The potential utility of liposomes for Neisseria vaccines.

INTRODUCTION: Species of the genus Neisseria are important global pathogens. Neisseria gonorrhoeae (gonococcus) causes the sexually transmitted disease gonorrhea and Neisseria meningitidis (meningococcus) causes meningitis and sepsis. Liposomes are self-assembled spheres of phospholipid bilayers enclosing a central aqueous space, and they have attracted much interest and use as a delivery vehicle for Neisseria vaccine antigens. AREAS COVERED: A brief background on Neisseria infections and the success of licensed meningococcal vaccines are provided. The absence of a gonococcal vaccine is highlighted. The use of liposomes for delivering Neisseria antigens and adjuvants, for the purposes of generating specific immune responses, is reviewed. The use of other lipid-based systems for antigen and adjuvant delivery is examined briefly. EXPERT OPINION: With renewed interest in developing a gonococcal vaccine, liposomes remain an attractive option for delivering antigens. The discipline of nanotechnology provides additional nanoparticle-based options for gonococcal vaccine development. Future work would be needed to tailor the composition of liposomes and other nanoparticles to the specific vaccine antigen(s), in order to generate optimal anti-gonococcal immune responses. The potential use of liposomes and other nanoparticles to deliver anti-gonococcal compounds to treat infections also should be explored further.

Gonococcal Colony Typing.

Gonococcal colony typing is part a science and part an art that has been central to studies which have identified crucial virulence antigens and also demonstrated the ability of the bacteria to undergo rapid phase and antigenic variation. Without this fundamental work, modern molecular biological studies of gonococcal pathogenesis would not have been possible. Indeed colony typing is still essential when performing biological experiments with clinical and laboratory isolates and for monitoring their outcome. In this chapter, the methods for performing colony typing and techniques to optimize the process are described.

Novel approaches to Neisseria meningitidis vaccine design.

A range of vaccines is available for preventing life-threatening diseases caused by infection with Neisseria meningitidis (meningococcus, Men). Capsule polysaccharide (CPS)-conjugate vaccines are successful prophylactics for serogroup MenA, MenC, MenW and MenY infections, and outer membrane vesicle (OMV) vaccines have been used successfully for controlling clonal serogroup MenB infections. MenB vaccines based on recombinant proteins identified by reverse vaccinology (Bexsero™) and proteomics (Trumenba™) approaches have recently been licensed and Bexsero™ has been introduced into the UK infant immunisation programme. In this review, we chart the development of these licensed vaccines. In addition, we discuss the plethora of novel vaccinology approaches that have been applied to the meningococcus with varying success in pre-clinical studies, but which provide technological platforms for application to other pathogens. These strategies include modifying CPS, lipooligosaccharide and OMV; the use of recombinant proteins; structural vaccinology approaches of designing synthetic peptide/mimetope vaccines, DNA vaccines and engineered proteins; epitope presentation on biological and synthetic particles; through vaccination with live-attenuated pathogen(s), or with heterologous bacteria expressing vaccine antigens, or to competitive occupation of the nasopharyngeal niche by commensal bacterial spp. After close to a century of vaccine research, it is possible that meningococcal disease may be added, shortly, to the list of diseases to have been eradicated worldwide by rigorous vaccination campaigns.

Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis.

The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3-14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a Δnmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on Δnmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3-14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the hSBA assay, which may correlate with variable surface exposure of CBF. Regardless, the attributes of amino acid sequence conservation and protein expression amongst different strains and the ability to induce cross-strain bactericidal antibodies indicates that rCBF could be a potential meningococcal vaccine antigen and merits further testing.

Vaccine potential of bacterial macrophage infectivity potentiator (MIP)-like peptidyl prolyl cis/trans isomerase (PPIase) proteins.

Peptidyl prolyl cis/trans isomerases (PPIases) are a superfamily of proteins ubiquitously distributed among living organisms, which function primarily to assist the folding and structuring of unfolded and partially folded polypeptide chains and proteins. In this review, we focus specifically on the Macrophage Infectivity Potentiator (MIP)-like PPIases, which are members of the immunophilin family of FK506-binding proteins (FKBP). MIP-like PPIases have accessory roles in virulence and are candidates for inclusion in vaccines protective against both animal and human bacterial pathogens. A structural vaccinology approach obviates any issues over molecular mimicry and potential cross-reactivity with human FKBP proteins and studies with a representative antigen, the Neisseria meningitidis-MIP, support this strategy. Moreover, a dual approach of vaccination and drug targeting could be considered for controlling bacterial infectious diseases of humans and animals.

