Enhancement of intestinal immune function in mice by ß-D-glucan from aureobasidium pullulans ADK-34.

ß-Glucans, glucose polymers that are the main constituents of the outer cell walls of micro-organisms such as fungi and yeast, are known to play an immunostimulatory role. We prepared ß-glucan (ß-(1-3),(1-6)-D-glucan) from an edible cultured fungus through fermentation techniques using a strain of Aureobasidium pullulans ADK-34. The purity of this ß-glucan preparation (AP-FBG) was demonstrated to be high through various instrumental analyses. We then examined the effects of AP-FBG on intestinal immune systems. We prepared Peyer's patch (PP) cells and measured interleukin (IL)-5, IL-6, and IgA production in culture media with AP-FBG. We found that both cytokines and IgA increased; furthermore, IL-6 secreted by PP dendritic cells (PPDCs) cultured in the presence of AP-FBG significantly increased. We tested IgA production after oral administration of AP-FBG for 2 weeks and found that AP-FBG tended to promote the production of IgA in the small intestine. Interestingly, we observed a significant increase in IgA production in the small intestines of mice treated with cyclophosphamide (CY; an immunosuppressant) after oral administration of AP-FBG diet compared with CY-treated and control diet mice. Production of IL-6 and IgA by PP cells and IL-6 production by PPDCs in AP-FBG-fed and CY-treated mice also increased. These results demonstrate that AP-FBG has the ability to activate PPDC and induce IL-6 production and IgA secretion in PP cells. These abilities were more clearly expressed when AP-FBG was orally administered in a CY-induced immunosuppressed condition. Therefore, AP-FBG may be a useful ingredient for preparing functional foods with immunomodulatory activities.

Differences in defining residues relevant to antibody binding by ELISA and proteolysis protection at the level of peptide antigenic determinants.

The interaction of monoclonal antibody (mAb) 1C3 with bovine beta-casein was analyzed by two methods. The competitive enzyme-linked immunosorbent assay in which mAb 1C3 bound competitively to a peptide in solution or beta-casein adsorbed to wells in microtitration plates showed that beta-casein (f194-200) (peptide of residues 194 to 200 of beta-casein) was the shortest peptide among the tested peptides that had binding ability for mAb 1C3. Protection against proteinase digestion was analyzed by incubating proline-specific endopeptidase, carboxypeptidase Y or aminopeptidase T with beta-casein (f193-202) in the presence of mAb 1C3 or nonspecific mAb 31A4. Proteolysis of peptide bonds between residues 200 and 201, and 201 and 202 was depressed in the presence of mAb 1C3. However, peptide bonds between 193 and 194, and 194 and 195 were cleaved in the presence of mAb 1C3 as easily as in the presence of mAb 31A4, suggesting that the region of residues 200 to 202 was obscured by, or within the antibody binding site, but that the region of residues 193 to 195 was not. The apparent antibody binding site shown from the protection against proteolysis by mAb was clearly not identical to the shortest antigen peptide, beta-casein (f194-200), indicated from the competitive binding assay.

Tryptophan-19 of beta-lactoglobulin, the only residue completely conserved in the lipocalin superfamily, is not essential for binding retinol, but relevant to stabilizing bound retinol and maintaining its structure.

Residue 19 of tryptophan in bovine beta-lactoglobulin (beta-LG) is the only invariant residue throughout the lipocalin superfamily having two characteristic features: binding ability for small hydrophobic molecules and the unique beta-barrel three-dimensional structure. In this study, we investigated whether this strictly conserved Trp-19 of beta-LG would be indispensable for its structure and function such as maintaining the molecular structure and biological activity of beta-LG. Spectroscopic and enzymatic oxidation experiments on retinol bound to W19Y, in which Tyr was substituted for Trp-19, showed that Trp-19 was not critical for this binding, but was important for stably maintaining the environment surrounding retinol and the bound retinol. An using four anti-beta-LG monoclonal antibodies as probes, revealed a structural change in region 20-29, but not in the reverse region of Trp-19. A guanidine hydrochloride-induced unfolding study showed that the conformational stability of W19Y was greatly reduced by 6.9 kcal/mol compared to that of wild-type beta-LG. These facts indicated that Trp-19 is one of the important residues in correctly maintaining the local structure of beta-LG and stably retaining its overall structure, thereby conserving the bound retinol molecule.

Accelerated secretion of mutant beta-lactoglobulin in Saccharomyces cerevisiae resulting from a single amino acid substitution.

