Nasal vaccination with the 40-kilodalton outer membrane protein of Porphyromonas gingivalis and a nontoxic chimeric enterotoxin adjuvant induces long-term protective immunity with reduced levels of immunoglobulin E antibodies

In this study, we demonstrated that the 40-kDa outer membrane protein of Porphyromonas gingivalis (40-kDa OMP) nasally administered with a nontoxic chimeric adjuvant that combines the A subunit of mutant cholera toxin E112K with the pentameric B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli (mCTA/LTB) elicited a long-term protective immune response. Immunization with the 40-kDa OMP and mCTA/LTB induced high levels of 40-kDa-OMP-specific immunoglobulin G (IgG) and IgA antibodies (Abs) in sera and elicited a significant IgA anti-40-kDa OMP Ab response in saliva. These Ab responses were maintained for at least 1 year after the immunization. Although using adjuvant mCTA/LTB gave Ab responses in the saliva comparable to those obtained using native cholera toxin (nCT) as the adjuvant, the levels of total IgE and 40-kDa-OMP-specific IgE Abs as well as interleukin-4 levels induced by the immunization with mCTA/LTB were lower than those induced by the immunization with nCT. Importantly, IgG Abs generated by nasal immunization with the 40-kDa OMP plus mCTA/LTB inhibited the coaggregation and hemagglutinin activities of P. gingivalis. Furthermore, the mice given nasal 40-kDa OMP plus mCTA/LTB showed a significant reduction of alveolar bone loss caused by oral infection with P. gingivalis even 1 year after the immunization compared to the loss in unimmunized mice. Because mCTA/LTB is nontoxic, nasally administered 40-kDa OMP together with mCTA/LTB should be an effective and safe mucosal vaccine against P. gingivalis infection in humans and may be an important tool for the prevention of chronic periodontitis.     

A human monoclonal antibody which inhibits the coaggregation activity of Porphyromonas gingivalis.

A B-cell line producing a human monoclonal antibody (HuMAb) against a recombinant 40-kDa outer membrane protein (OMP) of Porphyromonas gingivalis was constructed by in vivo immunization of a severe combined immunodeficiency C.B.-17/Icr mouse, which had been injected with human peripheral blood lymphocytes, with recombinant 40-kDa OMP and subsequent Epstein-Barr virus immortalization of B cells isolated from the spleen of the mouse. This HuMAb inhibited coaggregation between P. gingivalis vesicles and Actinomyces naeslundii cells.     

Role for Porphyromonas gingivalis in the progression of atherosclerosis

An association between atherosclerosis and Porphyromonas gingivalis (P. gingivalis), a major periodontopathogen, has been shown. However, the question of whether this relationship is causal or coincidental still exist. Many individuals with evidence of atherosclerosis demonstrate seropositivity to this pathogen. Both in vitro studies and in animal models document that P. gingivalis can accelerate atheroma deposition. Recently, evidence from diverse sources has suggested P. gingivalis can activate host innate immune responses associated with atherosclerosis. These studies confirm that the inflammatory response to P. gingivalis could exacerbate vascular inflammation via secreted cytokines and/or chemokines that ultimately modulate early atherogenesis that spontaneously develop atherosclerosis. Meanwhile, the cytokine and chemokines interact in the progression of atherosclerosis. Moreover, the reaction of endothelial cells in response to P. gingivalis and its various virulence factors are diverse, the expression of chemokine differ through different signal transduction pathways accordingly. Results from these studies reinforce the connection between P. gingivalis and atherosclerosis. Given the role of P. gingivalis in the initiation and progression of atherosclerosis, attaching importance to periodontal disease may offer new therapeutic options for the management of patients with atherosclerosis.     