A putative amino acid ABC transporter substrate-binding protein, NMB1612, from Neisseria meningitidis, induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins.

The nmb1612 (NEIS1533) gene encoding the ~27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)3 and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre <4) in a human serum bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same protein was killed (median titres of 16-64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines.

Recombinant protein truncation strategy for inducing bactericidal antibodies to the macrophage infectivity potentiator protein of Neisseria meningitidis and circumventing potential cross-reactivity with human FK506-binding proteins.

A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (-LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmip mutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines.

Immuno-proteomic analysis of human immune responses to experimental Neisseria meningitidis outer membrane vesicle vaccines identifies potential cross-reactive antigens.

Human volunteers were vaccinated with experimental Neisseria meningitidis serogroup B vaccines based on strain H44/76 detoxified L3 lipooligosaccharide (LOS)-derived outer membrane vesicles (OMV) or the licensed Cuban vaccine, VA-MENGOC-BC. Some volunteers were able to elicit cross-bactericidal antibodies against heterologous L2-LOS strain (760676). An immuno-proteomic approach was used to identify potential targets of these cross-bactericidal antibodies using an L2-LOS derived OMV preparation. A total of nine immuno-reactive spots were detected in this proteome: individuals vaccinated with the detoxified OMVs showed an increase in post-vaccination serum reactivity with Spots 2-8, but not with Spots 1 and 9. Vaccination with VA-MENGOC-BC induced sera that showed increased reactivity with all of the protein spots. Vaccinees showed increases in serum bactericidal activity (SBA) against the heterologous L2-LOS expressing strain 760676, which correlated, in general, with immunoblot reactivity. The identities of proteins within the immuno-reactive spots were determined. These included not only well-studied antigens such as Rmp, Opa, PorB and FbpA (NMB0634), but also identified novel antigens such as exopolyphosphatase (NMB1467) and γ-glutamyltranspeptidase (NMB1057) enzymes and a putative cell binding factor (NMB0345) protein. Investigating the biological properties of such novel antigens may provide candidates for the development of second generation meningococcal vaccines.

Immunization with recombinant Chaperonin60 (Chp60) outer membrane protein induces a bactericidal antibody response against Neisseria meningitidis.

Sera from individuals colonized with Neisseria meningitidis and from patients with meningococcal disease contain antibodies specific for the neisserial heat-shock/chaperonin (Chp)60 protein. In this study, immunization of mice with recombinant (r)Chp60 in saline; adsorbed to aluminium hydroxide; in liposomes and detergent micelles, with and without the adjuvant MonoPhosphoryl Lipid A (MPLA), induced high and similar (p>0.05) levels of antibodies that recognized Chp60 in outer membranes (OM). FACS analysis and immuno-fluorescence experiments demonstrated that Chp60 was surface-expressed on meningococci. By western blotting, murine anti-rChp60 sera recognized a protein of Mr 60kDa in meningococcal cell lysates. However, cross-reactivity with human HSP60 protein was also observed. By comparing translated protein sequences of strains, 40 different alleles were found in meningococci in the Bacterial Isolate Genome Sequence database with an additional 5 new alleles found in our selection of 13 other strains from colonized individuals and patients. Comparison of the non-redundant translated amino acid sequences from all the strains revealed ≥97% identity between meningococcal Chp60 proteins, and in our 13 strains the protein was expressed to high and similar levels. Bactericidal antibodies (median reciprocal titres of 32-64) against the homologous strain MC58 were induced by immunization with rChp60 in liposomes, detergent micelles and on Al(OH)3. Bactericidal activity was influenced by the addition of MPLA and the delivery formulation used. Moreover, the biological activity of anti-Chp60 antisera did not extend significantly to heterologous meningococcal strains. Thus, in order to provide broad coverage, vaccines based on Chp60 would require multiple proteins and specific bactericidal epitope identification.

The adhesin complex protein (ACP) of Neisseria meningitidis is a new adhesin with vaccine potential.