Transformed yeasts producing a mutant form of bovine beta-lactoglobulin (beta-LG), W19Y, in which Trp(19) was replaced with Tyr, were shown to secrete 6 times more than those producing wild type beta-LG. Northern blot analysis suggested that the enhanced level of secretion was not the result of upregulated transcription of W19Y. The ratio of the amount of W19Y secreted into the supernatant to the amount of W19Y remaining inside the cells was much larger than that in the case of wild type beta-LG as shown by immunoblot analysis. A pulse/chase experiment revealed that the speed of secretion of W19Y was significantly accelerated, compared to wild type beta-LG. These results indicated that W19Y was more efficiently and rapidly transported in the course of secretion than wild type beta-LG. Our previous study showed that the DeltaG of unfolding of W19Y in water is 6.9 kcal/mol smaller than that of wild type beta-LG. Furthermore, immunoblot analysis of intracellular beta-LG under non-reducing conditions indicated that W19Y as well as wild type beta-LG maintained a specific folded structure inside the yeast cells, whereas other non-secretable mutant beta-LGs with Phe or Ala at position 19 (W19F and W19A, respectively) did not. These data suggest that low molecular stability and the maintenance of a specific folded structure inside the yeast cells are prerequisites for efficient and rapid secretion. W19Y was more efficiently secreted than wild type beta-LG also in transformed ern1 mutant yeast cells expressing only a basal level of BiP which is considered to function in quality control in the endoplasmic reticulum (ER) by playing an important role in determining the secretion efficiency of secretory proteins. Thus, the reason for the enhanced secretion of W19Y is considered to be that the improved folding ability of W19Y can allow the half-life of the W19Y-BiP complex to become shorter than that of the wild type beta-LG-BiP complex, leading to faster translocation of W19Y into transport vesicles, or that W19Y can fold in a BiP-independent manner in the ER of the yeast cells. Our findings demonstrate that the amount of protein secreted can be improved by alteration of a single amino acid residue crucial for its structure.

Monoclonal antibodies as probes for monitoring the denaturation process of bovine beta-lactoglobulin.

Five monoclonal antibodies (MAbs) of different idiotypes were produced against bovine beta-lactoglobulin (beta-LG). Among them, MAbs 61B4 and 62A6 reacted preferentially to native beta-LG, while MAbs 21B3 and 31A4 reacted more strongly to the reduced carboxymethylated (denatured) beta-LG than to the native material. These two types of MAb were used to analyze the denaturation process of a beta-LG molecule during heating. The binding affinity of MAbs 21B3 and 31A4 with beta-LG was increased by increasing the heating temperature, the transition temperature being 67-68 degrees C, while that of MAbs 61B4 and 62A6 was reduced by increasing the temperature, this transition temperature being about 80 degrees C. Epitopes recognized by MAbs 31A4 and 61B4 were shown to be included in the segments, Lys8-Trp19 (mostly in the random-coil region) and Thr125-Lys135 (helical region), respectively. The heat-induced conformational change of the beta-LG molecule is, therefore, likely to start in random-coil region as Lys8-Trp19, and to be followed by a structural change in a helical region as Thr125-Lys135. This study demonstrates that MAb is a useful probe to monitor local conformational changes of a protein molecule during denaturation.

Antibodies raised against peptide fragments of bovine alpha s1-casein cross-react with the intact protein only when the peptides contain both B and T cell determinants.

In order to compare the immune response to peptides with that to the native protein, we used bovine alpha s1-casein (alpha s1-CN), a major milk protein whose conformation seems to be in a highly disordered state in solution, as the model antigen. Firstly, the T and B cell determinants on this protein were localized by a T cell proliferation assay and an enzyme-linked immunosorbent assay (ELISA), using 13 synthetic overlapping peptides encompassing the entire sequence of alpha s1-CN. T cells from alpha s1-CN-primed C3H/He mice, a high responder to alpha s1-CN, strongly proliferated in the presence of peptides 91-110, 61-80 and 46-65. In addition, peptides 151-170, 136-155 and 106-125 were also found to contain T cell determinants. On the other hand, peptides 181-199, 46-65, 76-95 and 106-125 were generally found to be immunodominant B cell determinants, while peptides 121-140, 136-155, 91-110 and 151-170 also had antibody-binding activity. The peptides were then tested for their ability to elicit a specific antibody. This revealed that only peptides 91-110, 106-125, 136-155 and 46-65 were able to produce specific antibodies that bound to the native protein as well as the peptides themselves. These peptides contained both B and T cell determinants on the intact protein. Thus, we confirmed that a peptide corresponding to both T and B cell determinants on alpha s1-CN was capable of eliciting a specific antibody that reacted with the protein as well as the peptide itself.