Impaired ATP-induced coronary blood flow and diminished aortic NTPDase activity precede lesion formation in apolipoprotein E-deficient mice

Intravascular ATP and ADP are important regulators of vascular tone, thrombosis, inflammation, and angiogenesis. This study was undertaken to evaluate the contribution of purinergic signaling to disturbed vasodilation and vascular remodeling during atherosclerosis progression. We used apolipoprotein E-deficient (Apoe(-/-)) mice as an appropriate experimental model for atherosclerosis. Noninvasive transthoracic Doppler echocardiography imaging with adenosine, ATP, and other nucleotides and nonhydrolyzable P2 receptor agonists and antagonists suggests that ATP regulates coronary blood flow in mice through activation of P2Y (most likely, endothelial ATP/UTP-selective P2Y(2)) receptors, rather than via its dephosphorylation to adenosine. Strikingly, compared to age-matched wild-type controls, young (10- to 15-week-old) Apoe(-/-) mice displayed diminished coronary reactivity in response to ATP but not adenosine. The impaired hyperemic response to ATP persisted in older (20- to 30-week-old) Apoe(-/-) mice, which were additionally characterized by mild atherosclerosis (as ascertained by aortic Oil Red O staining) and a systemic increase in plasma ATP and ADP levels. Concurrent thin-layer chromatographic analysis of nucleoside triphosphate diphosphohydrolase (NTPDase) and ecto-5'-nucleotidase/CD73 activities in thoracic aortas, lymph nodes, spleen, and serum revealed that aortic NTPDase was decreased by 40% to 50% in a tissue-specific manner both in young and mature Apoe(-/-) mice. Collectively, disordered purinergic signaling in Apoe(-/-) mice may serve as important prerequisite for impaired blood flow, local accumulation of ATP and ADP at sites of atherogenesis, and eventually, the exacerbation of atherosclerosis.     

Nasal immunization with a fusion protein consisting of the hemagglutinin A antigenic region and the maltose-binding protein elicits CD11c(+) CD8(+) dendritic cells for induced long-term protective immunity

We assessed the efficacy of a fusion protein consisting of the 25-kDa antigenic region of Porphyromonas gingivalis hemagglutinin A and the Escherichia coli maltose-binding protein (25k-hagA-MBP) as a nasal vaccine for the prevention of oral infection with P. gingivalis. Nasal immunization with 25k-hagA-MBP induced high levels of 25k-hagA-specific serum IgG, serum IgA, and salivary IgA antibodies in a Toll-like receptor 4 (TLR4)-dependent manner. These antibody responses were maintained for at least 1 year after immunization. Analysis of cytokine responses showed that nasal administration of 25k-hagA-MBP induced antigen-specific CD4(+) T cells producing interleukin 4 (IL-4) and IL-5, but not gamma interferon (IFN-γ), in the spleen and cervical lymph nodes (CLNs). Furthermore, increased numbers of CD11c(+) CD8α(+), but not CD11c(+) CD11b(+) or CD11c(+) B220(+), dendritic cells with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) molecules were noted in the spleen, CLNs, and nasopharynx-associated lymphoreticular tissues (NALT). Interestingly, when 25k-hagA-MBP or cholera toxin (CT) was given intranasally to enable examination of their presence in neuronal tissues, the amounts of 25k-hagA-MBP were significantly lower than those of CT. Importantly, mice given 25k-hagA-MBP nasally showed a significant reduction in alveolar bone loss caused by oral infection with P. gingivalis, even 1 year after the immunization. These results suggest that 25k-hagA-MBP administered nasally would be an effective and safe mucosal vaccine against P. gingivalis infection and may be an important tool for the prevention of chronic periodontitis in humans.     

Protective ability of antibodies against 78- and 40-kilodalton outer membrane antigens of Haemophilus somnus.

