobulins as compared with the non‐treated group. To evaluate the effect of a single subcutaneous dose, groups of mice were treated with OβG 2 days before E. vermiformis infection, at the time of infection and at 2 or 6 days after infection. From day 11 post‐infection the oocyst discharge was significantly diminished (P< 0.05‐0.01) in the OβG‐treated groups, except in those treated 6 days after infection, as compared to the non‐treated control group. The proliferative responses to E. vermiformis sporozoite antigen of lymphocytes isolated from the spleen were significantly increased (P < 0.05) when OβG was administered 2 days before or at the time of E. vermiformis infection. Lymphocyte proliferative responses to merozoite antigen were not influenced by treatment. In conclusion, OβG appeared to up‐regulate immune mechanisms and provide enhanced resistance against eimerian coccidiosis in mice.

β-(1→3, 1→4) Oat glucan enhances resistance to Eimeria vermiformis infection in immunosuppressed mice

The effect of intragastrically or parenterally administered beta-glucan, extracted from oats, on the enhancement of disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Groups of mice were immunosuppressed with dexamethasone (DXM), infected with oocysts of E. vermiformis and treated with oat beta-glucan by the intragastric (i.g.) or subcutaneous (s.c.) routes. Faecal oocyst shedding was reduced in the beta-glucan-treated groups compared to the non-treated group. Immunosuppressed mice which received no beta-glucan treatment showed more severe clinical signs of the disease and a 50% mortality, while minimal clinical signs and no mortality were recorded in the beta-glucan-treated groups. Total IgG, IgG1, IgG2a, IgM and IgA immunoglobulins in the serum of beta-glucan-treated groups were overall higher than those in the non-treated group. Specific IgG anti-sporozoite and merozoite immunoglobulins in serum were significantly higher in the beta-glucan-treated groups than in the non-treated animals. No significant differences were found in the levels of intestinal IgA anti-sporozoite and anti-merozoite immunoglobulins. IFN-gamma- and IL-4-secreting cells, in response to sporozoite antigen, were detected in the spleen and mesenteric lymph nodes of the beta-glucan-treated groups only. In conclusion, the i.g. and s.c. oat beta-glucan treatment increased the resistance to E. vermiformis infection in immunosuppressed mice.

Adjuvant action of Chenopodium quinoa saponins on the induction of antibody responses to intragastric and intranasal administered antigens in mice

Saponins extracted from the seed of Chenopodium quinoa (quinoa) were studied for their ability to act as mucosal adjuvants upon their intragastric or intranasal administration together with model antigens in mice. Quinoa saponins, co-administered intragastrically or intranasally with cholera toxin or ovalbumin, potentiated specific IgG and IgA antibody responses to the antigens in serum, intestinal and lung secretions. The potentiating effect of the saponins appeared, to some extent, mediated by increased permeability of the mucosa, allowing increased uptake of the antigen. The intragastric administration of 99mTc-radio-labeled human serum albumin together with quinoa saponins revealed an increased presence of the radiolabeled protein in blood, liver, spleen and lungs of mice. This study indicates the potential of quinoa saponins as adjuvants for mucosally administered vaccines.

Effect of dietary cereal on intestinal bacterial populations in weaned pigs

Diets containing corn, barley or wheat as the main carbohydrate source were formulated to a similar nutrient content and fed for 3 wk to weaned pigs. Gut bacterial populations differed significantly between the three diets and these differences were correlated with the fibre contents of the diets. Key words: Corn, wheat, barley, intestinal bacteria, pigs

Effect of Endotoxin on Cytokine Production and Cell Dynamics in Mice

Previous studies have shown that endotoxin potentiates immune responses by direct stimulation of B cells and macrophages. In the present study, we assessed the ability of endotoxin to stimulate cells from different lymphoid tissue compartments to release cytokines. The in vitro stimulation of macrophages with endotoxin resulted in the production of IL-1 and TNF-alpha in a dose and time-dependent manner. Endotoxin also induced the production of IL-2, IFN-gamma and IL-4 secretion in a dose dependent manner in cultured spleen, mesenteric lymph nodes and Peyers patches cells. The intraperitoneal administration of endotoxin in mice resulted in the accumulation of leucocytes in the peritoneal cavity and in the increase of IL-1, IL-6, TNF-alpha and IFN-gamma concentration in serum. In conclusion, this study confirmed that endotoxin possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.

Induction of Systemic and Mucosal Immune Responses Following Immunization with Somatostatin-Avidin Complexes Incorporated Into Iscoms

Synthetic, biotinylated somatostatin-14 (Somatotropin Release-Inhibiting Factor; SRIF) was conjugated to avidin, and the resulting complex incorporated into immune-stimulating complexes (ISCOMS). The ISCOMS were used to study the systemic and mucosal immune responses induced by parenteral and gastrointestinal vaccination. Mice were immunized by intraperitoneal (IP) and intragastric (IG) routes and subsequently by either IP or IG secondary immunizations (groups-IP/IP; IP/IG; IG/IG). Antigen specific IgG and IgA antibody secreting cells (ASC) from the spleen, mesenteric lymph nodes (MLN) and Peyers patches (PPs) were studied by an enzyme-linked immunospot assay (ELISPOT). Specific proliferative responses of spleen cells to avidin and to SRIF were measured. Immunization IP/IP evoked the highest serum IgG levels to avidin and to SRIF as well as the highest numbers of splenic IgG isotype ASC. The greatest IgA response in MLN and PPs was induced by IP/IG immunization. Only marginal mucosal immunity and no splenic cell specific proliferative responses were found by IG/IG immunization. These results indicate that ISCOMS are an effective delivery system for protein-peptide antigens. The ISCOMS system described elicited systemic and mucosal antibody immune responses, and primed specific proliferative response when administered IP/IG. This offers another approach for the design and delivery of mucosally administered peptide vaccines.