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PubMed1997

Immunization with meningococcal membrane-bound lipooligosaccharide accelerates granulocyte recovery and enhances lymphocyte proliferation in myelosuppressed mice.

Protective effects of detergent-treated outer membrane vesicles (D-OMVs) prepared from the parent group B M986 strain and the nonencapsulated M986-NCV mutant in myelosuppressed mice were investigated in models of experimental septic shock. The effects of D-OMVs on expansion of the myeloid compartment, on spleen cell proliferation to mitogen stimulation, and on cytokines induced during this period were investigated. On 3 consecutive days, mice were injected with 1 microgram/kg of lipooligosaccharide (LOS) or lipopolysaccharide, or 75 micrograms/kg D-OMV followed by a single dose of cyclophosphamide (200 mg/kg) 24 h later. Eight weeks after the last injection, animals were challenged with a combination of galactosamine (400 mg/kg) and live Neisseria meningitidis. More than 90% of control mice died within 24 h when challenged with 10(5) CFU of bacteria. Mice immunized with LOS or D-OMV were rendered neutropenic but were protected against bacterial challenge of at least 10(7) CFU. At different time intervals, peripheral blood samples were obtained to characterize changes in circulating blood cells. The rise in absolute granulocyte numbers occurred 24 h earlier with peak cell counts about 3 times higher than those seen in the placebo groups. Peripheral blood cells from D-OMV-treated animals expressed about twofold more Gr-1 antigen (myeloid surface cell marker) than placebo-treated controls. The proliferative responses to both B and T cells were reduced in all treatment groups due to the effects of cyclophosphamide. D-OMV treatment afforded the greatest protection for mitogen-activated lymphocytes from the lethal effects of cyclophosphamide and also enhanced T and B cell proliferation. Low IL-1 beta levels and increases in serum IL-6 were detected in all treatment groups. In contrast, significant IFN-gamma and IL-3 levels were only detected in D-OMV-treated groups. These results indicate that D-OMVs, which have reduced toxicity, have prophylactic potential in inducing specific cytokines that accelerate granulocyte recovery following cytoreductive therapy by promoting both proliferation and maturation of myeloid precursors as well as augmenting the immune system.

Animals↗
PubMed2004

Teasing apart structural determinants of 'toxicity' and 'adjuvanticity': implications for meningococcal vaccine development.

The use of lipopolysaccharide (LPS) as an adjuvant is limited by its high endotoxic activity. In particular, the fatty-acyl pattern of the lipid A part of LPS has been demonstrated to determine its biological activity. By genetic modification of the lipid A biosynthesis pathway in Neisseria meningitidis, a panel of recombinant strains with specific alterations in the lipid A acylation pattern, as well as a strain completely lacking LPS were isolated. Whereas all variations in the fatty-acyl pattern resulted in reduced endotoxic activity, as measured by TNF-alpha induction in the human macrophage cell line MM6, the adjuvant activity of the modified LPS was, in most cases, barely affected. The in vivo adjuvant properties of N. meningitidis wild-type and mutant LPS was found to correlate with induction of co-stimulatory molecules, in particular CD80 and CD40, and with IL-12 production by LPS-stimulated bone marrow-derived BALB/c dendritic cells in vitro. Our results suggest that the ability of LPS to stimulate pro-inflammatory cytokine induction is not necessarily linked to its adjuvant activity. The availability of this novel set of lipid A variants with improved pharmacological properties will be of great importance for the improvement of future outer membrane vesicle vaccines against N. meningitidis.

Animals↗
PubMed2005

Towards an improved serogroup B Neisseria meningitidis vaccine.

Meningococcal disease, presenting primarily as septicaemia and meningitis, continues to be a devastating problem around the world. Over the last century, vaccine development has been undertaken in earnest for the prevention of this disease. Polysaccharide vaccines have been available for almost 40 years, yet they are poorly immunogenic in young children who are at the highest risk. Since their introduction into some routine immunisation schedules in 1999, polysaccharide-protein conjugate vaccines for the prevention of serogroup C meningococcal infection have proven efficacious. A quadrivalent polysaccharide-protein conjugate vaccine against serogroups A, C, W135 and Y, which is being introduced in the US this year, is hoped to control disease caused by these serogroups. To date, however, the development of a universally safe, immunogenic and effective serogroup B Neisseria meningitidis vaccine has remained a challenge. This review details the many conventional vaccine strategies and the more recent genome-derived technological approaches being used in serogroup B vaccine development. The future prevention of serogroup B disease will rely on both outer membrane vesicle vaccines being used for serosubtype-specific outbreaks and new vaccines containing multiple other antigens. Investment by the pharmaceutical industry in preclinical research and development provides hope that an efficacious serogroup B meningococcal vaccine can be developed.

