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Pilot study of human experimental challenge with neisseria lactamica
424
Abstracts
cytochrome C translocation was observed in D39 and DR6, but not SC infected cells. These data were corroborated by nuclear morphology, which showed significantly higher levels of apoptosis in D39 and DR6 than in SC infected or MI THP-1s or MDMs. Infection with D39 resulted in the translocation and activation of the lysosomal protease, cathepsin D. Treatment of cells with the acid protease inhibitor pepstatin A reduced MMP and cytochrome C translocation. These data imply that the pore forming cytolytic activity of PLY is not essential for the induction of macrophage apoptosis, and that protein-protein interactions may be of more relevance, in particular, any PLY interactions with lysosomal factors which may serve to drive apoptosis through novel pathways such as cathepsin activation. Identification of these early events is essential before therapeutic manipulation of host-mediated apoptosis can be carried out.
a response to at least one strain of meningococci, it is likely that individual exposure to N. lactamica is resulting in a booster response of one or more specific antigens which are found on the outer membrane of these different strains of meningococci. These results support the hypothesis that colonisation by Neisseria lactamica does result in cross protection against meningococci.
PILOT STUDY OF HUMAN EXPERIMENTAL CHALLENGE WITH NEISSERIA LACTAMICA
Abstract Introduction: Involvement of the central nervous system (CNS) is the most serious manifestation of extra pulmonary tuberculosis (TB). Matrix metalloproteinases (MMPs) are upregulated in CNS TB and are implicated in the breakdown of the blood brain barrier. The inflammatory phenotype of microglia, resident CNS macrophages, is distinct from that of infiltrating monocytes/macrophages. The transcription factor NFjB has a central role in the up-regulation of many inflammatory genes, thus we hypothesised that NFjB differentially controls secretion of MMPs and their specific tissue inhibitors (TIMPs). Methods: In a cellular model of CNS TB human microglial cells were stimulated with conditioned media from Mycobacterium tuberculosis (M. tb)-infected human monocytes (CoMTB). Cells were pre-incubated with NFjB blocking chemicals and secretion of MMP-1 was measured by casein zymography and western blotting. MMP-3 and TIMP-1 were measured by ELISA. Gene expression and regulation were studied by real time RT-PCR and dual luciferase promoter reporter assays. NFjB subunit kinetics were studied using oligonucleotide based transcription factor ELISAs and by western blotting. Results: CoMTB increased MMP-1 secretion 13-fold and MMP-3 secretion 5-fold compared to controls (P<0.05). Blockade of the NFjB p65 subunit by helenalin and upstream IKK-2 inhibition by SC-514 demonstrated dose-dependent decreases in the secretion of MMP-1 and MMP-3 (P<0.0001) with no associated change in the expression of TIMP-1. Blockade of NFjB also inhibited MMP-1 and MMP-3 mRNA accumulation. Dual luciferase promotor reporter assays, using specific constructs with a mutated NFjB binding site, further established the critical importance of the consensus NFjB site in the MMP-1 promoter. CoMTB rapidly upregulated p65-p50 heterodimer recruitment to the nucleus at 30 minutes, with concomittant IjB-a degradation. However, the p65 signal only returned to baseline at 24 hours despite resynthesis of IjB-a by 60 minutes. Discussion: M. tb driven monocyte networks up-regulate MMP-1 and 3 secretion in human microglia. Early and sustained NFjB activity appears to be a key early step in this process. Better understanding of the mechanisms involved in CNS TB tissue degradation could identify novel therapeutic targets for this devastating disease.
C. Evans 1, A. Gorringe 2, M. Matheson 2, C. Pratt 2, R. Borrow 3, J. Findlow 3, R.C. Read 1 1 University of Sheffield, Sheffield, United Kingdom 2 Health Protection Agency, Porton Down, United Kingdom 3 Health Protection Agency, Manchester, United Kingdom Abstract Introduction: Neisseria lactamica is a non pathogenic commensal bacteria commonly found in the upper respiratory tract of infants. Its carriage dynamics have been extensively studied in young children and a clear correlation has been documented between colonisation by N. lactamica and protection from meningococcal disease. This is thought to be due to common antigens resulting in a cross protective immune response. Objectives: The aim of this study was to inoculate Neisseria negative healthy adult volunteers, with live N. lactamica and monitor their specific mucosal and systemic immune response, then analyse their serum bactericidal antibody response against 6 UK prevalent meningococcal B strains. Methodology: 9 individuals were inoculated with live N. lactamica, resulting in 5 successful colonisations. Colonisation was confirmed using nasopharyngeal swabs and gargles. Saliva and blood samples were taken at 0 weeks, 2, 4, 7, and 12 weeks. Findings: Results demonstrated a specific mucosal IgA and systemic IgG immune response to colonisation by N. lactamica. The specific mucosal IgA response from baseline to 12 weeks, comparing non colonised and colonised individuals was significant, p value Z0.0159. Baseline specific IgG and peak fold rise for each individual, using a Mann Whitney test, demonstrates a value of pZ0.063 in colonised individuals. Discussion: Most interestingly colonised individuals demonstrate a greater range of SBA responses to different meningococcal strains, than the non colonised group. Within the colonised individuals 2 volunteers had a significant (greater than 4 fold rise) response to 4 different meningococcal strains. Overall, all volunteers mounted
MATRIX METALLOPROTEINASE DRIVEN TISSUE DESTRUCTION IN CENTRAL NERVOUS SYSTEM TUBERCULOSIS IS REGULATED BY NFjB Justin Green, Shruti Dholakia, Rachel Moores, Lucinda Rand, Paul Ekington, Jon Friedland Imperial College, London, United Kingdom
Abstracts
cytochrome C translocation was observed in D39 and DR6, but not SC infected cells. These data were corroborated by nuclear morphology, which showed significantly higher levels of apoptosis in D39 and DR6 than in SC infected or MI THP-1s or MDMs. Infection with D39 resulted in the translocation and activation of the lysosomal protease, cathepsin D. Treatment of cells with the acid protease inhibitor pepstatin A reduced MMP and cytochrome C translocation. These data imply that the pore forming cytolytic activity of PLY is not essential for the induction of macrophage apoptosis, and that protein-protein interactions may be of more relevance, in particular, any PLY interactions with lysosomal factors which may serve to drive apoptosis through novel pathways such as cathepsin activation. Identification of these early events is essential before therapeutic manipulation of host-mediated apoptosis can be carried out.
