The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis

PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. We used the Cre-loxP system to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePten(flox/+) mice displayed enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect was partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePten(flox/+) endothelial cells showed enhanced proliferation/migration. Tie2CrePten(flox/flox) mice died before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and were associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis.     

Depression of delayed-type hypersensitivity in mice with hypothalamic lesion induced by monosodium glutamate: involvement of neuroendocrine system in immunomodulation.

Delayed-type hypersensitivity (DTH) was depressed in mice that had been treated with monosodium glutamate (MSG) in their suckling period. Analysis of the DTH depression by use of the macrophage migration inhibition assay showed dysfunction of DTH effector T cells. The neuronal loss of nuclei in the hypothalamus, which elaborates the corticotropin-releasing factor and the hypersecretion of adrenocorticotrophic hormone, was observed in the MSG-treated mice. Therefore, DTH response may be modulated by the neuroendocrine system.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Administration of superantigens protects mice from lethal Listeria monocytogenes infection by enhancing cytotoxic T cells

Superantigens stimulate T-cell-receptor Vbeta-selective T-cell proliferation accompanying the release of cytokines, which may eventually protect the host from microbial infections. We investigated here whether superantigens can rescue the host from lethal bacterial infection. Mice were pretreated with Staphylococcus aureus enterotoxin B (SEB) 1 and 2 days before bacterial infection, and the mortality of infected mice was assessed. SEB pretreatment protected mice from lethal infection with Listeria monocytogenes but not from lethal infection with Streptococcus pyogenes. This enhanced protection was also observed upon pretreatment with recombinant streptococcal pyrogenic exotoxin A. Furthermore, L. monocytogenes-specific delayed-type hypersensitivity (DTH) due to type 1 helper T (Th1) cells and the cytotoxicity of CD8(+) T cells were significantly enhanced after SEB administration and bacterial infection. Depletion of either CD4(+) T cells or CD8(+) T cells in SEB-pretreated mice completely abolished this protection. This phenomenon was ascribed to the elimination of L. monocytogenes-specific CD8(+) cytotoxic T lymphocytes (CTL). It was found that CD4(+) T cells contributed to the induction of the CTL populations. Furthermore, SEB pretreatment of heat-killed L. monocytogenes-immunized mice enhanced the protection from challenge of L. monocytogenes. Taken together, these results indicated that administrations of superantigens protected mice from infection with L. monocytogenes, which was dependent on the enhanced L. monocytogenes-specific CTL activity in the presence of CD4(+) T cells, and superantigens exhibited adjuvant activity in the immunization against intracellular pathogens.     

Mucosal and systemic immune responses in BALB/c mice to Bacteroides gingivalis fimbriae administered orally

A 41,000-molecular-weight fimbrial protein was isolated from freshly cultivated whole cells of Bacteroides gingivalis 381 and purified chromatographically. Salivary and serum antibody responses to the fimbriae, which had been orally administered in the presence of an acyl derivative of muramylpeptides, i.e., either N2-[(N-acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine [MDP-Lys(L18)] or sodium beta-N-acetyl-glucosaminyl-(1----4)-N-acetylmuramyl-L-alanyl-D-isoglu tam inyl- (L)-stearoyl-(D)-meso-2,6-diaminopimelic acid-(D)-amine-D-alanine (GM-53), or in the absence of adjuvant, were examined in BALB/c mice when administered by gastric intubation on days 0 and 1 as primary immunizations and on days 27 and 28 as booster immunizations. Gastric intubation of the fimbriae with an adjuvant significantly enhanced the production of anti-fimbria immunoglobulin A (IgA) in saliva. Subcutaneous injection of fimbriae along with an adjuvant also raised anti-fimbria IgA levels, as well as IgG levels, in saliva. Both immunization procedures enhanced the levels of anti-fimbria IgG, IgA, and IgM in serum, and the major class of fimbria-specific antibody was IgG, followed by IgA and IgM. However, subcutaneous injection was more effective than gastric intubation to enhance the production of serum antibody in mice. The subclasses of IgG antibody specific for fimbriae in serum were mainly IgG1, followed by IgG2a, IgG2b, and IgG3. These results demonstrated that the combined use of B. gingivalis fimbrial antigen and either GM-53 or MDP-Lys(L18) resulted in a sharply increased IgA antibody response in saliva and a predominantly stimulated IgG antibody response in serum, respectively. Both antibodies were found to be specific for the fimbriae used for immunization.     