Animals↗
PubMedDate not supplied

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium.

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30 degrees C after growing for 16 h at 37 degrees C supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30 degrees C. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30 degrees C than from the cells after 16 h at 37 degrees C. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30 degrees C as from the cells after 16 h at 37 degrees C.

Bacterial Proteins↗
PubMed2001

Role for periodontal bacteria in cardiovascular diseases.

BACKGROUND: Several epidemiological studies as well as a recent animal model approach have suggested a role for periodontal diseases in the development of cardiovascular disease (CVD). This relationship could be mediated by inflammatory responses induced by periodontal pathogens as well as direct interaction of these organisms with cardiac tissue. METHODS: In order to explore these possibilities, the effects of the periodontal pathogen Porphyromonas gingivalis on cellular events proposed to play a role in CVD were investigated. RESULTS: P. gingivalis, as well as its outer membrane vesicles (OMV), was able to induce foam cell formation (an important characteristic of CVD) in the murine macrophage cell line J774 A.1. This property appears to be mediated by the lipopolysaccharide (LPS) fraction of the cells. Several other oral bacteria were also able to induce foam cell formation. Furthermore, since the rupture of the fibrous cap of plaque appears to be an important factor in acute coronary syndrome, it was demonstrated that P. gingivalis 381 degraded fibrous caps isolated from autopsy samples. In addition, it was observed that strain 381 strongly induced matrix metalloproteinase (MMP)-9 protease activity, implicated in plaque rupture, from the J774 A.1 macrophages. Finally, strain 381 was able to enhance monocyte chemoattractant protein-1 (MCP-1) and NADH oxidase expression from endothelial cells. CONCLUSIONS: Therefore, P. gingivalis exhibits several properties which could play a role in CVD as mediators of LDL oxidation, foam cell formation, and rupture of atherosclerotic plaque.

Animals↗
PubMed2002

Cellular responses to oral pathogens.

BACKGROUND: Several previous epidemiological studies, along with the results of more recent animal model approaches, have suggested a role for periodontitis in atherosclerosis. Such an association could be mediated by direct interactions of periodontopathic bacteria with host vascular tissues. METHODS: The interactions of Porphyromonas gingivalis with endothelial cells and macrophages in vitro were investigated relative to modification of low-density lipoproteins (LDL). RESULTS: P. gingivalis 381, its outer membrane vesicles, and the lipopolysaccharide (LPS) derived from these organisms were all shown to induce modification of LDL in the presence of the murine macrophage J774.A.1. Such alterations led to an increase in the migration of the particles through agarose gels. In addition, direct modification of LDL by strain 381 was demonstrated in the absence of macrophages. This latter property appears to be related to the potent protease activities of the bacterium. These properties may contribute to modification of LDL to forms which have been strongly implicated in cholesterol lipid accumulation in vascular tissues. P. gingivalis 381 also appears to induce cyclooxygenase-2 expression in endothelial cells as determined with human umbilical vascular endothelial cells (HUVEC). CONCLUSIONS: These in vitro results with vascular cells in culture suggest a molecular basis for a potential role for periodontopathic bacteria such as P. gingivalis in augmenting foam cell formation characteristic of atherosclerotic lesions.

Animals↗
PubMed1988

Interaction of bacteria with the immune system of Waldeyer's ring in otitis media with effusion.