UNLABELLED: The acp gene encoding the 13-kDa adhesin complex protein (ACP) from Neisseria meningitidis serogroup B strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant ACP (rACP) was used for immunization studies. Analysis of the ACP amino acid sequences from 13 meningococcal strains, isolated from patients and colonized individuals, and 178 strains in the Bacterial Isolate Genome Sequence (BIGS) database showed the presence of only three distinct sequence types (I, II, and III) with high similarity (> 98%). Immunization of mice with type I rACP in detergent micelles and liposomes and in saline solution alone induced high levels of serum bactericidal activity (SBA; titers of 1/512) against the homologous strain MC58 and killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/512). Levels of expression of type I, II, or III ACP by different meningococcal strains were similar. ACP functioned as an adhesin, as demonstrated by reduced adherence of acp knockout (MC58 ΔACP) meningococci to human cells in vitro and the direct surface binding of rACP and by the ability of anti-rACP sera to inhibit adherence of wild-type bacteria. ACP also mediated the invasion of noncapsular meningococci into human epithelial cells, but it was not a particularly impressive invasin, as the internalized bacterial numbers were low. In summary, the newly identified ACP protein is an adhesin that induces cross-strain bactericidal activity and is therefore an attractive target antigen for incorporation into the next generation of serogroup B meningococcal vaccines. IMPORTANCE: Infections caused by Neisseria meningitidis serogroup B are still significant causes of mortality and morbidity worldwide, and broadly protective vaccines of defined antigen composition are not yet licensed. Here, we describe the properties of the adhesin complex protein (ACP), which we demonstrate is a newly recognized molecule that is highly conserved and expressed to similar levels in meningococci and facilitates meningococcal interactions with human cells. We also report that a recombinant ACP protein vaccine induces murine antibodies that significantly kill meningococci expressing different ACP. Taken together, these properties demonstrate that ACP merits serious consideration as a component of a broadly protective vaccine against serogroup B meningococci.

Group B Streptococcus interactions with human meningeal cells and astrocytes in vitro.

BACKGROUND: Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of life-threatening neonatal meningitis and survivors often suffer permanent neurological damage. How this organism interacts with the meninges and subsequently with astrocytes that constitute the underlying cortical glia limitans superficialis is not known. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate dose-dependent adherence of GBS over time to human meningioma cells and fetal astrocytes in vitro, which was not influenced by expression of either ß-haemolysin/cytolysin (ß-h/c) toxin, different capsule serotypes or by absence of capsule (p>0.05). Internalization of GBS by both cell types was, however, a slow and an infrequent event (only 0.02-0.4% of associated bacteria were internalised by 9 h). Expression of ß-h/c toxin did not play a role in invasion (p>0.05), whereas capsule expression lead to a reduction (p<0.05) in the numbers of intracellular bacteria recovered. GBS strains induced cytotoxicity as demonstrated by the measurement of lactate dehydrogenase (LDH) enzyme release by 9 h and by viable staining. Increasing levels of meningioma cell death correlated with bacterial growth and the phenotype of ß-h/c toxin production, i.e. from weakly, to normo- to hyper-haemolytic. However, cytotoxicity was significantly greater (p<0.05) towards astrocytes, and infection with initial MOI≥0.003 induced 70-100% LDH release. By comparing wild-type (ß-h/c(+)) and mutant (ΔcylE ß-h/c(-)) strains and ß-h/c toxin extracts and by using the surfactant dipalmitoylphosphatidylcholine in cytotoxicity inhibition experiments, ß-h/c toxin was demonstrated as principally responsible for cell death. CONCLUSIONS/SIGNIFICANCE: This study has described key events in the interactions of GBS with meningeal cells and astrocytes in vitro and a major virulence role for ß-h/c toxin. Understanding the mechanisms involved will help to identify potential therapies for improving patient survival and for reducing the incidence and severity of neurological sequelae.

Coadministration of the cyanobacterial lipopolysaccharide antagonist CyP with antibiotic inhibits cytokine production by an in vitro meningitis model infected with Neisseria meningitidis.

OBJECTIVES: In this study, the objective was to determine the anti-inflammatory properties of CyP, a cyanobacterial lipopolysaccharide (LPS) antagonist, used in combination with antibiotic chemotherapy during infection of an in vitro meningitis model infected with Neisseria meningitidis (meningococcus). METHODS: Monocultures of human meningioma cells and meningioma-primary human macrophage co-cultures were infected with meningococci (10(2)-10(8) cfu/monolayer) or treated with isolated outer membranes or purified LPS (0.1-100 ng/monolayer) from N. meningitidis. CyP (1-20 µg/monolayer) was added at intervals from t = 0 to 4 h, with and without benzylpenicillin (1-20 µg/monolayer). The antagonistic effect of CyP and its adjunctive properties to benzylpenicillin administration was determined by measuring cytokine levels in culture supernatants after 24 h. RESULTS: CyP significantly inhibited (P < 0.05) the secretion of interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 and RANTES ('regulated upon activation, normal T cell expressed and secreted') (overall reduction levels from 50% to >95%) by meningioma cell lines and meningioma-macrophage co-cultures challenged with either live meningococci or bacterial components. Inhibition was effective when CyP was added within 2 h of challenge (P < 0.05) and was still pronounced by 4 h. In the co-culture model, CyP alone partially inhibited IL-1ß secretion, but did not prevent tumour necrosis factor (TNF)-α secretion, whereas penicillin alone inhibited IL-1ß and TNF-α but conversely did not reduce MCP-1 and RANTES secretion. However, coadministration of CyP and penicillin in both models had an additive effect and restored the overall inhibitory profile. CONCLUSIONS: CyP inhibits cytokine production in an in vitro meningitis model and augments the anti-inflammatory response when combined with benzylpenicillin. Administration of an LPS antagonist with antibiotic merits consideration in the emergency treatment of patients presenting with meningococcal infection.