Distribution of Porphyromonas gingivalis strains with fimA genotypes in periodontitis patients

Fimbriae (FimA) of Porphyromonas gingivalis are filamentous components on the cell surface and are thought to play an important role in the colonization and invasion of periodontal tissues. We previously demonstrated that fimA can be classified into four variants (types I to IV) on the basis of the nucleotide sequences of the fimA gene. In the present study, we attempted to detect the four different fimA genes in saliva and plaque samples isolated from patients with periodontitis using the PCR method. Four sets of fimA type-specific primers were designed for the PCR assay. These primers selectively amplified 392-bp (type I), 257-bp (type II), 247-bp (type III), and 251-bp (type IV) DNA fragments of the fimA gene. Positive PCR results were observed with reference strains of P. gingivalis in a type-specific manner. All other laboratory strains of oral and nonoral bacteria gave negative results. The sensitivity of the PCR assay for fimA type-specific detection was between 5 and 50 cells of P. gingivalis. Clinical samples were obtained from saliva and subgingival plaque from deep pockets (>/=4 mm) of 93 patients with periodontitis. Bacterial genomic DNA was isolated from the samples, and the targeted fragments were amplified by PCR. The presence of P. gingivalis was demonstrated in 73 patients (78.5%), and a single fimA gene was detected in most patients. The distribution of the four fimA types among the P. gingivalis-positive patients was as follows: type I, 5.4%; type II, 58.9%; type III, 6. 8%; type IV, 12.3%; types I and II, 6.8%; types II and IV, 2.7%; and untypeable, 6.8%. P. gingivalis with type II fimA was detected more frequently in the deeper pockets, and a significant difference of the occurrence was observed between shallow (4 mm) and deep (>/=8 mm) pockets. These results suggest that P. gingivalis strains that possess type II fimA are significantly more predominant in periodontitis patients, and we speculate that these organisms are involved in the destructive progression of periodontal diseases.     

Construction and characterization of a fimA mutant of Porphyromonas gingivalis.

Although fimbriae of Porphyromonas gingivalis have been implicated as playing a major role in adherence to gingival tissue surfaces, no conclusive genetic evidence has yet been obtained. The fimA gene, the determinant for the major fimbrial subunit protein, was cloned and sequenced (D. P. Dickinson, M. A. Kubiniec, F. Yoshimura, and R. J. Genco, J. Bacteriol. 170:1658-1665, 1988). We undertook to inactivate the fimA gene by a homologous recombination technique and examined the fimA mutant for changes in surface properties, including production of fimbriae, adherence to human gingival fibroblasts and epithelial cells, hemagglutinating activity, and surface hydrophobicity. To inactivate the fimA gene, we disrupted a fimA clone by insertion of a DNA segment containing an erythromycin resistance (Emr) gene. This was then delivered into P. gingivalis ATCC 33277 from an Escherichia coli K-12 strain, SM10 lambda pir, by using a mobilizable suicide vector, pGP704; recombination at the fimA locus led to the isolation of a fimA mutant. Disruption of the fimA locus and disappearance of FimA production were confirmed by Southern hybridization with a fimA-specific DNA probe and Western immunoblotting with a monoclonal antibody against the FimA protein, respectively. The fimA mutant constructed failed to express long (0.5- to 1.0-micron) fimbriae from the bacterial surface and had a diminished adhesive capacity to tissue-cultured human gingival fibroblasts and epithelial cells. Observation of the bacteria adhering to human gingival fibroblasts by scanning electron microscopy revealed that the wild-type strain had dramatic local changes in the appearance of the microvilli at the point of contact with large bacterial clumps, whereas the fimA mutant did not. In contrast, neither the hemagglutinating activity nor the surface hydrophobicity was changed in the fimA mutant. These data thus constitute the first direct genetic evidence demonstrating that the FimA protein of P. gingivalis is essential for the interaction of the organism with human gingival tissue cells through a function(s) encoded by the fimA gene.     