An in vitro assay was used to study the adherence of Haemophilus influenzae (HI) to human nasal, nasopharyngeal, and buccal mucosal cells from patients with otitis media with effusion (OME). Adherence of unencapsulated HI was stronger than that of type b HI. HI adherence to both nasal and nasopharyngeal mucosal cells is stronger than that of buccal ones, indicating the presence of an important route of HI infection from the nose to the nasopharynx in OME. Specific antibodies against outer membrane vesicles (OMV) of HI were also examined using enzyme-linked immunosorbent assay. No significant correlation could be observed between specific secretory IgA or IgG titers and HI adherence to the nasopharyngeal mucosal cells, which suggests that HI adherence to the mucosal cells seems to depend on various factors including the local immunological defense system in OME patients. Anti-OMV IgA antibody producing cells were identified in the adenoid using the three-step immunoperoxidase method. These results suggest that non-typable strains of HI adhere more readily to epithelial cells and consequently are better suited to colonize the nasopharynx or to cause local infections.

Adolescent↗
PubMed2002

Neutrophil-mediated host response to Porphyromonas gingivalis.

Periodontal diseases are infections initiated by specific species of microorganisms and are among the most common human infections. The pathogenesis of periodontitis is mediated by interactions between host and microbial factors, complicated by genetic and environmental risk factors. Periodontal disease also represents a unique model in which to study the roles of bacterial and host-related factors, a model in which patients do not suffer from life-threatening disease. The aim of this paper is to focus on recent findings relating to neutrophil-mediated host response mechanisms in Porphyromonas gingivalis-induced periodontal disease. Virulence factors of Porphyromonas gingivalis such as the gingipains, fimbrillin peptides, capsule polysaccharides, lipopolysaccharides, haemagglutinating and haemolysing activities, toxic products of metabolism, outer membrane vesicles, and other enzymes have important roles in eliciting host responses in various ways. These factors significantly affect epithelial/endothelial cells, but their major effect is observed on the modulation of neutrophil response. Periodontitis represents an important model for neutrophil-mediated host tissue injury. In this model, neutrophils, primed or stimulated by the presence or persistence of infection, express an elevated and excessive response. This, in turn, leads to tissue destruction mediated by neutrophil activity. It is essential to understand the mechanisms underlying the interactions between the neutrophils and the microbial virulence factors to be able to develop rational, novel treatment strategies.

Adhesins, Bacterial↗
PubMed2003

Expression and involvement of Toll-like receptors (TLR)2, TLR4, and CD14 in monocyte TNF-alpha production induced by lipopolysaccharides from Neisseria meningitidis.

BACKGROUND: The present study was undertaken to examine the ability of lipopolysaccharide-containing outer membrane vesicles (OMV-LPS) and purified LPS (P-LPS) from the same meningococcal strain to induce the expression of Toll-like receptors (TLR2 and TLR4) and TNF-alpha production in leukocytes, and further to study the involvement of TLRs, and CD14 in monocyte TNF- alpha production in an ex vivo human whole blood system. MATERIAL/METHODS: OMV-LPS or P-LPS were added to human whole blood and expression of TLR2/4 and production of TNF- alpha in leukocytes were measured by flow cytometry. To study involvement of TLRs and CD14 in monocyte cytokine production we used monoclonal antibodies against TLR2/4 and CD14. RESULTS: OMV-LPS and P-LPS induced surface expression (maximal at 120 min) of TLR2 and TLR4 on granulocytes and monocytes. LPS incorporated in OMV was less potent (weight basis) than P-LPS in inducing monocyte TNF- alpha production. When inducing monocyte TNF-alpha by OMV-LPS, antibodies directed against TLR2 and TLR4 caused 45 and 78% inhibition, respectively. When inducing TNF- alpha by P-LPS, antibodies against TLR2 had no effect, whereas anti-TLR4 antibodies caused 63% inhibition. Antibodies against CD14 inhibited nearly completely the monocyte TNF- alpha response induced by meningococcal LPS irrespective of whether LPS was presented in purified form or incorporated in membrane vesicles. CONCLUSIONS: OMV-LPS and P-LPS from the same meningococcal strain induced expression of TLR2/4 on monocytes and granulocytes. Surface receptors TLR2/4 and CD14 are essential for in vitro cellular activation induced by OMV-LPS and P-LPS, but the functional significance of these receptors during meningococcal infections remains elusive.

Humans↗
PubMed2004

The New Zealand Meningococcal Vaccine Strategy: a tailor-made vaccine to combat a devastating epidemic.

