Biomedical subjects

Myron Christodoulides

Publications and source records attributed to Myron Christodoulides.

16 recordsLinked to original sources

PubMed2006

Proteomic analysis of outer membranes and vesicles from wild-type serogroup B Neisseria meningitidis and a lipopolysaccharide-deficient mutant.

Current experimental vaccines against serogroup B Neisseria meningitidis are based on meningococcal outer membrane (OM) proteins present in outer membrane vesicles (OMV) in which toxic lipopolysaccharide is depleted by detergent extraction. Knowledge of the composition of OM and OMV is essential for developing new meningococcal vaccines based on defined antigens. In the current study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nanocapillary liquid chromatography-tandem mass spectrometry were used to investigate the proteomes of OM and OMV from meningococcal strain MC58 and OM from a lipopolysaccharide-deficient mutant. The analysis of OM revealed a composition that was much more complex than the composition that has been reported previously; a total of 236 proteins were identified, only 6.4% of which were predicted to be located in the outer membrane. The most abundant proteins included not only the well-established major OM proteins (PorA, PorB, Opc, Rmp, and Opa) but also other proteins, such as pilus-associated protein Q (PilQ) and a putative macrophage infectivity protein. All of these proteins were also present in OMV obtained by extraction of the OM with deoxycholate. There were markedly increased levels of some additional proteins in OM from the lipopolysaccharide-deficient mutant, including enzymes that contribute to the tricarboxylic acid cycle. In all the preparations, the proteins not predicted to have an OM location were predominantly periplasmic or cytoplasmic or had an unknown location, and relatively few cytoplasmic membrane proteins were detected. However, several proteins that have previously been identified as potential vaccine candidates were not detected in either OM preparations or in OMV. These results have important implications for the development and use of vaccines based on outer membrane proteins.

Bacterial Outer Membrane Proteins↗

PubMed2006

Comparison of the inflammatory responses of human meningeal cells following challenge with Neisseria lactamica and with Neisseria meningitidis.

The rationale for the present study was to determine how different species of bacteria interact with cells of the human meninges in order to gain information that would have broad relevance to understanding aspects of the innate immune response in the brain. Neisseria lactamica is an occasional cause of meningitis in humans, and in this study we investigated the in vitro interactions between N. lactamica and cells derived from the leptomeninges in comparison with the closely related organism Neisseria meningitidis, a major cause of meningitis worldwide. N. lactamica adhered specifically to meningioma cells, but the levels of adherence were generally lower than those with N. meningitidis. Meningioma cells challenged with N. lactamica and N. meningitidis secreted significant amounts of the proinflammatory cytokine interleukin-6 (IL-6), the C-X-C chemokine IL-8, and the C-C chemokines monocyte chemoattractant protein 1 (MCP-1) and RANTES, but it secreted very low levels of the cytokine growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). Thus, meningeal cells are involved in the innate host response to Neisseria species that are capable of entering the cerebrospinal fluid. The levels of IL-8 and MCP-1 secretion induced by both bacteria were essentially similar. By contrast, N. lactamica induced significantly lower levels of IL-6 than N. meningitidis. Challenge with the highest concentration of N. lactamica (10(8) CFU) induced a small but significant down-regulation of RANTES secretion, which was not observed with lower concentrations of bacteria. N. meningitidis (10(6) to 10(8) CFU) also down-regulated RANTES secretion, but this effect was significantly greater than that observed with N. lactamica. Although both bacteria were unable to invade meningeal cells directly, host cells remained viable on prolonged challenge with N. lactamica, whereas N. meningitidis induced death; the mechanism was overwhelming necrosis with no significant apoptosis. It is likely that differential expression of modulins between N. lactamica and N. meningitidis contributes to these observed differences in pathogenic potential.

Cells, Cultured↗

PubMed2006

Infection of human fallopian tube epithelial cells with Neisseria gonorrhoeae protects cells from tumor necrosis factor alpha-induced apoptosis.

Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-alpha). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-alpha was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-alpha antibodies; and (iii) the addition of exogenous TNF-alpha induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-alpha-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-alpha-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease.

Apoptosis↗

PubMed2005

Multivalent liposome-based vaccines containing different serosubtypes of PorA protein induce cross-protective bactericidal immune responses against Neisseria meningitidis.