The ability of concentrated antibody against the 78- or 40-kilodalton (kDa) outer membrane protein (OMP) of Haemophilus somnus to passively protect calves against H. somnus-induced pneumonia was determined. The 78- and 40-kDa OMPs were evaluated in passive protection experiments, because results of previous studies demonstrated their (i) immunogenicity for cattle, (ii) intense reactivity with convalescent-phase sera which passively protected calves against experimental H. somnus pneumonia, (iii) surface location and accessibility to antibody, and (iv) conservation among a wide range of H. somnus isolates obtained from animals with different diseases and from different geographic locations. The specificity of the two antisera evaluated in this study was verified by (i) immunoblots in which reactivity against the 78- or 40-kDa OMP was present in postimmunization but not preimmunization serum and (ii) immunoblots in which affinity-purified, surface-reactive antibodies in each antisera were used, which demonstrated that essentially only antibody to the 78- or 40-kDa OMP was reactive with the surface of H. somnus. In enzyme-linked immunosorbent assays, the antiserum against the 40-kDa OMP contained immunoglobulin G1 (IgG1), IgG2, and IgM against H. somnus, while the antiserum against the 78-kDa OMP contained IgG1 and IgM but no IgG2 against H. somnus. The antiserum against the 40-kDa OMP contained IgG1 and IgG2 specific for the 40-kDa OMP, as determined by Western blot analysis. Slight reactivity against H. somnus lipopolysaccharide was detected by enzyme-linked immunosorbent assay but not by Western blot analysis. In passive protection experiments, preincubation of bacteria with antibody against the 40-kDa OMP protected calves (P less than 0.025) against H. somnus pneumonia, while antibody against the 78-kDa OMP failed to protect calves against H. somnus pneumonia. Determination of the potential protective capacity of the 78-kDa OMP awaits resolution of the role of anti-78-kDa IgG2 in protection against H. somnus pneumonia. The 40-kDa OMP is, however, a good candidate antigen for evaluation of protective ability against H. somnus pneumonia following active immunization.     

Dietary antioxidants decrease serum soluble adhesion molecule (sVCAM-1, sICAM-1) but not chemokine (JE/MCP-1, KC) concentrations, and reduce atherosclerosis in C57BL but not apoE*3 Leiden mice fed an atherogenic diet

Dietary antioxidants are reported to suppress cellular expression of chemokines and adhesion molecules that recruit monocytes to the artery wall during atherosclerosis. In the present study we measured the effect of feeding apoE*3 Leiden mice or their non-transgenic (C57BL) littermates with atherogenic diets either deficient in, or supplemented with, dietary antioxidants (vitamin E, vitamin C and beta-carotene) for 12 weeks, on serum levels of CC (JE/MCP-1) and CXC (KC) chemokines and soluble adhesion molecules (sVCAM-1, sICAM-1) and atherosclerotic lesion size. ApoE*3 Leiden mice developed gross hypercholesterolaemia, and markedly accelerated (10-20 fold; P < 0.0001) atherogenesis, compared with non-transgenic animals. Antioxidant consumption reduced lesion area in non-transgenic, but not apoE*3 Leiden, mice. Serum sVCAM-1 and sICAM-1 levels were significantly (P<0.0001) increased (sVCAM-1 up to 3.9 fold; sICAM-1 up to 2.4 fold) by 4-8 weeks in all groups, and then declined. The initial increase in the concentration of adhesion molecules was reduced by 38%-61% (P < 0.05) by antioxidant consumption, particularly in non-transgenic mice. By contrast, serum chemokine levels tended to increase more rapidly from baseline in apoE*3 Leiden mice, compared with non-transgenic animals, but were unaffected by dietary antioxidants. We conclude that dietary antioxidants reduce circulating soluble adhesion molecules and atherosclerosis in C57BL mice.     

Effect of porphyrins and host iron transport proteins on outer membrane protein expression in Porphyromonas (Bacteroides) gingivalis: identification of a novel 26 kDa hemin-repressible surface protein.

Porphyromonas gingivalis is capable of in vitro growth when iron sources are either complexed to hemin or host iron transport proteins, or exist in an inorganic form. This study examined the effect of these iron sources on outer membrane protein (OMP) expression in P. gingivalis W50. Hemin (iron) starved P. gingivalis was transferred into growth medium containing hemin, hemoglobin, hemin-saturated human serum albumin, hemin-free human serum albumin, transferrin, lactoferrin, or inorganic iron. Surface proteins were identified by 125I-labeling and resolved by SDS-PAGE and autoradiography. When grown under hemin starved conditions, P. gingivalis W50 and related strains expressed a major 26 kDa OMP, as revealed by 125I-autoradiography. Autoradiographic analysis demonstrated the absence of this 26 kDa OMP from the P. gingivalis surface in hemin-containing environments. Growth of P. gingivalis W50 in the presence of host iron transport proteins (hemin-free) or inorganic iron resulted in surface expression of a 26 kDa OMP. The presence of protoporphyrin IX or substitution of hemin-associated iron with zinc, resulted in continued surface expression of the 26 kDa OMP, indicating that repressibility of this OMP required the coordination of iron to the protoporphyrin IX molecule (i.e. hemin). A survey of 125I-labeled OMPs from several hemin starved P. gingivalis and related strains, demonstrated that a hemin-repressible 26 kDa OMP occurred only in P. gingivalis. We report here a newly described 26 kDa hemin-regulated surface protein occurring in several strains of P. gingivalis which is expressed on the cell surface in hemin starved conditions and is lost from the cell surface in response to an environment containing iron coordinated specifically to protoporphyrin IX (i.e. hemin).     

