Oral λ-carrageenan intake alleviates skin symptoms in a hapten induced atopic dermatitis-like model.

Lambda carrageenan is a widely used food additive. It has been shown that its oral intake induces suppression of T cell proliferation and antibody-mediated and cell-mediated immune response in experimental animals. In this study, we estimated the effect of oral ingestion of 0.001% λ-carrageenan on trinitrochlorobenzen-induced atopic dermatitis model mouse. Oral carrageenan ingestion alleviated ear swelling of hapten challenged mice and significantly suppressed mast cell hyperplasia in the topical skin. Serological analysis revealed that the treatment suppressed total IgE and antigen-specific IgG, and also suppressed both allergy driving cytokine interleukin-4 and counter-acting cytokine interferon-γ levels. It is suggested that the oral ingestion of λ-carrageenan may suppress the immunological response to the allergen and might be useful to treat atopic dermatitis.

Evaluation of pH-sensitive fusogenic polymer-modified liposomes co-loaded with antigen and α-galactosylceramide as an anti-tumor vaccine.

pH-Sensitive fusogenic polymer-modified (pH-sensitive) liposomes co-loaded with tumor model antigen, ovalbumin (OVA), and adjuvant, α-galactosylceramide (α-GalCer) were fabricated and administered subcutaneously into mice. The ability of pH-sensitive liposomes containing OVA and α-GalCer to stimulate cellular and humoral immune responses in vivo was compared with OVA-encapsulating pH-sensitive liposomes as well as with OVA alone. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, antigen-specific IgG1 antibody responses were noted in mice immunized with OVA alone, whereas immunization with OVA-containing pH-sensitive liposomes and with pH-sensitive liposomes containing OVA and α-GalCer resulted in the induction of OVA-specific IgG1 and IgG2b antibody responses. Moreover, more substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from mice immunized with pH-sensitive liposomes having OVA and α-GalCer than OVA-containing pH-sensitive liposomes in vitro. Spleen cells from the immunized mice showed strong cytotoxic activity against E.G7-OVA tumor cells. In addition, prophylactic vaccination efficacy against tumor formation was evaluated. In all mice immunized with pH-sensitive liposomes having OVA and α-GalCer, immunization provided substantial protection from tumor formation. The therapeutic efficacy of pH-sensitive liposomes containing OVA and α-GalCer against already established E.G7-OVA tumors was also investigated. Tumor growth was reduced significantly in all mice treated with pH-sensitive liposomes having OVA and α-GalCer. The provided evidence on the advantage of antigen and α-GalCer co-encapsulation into pH-sensitive liposomes should be considered in the design of future cancer vaccines for prophylactic and therapeutic purposes.

Induction of antibody response in the oral cavity of dogs following intraocular (eye drop) immunization with Porphyromonas gingivalis cell lysate incorporated in pH-sensitive fusogenic polymer-modified liposomes.

Induction of mucosal immune responses against Porphyromonas gingivalis within the oral cavity of dogs was studied by immunizing with pH-sensitive fusogenic polymer (MGluPG)-modified liposome-associated cell lysate. Dogs immunized with P. gingivalis cell lysate-containing MGluPG-modified liposomes by intraocular (eye drop) route displayed significant levels of P. gingivalis cell lysate-specific serum IgG and IgA as well as mucosal IgA antibodies in saliva secretion. Serum and salivary antibodies generated by intraocularly immunized with MGluPG-modified liposome-associated P. gingivalis cell lysate revealed a significant aggregation activity against P. gingivalis, whereas serum and saliva from dogs receiving MGluPG-modified liposomes unentrapping P. gingivalis cell lysate did not show the aggregation activity against P. gingivalis. Furthermore, P. gingivalis-specific antibodies in saliva of immunized dogs inhibited the adherence of P. gingivalis to cultured HeLa cells. More importantly, salivary antibodies induced by intraocular immunization with P. gingivalis cell lysate-containing MGluPG-modified liposomes significantly inhibited the coaggregation of P. gingivalis with Actinomyces naeslundii and the cell damage activity of P. gingivalis against FaDu cells, an oral epithelial cell. These results suggest that intraocularly administered P. gingivalis cell lysate-containing MGluPG-modified liposomes should be an effective mucosal vaccine against P. gingivalis infection in dogs and may be an important tool for the prevention of periodontitis.

Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus.