Four serosubtypes (P1.7, 16, P1.7-2, 4, P1.19, 15 and P1.5-1, 10-4) of the PorA outer-membrane protein of Neisseria meningitidis were purified as recombinant proteins and incorporated into liposomes to investigate their immunogenicity. Each serosubtype induced high levels of bactericidal activity against the homologous strain. In addition, liposome preparations containing multiple serosubtypes induced high levels of bactericidal activity against each of the four strains. Significantly, antisera raised against monovalent and multivalent liposomes also showed cross-reactive bactericidal activity against heterologous strains. These data demonstrate that multivalent liposome vaccines, containing multiple PorA serosubtypes, have the potential to provide protection against a broad range of meningococcal strains.

Animals↗

PubMed2005

Activation of human meningeal cells is modulated by lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis and is independent of Toll-like receptor (TLR)4 and TLR2 signalling.

The interactions of Neisseria meningitidis with cells of the meninges are critical to progression of the acute, compartmentalized intracranial inflammatory response that is characteristic of meningococcal meningitis. An important virulence mechanism of the bacteria is the ability to shed outer membrane (OM) blebs containing lipopolysaccharide (LPS), which has been assumed to be the major pro-inflammatory molecule produced during meningitis. Comparison of cytokine induction by human meningeal cells following infection with wild-type meningococci, LPS-deficient meningococci or after treatment with OM isolated from both organisms, demonstrated the involvement of non-LPS bacterial components in cell activation. Significantly, recognition of LPS-replete OM did not depend on host cell expression of Toll-like receptor (TLR)4, the accessory protein MD-2 or CD14, or the recruitment of LPS-accessory surface proteins heat shock protein (HSP)70, HSP90alpha, chemokine receptor CXCR4 and growth differentiation factor (GDF)5. In addition, recognition of LPS-deficient OM was not associated with the expression of TLR2 or any of these other molecules. These data suggest that during meningococcal meningitis innate recognition of both LPS and non-LPS modulins is dependent on the expression of as yet uncharacterized pattern recognition receptors on cells of the meninges. Moreover, the biological consequences of cellular activation by non-LPS modulins suggest that clinical intervention strategies based solely on abrogating the effects of LPS are likely to be only partially effective.

Antigens, Surface↗

PubMed2004

Recombinant meningococcal PorA protein, expressed using a vector system with potential for human vaccination, induces a bactericidal immune response.

The PorA protein of Neisseria meningitidis (subtype P1.7,16) was expressed as a recombinant protein using three vectors; pTWIN, pQE30 and pRSETA, which introduce different sized N-terminal leader sequences to the mature protein. The immunogenicity of these proteins was compared following incorporation into liposomes and ZW-micelles. All of the recombinant PorA (rPorA) preparations induced high titres of antibody that recognised the homologous PorA within the outer membrane (OM) and on the surface of meningococci. Antisera raised against liposomes and micelles containing the different rPorA proteins induced high and comparable levels of complement-mediated killing of the homologous, but not heterologous, strain. Furthermore, the bactericidal effect was greater when rPorA were incorporated into liposomes rather than detergent micelles. The minimal addition of three N-terminal amino acids in rPorA purified from the pTWIN vector represents a significant improvement over rPorA purified from vectors pQE30 and pRSETA, plus other previously purified rPorA, when considering use of these proteins in vaccines for human use.

Animals↗

PubMed2004

Different meningitis-causing bacteria induce distinct inflammatory responses on interaction with cells of the human meninges.

The interactions of bacterial pathogens with cells of the human leptomeninges are critical events in the progression of meningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the interactions of the meningeal pathogens Escherichia coli K1, Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. A rank order of association with meningioma cells was observed, with N. meningitidis showing the highest levels of adherence, followed by E. coli, S. pneumoniae and H. influenzae. Neisseria meningitidis and H. influenzae did not invade meningioma cells or induce cell death, but induced a concentration-dependent secretion of inflammatory mediators. Neisseria meningitidis induced higher levels of IL-6, MCP-1, RANTES and GM-CSF than H. influenzae, but there was no significant difference in the levels of IL-8 induced by both pathogens. Streptococcus pneumoniae was also unable to invade meningioma cells, but low concentrations of bacteria failed to stimulate cytokine secretion. However, higher concentrations of pneumococci led to cell death. By contrast, only E. coli K1 invaded meningioma cells directly and induced rapid cell death before an inflammatory response could be induced. These data demonstrate that the interactions of different bacterial pathogens with human meningeal cells are distinct, and suggest that different intervention strategies may be needed in order to prevent the morbidity and mortality associated with bacterial meningitis.

