Akio Ametani

Lactobacillus casei Inhibits Antigen-Induced IgE Secretion through Regulation of Cytokine Production in Murine Splenocyte Cultures

Background:Lactobacillus casei is a nonpathogenic gram-positive bacterium widely used in dairy products and has been shown to enhance the cellular immunity of the host. Methods: To examine the inhibitory effect of L. casei on IgE production, splenocytes obtained from ovalbumin (OVA)-primed BALB/c mice were restimulated in vitro with the same antigen in the presence of heat-killed L. casei. The effect of this bacterium on T helper (Th) phenotype development was also examined with naive T cells from OVA-specific T cell receptor-transgenic mice. Results:L. casei induced IFN-γ, but inhibited IL-4 and IL-5 secretion, and markedly suppressed total and antigen-specific IgE secretion by OVA-stimulated splenocytes. The inhibitory effect of L. casei on IgE, IL-4, and IL-5 production was partially abrogated by addition of neutralizing antibody to IFN-γ. Augmented IL-12 production was also observed in the cell cultures containing L. casei, and anti-IL-12 monoclonal antibody completely restored the IgE, IL-4, and IL-5 production to the control levels. The IL-12 augmentation by L. casei was macrophage-dependent. The Th cell development assay showed the ability of L. casei to induce Th1 development preferentially. This effect was also completely blocked by anti-IL-12 antibody. Conclusions: This is the first demonstration that a nonpathogenic microorganism, L. casei, can inhibit antigen-induced IgE production through induction of IL-12 secretion by macrophages. The findings suggest a potential use of this organism in preventing IgE-mediated allergy.

Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin.

In an effort to demonstrate the potential usefulness of Bacillus subtilis (natto) as a probiotic, we examined the effect of this organism on the growth of three strains of lactobacilli co-cultured aerobically in vitro. Addition of B. subtilis (natto) to the culture medium resulted in an increase in the number of viable cells of all lactobacilli tested. Since B. subtilis (natto) can produce catalase, which has been reported to exhibit a similar growth-promoting effect on lactobacilli, we also examined the effect of bovine catalase on the growth of Lactobacillus reuteri JCM 1112 and L. acidophilus JCM 1132. Both catalase and B. subtilis (natto) enhanced the growth of L. reuteri JCM 1112, whereas B. subtilis (natto) but not catalase enhanced the growth of L. acidophilus JCM 1132. In a medium containing 0.1 mM hydrogen peroxide, its toxic effect on L. reuteri JCM 1112 was abolished by catalase or B. subtilis (natto). In addition, a serine protease from B. licheniformis, subtilisin, improved the growth and viability of L. reuteri JCM 1112 and L. acidophilus JCM 1132 in the absence of hydrogen peroxide. These results indicate that B. subtilis (natto) enhances the growth and (or) viability of lactobacilli, possibly through production of catalase and subtilisin.

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

Naive CD4+ T Cells Exhibit Distinct Expression Patterns of Cytokines and Cell Surface Molecules on Their Primary Responses to Varying Doses of Antigen

The amount of an Ag used for stimulation affects the type and magnitude of T cell responses. In this study we have investigated the primary response of naive CD4+ T cells derived from OVA-specific TCR-transgenic mice (OVA23-3) upon stimulation with varying doses of the antigenic peptide, OVA323–339. IL-4 expression was maximal with 50 nM Ag and decreased significantly with increasing doses. In contrast, IFN-γ expression, which was also detected at 50 nM Ag, increased with increasing doses. The expression patterns of mRNA for the Th2-specific transcription factors GATA-3 and c-Maf were parallel to that of IL-4. These expression profiles were not altered by the addition of anti-IL-4 plus anti-IL-12 mAbs, suggesting that cytokine receptor signaling is not essential. Naive CD4+ T cells stimulated with 5 nM Ag elicited IgM secretion from cocultured B cells, whereas those stimulated with 50 nM Ag or more elicited apoptosis of B cells. This may be because at lower doses of Ag (5 nM), naive CD4+ T cells express CD40 ligand and OX40, whereas at higher doses (50 nM), they express Fas ligand. Clearly, the expression of each type of molecule depends on the Ag dose, and different molecules had different expression patterns. Thus, in the primary response, naive CD4+ T cells can exhibit different functions depending on the dose of Ag.

Cutting Edge: Introduction of an Endopeptidase Cleavage Motif into a Determinant Flanking Region of Hen Egg Lysozyme Results in Enhanced T Cell Determinant Display

The choice of which determinants of a whole Ag will be presented on cell surface MHC class II molecules after uptake and processing by APC is the result of the interplay between structural characteristics of the Ag and the processing machinery of the APC. In this study, we demonstrate that introduction of a dibasic motif adjacent to a subdominant determinant enhances the presentation of this determinant from the whole molecule. This is the first report showing that a single amino acid substitution in a whole Ag, designed to introduce an endopeptidase recognition site, enhances display of class II-restricted determinants, most likely by creating a peptide chain cleavage in the antigenic molecule. Our findings have important implications for the understanding of immunodominance and for vaccine design.

