Teruo Ikeda

Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection.

The FimH subunit of type 1-fimbriated Escherichia coli has been implicated as an important determinant of bacterial adherence and colonization of the urinary tract. Here, we sought to localize the functionally important domain(s) within the FimH molecule and to determine if antibodies against this domain would block adherence of type 1-fimbriated E. coli to the bladder mucosa in situ and in vivo in an established mouse model of cystitis. We generated translational fusion proteins of disparate regions of the FimH molecule with an affinity tag MalE, and tested each of the fusion products in vitro for functional activity. The minimum region responsible for binding mouse bladder epithelial cells and a soluble mannoprotein, horseradish peroxidase, was contained within residues 1-100 of the FimH molecule. We validated and extended these findings by demonstrating that antibodies directed at the putative binding region of FimH or at synthetic peptides corresponding to epitopes within the binding domain could specifically block type 1 fimbriae-mediated bacterial adherence to bladder epithelial cells in situ and yeast cells in vitro. Next, we compared the ability of mice passively immunized intraperitoneally with antisera raised against residues 1-25 and 253-264 of FimH or 1-13 of FimA to resist bladder colonization in vivo after intravesicular challenge with type 1-fimbriated E. coli. Only the antibody directed at the putative binding region of FimH (anti- s-FimH1-25) significantly reduced E. coli bladder infections in the experimental mouse model of urinary tract infections. Similar results were obtained when the mice were actively immunized with synthetic peptides corresponding to residues 1-25 and 253-264 of FimH or 1-13 of FimA. The mechanism of protection was attributed, at least in part, to inhibition of bacterial adherence to the bladder surface by s-FimH1-25-specific antibody molecules that had filtered through the kidneys into the urine. The level of FimH antibodies entering the bladder from the circulatory system of the immunized mice was found to be markedly enhanced upon bacterial challenge. The potential broad spectrum activity of the protective FimH antibody was indicated from its serologic cross-reactivity with various urinary tract bacterial isolates bearing type 1 fimbriae. These findings could be relevant in the design of an efficacious and broadly reactive FimH vaccine against urinary tract infections.

Altered function of murine mast cells in response to lipopolysaccharide and peptidoglycan

Toll-like receptors (TLRs) recognize and signal the presence of bacterial components such as lipopolysaccharide (LPS) and peptidoglycan (PG) as a part of innate immunity. Our previous studies revealed that mast cells function as effector cells in the protection of mice against lethal enterobacterial infections. In this study, we examined both the gene expression of molecules involved in TLR signaling and the effects of LPS and PG in bone marrow-derived cultured mast cells (BMCMCs). The mRNA expression of TLR2, TLR4 and TLR6 was detected in BMCMCs. CD14, MD-2 and MyD88, which are also involved in TLR pathway, were also expressed. Neither LPS nor PG affected degranulation in BMCMCs, but release of tumor necrosis factor increased slightly in response to LPS and PG. Both LPS and PG enhanced expression of pro-matrix metalloproteinase 9 (pro-MMP-9) in a dose-dependent manner, and DNA fragmentation was induced by LPS, but not by PG. These results suggest that mast cells are the targets of LPS and PG, and that the functions of these molecules produced exclusively by bacteria partly overlap, but are distinct.

Role of activin A in murine mast cells: modulation of cell growth, differentiation, and migration

Activins, members of the transforming growth factor‐β (TGF‐β) superfamily, are potent growth and differentiation factors. Our previous studies revealed that activin A, a homodimer of inhibin/activin βA, was induced in mast cells and peritoneal macrophages in response to their activation. In the present study, we examined the roles of activin A in murine bone marrow‐derived, cultured mast cell progenitors (BMCMCs), which expressed gene transcripts for molecules involved in activin signaling, suggesting that BMCMCs could be target cells of activin A. Treatment of activin A inhibited 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide uptake into BMCMCs in a dose‐dependent manner. The IC50 concentration was 2.1 nM, which was less potent than 185 pM TGF‐β1. Activin A treatment caused morphological changes toward the differentiated cells at 2 nM and up‐regulated mRNA of mouse mast cell protease‐1 (mMCP‐1), a marker enzyme of mature mucosal mast cells, at 1 nM. Activin A also showed activity in inducing migration of BMCMCs; the optimal concentration for maximal migration was 10 pM, which was much lower than the concentrations to inhibit cell growth and to activate the mMCP‐1 gene. Taking the present results together with our previous results, it is suggested that activin A secreted from activated immune cells recruits mast cell progenitors to sites of inflammation and that with increasing activin A concentration, the progenitors differentiate into mature mast cells. Thus, activin A may positively regulate the functions of mast cells as effector cells of the immune system.

Transcriptional Activation of Mouse Mast Cell Protease-7 by Activin and Transforming Growth Factor-β Is Inhibited by Microphthalmia-associated Transcription Factor

Previous studies have revealed that activin A and transforming growth factor-β1 (TGF-β1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-β1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-β pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-β signaling in a tissue-specific manner.