Bacterial Adhesion↗

PubMed2004

Activation of human dendritic cells by the PorA protein of Neisseria meningitidis.

The major porin proteins present in the outer membrane of Neisseria meningitidis, the causative agent of life-threatening meningitis and septicaemia, are believed to have potent immunostimulatory effects. In this study, the interactions between human monocyte-derived dendritic cells (mo-DC) and the PorA porin were investigated, in order to reveal the role of this protein in promoting innate and adaptive immune responses. Recombinant (r)PorA induced mo-DC maturation, as reflected by reduced receptor-mediated endocytosis, increased production of the chemokines IL-8, RANTES, MIP-1 alpha and MIP-1 beta and augmented expression of the surface markers CD40, CD54, CD80, CD86 and major histocompatibility complex class II molecules. However, rPorA induced either low level or no significant secretion of pro-inflammatory cytokines from mo-DC. The protein potently augmented the capacity of mo-DC to activate both allogeneic CD4(+) memory T-cells and CD4(+)RA(+) naïve T-cells. In addition, rPorA appeared to inhibit the production of IL-12p70 that follows from the interaction between CD40 on the mo-DC and CD40-ligand on T-cells, thereby directing T-cell differentiation towards a Th2 type response. These data demonstrate that PorA is involved in DC activation and in influencing the nature of the T-helper immune response, which are important properties for generating antibody responses required for protective immunity against meningococci and for determining the immuno-adjuvant effects of this protein.

Antigens, CD↗

PubMed2004

Development of immunity to serogroup B meningococci during carriage of Neisseria meningitidis in a cohort of university students.

Understanding the basis of protective immunity is a key requirement for the development of an effective vaccine against infection with Neisseria meningitidis of serogroup B. We have conducted a longitudinal study into the dynamics of meningococcal acquisition and carriage in first-year university students. The detection of carriage of serogroup B meningococci correlated with an increase in detection of serum bactericidal activity (SBA) against both colonizing and heterologous serogroup B strains. Once induced, SBA remained high throughout the study. Although students showed increases in antibodies reactive with capsular polysaccharide and lipopolysaccharide (LPS), these antibody responses were transitory, and their decline was not accompanied by a corresponding decline in SBA. In contrast, there was a significant correlation between the presence of antibodies to the PorA outer membrane protein and SBA against both homologous and heterologous strains. SBA induced by a PorA-negative mutant confirmed the contribution of PorA to heterologous activity. Increases in SBA against a range of serogroup B strains were also observed in students in whom no meningococcal carriage was detected. This heterologous protection could not be associated with the presence of antibodies reacting with capsule, LPS, PorA, PorB, Rmp, Opa, Opc, or pilin, demonstrating that other, as yet unidentified, antigens contribute to the development of immunity to serogroup B meningococci. Identification of such antigens with the ability to induce an effective cross-reactive bactericidal response to a range of strains would be a major step in the production of a universally effective vaccine against infections caused by serogroup B meningococci.

Adolescent↗

PubMed2003

Serological correlates of protection against meningococci in a cohort of university students, before and during an outbreak of serogroup C infection.

The association between individual meningococcal antigens and the development of protective immunity to both serogroup C and B meningococci was studied before and during an outbreak of serogroup C infection among university students. Persons who became infected showed, in serum taken either before infection or on admission to the hospital, low levels of bactericidal activity against the outbreak strain; patients who survived infection developed bactericidal activity that correlated with production of antibodies to serogroup C capsular polysaccharide but not to either lipopolysaccharide or major outer-membrane proteins. Uninfected classmates also showed a strong correlation between bactericidal activity and the presence of anti-capsular antibodies. In contrast, bactericidal activity against serogroup B did not correlate with the presence of antibodies to capsular polysaccharide but did correlate with antibodies reacting with the porin proteins PorA and PorB. These studies support the introduction of conjugate MenC vaccines, validate strategies for prevention of serogroup B infection that are based on vaccines containing PorA, and suggest that PorB may also be an important component of such vaccines.