Selective induction of CD8+ T cell functions by single substituted analogs of an antigenic peptide: distinct signals for IL-10 production.

The CD8+ T cell clone 5F1 produces interleukin 10 (IL-10) and interferon gamma(IFN-gamma) in response to stimulation with a peptide corresponding to region 142-149 of bovine alpha(s1)-casein (p142-149). Ninety analog peptides derived from p142-149 with single amino acid substitutions of putative T cell receptor contact residues were prepared to examine whether production of IL-10 and IFN-gamma by 5F1 can be altered by stimulation with these peptides. We found that some peptides triggered only IL-10 production whereas others induced production of IFN-gamma alone or both of these cytokines. Peptides inducing IFN-gamma production triggered both cytotoxicity and a proliferative response, whereas peptides inducing production of IL-10 but not IFN-gamma triggered neither of these responses. Our results clearly demonstrate that the signaling pathway required for IL-10 production in CD8+ T cells differs from that required for IFN-gamma production. The distinct cellular signals for IL-10 production appear to be independent of those for cytotoxicity and the proliferative response of CD8+ T cells.

Primary response of naive CD4(+) T cells to amino acid-substituted analogs of an antigenic peptide can show distinct activation patterns: Th1- and Th2-type cytokine secretion, and helper activity for antibody production without apparent cytokine secretion.

Naive CD4(+) T cells differentiate into two types of helper T cells showing an interferon-gamma-predominant (Th1) or an interleukin-4-predominant (Th2) cytokine secretion profile after repeated antigenic stimulation. Their differentiation can be influenced by slight differences in the interaction between the T cell receptor (TCR) and its ligand at the time of primary activation. However, the primary response of freshly isolated naive CD4(+) T cells to altered TCR ligands is still unclear. Here, we investigated the primary response of splenic naive CD4(+) T cells derived from transgenic mice expressing TCR specific for residues 323-339 of ovalbumin (OVA323-339) bound to I-A(d) molecules. Naive CD4(+) T cells secreted either Th1- or Th2-type cytokines immediately after stimulation with OVA323-339 or its single amino acid-substituted analogs. Helper activity for antibody secretion by co-cultured resting B cells was also found in the primary response, accompanied by either low-level Th2-type cytokine secretion or no apparent cytokine secretion. Our results clearly indicate that dichotomy of the Th1/Th2 cytokine secretion profile can be elicited upon primary activation of naive CD4(+) T cells. We also demonstrate that the helper activity of naive CD4(+) T cells for antibody production does not correspond to the amounts of the relevant cytokines secreted.

Autocrine B-cell stimulation by interleukin-2 during a cognate interaction with T-cells.

Interleukin-2 (IL-2) secretion as well as expression of IL-2 receptor has been demonstrated for B-cells in response to several activating stimuli. However, the exact role of B-cell-derived IL-2 in the T-cell-dependent antibody response remains to be determined. Here, we have examined the autocrine regulatory roles of IL-2 secreted from B-cells. Splenic resting B-cells were stimulated with a fixed pre-activated Th1 clone, G1.19, in the presence of a single amino acid-substituted peptide (pD129A; Ala-129 substituted for Asp-129), an analog of the original ligand (p119-133, derived from bovine beta-lactoglobulin) recognized by G1.19 cells. pD129A allowed a cognate interaction between B-cells and fixed pre-activated G1.19 T-cells, but pD129A had no agonistic activity against G1.19 T-cells. Thus, the level of expression of B-cell-activating molecules on T-cells remained unchanged after stimulation with pD129A. Regardless of the lack of ability to induce IL-2 secretion in the case of T-cells, pD129A significantly enhanced antibody secretion from B-cells, and this was partially blocked by anti-IL-2 antibody. Furthermore, IL-2 secretion from B-cells was modestly upregulated in response to added pD129A. Taken together, these data suggest that helper signals from interacting cognate T-cells induce IL-2 secretion by B-cells, which can enhance antibody secretion in an autocrine manner.

Localization of T-cell determinants on bovine beta-lactoglobulin.

