Yoshikazu Yuki

Identification of low‐abundance proteins of bovine colostral and mature milk using two‐dimensional electrophoresis followed by microsequencing and mass spectrometry

We identified several low‐abundance proteins of bovine colostrum and mature milk using the immunoabsorption technique and two‐dimensional electrophoresis (2‐DE) followed by microsequencing and mass spectrometry. Two major milk proteins, β‐casein and immunoglobulin G (IgG), were effectively removed from the milk using immunoabsorbents. Milk samples before and after immunoabsorption were separated by 2‐DE. Protein identification of the spots on 2‐DE was performed by either gel comparison, microsequencing, matrix‐assisted laser desorption/ionization‐time of flight mass‐spectrometry (MALDI‐TOF‐MS), peptide mass fingerprinting or peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight‐MS (Q‐TOF). Significant differences in protein patterns were observed between the low‐abundance proteins of colostrum and mature milk. In addition, several low‐abundance proteins including fibrinogen β‐chain, chitinase 3‐like 1, α‐antitrypsin, complement C3 α‐chain, gelsolin and apolipoprotein H were observed only in colostrum. However, the level of β‐casein fragments increased significantly during this lactation period. α‐Lactalbumin and β‐lactoglobulin as well as some low‐abundance proteins including bovine serum albumin, serotransferrin and lactoferrin were identified in both colostral and mature milk. Low‐abundance proteins in bovine colostrum may have special physiologic relevance to the health and development of calves early in lactation.

Transcription factor Spi-B–dependent and –independent pathways for the development of Peyer’s patch M cells

Although many of the biological features of microfold cells (M cells) have been known for many years, the molecular mechanisms of M-cell development and antigen recognition have remained unclear. Here, we report that Umod is a novel M-cell–specific gene, the translation products of which might contribute to the uptake function of M cells. Transcription factor Spi-B was also specifically expressed in M cells among non-hematopoietic lineages. Spi-B–deficient mice showed reduced expression of most, but not all, other M-cell–specific genes and M-cell surface markers. Whereas uptake of Salmonella Typhimurium via M cells was obviously reduced in Spi-B–deficient mice, the abundance of intratissue cohabiting bacteria was comparable between wild-type and Spi-B–deficient mice. These data indicate that there is a small M-cell population with developmental regulation that is Spi-B independent; however, Spi-B is probably a candidate master regulator of M-cell functional maturation and development by another pathway.

Nasal immunization with E. coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.

Nasal administration of cholera toxin (CT) suppresses clinical signs of experimental autoimmune encephalomyelitis (EAE).

Cholera toxin (CT), a major enterotoxin produced by Vibrio cholerae, elicits mucosal adjuvant activities by inducing antigen-specific CD4+ T cells secreting T helper type 2 (Th2) cytokines. Experimental autoimmune encephalomyelitis (EAE) is induced by Th1 cells specific for myelin-derived antigens. We induced EAE in C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG) 35-55 and CT was nasally administered as an immunomodulator on day 7 following MOG challenge. Clinical severity in the CT-treated mice was milder when compared to PBS-treated mice, while the levels of expression of interleukin (IL)-12 and interferon (IFN)-gamma in the central nervous system (CNS) of CT-treated mice were lower than PBS-treated mice. Thus, nasal administration of the mucosal immunomodulator CT ameliorated the severity of EAE, which was associated with the suppression of Th1 cell responses.

Critical role of TSLP-responsive mucosal dendritic cells in the induction of nasal antigen-specific IgA response

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine involved in T helper 2 type immune responses. The primary target of TSLP is myeloid dendritic cells (DCs), however, little is known about the mechanism by which TSLP elicits respiratory IgA immune responses upon mucosal immunization. Here, we found that the levels of TSLP and TSLPR were upregulated in the mucosal DCs of mice nasally immunized with pneumococcal surface protein A (PspA) plus cholera toxin (CT) compared with those immunized with PspA alone. PspA-specific IgA responses, but not IgG Ab responses were significantly reduced in both serum and mucosal secretions of TSLPR knockout mice compared with wild-type mice after nasal immunization with PspA plus CT. Furthermore, CD11c+ mucosal DCs isolated from TSLPR knockout mice nasally immunized with PspA plus CT were less activated and exhibited markedly reduced expression of IgA-enhancing cytokines (e.g., APRIL, BAFF, and IL-6) compared with those from equivalently immunized wild-type mice. Finally, exogenous TSLP promoted production of IgAs in an in vitro DC–B cell co-culture system as exhibited by enhanced IL-6 production. These results suggest that TSLP–TSLPR signaling is pivotal in the induction of nasal respiratory immunity against pathogenic pneumococcal infection.