Adolescent↗

PubMed2003

Expression of proinflammatory cytokines and receptors by human fallopian tubes in organ culture following challenge with Neisseria gonorrhoeae.

Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae can lead to acute salpingitis, an inflammatory condition, which is a major cause of infertility. Challenge of explants of human FT with gonococci induced mRNA expression and protein secretion for the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, and tumor necrosis factor alpha (TNF-alpha) but not for granulocyte-macrophage colony-stimulating factor. In contrast, FT expression of IL-6 and of the cytokine receptors IL-6R, TNF receptor I (TNF-RI), and TNF-RII was constitutive and was not increased by gonococcal challenge. These studies suggest that several proinflammatory cytokines are likely to contribute to the cell and tissue damage observed in gonococcal salpingitis.

Cytokines↗

PubMed2003

Activation of human dendritic cells is modulated by components of the outer membranes of Neisseria meningitidis.

Neisseria meningitidis serogroup B is a major cause of life-threatening meningitis and septicemia worldwide, and no effective vaccine is available. Initiation of innate and acquired immune responses to N. meningitidis is likely to be dependent on cellular responses of dendritic cells (DC) to antigens present in the outer membrane (OM) of the meningococcus. In this study, the responses of human monocyte-derived DC (mo-DC) to OM isolated from parent (lipopolysaccharide [LPS]-replete) meningococci and from a mutant deficient in LPS were investigated. Parent OM selectively up-regulated Toll-like receptor 4 (TLR4) mRNA expression and induced mo-DC maturation, as reflected by increased production of chemokines, proinflammatory cytokines, and CD83, CD80, CD86, CD40, and major histocompatibility complex (MHC) class II molecules. In contrast, LPS-deficient OM selectively up-regulated TLR2 mRNA expression and induced moderate increases in both cytokine production and expression of CD86 and MHC class II molecules. Preexposure to OM, with or without LPS, augmented the allostimulatory properties of mo-DC, which induced proliferation of naive CD4+ CD45RA+ T cells. In addition, LPS-replete OM induced a greater gamma interferon/interleukin-13 ratio in naive T cells, whereas LPS-deficient OM induced the reverse profile. These data demonstrate that components of the OM, other than LPS, are also likely to be involved in determining the levels of DC activation and the nature of the T-helper immune response.

Antigens, CD↗

PubMed2002

Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag.

The class 1 protein (PorA) is a major component of the outer membrane of Neisseria meningitidis and functions as a cationic porin. The protein is particularly effective in generating a bactericidal immune response following infection and is therefore under investigation as a potential antigen for inclusion in new meningococcal vaccines. Studies on the vaccine potential of PorA would be facilitated by the production of pure protein, free from other components of the meningococcal outer membrane. In the current study, PorA was expressed from the heterologous host Escherichia coli as a C-terminal fusion to an inducible protein-splicing element (intein) with an N-terminal chitin-binding domain (CBD) (IMPACT-TWIN system). The CBD acted as an affinity tag and allowed binding of the fusion protein to a chitin bead column, after which self-cleavage of the intein at its C-terminus was induced, resulting in the release of mature PorA. Cleavage of the fusion protein was temperature- and time-dependent, and was optimal at pH 7.0 after 5 days of storage at 4 degrees C. Efficient cleavage was also dependent on the addition of a minimal amino acid sequence (Gly-Arg-Ala) to the N-terminus of the mature PorA protein. This represented a significant improvement on the large N-terminal sequences introduced by other expression systems previously used to prepare recombinant PorA, and the yields of PorA purified with the IMPACT-TWIN system were similar. Thus, the IMPACT-TWIN system provides a facile method for producing recombinant PorA and may also be useful for the production of other bacterial outer-membrane proteins for vaccine studies.

Affinity Labels↗

PubMed2002

Immunization with the recombinant PorB outer membrane protein induces a bactericidal immune response against Neisseria meningitidis.