T-cell determinants of bovine beta-lactoglobulin (beta-LG) in BALB/c(H-2d), C57BL/6(H-2b) and C3H/He(H-2k) mice were identified using a set of overlapping synthetic peptides encompassing the entire primary structure of the protein. Lymph node cells from mice immunized with beta-LG were subjected to cell proliferation assay in the presence of these peptides and uptake of 3H-labeled thymidine was measured. Determinant regions were indicated to lie in residues 42-56, 62-76 and 139-153 in BALB/c mice, residues 11-26, 72-86, 100-113 and 119-133 in C57BL/6 mice, residues 72-86, 91-104, 129-143 and 139-153 in C3H/He mice. Some of these fragments included the antigenic motifs predicted by hypotheses according to amphipathicity and sequential patterns of peptides. We reported elsewhere that residues 42-56 and 72-86 represent one of the B-cell antigenic determinants in BALB/c and C3H/He, respectively. These peptides serve as good models of colinear T- and B-cell determinants as they contain both of T- and B-cell determinants.

MHC class II/T-cell receptor interactions potentiate secretion of IgG but not IgM in response to T-dependent antigens.

We have examined whether the interaction of peptide-loaded MHC molecules on the surface of B-cells with antigen-specific T-cell receptors (TCRs) enhances Ig secretion in the presence of other antigen-independent interactions in vitro. B-cells specific for region 25-40 of beta-lactoglobulin (beta-LG) were stimulated in a T-cell dependent manner using plasma membranes (PM) derived from two different T-helper (Th) clones, culture supernatants of activated Th2 cells and beta-LG as a specific antigen. PM were obtained from either the beta-LG-specific T-cell clone H1.1 which can mediate specific TCR/MHC class II interactions as well as antigen-independent ones or from the D10 clone which bears a TCR of an irrelevant specificity and thus, can only mediate antigen-independent interactions. IgG, but not IgM, secretion was specifically enhanced by H1.1 PM, but not D10 PM in the presence of beta-LG. Furthermore, a blockade of TCR/MHC class II interactions using either anti-T-cell receptor, beta or anti-CD4 monoclonal antibodies inhibited this enhanced IgG secretion in response to beta-LG. The results show that while antigen-independent interactions between T- and B-cells can enhance secretion of IgM antibodies, specific interactions between TCRs and peptide:MHC complexes stimulate B-cells to enhance secretion of IgG but not IgM antibodies. This mechanism may contribute to antibody secretion only from B-cells activated through cognate interaction in vivo.

Rapid screening of antigenically reactive fragments of alpha s1-casein using HPLC and ELISA.

Screening of antigenically reactive fragments of alpha S1-casein (alpha S1-CN), the major casein in bovine milk, was done by using HPLC and enzyme-linked immunosorbent assay (ELISA). BALB/c mice (6-week-old) were injected intraperitoneally with alpha S1-CN and complete Freund's adjuvant, and 14 days later, all the mice were boosted with alpha S1-CN and incomplete Freund's adjuvant. Twenty-one days after the 1st immunization, the mice were bled and antiserum was separated. Anti alpha S1-CN antibody fraction was obtained by precipitation from the antiserum with 50% saturated ammonium sulfate. alpha S1-CN was digested with trypsin and chymotrypsin, and 35 peptides were purified from the digests by reversed-phase HPLC with ODS (octadecylsilica) columns. Reactivity of peptides with the antibody were examined by ELISA. The solid phase in the wells of the polystyrene microtiter plate was coated with peptides, and the plate was successively incubated with anti alpha S1-CN antibody, conjugate of anti mouse immunoglobulin with alkaline phosphatase (ALP) and substrate of ALP. Two tryptic fragments (the residues 104-119 and 133-151) and three chymotryptic fragments (33-54, 105-121, and 174-199) were positive in an ELISA test. These five fragments would correspond to four antigenic sites. We could thus find antigenically reactive fragments of alpha S1-CN by the direct and simple detection of specific antigen-antibody interaction.

Monoclonal antibodies against hen's egg ovomucoid.