Determinant analysis of IgE and IgG4 antibodies and T cells specific for bovine αs1-casein from the same patients allergic to cow's milk: Existence of αs1-casein–specific B cells and T cells characteristic in cow's-milk allergy

In an effort to clarify the etiology of milk allergy from the standpoint of allergen-specific immune reactions, we investigated the determinants of IgE, IgG4, and T cells specific for bovine alpha(s)1-casein from the same individual patients by using its synthetic peptides and cyanogen bromide-digested fragments. Alpha(s)1-casein is a major allergen in cows milk, and its unique conformation enabled us to investigate the determinants of antibodies without consideration about missing the reactivities because of conformational changes. Nine patients were selected as subjects from among 129 milk-sensitive infants screened by ELISA to assess the anti-alpha(s)1-casein IgE levels in their sera. By using ELISA for epitope mapping, a C-terminal region of alpha(s)1-casein was identified as a common binding site for IgE from all of these patients, whereas those for anti-alpha(s)1-casein IgG4 were located in multiple regions of alpha(s)1-casein. We determined the specificities of seven alpha(s)1-casein-specific T-cell lines established from peripheral blood mononuclear cells of two of the patients. These T cells have been shown to secrete IL-4. All of the T-cell lines had different specificities to alpha(s)1-casein. However, a common amino acid residue use was found among the determinants of various T-cell lines from each patient. The results suggest that patients allergic to cows milk have characteristic B cells recognizing a limited region of alpha(s)1-casein and secreting alpha(s)1-casein-specific IgE. These B cells may interact particularly with T cells recognizing determinants with a common structure.

High-Resolution Analysis of the 5′-End Transcriptome Using a Next Generation DNA Sequencer

Massively parallel, tag-based sequencing systems, such as the SOLiD system, hold the promise of revolutionizing the study of whole genome gene expression due to the number of data points that can be generated in a simple and cost-effective manner. We describe the development of a 5′–end transcriptome workflow for the SOLiD system and demonstrate the advantages in sensitivity and dynamic range offered by this tag-based application over traditional approaches for the study of whole genome gene expression. 5′-end transcriptome analysis was used to study whole genome gene expression within a colon cancer cell line, HT-29, treated with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5Aza). More than 20 million 25-base 5′-end tags were obtained from untreated and 5Aza-treated cells and matched to sequences within the human genome. Seventy three percent of the mapped unique tags were associated with RefSeq cDNA sequences, corresponding to approximately 14,000 different protein-coding genes in this single cell type. The level of expression of these genes ranged from 0.02 to 4,704 transcripts per cell. The sensitivity of a single sequence run of the SOLiD platform was 100–1,000 fold greater than that observed from 5′end SAGE data generated from the analysis of 70,000 tags obtained by Sanger sequencing. The high-resolution 5′end gene expression profiling presented in this study will not only provide novel insight into the transcriptional machinery but should also serve as a basis for a better understanding of cell biology.

Tryptophan-19 of β-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.

Autoreactive T cells can be protected from tolerance induction through competition by flanking determinants for access to class II MHC

It is not clear why the N-terminal autoantigenic determinant of myelin basic protein (MBP), Ac1–9, is dominant in the B1O.PL (H-2u) mouse, given its weak I-Au-MHC binding affinity. Similarly, how do high-affinity T cells specific for this determinant avoid negative selection? Because the MBP:1–9 sequence is embryonically expressed uniquely in the context of Golli-MBP, determinants were sought within the contiguous N-terminal “Golli” region that could out-compete MBP:1–9 for MHC binding, and thereby prevent negative selection of the public response to Ac1–9, shown here to be comprised of a Vβ8.2Jβ2.7 and a Vβ8.2Jβ2.4 expansion. Specifically, we demonstrate that Ac1–9 itself can be an effective inducer of central tolerance induction; however, in the context of Golli-MBP, Ac1–9 is flanked by determinants which prevent its display to autoreactive T cells. Our data support competitive capture as a means of protecting high-affinity, autoreactive T cells from central tolerance induction.

The topography of αs1-casein adsorbed to an oil/water interface: an analytical approach using proteolysis

Abstract An α s1 -casein solution and lipid were mixed and homogenized so that an oil/water emulsion was prepared. The topography of an α s1 -casein chain adsorbed to oil droplets in the emulsion was analyzed. Trypsin and α-chymotrypsin were allowed to act on α s1 -casein in the emulsion (the emulsion system) and that dissovled in an aqueous solution (the solution system). The digests were separated by reversed-phase high performance liquid chromatography (reversed-phase HPLC), and subjected to amino acid analyses. The peaks of the HPLC patterns were assigned to peptides in the total sequence of α s1 -casein. Cleavage of thirteen peptide bonds in an α s1 -casein chain in the emulsion system was difficult, as compared with those in the solution system. The results suggest that these peptide bonds were among definite regions inaccessible to proteinases, regions which could be some definite adsorption sites of α s1 -casein to oil. A preliminary model is proposed for the structure of α s1 -casein at an oil/water interface, and it is shown that some hydrophobic regions of an α s1 chain. The neighborhood of the residues 21–25, 94, 135 and 142 from the N-terminal, might interact strongly with oil, while the other hydrophobic region, the neighborhood of the residue 165, might not.