To rapidly and simply determine whether or not bacterial colonies growing on agar were Vibrio parahaemolyticus, we developed an immunochromatographic assay (VP-ICA) using two different monoclonal antibodies (designated mAb-VP34 and mAb-VP109) against the delta subunit of V. parahaemolyticus-F0F1 ATP synthase. The epitopes recognized by mAb-VP34 and mAb-VP109 were mapped to sequences of eight ((47)LLTSSFSA(54)) and six amino acid residues ((16)FDFAVD(21)), respectively. An amino acid sequence similarity search of the NCBI database using BLASTP showed that both epitopic amino acid sequences were present together only in V. parahaemolyticus. When 124 V. parahaemolyticus strains and 94 strains of 27 other Vibrio species or 35 non-Vibrio species were tested using the VP-ICA, the VP-ICA identified V. parahaemolyticus with 100% accuracy. The VP-ICA rapidly and simply identified the pathogen directly from a single agar colony within 30 min, indicating that VP-ICA will greatly reduce labor and time required to identify V. parahaemolyticus compared with conventional biochemical tests.

Production and characterization of a novel monoclonal antibody against Vibrio parahaemolyticus F0F1 ATP synthase's delta subunit and its application for rapid identification of the pathogen.

We raised monoclonal antibodies (MAbs) against Vibrio parahaemolyticus cell extracts. One of the MAbs, designated MAb-VP34, reacted in enzyme-linked immunosorbent assays (ELISAs) with 140 V. parahaemolyticus strains, regardless of serotype or origin. MAb-VP34 did not detectably react with 96 strains belonging to 27 other Vibrio species (except for Vibrio natriegens) or with 29 non-Vibrio species. These results show that MAb-VP34 is highly specific for V. parahaemolyticus. Western blotting and mass spectrometry analyses revealed that MAb-VP34 recognized V. parahaemolyticus F(0)F(1) ATP synthase's delta subunit. Using MAb-VP34, a rapid and simple immunodot blotting assay (VP-Dot) was developed to determine whether bacterial colonies growing on selective agar, represented V. parahaemolyticus. To evaluate VP-Dot, 20 V. parahaemolyticus strains and 19 non-related strains were tested. The results indicated that VP-Dot is a reliable tool for identification of V. parahaemolyticus colonies. The simple VP-Dot procedure took 40min, indicating that the MAb-VP34 based immunological method will greatly reduce labor, time, and costs required to verify V. parahaemolyticus colonies as compared with the conventional biochemical test.

Protection against atypical Aeromonas salmonicida infection in common carp, Cyprinus carpio L., by oral administration of a mixed microbial culture of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae.

A microbial culture was prepared by co-cultivation of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae for 48 hr at 30°C in rice bran extract medium, supplemented with dextrose. Oral administration of the resulting non-viable heat-inactivated microbial culture to common carp, Cyprinus carpio L., delivered in feed for four weeks, induced effective protection against experimental atypical Aeromonas salmonicida infection which causes "ulcer disease". After challenge of the carp by immersion, fish mortality and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with mixed microbial culture adsorbed on dry pellets relative to carp treated with medium or without extract. Atypical A. salmonicida was re-isolated from ulcerative lesions in parts of dead and surviving fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying microbial population changes during the progression of skin lesions. Among interleukin-1ß (IL-1ß), tumor necrosis factor-α, as well as CXC-α and CXC-ß chemokines, gene expression of IL-1ß was up regulated in the spleen and head kidney three weeks after administration of the mixed microbial culture. These results clearly show that this mixed microbial culture, delivered in feed, is effective in preventing A. salmonicida disease in carp.

Protection against Flavobacterium psychrophilum infection (cold water disease) in Ayu fish (Plecoglossus altivelis) by oral administration of humus extract.

Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms have been present. In the present study, oral administration of humus extract to ayu fish (Plecoglossus altivelis) induced effective protection against experimental Flavobacterium psychrophilum infection (cold water disease). Mortality of fish and development of skin lesions, such as erosion and hemorrhages on the skin, gill cover or mouth, were significantly suppressed in fish treated with 10%, 5% or 1% humus extract adsorbed on dry pellets. Although F. psychrophilum was not re-isolated from gills and erosion lesions of the skin of dead fish, bacterial gyrB DNA could be amplified in these specimens from dead fish and surviving control fish using the polymerase chain reaction. The protective effect of the extract was not the results of direct killing of bacteria or antibiotic activity of the extract since no obvious reduction in the bacterial number was observed at 5 times to 5,000 times dilution of the humus extract having pH 5.45 to 7.40. These results clearly show that treating fish with humus extract is effective in preventing cold water disease.