Two monoclonal antibodies (mAb 23E5 and 32A8) to hen's egg ovomucoid (OM), which causes hen's egg allergy and has trypsin inhibitory activity, were prepared and purified. Their affinity to the three separate domains of the ovomucoid, which are homologous in primary structure and are designated as DI, DII, and DIII, was studied by a competitive radioimmunoassay. MAb 23E5 bound to OM more efficiently than to DI, DII, or DIII-2 (with carbohydrate), but reacted with DIII-1 (free from carbohydrate) more efficiently than with OM. Except for the binding to OM, mAb 32A8 bound to DIII-2 most efficiently and to DIII-1 least efficiently, suggesting that this antibody recognized the carbohydrate moiety of DIII. MAb 32A8 inhibited the trypsin inhibitory activity of OM, whereas mAb 23E5 had no effect on it. These monoclonal antibodies should be useful for analyzing the antigenic determinants and trypsin inhibitory activity of ovomucoid.

Isolation and properties of human kappa-casein.

Human kappa-casein was isolated from human whole casein by gel filtration with Sephadex G-200 and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate (SDS). The kappa-casein was calcium-insensitive and did stabilize human beta-casein and bovine alpha s1-casein against precipitation by calcium ions. Formation of micelles from human beta- and kappa-caseins, and calcium ions was confirmed by electron microscopic observation. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single band was obtained. The formation of para-kappa-caseins by chymosin was confirmed by SDS-PAGE. Two para-kappa-caseins with apparent molecular weights of 13,000 and 11,000 appeared. The molecular weight of intact human kappa-casein was estimated to be approximately 33,000. The human kappa-casein contained about 40% carbohydrate (15% galactose, 3% fucose, 15% hexosamines, and 5% sialic acid) and 0.10% (1 mol/mol) phosphorus. Its amino acid composition was similar to that of bovine kappa-casein except for serine, glutamic acid, and lysine contents.

NMDA receptor mediated Ca2+ responses in neurons differentiated from p53-/- immortalized Murine neural stem cells.

NMDA receptors play important roles in brain formation by taking part in synaptogenesis and apoptosis. In the present study, the expression of NMDA receptors was analyzed in a neural stem cell line, MSP-1, which lacks p53. p53 is a transcription factor involved in excitotoxic neuronal apoptosis. It is quite likely that p53-mediated transcription control affects the expression of NMDA receptors inducing intracellular Ca2+ signaling after neuronal differentiation and is essential for neural development. By means of calcium digital imaging, NMDA receptor-mediated Ca2+ responses were detected from cultured neurons differentiated from neural stem cells which lack p53. This result implies that p53-related apoptosis is not due to NMDA receptor expression.

The direct cloning of the immunoglobulin VH genes from primary cultured B cells specific for a short peptide.

A new and simple method was devised to obtain immunoglobulin VH genes directly from primary cultured B cells specific for a short peptide. Peptide-specific B cells were separated from splenocytes of peptide-immunized BALB/c mice with antigen-coated magnetic beads, and were cloned by a limitedly diluted culture in the presence of lipopolysaccharide, recombinant interleukin (rIL) -2, rIL-4 and rIL-5, and 3T3 fibroblasts as filler cells for 7 days. Seventeen clones were obtained from 3 x 10(3) fractionated cells by screening the positive wells containing anti-peptide antibody-secreting cells by an enzyme-linked immunosorbent assay (ELISA). The VH cDNAs of these clones were amplified by a set of primers; a primer complementary to the mu-chain constant region gene and the other with high complementarity to most of the VH genes. This is the first report of success in obtaining unknown VH genes directly from primary B cell clones, after their antigen-specificity has been confirmed by ELISA. This new method will provide a powerful tool for designing specific recombinant antibodies.

Monoclonal antibody raised against tetrodonic acid, a derivative of tetrodotoxin.

Tetrodonic acid, a relatively non-toxic derivative of tetrodotoxin, was conjugated with bovine serum albumin and injected i.p. to BALB/c mice. After several injections, spleen cells were isolated, fused with myeloma cells X63-Ag8-6.5.3. and cloned by the limiting dilution method. The monoclonal antibody produced in ascites fluid in the mouse by the cloned cell showed an increasing reactivity with tetrodotoxin at concentrations ranging from 0.03 to 100 micrograms per well.

Exopeptidase profiles of bifidobacteria.

The exopeptidase activities of five different strains of bifidobacteria occurring habitually in healthy human intestinal canal were measured on 61 synthetic substrates. The cluster analysis, based on the results, indicates that four strains, with the exception of Bifidobacterium adolescentis a M101-4, have similar exopeptidase profiles. All CFE from these five strains contained at least three kinds of aminopeptidases (aminopeptidase with broad substrate specificity, aminopeptidase hydrolyzing selectively X-Pro type and aminopeptidase hydrolyzing selectively Pro-X type) and carboxypeptidase.