The New Zealand Meningococcal Vaccine Strategy aims to end the devastating 14-year epidemic of B:4:P1.7b,4 group B meningococcal disease in New Zealand through a mass immunisation programme to all under 20 year olds using a tailor-made vaccine (MeNZB). This paper describes the scientific rationale, development, and key components of the New Zealand Meningococcal Vaccine Strategy. A summary of the efficacy and safety data of existing outer membrane vesicle group B meningococcal vaccines is included as these data critically support the Strategy.

Disease Outbreaks↗
PubMed2004

Meningococcal vaccines.

BACKGROUND: The 5 major pathogenic serogroups of the Gram-negative encapsulated bacterium Neisseria meningitidis are A, B, C, Y, and W135. In the 1960s, vaccines consisting of purified capsular polysaccharide antigens were developed against serogroups A, C, Y, and W135. These vaccines were highly effective among adults but were not efficacious among infants and young children. Capsular polysaccharide-protein conjugate vaccines were subsequently developed. METHODS: The development of second-generation capsular polysaccharide glycoconjugate vaccines that are effective among infants and children is reviewed. The approaches for development of a broadly protective serogroup B vaccine, including attempts toward optimization of outer membrane vesicle vaccines and identification of conserved antigens, are discussed. CONCLUSIONS: Monovalent serogroup C conjugate vaccines have been successful in reducing the burden of serogroup C meningococcal disease among infants, older children, and adults in the United Kingdom and Canada. Tetravalent serogroup A/C/W/Y conjugate vaccines are in late stage development. The use of serogroup B capsular polysaccharide as the basis for a vaccine for prevention of serogroup B meningococcal disease has proved problematic. The recent sequencing of the serogroup B genome led to the identification of additional, genome-derived, neisserial antigens, through a process called "reverse vaccinology."

Bacterial Capsules↗
PubMed2006

Prolonged outbreak of B meningococcal disease in the Seine-Maritime department, France, January 2003 to June 2005.

Between January 2003 and June 2005, an outbreak of meningococcal disease occurred in the department of Seine-Maritime in northern France. Eighty six cases were notified, giving an average annual incidence of 2.7 cases per 100,000 inhabitants, compared with 1.6 in France. An especially affected area was defined as the city of Dieppe and its surrounding area (26 cases, giving an annual incidence of 12 cases per 100,000). This outbreak was due to N. meningitidis phenotype B:14:P1.7,16 belonging to the clonal complex ST-32/ET-5. Over the 31 B14:P1.7,16 cases confirmed by phenotyping methods at the national reference centre for meningococci (CNR, Centre National de Reference des meningocoques) the case-fatality rate (19%) and the proportion of purpura fulminans (42%) were especially high. Teenagers aged between 15 and 19 years and children aged 1 to 9 years were the most affected. In 2003, health authorities put in place enhanced epidemiological surveillance and informed practitioners and population about the disease. In 2004, the national vaccination advisory board studied the opportunity of using a non licensed outer membrane vesicle vaccine developed in Norway which may be effective against the B14:P1.7,16 strain. The Ministry of health decided in 2006 to offer vaccination with this vaccine to people aged 1 to 19 years in Seine-Maritime.

Adolescent↗
PubMedDate not supplied

Bacterial toxins.

Many bacterial toxins are proteins, encoded by the bacterial chromosomal genes, plasmids or phages. Lysogenic phages form part of the chromosome. The toxins are usually liberated from the organism by lysis, but some are shed with outer membrane proteins in outer membrane vesicles. An important non-protein toxin is lipopolysaccharide or endotoxin, which is a constituent of the cell wall of gram negative bacteria. Toxins may damage the eukaryotic cell membrane by combining with some structural component, or otherwise alter its function. Many toxins combine with specific receptors on the surface membrane, frequently glycoproteins or gangliosides, and penetrate the cell to reach their intracellular target. A common mechanism of entry is absorptive endocytosis. Many protein toxins have an A-B structure, B being a polypeptide which binds to the receptor and A being an enzyme. Many toxins are activated, either when produced by the bacterium or when bound to the membrane receptor, by proteases (nicking). An enzymatic process common to many toxins is adenosine diphosphate (ADP)-ribosylation of the adenylate cyclase regulatory proteins, leading to an increase in intracellular cyclic adenosine monophosphate (cAMP). This is the mechanism of action of cholera toxin. Diphtheria toxin catalyzes the transfer of ADP-ribose to elongation factor-2, inhibiting protein synthesis. Most toxins act on the target cells to which they bind, but tetanus toxin, and, to a lesser degree, botulinum toxin, ascend axons and affect more distant structures. Although many toxin effects caused by bacteria have been described, only a few toxins have been identified, characterized, and their mode of action determined at the molecular level. The best known of these are discussed.