Degranulation in RBL-2H3 cells: regulation by calmodulin pathway

Involvement of the calmodulin pathway in Ca2+‐induced degranulation was evaluated in RBL‐2H3 mast cells. Pretreatment of RBL‐2H3 cells with a calmodulin antagonist, W‐13, blocked ionomycin‐dependent release of β‐hexosaminidase into the supernatant, although W‐13 treatment alone slightly but significantly increased the release. Ca2+/calmodulin activates various protein kinases and phosphatases including myosin‐light chain kinase (MLCK), calmodulin‐dependent protein kinases (CaMKs), and calcineurin. When RBL‐2H3 cells were pretreated with a MLCK inhibitor, ML‐7, or a CaMKs inhibitor, KN‐93, the ionomycin‐dependent release of β‐hexosaminidase into the supernatant was inhibited. In addition, pretreatment with calcineurin inhibitors, cyclosporin A and FR901725, resulted in blockage of the ionomycin‐dependent release of β‐hexosaminidase into the supernatant. Our results indicate that Ca2+/calmodulin, activated calmodulin, is indispensable for Ca2+‐induced degranulation, and that within the calmodulin pathways, at least MLCK, CaMKs and calcineurin positively regulate the release of granules initiated by increasing cytosolic Ca2+concentrations in RBL‐2H3 cells.

Calcium-regulated expression of activin A in RBL-2H3 mast cells.

The present study examined the regulatory expression of activin A, a potent growth and differentiation factor, in rat basophilic leukemia (RBL-2H3) mast cells. Treatment of RBL-2H3 cells sensitized with anti-dinitrophenyl IgE with multivalent dinitrophenyl led to a clear increase in RT-PCR products of inhibin/activin beta(A). The steady-state mRNA of inhibin/activin beta(A) was also induced by increasing cytosolic Ca(2+) concentration with ionomycin, which required de novo protein synthesis, and was regulated at the transcriptional level. Pretreatment of RBL-2H3 cells with antagonists or inhibitors for the calmodulin pathway blocked ionomycin-dependent inhibin/activin beta(A) transcription and mRNA induction, suggesting the involvement of calmodulin-dependent kinase (CaMK) and calcineurin. The ionomycin-dependent inhibin/activin beta(A) induction was also partially blocked by preincubation with c-Jun NH(2)-terminal kinase (JNK) and p38 kinase inhibitors, but not with MEK1 inhibitor. These results suggest that inhibin/activin beta(A) gene activation is achieved by the JNK and p38 kinase activation through the calmodulin pathway in mast cells.

Detection of feline herpesvirus 1 DNA by the nested polymerase chain reaction.

The thymidine kinase region of feline herpesvirus 1 (FHV 1) genome in ocular/nasal swabs from cats with clinical manifestations of upper respiratory disease was amplified by nested polymerase chain reaction (nested PCR). Two primer pairs were prepared for nested PCR. FHV 1 DNA in ocular/nasal swabs was extracted using instaGene-DNA purification matrix. Nested PCR for the FHV 1 culture supernatants was ten times as sensitive as single PCR. On comparing viral isolation with single PCR and nested PCR for the detection of FHV 1 in ocular/nasal secretions, of 5 samples that yielded infectious virus in cell culture, 3 (60%) were positive in single PCR and 5 (100%) were positive in nested PCR. When 22 ocular/nasal swabs that did not yield FHV 1 were assayed, 3 were negative in both single PCR and nested PCR, 2 were positive in both single and nested PCR and 17 were positive in only nested PCR. Thus, FHV 1 was detected in 19/22 (86.4%) by the nested PCR and in 2/22 (9%) by single PCR. These results show that nested PCR is 4.8 (24 positive samples/5 positive samples) times as sensitive as single PCR.

Plasma (1→3)‐β‐D‐glucan assay and immunohistochemical staining of (1→3)‐β‐D‐glucan in the fungal cell walls using a novel horseshoe crab protein (T‐GBP) that specifically binds to (1→3)‐β‐D‐glucan

A highly sensitive enzyme‐linked immunosorbent assay specific to (1→3)‐β‐D‐glucans (GBP‐ELISA) has been developed using a novel (1→3)‐β‐D‐glucan‐binding protein (T‐GBP), which was purified from the amebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus. This method allowed quantitation of the glucans in a concentration range of 0.1–1,000 ng/ml, regardless of linear and branched structures, and was applied to determine the amounts of (1→3)‐β‐D‐glucan in human and animal plasmas for diagnosis of fungemia. High levels of plasma glucan contents in clinical samples were found to be correlated closely with the severity of fungal infection. T‐GBP was successfully utilized for indirect immunofluorescence staining of (1→3)‐β‐D‐glucan in Candida albicans cell walls. J. Clin. Lab. Anal. 11:104–109.

Bacteria--Mast Cell Interactions in Inflammatory Disease.

Chronic inflammatory diseases of the gastrointestinal tract such as ulcerative colitis and Crohns disease are characterized by mast cell proliferation and secretion of inflammatory mediators. The determinant(s) responsible for stimulating mast cells in the intestinal mucosa is not known. We investigated the interaction of mast cells with type 1 fimbriated Escherichia coli, an opportunistic pathogen and a constituent of the normal indigenous microflora of the gut. Unlike a mutant derivative deficient in the FimH subunit of the fimbriae or nonfimbriated E. coli, type 1 fimbriated E. coli adhered avidly to mast cells. As a consequence of this interaction, the mast cells phagocytozed and killed adherent bacteria. The mast cell bactericidal activity involved generation of superoxide anion and acidification of phagocytic vacuoles. In addition, many of the mast cells had degranulated and released inflammatory mediators such as histamine. These observations have implications both for normal host defense and for the initiation and perpetuation of inappropriate inflammatory responses in the gastrointestinal tract.

Diagnostic Exercise: Tyzzer's Disease, Distemper, and Coccidiosis in a Pup

A 2-month-old mongrel dog had multifocal necrotizing hepatitis, interstitial pneumonia, and hemorrhagic enteritis. Immunohistochemistry detected antigens of Clostridium piliforme in the intestine and liver, and antigens of canine distemper virus within the lung, urinary bladder, brain, spleen, and liver. Furthermore, uncharacterized intralesional coccidian protozoa were observed within the intestine.