Purification and high-resolution top-down mass spectrometric characterization of human salivary α-amylase.

Human salivary α-amylase (HSAMY) is a major component of salivary secretions, possessing multiple important biological functions. Here we have established three methods to purify HSAMY in human saliva for comprehensive characterization of HSAMY by high-resolution top-down mass spectrometry (MS). Among the three purification methods, the affinity method based on the enzyme-substrate specific interaction between amylase and glycogen is preferred, providing the highest purity HSAMY with high reproducibility. Subsequently, we employed Fourier transform ion cyclotron resonance MS to analyze the purified HSAMY. The predominant form of α-amylase purified from saliva of various races and genders is nonglycosylated with the same molecular weight of 55,881.2, which is 1885.8 lower than the calculated value based on the DNA-predicted sequence. High-resolution MS revealed the truncation of the first 15 N-terminal amino acids (-1858.96) and the subsequent formation of pyroglutamic acid at the new N-terminus Gln (-17.03). More importantly, five disulfide bonds in HSAMY were identified (-10.08) and effectively localized by tandem MS in conjunction with complete and partial reduction by tris (2-carboxyethyl) phosphine. Overall, this study demonstrates that top-down MS combined with affinity purification and partial reduction is a powerful method for rapid purification and complete characterization of large proteins with complex and overlapping disulfide bond patterns.

Immunomodulatory effect of halophilic lactic acid bacterium Tetragenococcus halophilus Th221 from soy sauce moromi grown in high-salt medium.

A halophilic lactic acid bacterium, Tetragenococcus halophilus, was found to possess an immunomodulatory activity that promotes T helper type 1 (Th1) immunity in addition to its important roles in soy sauce brewing. Strain Th221 was selected from 151 strains isolated from soy sauce (shoyu) moromi, since it induced strong interleukin (IL)-12 production by mouse peritoneal macrophages in vitro. The relationship between the salt concentration in the medium and the IL-12 production-inducing activity of this strain was investigated, and the activity was found to be strong when the bacteria were grown in medium containing > or =10% (w/v) salt. The Th1-promoting activity was also manifested in an in vivo mouse study, since Th1-dependant contact sensitivity was augmented and Th2 immunity, as evaluated by specific immunoglobulin E production, was suppressed following oral ingestion of Th221. Based on these findings, Th221 administration may be useful for improving allergic symptoms.

Soy isoflavone aglycone modulates expression of cell surface antigens in vitro and in vivo.

Soy isoflavone aglycones (IFAs) have a wide range of biological actions. We investigated in this study the effect of IFAs on myeloid cells. The cell surface expression of both CD80 and CD86 was up-regulated by treating myeloid cells with IFAs in vitro and in vivo. The findings suggest that IFAs could modulate the myeloid cell function.

Airway hyper-reactivity mediated by B-1 cell immunoglobulin M antibody generating complement C5a at 1 day post-immunization in a murine hapten model of non-atopic asthma.

Contact skin immunization of mice with reactive hapten antigen and subsequent airway challenge with the same hapten induces immediate airflow obstruction and subsequent airway hyper-reactivity (AHR) to methacholine challenge, which is dependent on B cells but not on T cells. This responsiveness to airway challenge with antigen is elicited as early as 1 day postimmunization and can be adoptively transferred to naïve recipients via 1-day immune cells. Responses are absent in 1-day immune B-cell-deficient JH(-/-) mice and B-1 B-cell-deficient xid male mice, as well as in recipients of 1-day immune cells depleted of cells with the B-1 cell phenotype (CD19(+) B220(+) CD5(+)). As B-1 cells produce immunoglobulin M (IgM), we sought and found significantly increased numbers of anti-hapten IgM-producing cells in the spleen and lymph nodes of 1-day immune wild-type mice, but not in xid mice. Then, we passively immunized naive mice with anti-hapten IgM monoclonal antibody and, following airway hapten challenge of the recipients, we showed both immediate airflow obstruction and AHR. In addition, AHR was absent in complement C5 and C5a receptor-deficient mice. In summary, this study of the very early elicited phase of a hapten asthma model suggests, for the first time, a role of B-1 cells in producing IgM to activate complement to rapidly mediate asthma airway reactivity only 1 day after immunization.

Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-gamma via CXCR3 chemokines.

We investigated the role of T helper (Th)1- and Th2-type cytokines in delayed-type hypersensitivity to soluble protein antigens elicited early postimmunization. Mice were sensitized by intradermal injection without adjuvants, or subcutaneously with complete Freund's adjuvant, and subsequently ear challenged intradermally. As soon as day 3, antigen-specific eosinophil-rich responses were elicited in wild-type mice, but not in T-cell receptor-alpha-/- mice without adjuvant. Draining lymph node T cells stimulated with antigen secreted interleukin (IL)-4, IL-5 and interferon-gamma (IFN-gamma). IFN-gamma-dependent specific immunoglobulin G (IgG)2a and IL-4-dependent IgG1 were also generated. Delayed-type hypersensitivity ear swelling and local eosinophil recruitment were decreased in IL-5-/-, IL-4-/- and signal transducer and activator of transcription-6 (STAT-6)-/- mice, and with anti-IL-4 treatment of wild-type mice, suggesting Th2 mechanisms. Interestingly, responses were also decreased in IFN-gamma-/- mice, and IFN-gamma protein and the IFN-gamma-inducible CXC chemokine, IP-10, were present in 24-hr ear tissue extracts, suggesting Th1 effects. Finally, ear swelling, total histology and eosinophils were decreased in mice deficient in CXCR3, the chemokine receptor for IP-10. These results suggest that both a Th2-like (IL-5, IL-4 and STAT-6) and a Th1-like (IFN-gamma, IP-10, CXCR3) pathway contribute to eosinophil recruitment in early delayed-type hypersensitivity.

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity.

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5(+), CD19(+), CD22(+), B220(+), Thy1(+), and Mac1(+), suggesting that they are B-1 B cells. DTH initiation is absent in micro MT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.

B cell-dependent T cell responses: IgM antibodies are required to elicit contact sensitivity.

Contact sensitivity (CS) is a classic example of in vivo T cell immunity in which skin sensitization with reactive hapten leads to immunized T cells, which are then recruited locally to mediate antigen-specific inflammation after subsequent skin challenge. We have previously shown that T cell recruitment in CS is triggered by local activation of complement, which generates C5a that triggers C5a receptors most likely on mast cells. Here, we show that B-1 cell-derived antihapten IgM antibodies generated within 1 day (d) of immunization combine with local challenge antigen to activate complement to recruit the T cells. These findings overturn three widely accepted immune response paradigms by showing that (a) specific IgM antibodies are required to initiate CS, which is a classical model of T cell immunity thought exclusively due to T cells, (b) CS priming induces production of specific IgM antibodies within 1 d, although primary antibody responses typically begin by day 4, and (c) B-1 cells produce the 1-d IgM response to CS priming, although these cells generally are thought to be nonresponsive to antigenic stimulation. Coupled with previous evidence, our findings indicate that the elicitation of CS is initiated by rapidly formed IgM antibodies. The IgM and challenge antigen likely form local complexes that activate complement, generating C5a, leading to local vascular activation to recruit the antigen-primed effector T cells that mediate the CS response.

Subunits of IgM reconstitute defective contact sensitivity in B-1 cell-deficient xid mice: kappa light chains recruit T cells independent of complement.

The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP kappa L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while kappa L chains act via another C-independent but mast cell-dependent pathway.

A simple method for enrichment of uninucleate conidia of Aspergillus oryzae.

Aspergillus oryzae produces multinucleate conidia, which makes the obtaining of homokaryons labor-intensive. Analysis of conidia by flow cytometry clarified the relationship that conidia of lower nuclear number were smaller in size. Based on this, we have developed a simple way to enrich uninucleate conidia with a membrane filter. Our results also suggest that the method is useful for elimination of heterokaryons.