Ssm

SPD (60-diluted SpHW) : Sterilized water

: Sterilized SPD(60-fold diluted SpHW)

: primary immunization with 400 ig of shrimp and inactivated Bordetella pertussis (1 x 1010cells/0.5ml), ip injection crude shrimp (1.0 mg/mouse), po, once

PBS 0.5 ml, ip injection, Jj, ; PBS(0.2ml/mouse), po, once

Collected spleen, mesenteric lymph node and Peyer's patch

FIGURE 10.7 Protective ingestion of Spirulina extract, SPD, before antigen administration Five mice per group were used in the experiments.

(Figure 10.7). Crude shrimp extract antigen with Bordetella adjuvant was intraperi-toneally injected as described in the former experiment and crude shrimp extract was orally administered once after the primary immunization. IgA antibody production in culture cells of some lymphoid organs from mice treated with Spirulina extract was examined.

IgA antibody levels in the culture supernatant of cells of lymphoid organs, especially in the spleen and mesenteric lymph node of the SPD-Ag group was significantly enhanced in comparison with Ag groups as shown in Figure 10.8, whereas neither antigen stimulation alone in Ag group nor administration of SPD alone in SPD group increased the IgA antibody in any lymphoid organ.

In conclusion, these results from the experiments in both concurrent and protective ingestion of Spirulina showed that Spirulina at least neither induced nor enhanced allergic reaction like food allergy dependent on IgE antibody, and that Spirulina ingested both concurrently with antigen and before antigen administration significantly enhanced IgA antibody production to protectively affect against infection or allergic reaction.

Secretory IgA antibodies exhibit synergistic interaction with antibacterial substances such as lysozyme and lactoferrin.20 Cholera toxin, bacterial lipopolysacchar-ides or lipid A, muramyl dipeptide, and a synthetic or nonbacteria1 1ipoidal amine, are known as mucosal adjuvants and have been found to potentiate secretory immune response to stimulate the production of IgA antibody.25 It has also been known that orally ingested lactic acid bacteria—Bifidobacterium longum, B. breve, and Lactobacillus casei—increased mucosal IgA response to antigen in in vitro or in vivo studies

Ag SPD SPD-Ag

FIGURE 10.8 Total IgA antibody in cell-culture supernatant of lymphoid organs from mice protectively ingested with Spirulina extract before antigen stimulation as shown in Figure 10.7 (Adapted from Hayashi, O., J. Nutr. Sci. Vitaminol, 44, 848, 1998. With permission.) Means ± SD of five mice **p < .01 compared to Ag group

Ag SPD SPD-Ag

FIGURE 10.8 Total IgA antibody in cell-culture supernatant of lymphoid organs from mice protectively ingested with Spirulina extract before antigen stimulation as shown in Figure 10.7 (Adapted from Hayashi, O., J. Nutr. Sci. Vitaminol, 44, 848, 1998. With permission.) Means ± SD of five mice **p < .01 compared to Ag group in animals and in humans.26,27 The role of cytokines in orchestrating the mucosal immune response has been greatly investigated for the potential of therapeutic applications to improve mucosal responses and to control systemic autoimmunity.28 Further experiments concerning the mechanisms of stimulating local immune response by Spirulina, in regard to cytokine production, are necessary, in addition to investigations of the enhancing effect of Spirulina on IgA production in humans.

DISTINCT EFFECTS OF PHYCOCYANIN INGESTION ON SECRETORY IgA AND ALLERGIC IgE ANTIBODY RESPONSES IN MICE

Spirulina contains phycocyanin, a blue, 270-kDa photosynthetic pigment protein, which accounts for approximately 15% of the dry weight of Spirulina.3 Previously we investigated the effect of phycocyanin ingestion on the immune response of the intestinal mucosa, and found that ingestion of phycocyanin promoted IgA antibody production in mice immunized with aqueous solution of ovalbumin (OVA) as an antigen.29 In Peyer's patches that are contained in the peripheral lymphoid tissues, production of total IgA antibody was also promoted by phycocyanin. However, the antigen-specific IgA antibodies in both Peyer's patches and mesenteric lymph nodes were undetected, probably because OVA, used as an antigen, was an aqueous solution. Aqueous antigen could be too easily degraded in the digestive tract to retain functional antigenicity. In order to solve this problem, we prepared OVA antigen-entrapped biodegradable microparticles made of poly (DL-lactide-co-glycolide), and poly (DL-lactide-co-glycolide)

poly (DL-lactide-co-glycolide)

FIGURE 10.9 (Water-in-oil)-in-water emulsion (left) and Scanning electron micrograph

(right) of OVA microparticles (Adapted from Nemoto-Kawamura, C., J. Nutr. Sci. Vitaminol.,

50, 132, 2004. With permission.)

Average diameter was 4 |im.

FIGURE 10.9 (Water-in-oil)-in-water emulsion (left) and Scanning electron micrograph

(right) of OVA microparticles (Adapted from Nemoto-Kawamura, C., J. Nutr. Sci. Vitaminol.,

50, 132, 2004. With permission.)

Average diameter was 4 |im.

used as a stimulating antigen for local antibody response in the mucous. Antigen that has been entrapped in biodegradable microparticles may circumvent the problem of antigen degradation.30-32 We focused on the study of immune responses in the intestinal mucosa, mesenteric lymph nodes, and Peyer's patches in mice that had ingested phycocyanin. The effect of phycocyanin on type I allergy was also studied by measuring serum IgE levels and its relation to inflammation.

OVA-entrapped biodegradable microparticles (OVA microparticles) were prepared using the (water-in-oil)-in-water (w/o/w) emulsion solvent evaporation technique.33 A mixture of OVA aqueous solution and poly (DL-lactide-co-glycolide) in dichloromethane (DCM) was homogenized at 8000 rpm for 10 s in a micro homo-genizer followed by addition of polyvinyl alcohol aqueous solution. Sediment of a (water-in-oil)-in-water (w/o/w) emulsion obtained by secondary homogenization similar to the first step was washed three times with sterile distilled water by centrifu-gation, and lyophilized to recover the resulted microparticles. To determine the shape of the microparticles and to determine the average value of diameter, more than 400 microparticles in each batch were observed by scanning electron microscopy (Figure 10.9). Average diameter of the microparticles was 4 /m. Protein contents of the microparticles were found to be 15% as a standard of bovine serum albumin, measured by using a bicinchoninic acid kit.

Antigen-entrapped microparticles may be a useful tool to study the mucosal immune responses. Some investigators have shown that microparticles of 3-4 /m diameters activated both mucosal and systemic immunity32 and resulted in the greatest increase in serum antigen-specific IgG1 antibody levels in mice.34 We reported as a preliminary experiment that microparticles having a diameter of approximately 4 /m also exhibited strong adhesion to Peyer's patches.35 These data were consistent with the idea that there was an appropriate particle size that renders the microparticles effective. As a result of using antigen-entrapped microparticles with 4 /m average diameters, OVA-specific IgA antibody was successfully induced. In addition to

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