Neisseria gonorrhoeae pilus attenuates cytokine response of human fallopian tube explants.

BACKGROUND: A role for pilus during attachment of Neisseria gonorrhoeae to epithelia of the female reproductive tract is currently assumed. However, Pil⁻ gonococci have been observed during infection of the reproductive tract, which prompted us to examine the effect of pili on the dynamics of infection and the inflammatory responses of mucosal explants of the human fallopian tube. METHODS: Mucosal explants were infected in vitro with Opa negative Pil⁻ and Pil⁺N. gonorrhoeae strains. RESULTS: Piliation enhanced gonococcal adherence to the epithelium within 3 h of infection (P < 0.05) but thereafter did not offer advantage to gonococci to colonize the epithelial cell surface (P > 0.05). No differences were found between the strains in numbers of gonococci inside epithelial cells. Pil⁻ bacteria induced higher levels (P < 0.05) of IL-1ß, TNF-α, GM-CSF, MCP-1, and MIP-1ß than Pil⁺ bacteria. There were no differences between both strains in LOS pattern, and Pil expression did not change after coincubation with mucosal strips. CONCLUSIONS: Results show that gonococcal invasion of the human fallopian tube can occur independently of pilus or Opa expression, and suggest that pilus, by inhibition of several key elements of the initial inflammatory response, facilitates sustained infection of this organ.

Analysis of the immune response to Neisseria meningitidis using a proteomics approach.

The availability of Neisseria genome sequences together with improvements in proteomic technologies provide the opportunity to study at high resolution the immune response to Neisseria meningitidis. In this chapter, we describe a protocol that combines two-dimensional (2D) SDS-PAGE of meningococcal outer membranes with western blotting of human antisera to identify proteins associated with the development of protective antibody responses. This methodology can identify putative vaccine candidates for incorporation in a multi-component serogroup B meningococcal vaccine.

The Neisseria meningitidis macrophage infectivity potentiator protein induces cross-strain serum bactericidal activity and is a potential serogroup B vaccine candidate.

A gene encoding a 29-kDa protein from Neisseria meningitidis serogroup B strain MC58 with homology to the macrophage infectivity potentiator (MIP) protein of Legionella pneumophila was cloned and expressed in Escherichia coli, and the purified soluble recombinant protein (rMIP) was used for immunization studies. Analysis of the predicted amino acid sequences of MIP from 13 well-characterized meningococcal strains, isolated from carriers or patients and differing in serogroup, serotype, and subtype, showed that the protein was highly conserved (98 to 100%), with only three distinct sequence types (designated I, II, and III) found. Western blotting showed that the MIP protein was expressed at similar levels by all of these strains. Immunization of mice with type I MC58 rMIP in detergent micelles and liposomes containing monophosphoryl lipid A (MPLA) induced high levels of surface-reactive antibodies with serum bactericidal activity (SBA) titers of 1/1,024 against the homologous strain. Bactericidal antibodies were also induced with the protein in saline alone and liposomes alone (titers, 1/128) but not following adsorption to Al(OH)(3). Significantly, antisera raised against type I rMIP administered in saline or liposomes killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/256). Taken together, these findings suggest that rMIP can provide cross-strain protection against meningococci and should be considered a potential antigen for inclusion in new vaccines against meningococcal infection.

Nitric oxide is not involved in Neisseria gonorrhoeae-induced cellular damage of human Fallopian tubes in vitro.