Infections with Neisseria meningitidis are characterized by life-threatening meningitis and septicemia. The meningococcal porin proteins from serogroup B meningococci have been identified as candidates for inclusion in vaccines to prevent such infections. In this study, we investigated the vaccine potential of the PorB porin protein free of other meningococcal components. The porB gene from a strain of Neisseria meningitidis expressing the class 3 outer membrane porin protein (PorB3) was cloned into the pRSETB vector, and the protein was expressed at high levels in a heterologous host Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and used for immunization after incorporation into liposomes and into micelles composed either of zwitterionic detergent or nondetergent sulfobetaine. The immunogenicity of these preparations was compared to recombinant PorB protein adsorbed to Al(OH)(3) adjuvant as a control. Although sera raised against the protein adsorbed to Al(OH)(3) reacted with the purified recombinant protein, sera raised against liposomes and micelles showed greater activity with native protein, as measured by enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Reactivity with native protein was considerably enhanced by incorporation of the adjuvant monophosphoryl lipid A into the liposome or micelle preparations. Recognition of the native protein was in a serotype-specific manner and was associated with the ability of the antisera to promote high levels of serotype-specific complement-mediated killing of meningococci. These results demonstrate that the PorB protein should be considered as a component of a vaccine designed to prevent serogroup B meningococcal infection.

Animals↗

PubMed2002

Interaction of Neisseria meningitidis with human meningeal cells induces the secretion of a distinct group of chemotactic, proinflammatory, and growth-factor cytokines.

The interactions of Neisseria meningitidis with cells of the leptomeninges are pivotal events in the progression of bacterial leptomeningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the role of the leptomeninges in the inflammatory response. Following challenge with meningococci, meningioma cells secreted specifically the proinflammatory cytokine interleukin-6 (IL-6), the CXC chemokine IL-8, the CC chemokines monocyte chemoattractant protein 1 (MCP-1) and regulated-upon-activation, normal-T-cell expressed and secreted protein (RANTES), and the cytokine growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). A temporal pattern of cytokine production was observed, with early secretion of IL-6, IL-8, and MCP-1 followed by later increases in RANTES and GM-CSF levels. IL-6 was induced equally by the interactions of piliated and nonpiliated meningococci, whereas lipopolysaccharide (LPS) had a minimal effect, suggesting that other, possibly secreted, bacterial components were responsible. Induction of IL-8 and MCP-1 also did not require adherence of bacteria to meningeal cells, but LPS was implicated. In contrast, efficient stimulation of RANTES by intact meningococci required pilus-mediated adherence, which served to deliver increased local concentrations of LPS onto the surface of meningeal cells. Secretion of GM-CSF was induced by pilus-mediated interactions but did not involve LPS. In addition, capsule expression had a specific inhibitory effect on GM-CSF secretion, which was not observed with IL-6, IL-8, MCP-1, or RANTES. Thus, the data demonstrate that cells of the leptomeninges are not inert but are active participants in the innate host response during leptomeningitis and that there is a complex relationship between expression of meningococcal components and cytokine induction.

Bacterial Capsules↗

PubMed1998

Immunization with recombinant class 1 outer-membrane protein from Neisseria meningitidis: influence of liposomes and adjuvants on antibody avidity, recognition of native protein and the induction of a bactericidal immune response against meningococci.

The porA gene from Neisseria meningitidis was cloned into the pRSETA vector and recombinant class 1 outer-membrane protein expressed at high levels in Escherichia coli. The protein was readily purified by affinity chromatography on a Ni2+ matrix and used for immunization of mice with conventional AI(OH)3 adjuvant, with experimental adjuvants which have the potential for human use, and with liposomes. The resulting sera were analysed for the magnitude, subclass distribution and antigenic specificity of the immune response. In addition, surface plasmon resonance (SPR) was used to quantify antibody avidity by analysis of the kinetics of binding to native class 1 protein. Immunization with conventional and experimental adjuvants induced antibodies of low avidity that did not recognize native class 1 protein. In contrast, immunization with recombinant protein in liposomes induced antibodies of high avidity which recognized native class 1 protein, as measured by their ability to label meningococcal cells in immunofluorescence assays and to inhibit the binding of a protective mAb. These properties were associated with the presence in sera of high levels of antibodies with the ability to induce complement-mediated killing of meningococci. These data show that liposomes containing recombinant class 1 protein represent a potential basis of future vaccines, of defined composition, designed for the prevention of group B meningococcal infections.

Adjuvants, Immunologic↗