A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy.

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.

In vitro enhancement by CpG ODN of phagocytic activity of rainbow trout head kidney phagocytes against fish pathogen Vibrio ordalii, but not against polystyrene particles.

Rainbow trout (Oncorhynchus mykiss) head kidney phagocytes precultured with a synthetic cytidine-phosphate-guanosine (CpG) oligodeoxynucleotide (ODN) displayed significantly higher phagocytic activity against Vibrio ordalii than phagocytes precultured with non-CpG ODN. However, head kidney phagocytes precultured with CpG ODN did not show enhanced phagocytic activity against polystyrene particles.

Induction of immune responses against glycosphingolipid antigens: comparison of antibody responses in mice immunized with antigen associated with liposomes prepared from various phospholipids.

The immune responses of mice against glycosphingolipid (GSL) antigens and the effect of the phospholipid composition of liposomes on the immunogenicity in mice of liposome-associated GSL antigens were examined. The immunization with GSL antigen alone was unable to induce any detectable anti-GSL antibody responses. On the other hand, the immune responses against GSL antigens were detected after immunization with liposomes composed of dipalmitoylphosphatidylcholine (DPPC) (0.5 micromol), cholesterol (Chol) (0.5 micromol), Salmonella minnesota R595 lipopolysaccharides (LPS) (10 microg) and GSL (0.05 micromol) (DPPC-liposome). However, the administration with liposome composed of dimyristoylphosphatidylcholine (DMPC) (0.5 micromol), Chol (0.5 micromol), S. minnesota R595 LPS (10 microg) and GSL (0.05 micromol) and with liposomes composed of distearylphosphatidylcholine (DSPC) (0.5 micromol), Chol (0.5 micromol), and S. minnesota R595 LPS (10 microg) and GSL (0.05 micromol) was ineffective for the induction of the immune responses against GSL antigens. These results suggest that DPPC-liposome would serve effectively as a delivery vehicle for inducing immune responses against GSL antigen.

Protection against experimental Aeromonas salmonicida infection in carp by oral immunisation with bacterial antigen entrapped liposomes.

Liposome-entrapped atypical Aeromonas salmonicida antigen was prepared to investigate the potential protective efficacy for A. salmonicida infection. Carp (Cyprinus carpio) were immunised orally with liposome-entrapped A. salmonicida antigen. After immunisation, significantly higher antigen-specific antibodies were detected in serum, intestinal mucus and bile than non-immunised control group. Furthermore, immunised carp were challenged by immersion with 1 x 10(6) cfu ml(-1) of A. salmonicida for 60 min. Of the eight non-immunised carp, three carp died (62.5% survival), whereas five out of six (83.5%) immunised survived. Furthermore, the development of skin ulcers was significantly inhibited in carp immunised with liposomes containing A. salmonicida antigen. These results suggest that liposomes containing A. salmonicida antigen have the potential for the induction of a protective immune response against atypical A. salmonicida infection and also suggest the possibility of developing a vaccine that may ultimately be used for the prevention of fish diseases.

Effect of turpentine oil on C-reactive protein (CRP) production in rainbow trout (Oncorhynchus mykiss).

The effect of turpentine oil on C-reactive protein (CRP) production was studied in rainbow trout (Oncorhynchus mykiss). Serum CRP concentration was estimated by sandwich enzyme-linked immunosorbent assay using anti-rainbow trout CRP monoclonal antibody (mAb) AC4 and polyclonal antibody. Intracellular CRP was demonstrated by flow cytometry using anti-trout CRP mAb. Hepatocytes, head kidney macrophages, spleen lymphocytes and peripheral blood lymphocytes showed reaction against AC4, but RTG-2 fibroblastic line cells, derived from rainbow trout gonad did not. This is the first report on the detection of intracellular CRP in fish. CRP levels decreased significantly 1 day after intramuscular injection of turpentine oil and remained low for 14 days. Significant decreases in the expression of CRP in hepatocytes, head kidney macrophages and spleen lymphocytes after injection of turpentine oil were found. The reduction of serum CRP concentration after turpentine oil injection may be attributed to decreases in intracellular CRP synthesis.

Changes of C-reactive protein levels in rainbow trout (Oncorhynchus mykiss) sera after exposure to anti-ectoparasitic chemicals used in aquaculture.