In the present study, we investigated whether cellular damage, as demonstrated by lactate dehydrogenase (LDH) release in the human fallopian tube (FT) infected by Neisseria gonorrhoeae (Ngo), correlated with high levels of nitric oxide synthase (NOS) mRNA and enzyme activity. Infection with Ngo induced a significant increase (~35-fold) in mRNA transcripts of the inducible isoform of NOS. Paradoxically, a reduction in NOS enzyme activity was observed in infected cultures, suggesting that gonococcal infection possibly influences translation of iNOS mRNA to the enzyme. In addition, treatment with the NOS inhibitor TRIM did not prevent gonococcal-induced cellular damage. In contrast, the addition of the inhibitor L-NAME induced a 40% reduction in LDH release, which correlated with a ~50% reduction in gonococcal numbers. Moreover, treatment of normal FT explants with an exogenous NO donor, SNAP, did not induce significant cellular damage. Taken together, our data suggest that NO does not contribute to cellular damage during infection of the human FT with Neisseria gonorrhoeae.

The influence of genomics and proteomics on the development of potential vaccines against meningococcal infection.

There is a particular need for an effective vaccine against life-threatening meningitis and septicemia caused by Neisseria meningitidis (meningococcus) serogroup B strains. Vaccine strategies incorporating capsular polysaccharide have proved effective against other meningococcal serogroups, but are not applicable to serogroup B. Attention has therefore focused on the subcapsular outer membrane protein antigens as potential vaccine components. The sequencing of genomes from three serogroups and the availability of the corresponding translated protein databases, combined with the development of sensitive proteomic techniques, have opened up new avenues of meningococcal vaccine research. This has resulted in the identification of potential candidate antigens for incorporation into multicomponent meningococcal vaccines.

Neuropeptide alpha-MSH exerts pro-inflammatory effects on Neisseria meningitidis infection in vitro.

OBJECTIVE AND DESIGN: alpha-Melanoycte stimulating hormone (alpha-MSH), a neuropeptide hormone with reported anti-microbial and immuno-modulatory properties in vitro, has previously been detected in the cerebrospinal fluid of children with bacterial meningitis. We investigated the therapeutic effects of alpha-MSH administration on Neisseria meningitidis infection of human meningeal cell cultures in vitro. MATERIALS AND METHODS: Meningeal cell lines (n = 2) were infected with meningococci (10(2)-10(8) cfu/monolayer), isolated bacterial outer membranes (OM; 1 microg/ml) or lipo-oligosaccharide (LOS; 1 microg/ml) with and without alpha-MSH (10(-5)-10 microM). Bacterial adherence was quantified at 6 h, and cytokine production and microbicidal activity of alpha-MSH for meningococci were assessed at 24 h. RESULTS: Compared with infection by meningococci alone, alpha-MSH (10 microM) up-regulated secretion of IL-6 and IL-8 (mean values increased from approximately 33 to 60 ng/ml), RANTES (mean values increased from approximately 26 to 105 ng/ml) and GM-CSF (mean values increased from approximately 0.3 to 1 ng/ml; P < 0.05). Upregulated secretion correlated with a neuropeptide-mediated rapid and >5-fold increase (P < 0.05) in bacterial adherence to cells and was dependent on OM components including LOS acting synergistically with alpha-MSH. Meningococci were resistant to the anti-microbial activity of alpha-MSH at all concentrations tested. CONCLUSIONS: Our study demonstrates that a potentially therapeutic neuropeptide exerts pro-inflammatory effects during meningococcal infection in vitro and its use in the treatment of meningitis is contra-indicated.

Nitric oxide is not involved in Neisseria gonorrhoeae-induced cellular damage of human Fallopian tubes in vitro

In the present study, we investigated whether cellular damage, as demonstrated by lactate dehydrogenase (LDH) release in the human fallopian tube (FT) infected by Neisseria gonorrhoeae (Ngo), correlated with high levels of nitric oxide synthase (NOS) mRNA and enzyme activity. Infection with Ngo induced a significant increase (~35-fold) in mRNA transcripts of the inducible isoform of NOS. Paradoxically, a reduction in NOS enzyme activity was observed in infected cultures, suggesting that gonococcal infection possibly influences translation of iNOS mRNA to the enzyme. In addition, treatment with the NOS inhibitor TRIM did not prevent gonococcal-induced cellular damage. In contrast, the addition of the inhibitor L-NAME induced a 40 percent reduction in LDH release, which correlated with a ~50 percent reduction in gonococcal numbers. Moreover, treatment of normal FT explants with an exogenous NO donor, SNAP, did not induce significant cellular damage. Taken together, our data suggest that NO does not contribute to cellular damage during infection of the human FT with Neisseria gonorrhoeae.