Monoclonal antibodies against the 70-kilodalton iron-regulated protein of Neisseria meningitidis are bactericidal and strain specific.

When grown under iron limitation, Neisseria meningitidis expresses a number of outer membrane proteins (OMPs), one of which is a 70-kilodalton (kDa) major OMP. After immunization of mice with outer membrane preparations of iron-depleted cells of strain H44/76 (B:15:P1.7,16), hybridoma cell lines producing monoclonal antibodies against the 70-kDa OMP were obtained. Some of these monoclonal antibodies demonstrated strong bactericidal activity against the homologous strain H44/76 in the presence of human complement, suggesting potential application of the 70-kDa OMP as a vaccine component. However, none of the 10 selected monoclonal antibodies was able to recognize the corresponding protein from five heterologous strains of various serosubtyping characteristics. A polyclonal anti-70-kDa OMP serum also did not react with the other strains. This result shows that immunodominant surface-exposed epitopes of the meningococcal 70-kDa iron-limitation-inducible OMP are strain specific.     

Polymicrobial infection with periodontal pathogens specifically enhances microRNA miR-146a in ApoE-/- mice during experimental periodontal disease

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are periodontal pathogens associated with the etiology of adult periodontitis as polymicrobial infections. Recent studies demonstrated that oral infection with P. gingivalis induces both periodontal disease and atherosclerosis in hyperlipidemic and proatherogenic ApoE(-/-) mice. In this study, we explored the expression of microRNAs (miRNAs) in maxillas (periodontium) and spleens isolated from ApoE(-/-) mice infected with P. gingivalis, T. denticola, and T. forsythia as a polymicrobial infection. miRNA expression levels, including miRNA miR-146a, and associated mRNA expression levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) were measured in the maxillas and spleens from mice infected with periodontal pathogens and compared to those in the maxillas and spleens from sham-infected controls. Furthermore, in response to these periodontal pathogens (as mono- and polymicrobial heat-killed and live bacteria), human THP-1 monocytes demonstrated similar miRNA expression patterns, including that of miR-146a, in vitro. Strikingly, miR-146a had a negative correlation with TNF-α secretion in vitro, reducing levels of the adaptor kinases IL-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6). Thus, our studies revealed a persistent association of miR-146a expression with these periodontal pathogens, suggesting that miR-146a may directly or indirectly modulate or alter the chronic periodontal pathology induced by these microorganisms.     

Surface exposure of outer membrane protein and lipopolysaccharide epitopes in Brucella species studied by enzyme-linked immunosorbent assay and flow cytometry.