Changes of serum C-reactive protein (CRP) levels in rainbow trout (Oncorhynchus mykiss) were studied after exposure to formalin, metriphonate or potassium permanganate, which are used in aquaculture as anti-ectoparasitic chemicals. The CRP level in normal trout sera is 88+/-5 microg ml(-1) according to sandwich enzyme-linked immunosorbent assay. CRP levels increased to a maximum at six or nine days after exposure to formalin for 3.5 h at 300 ppm or 9.5 h at 30 ppm, respectively; these levels are 4.3 and 18 times higher than normal. At 18 days after treatment, the CRP level had decreased to significantly below the normal level. After exposure to metriphonate (0.4 ppm for 30 min), the CRP level increased significantly to a maximum at three days after exposure (9.9 times higher than normal), then decreased to below normal. With exposure to potassium permanganate at 40 ppm for 45 min, fish showed significantly lower CRP levels than the normal level at 14 days after exposure. Fish reared at a water temperature of 16.5-19.5 degrees C showed significantly higher CRP levels than those reared at 13 degrees C. Measurement of CRP levels in trout serum can be used as a bioindicator of the health condition of the fish.

Binding of Vibrio anguillarum to neutral glycosphingolipids from intestinal mucosa of rainbow trout (Oncorhynchus mykiss).

To test whether glycosphingolipids (GSLs) on the intestinal mucosa of rainbow trout (Oncorhynchus mykiss) serve as a binding receptor for Vibrio anguillarum, we analyzed neutral GSLs from rainbow trout intestinal mucosa and investigated the binding of bacteria to neutral GSLs. Two kinds of neutral GSLs, designated N-1 and N-2, were identified on high-performance thin-layer chromatography (TLC) plates. In TLC immunostaining tests, V. anguillarum bound only to galactosylceramide (GalCer), lactosylceramide and N-1 having the same TLC mobility as GalCer, but neither to glucosylceramide nor to N-2. These results suggest that N-1 is GalCer (Gal beta 1-1Cer) and also that N-1 (GalCer) on rainbow trout intestinal mucosa act as a receptor for V. anguillarum.

An autocrine function of nerve growth factor for cell cycle regulation of vascular endothelial cells.

Nerve growth factor (NGF) regulates maintenance, survival, and function of not only neuronal cells but also various kinds of non-neuronal cells. Here we clearly demonstrated that mouse aortic endothelial cells (AEC) produced bioactive NGF, and the production was enhanced by a proinflammatory cytokine, interleukin (IL)-1beta. AEC expressed both high affinity (TrkA) and low affinity (p75(NGFR)) receptors for NGF. Exogenously added NGF induced rapid phosphorylation of TrkA tyrosine kinase. Addition of anti-NGF neutralizing antibody resulted in an increase in the proportion of AEC in S and G(2)/M phases and in a hypodiploid range. Since the vascular endothelium plays a pivotal role in inflammatory conditions, these results strongly suggest that NGF, whose production is enhanced at the affected site, may contribute to maintenance, survival, and function of vascular endothelial cells by autocrine and/or paracrine mechanisms.

Suppression of Salmonella enterica serovar Enteritidis excretion by intraocular vaccination with fimbriae proteins incorporated in liposomes.

Liposome-associated fimbriae antigens (SEF14 and SEF21) were prepared for intraocular immunization to seek protective efficacy for intestinal infection with Salmonella enterica serovar Enteritidis. Chickens were immunized intraocularly with the antigens at 8 and 10 weeks of age. Evidence of an IgA and IgG responses were found in the intestinal tract and in sera of these chickens. Antibody-secreting lymphocytes were detected in the Harderian gland of immunized chickens as determined by enzyme-linked immunospot assay. Two weeks after the booster immunization, the chickens were challenged orally with 1x10(7) live Salmonella Enteritidis, and fecal samples were examined for bacterial excretion from the intestinal tract. Significantly less fecal excretion of bacteria was observed in immunized chickens for 15 days after challenge. The numbers of bacteria in the intestinal contents (caecum and rectum) were also significantly lower in immunized chickens than in unimmunized controls. Detection of S. Enteritidis-specific DNA by the polymerase chain reaction was consistent with the bacterial observations. Intraocular immunization with liposome-associated SEF14 and SEF21 therefore elicits both systemic and mucosal antibody responses, so that bacterial colonization in the intestinal tract and excretion of S. Enteritidis in the feces are suppressed by immunization.