Biochemical and immunobiological properties of lipopolysaccharide (LPS) from Bacteroides gingivalis and comparison with LPS from Escherichia coli.

Lipopolysaccharides (LPSs) were isolated from Bacteroides gingivalis and Escherichia coli by the phenol-water and butanol-water procedures. The phenol-water-extracted LPS from B. gingivalis 381 was composed of 46% carbohydrate, 23% hexosamine, 18% fatty acid, and 5% protein. The major component sugars of this preparation were glucose, glucosamine, rhamnose, galactose, galactosamine, and mannose, and their molecular ratio was 1:0.9:0.7:0.6:0.6:0.4, respectively. Neither heptose nor 2-keto-3-deoxyoctonate was detected. The butanol-water-extracted LPS from this strain was composed of 76% glucose, 7% fatty acid, and 13% protein, and it was associated with a number of polypeptides (13 to 56 kilodaltons). The main fatty acid of both LPS preparations was palmitic acid. It was found that biological activities of LPS from B. gingivalis were comparable to those of LPS from E. coli in terms of activation of the clotting enzyme of Limulus amebocyte lysate, mitogenicity, polyclonal B cell activation, and stimulation of interleukin 1 production in BALB/c mice. Furthermore, LPS-nonresponsive C3H/HeJ spleen cells were found to yield good mitogenic responses to both phenol-water-extracted LPS and butanol-water-extracted LPS from B. gingivalis or butanol-water-extracted LPS from E. coli. On the other hand, spleen cells from LPS-responsive C3H/HeN mice responded well to all these LPS preparations.     

Cysteine protease of Porphyromonas gingivalis 381 enhances binding of fimbriae to cultured human fibroblasts and matrix proteins.

It has been shown that Porphyromonas gingivalis 381, a suspected periodontopathogen, possesses fimbriae on its cell surface. The organism is also known to produce proteases which can degrade the host cell surface matrix proteins. In this study, we investigated the effect of protease on the binding of the purified P. gingivalis fimbriae to cultured fibroblasts or matrix proteins. A protease that can hydrolyze benzoyl-L-arginine p-nitroanilide was obtained from P. gingivalis 381 cells by sonication in phosphate-buffered 0.2% Triton X-100 and was purified by column chromatography. The molecular size of the protease was estimated to be 55 kDa by gel filtration or 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity was markedly inhibited by sulfhydryl reagents, antipain, and leupeptin. The protease degraded various host proteins, including collagen and fibronectin, and cleaved the COOH terminus of the arginine residue in peptides such as benzoyl-L-arginine p-nitroanilide. However, P. gingivalis fimbriae were not degraded by protease activity. The enzyme activity was enhanced in the presence of reducing agents or CaCl2. When cultured fibroblasts were partially treated with the protease, the binding of the purified P. gingivalis fimbriae to the fibroblast monolayer was increased significantly. However, this enhancing effect was suppressed upon the addition of antipain and leupeptin. Similarly, binding of the fimbriae to the collagen or fibronectin immobilized on the microtiter wells was also enhanced. Addition of these host matrix proteins efficiently inhibited the binding of fimbriae to the fibroblast monolayer. The binding assay of fimbriae using dipeptidyl ligand affinity column chromatography demonstrated a clear interaction between fimbriae and the arginine residue. Taken together, these results indicate that the P. gingivalis protease at least partially degrades the host matrix proteins, which, in turn, may lead to an increased exposure of the cryptic ligands that can result in enhanced fimbria-mediated binding of this organism to periodontal tissues.