Seven surface-exposed outer membrane proteins (OMPs) in Brucella supp. have been previously described (A. Cloeckaert, P. de Wergifosse, G. Dubray, and J. N. Limet, Infect. Immun. 58:3980-3987, 1990). OMPs were shown to be more accessible to monoclonal antibodies (MAbs) on rough (R) Brucella melitensis and B. abortus strains than to MAbs on their smooth (S) counterparts. In this work, we have extended this study to representatives of the main Brucella species, using MAbs specific for OMPs and S and R lipopolysaccharides (S-LPS and R-LPS). Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoelectron microscopy showed important differences between strains in the binding of OMP- and R-LPS-specific MAbs which were in part related to the particular expression of S-LPS, irrespective of the species. Results indicated that both the amount and the length of O polysaccharide on S-LPS greatly influenced the accessibility of OMP and R-LPS epitopes to MAbs. S-R B. melitensis EP and S B. suis 40, for instance, which express O-polysaccharide chains in small amounts and with short mean length, respectively, bound a greater number of OMP- and R-LPS-specific MAbs than the other S Brucella strains. The major 31- to 34-kDa OMP was the most exposed OMP on S strains of B. melitensis and B. suis. In most cases, flow cytometry results agreed with those of ELISA and supplied additional data, such as the homogeneity or heterogeneity of OMP expression at the strain level. However, there were some discordances between flow cytometry and ELISA results concerning the surface exposure of the 25- to 27-kDa and 31- to 34-kDa OMPs on S strains and that of minor OMPs in vaccine strain B. melitensis Rev.1. Immunoelectron microscopy confirmed the poor accessibility of OMPs to MAbs on the surface of S Brucella strains. The naturally R pathogenic species B. ovis and B. canis bound the majority of OMP-specific MAbs as well as the R-LPS-specific MAbs. Therefore, the conserved OMP and R-LPS epitopes could play a role as targets of protective antibody-mediated immunity in infections caused by naturally R B. ovis and B. canis.     

高脂饲料喂养加静脉注射小牛血清白蛋白建立兔动脉粥样硬化模型**

背景：目前单纯饲喂高脂饲料建立动脉粥样硬化狭窄模型较常见。 目的：采用高脂饲料喂养加静脉注射小牛血清白蛋白建立兔动脉粥样硬化模型。 方法：分别单纯高脂饮食、高脂饲料+脂肪乳灌胃以及高脂饲料+小牛血清白蛋白3种不同的方法喂养兔构建动脉硬化模型,设立正常对照组,予普通饲料喂养。 结果与结论：各组兔血清胆固醇、三酰甘油、低密度脂蛋白和高密度脂蛋白水平较正常对照组显著升高(P < 0.01)。单纯高脂组可出现高脂血症,未发现典型的动脉粥样硬化病变;高脂+脂肪乳灌胃组可形成纤维斑块;高脂+小牛血清白蛋白组可形成较成熟的动脉粥样硬化斑块。结果证实,高脂饲料加静脉注射小牛血清白蛋白可形成较成熟的动脉粥样硬化斑块,可成功建立动脉粥样硬化兔模型。关键词：动脉粥样硬化;小牛血清白蛋白;高脂饲料;兔;动物模型;组织工程 doi:10.3969/j.issn.1673-8225.2012.20.018     

Experimental obstructive coronary atherosclerosis in the hyperlipidemic hamster

The evolution of coronary atherosclerotic lesions induced by a hyperlipidemic diet was examined in male hamsters subjected for up to 40 weeks to a standard chow supplemented with 3% cholesterol and 15% butter. Control animals were fed standard chow only. Five to seven hamsters were monthly sacrificed and investigated for serum lipids and coronary artery lesions. As compared with control animals, the hamsters fed the fat diet showed a progressive increase in serum cholesterol which reached maximum values up to 17 fold in the 10th month. The serum of the hyperlipidemic hamster examined by agarose electrophoresis, Laurell immunoelectrophoresis and cross-immunoelectrophoresis showed at most a 14 fold increase in low density lipoproteins after 10 months diet. The examination of coronary arteries revealed morphologic changes already detectable at 2 weeks of diet. The earliest modifications observed were characterized by proliferation of the subendothelial matrix or/and the appearance of liposome-like structures in the intima. After 2-3 weeks of diet, smooth muscle cells appeared occasionally in the intima and monocytes adhered and penetrated through the endothelium. Later on, smooth muscle cells and macrophage displayed lipid deposits. Focally, in areas of intimal proliferation and foam cells, endothelial cells were also lipid-loaded. Like in human atherosclerotic plaque, in the late stages of hamster coronary lesions, there was a progressive accumulation of extracellular unesterified cholesterol, calcium deposition and necrosis. Lesions evolved to a progressive narrowing of the coronary branches affected, with complete obstruction of some small arterial ramifications. Hamster appears to be a suitable model for studying the molecular and cellular events leading to obstructive coronary atherosclerosis.     

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.     

Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia

Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis.     