Bacteria↗
PubMed1998

Towards a nasal vaccine against meningococcal disease, and prospects for its use as a mucosal adjuvant.

A Norwegian outer membrane vesicle (OMV) vaccine against group B meningococcal disease proved to be strongly immunogenic when administered intranasally in mice. The OMV preparation, made from Neisseria meningitidis and intended for parenteral use, was therefore given without adjuvant to human volunteers (n = 12) in the form of nose drops or nasal spray. Such immunizations, which were carried out at weekly intervals during a three-week period, were able to induce systemic antibodies with bactericidal activity in more than half of the individuals. In addition, all vaccinees developed marked increases in OMV-specific IgA antibodies in nasal secretions. The potential of the OMV particles as carriers for other less immunogenic antigens were elucidated in mice with use of whole inactivated influenza virus. Even though influenza virus alone did induce some systemic and salivary antibody responses after being administered intranasally, these responses were greatly augmented when the virus was presented together with OMVs. Thus, it is possible that a nasal OMV vaccine may induce protection against invasive meningococcal disease, and also that it might be used as a vehicle for nasal vaccines against other diseases.

Adjuvants, Immunologic↗
PubMed2002

The ultrastructure of Ignicoccus: evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon.

A novel genus of hyperthermophilic, strictly chemolithotrophic archaea, Ignicoccus, has been described recently, with (so far) three isolates in pure culture. Cells were prepared for ultrastructural investigation by cultivation in cellulose capillaries and processing by high-pressure freezing, freeze-substitution and embedding in Epon. Cells prepared in accordance with this protocol consistently showed a novel cell envelope structure previously unknown among the Archaea: a cytoplasmic membrane; a periplasmic space with a variable width of 20 to 400 nm, containing membrane-bound vesicles; and an outer sheath, approximately 10 nm wide, resembling the outer membrane of gram-negative bacteria. This sheath contained three types of particles: numerous tightly, irregularly packed single particles, about 8 nm in diameter; pores with a diameter of 24 nm, surrounded by tiny particles, arranged in a ring with a diameter of 130 nm; and clusters of up to eight particles, each particle 12 nm in diameter. Freeze-etched cells exhibited a smooth surface, without a regular pattern, with frequent fracture planes through the outer sheath, indicating the presence of an outer membrane and the absence of an S-layer. The study illustrates the novel complex architecture of the cell envelope of Ignicoccus as well as the importance of elaborate preparation procedures for ultrastructural investigations.

Cell Division↗
PubMed1989

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins.

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.

Adenosine Triphosphate↗
PubMed1983

The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles.

The yeast mitochondrial outer membrane was isolated and 10 of its major polypeptides were identified (mol. wts. 109, 70, 57, 45, 45, 42, 33, 29, 25 and 14 kd). The membrane has no major polypeptide in common with either mitochondrial inner membrane or rough microsomes. Protease treatment and immunochemical techniques showed that virtually all of the isolated outer membrane vesicles are sealed and display the same surface orientation as in the intact mitochondrion.

Journal Article↗
PubMed1989

Characterization of chloride channels in membrane vesicles from the kidney outer medulla.

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na+/K+ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl-. In order to study the Cl- conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl- channel activity was determined by a 36Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The accumulation of 36Cl- inside the vesicles was found to be dependent on the intravesicular Cl- concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl- channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl- gradient. The anion selectivity of the channels was Cl- greater than NO3- = I- much greater than gluconate. Among the Cl- transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4'-diisothiocyano-stilbene-2,2'-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC50 of 110, 200 and 550 microM, respectively. Inhibition of 36Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 microM). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