Characterization of heat-inducible expression and cloning of HtpG (Hsp90 homologue) of Porphyromonas gingivalis

Porphyromonas gingivalis is implicated in the etiology of periodontal disease. Associations between microbial virulence and stress protein expression have been identified in other infections. For example, Hsp90 homologues in several microbial species have been shown to contribute to virulence. We previously reported that P. gingivalis possessed an Hsp90 homologue (HtpG) which cross-reacts with human Hsp90. In addition, we found that elevated levels of serum antibody to Hsp90 stress protein in individuals colonized with this microorganism were associated with periodontal health. However, the role of HtpG in P. gingivalis has not been explored. Therefore, we cloned the htpG gene and investigated the characteristics of HtpG localization and expression in P. gingivalis. htpG exists as a single gene of 2,052 bp from which a single message encoding a mature protein of approximately 68 kDa is transcribed. Western blot analysis revealed that the 68-kDa polypeptide was stress inducible and that a major band at 44 kDa and a minor band at 40 kDa were present at constitutive levels. Cellular localization studies revealed that the 44- and 40-kDa species were associated with membrane and vesicle fractions, while the 68-kDa polypeptide was localized to the cytosolic fractions.     

Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial cytokeratins may act as receptor(s) for P. gingivalis fimbriae. We hypothesize that adherence of P. gingivalis fimbriae to cytokeratin may be important for colonization of oral mucous membranes and possibly also for activation of epithelial cells.     

Reduction of atherosclerosis despite hypercholesterolemia in lyn-deficient mice fed a high-fat diet

Oxidation and other modifications of serum low-density lipoprotein (LDL) are associated with the development of atherosclerosis, and a scavenger receptor and CD40 signalling are also known to play important roles in the process. We previously showed that the Src family protein-tyrosine kinase Lyn is physically and/or functionally associated with macrophage type-I and type-II class-A scavenger receptors (MSR-A) and CD40. In this study, we addressed whether Lyn is involved in the build-up of serum lipid levels and in atherosclerotic changes. When fed a normal diet, lyn-deficient mice had serum lipid levels that were no different from those of wild-type mice. By contrast, lyn-deficient mice fed a high-fat diet showed serum lipid levels that were much higher than those seen in wild-type mice. Curiously, however, the lyn-deficient mice fed either diet showed no increase in incidence of atherosclerotic lesions compared with wild-type mice. This may be partly explained by our data showing suppression of proliferation of peritoneal macrophages in response to oxidized LDL in the absence of Lyn, and failure of stimulation of the CD40 pathway in lyn-deficient macrophages to induce expression of monocytic chemoattractant protein-1 (MCP-1), which is related to atherosclerosis. These results suggest that Lyn plays an important role in the metabolism of serum lipids and in the development of atherosclerotic lesions on high-fat diets.     

构建不同周龄载脂蛋白E基因敲除动脉粥样硬化模型小鼠血脂和病理学观察

背景：载脂蛋白E基因敲除小鼠形成的动脉粥样硬化病变与人类全身动脉粥样硬化好发处相近,是目前建立动脉粥样硬化理想的动物模型。 目的：研究载脂蛋白E基因敲除小鼠不同周龄动脉粥样硬化的病理进程,探讨不同饮食对载脂蛋白E基因敲除小鼠动脉粥样硬化发生发展的影响。 方法：将8周龄雄性载脂蛋白E基因敲除小鼠,随机分为2组,分别给予高脂饮食和普通饮食喂养8,12,16,20,24周。 结果与结论：血清学指标检测显示,不同周龄的高脂饮食组血清中总胆固醇、三酰甘油和低密度脂蛋白胆固醇水平显著高于普通饮食组(P < 0.05),呈时间依赖性。大体和冰冻切片油红O染色结果显示,高脂饮食组动脉粥样硬化管腔斑块面积显著高于普通饮食组(P < 0.05),呈时间依赖性,此时两组各周龄小鼠管腔斑块面积相比均有显著性意义(P < 0.05),小鼠在高脂饮食16周时主动脉可见明显的脂质斑块。结果表明,实验成功构建了载脂蛋白E基因敲除动脉粥样硬化模型小鼠,此模型形成脂质条纹和纤维增生病变的时间较普通饮食组更快。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：