a response to at least one strain of meningococci, it is likely that individual exposure to N. lactamica is resulting in a booster response of one or more specific antigens which are found on the outer membrane of these different strains of meningococci. These results support the hypothesis that colonisation by Neisseria lactamica does result in cross protection against meningococci.
PILOT STUDY OF HUMAN EXPERIMENTAL CHALLENGE WITH NEISSERIA LACTAMICA
Abstract Introduction: Involvement of the central nervous system (CNS) is the most serious manifestation of extra pulmonary tuberculosis (TB). Matrix metalloproteinases (MMPs) are upregulated in CNS TB and are implicated in the breakdown of the blood brain barrier. The inflammatory phenotype of microglia, resident CNS macrophages, is distinct from that of infiltrating monocytes/macrophages. The transcription factor NFjB has a central role in the up-regulation of many inflammatory genes, thus we hypothesised that NFjB differentially controls secretion of MMPs and their specific tissue inhibitors (TIMPs). Methods: In a cellular model of CNS TB human microglial cells were stimulated with conditioned media from Mycobacterium tuberculosis (M. tb)-infected human monocytes (CoMTB). Cells were pre-incubated with NFjB blocking chemicals and secretion of MMP-1 was measured by casein zymography and western blotting. MMP-3 and TIMP-1 were measured by ELISA. Gene expression and regulation were studied by real time RT-PCR and dual luciferase promoter reporter assays. NFjB subunit kinetics were studied using oligonucleotide based transcription factor ELISAs and by western blotting. Results: CoMTB increased MMP-1 secretion 13-fold and MMP-3 secretion 5-fold compared to controls (P<0.05). Blockade of the NFjB p65 subunit by helenalin and upstream IKK-2 inhibition by SC-514 demonstrated dose-dependent decreases in the secretion of MMP-1 and MMP-3 (P<0.0001) with no associated change in the expression of TIMP-1. Blockade of NFjB also inhibited MMP-1 and MMP-3 mRNA accumulation. Dual luciferase promotor reporter assays, using specific constructs with a mutated NFjB binding site, further established the critical importance of the consensus NFjB site in the MMP-1 promoter. CoMTB rapidly upregulated p65-p50 heterodimer recruitment to the nucleus at 30 minutes, with concomittant IjB-a degradation. However, the p65 signal only returned to baseline at 24 hours despite resynthesis of IjB-a by 60 minutes. Discussion: M. tb driven monocyte networks up-regulate MMP-1 and 3 secretion in human microglia. Early and sustained NFjB activity appears to be a key early step in this process. Better understanding of the mechanisms involved in CNS TB tissue degradation could identify novel therapeutic targets for this devastating disease.
C. Evans 1, A. Gorringe 2, M. Matheson 2, C. Pratt 2, R. Borrow 3, J. Findlow 3, R.C. Read 1 1 University of Sheffield, Sheffield, United Kingdom 2 Health Protection Agency, Porton Down, United Kingdom 3 Health Protection Agency, Manchester, United Kingdom Abstract Introduction: Neisseria lactamica is a non pathogenic commensal bacteria commonly found in the upper respiratory tract of infants. Its carriage dynamics have been extensively studied in young children and a clear correlation has been documented between colonisation by N. lactamica and protection from meningococcal disease. This is thought to be due to common antigens resulting in a cross protective immune response. Objectives: The aim of this study was to inoculate Neisseria negative healthy adult volunteers, with live N. lactamica and monitor their specific mucosal and systemic immune response, then analyse their serum bactericidal antibody response against 6 UK prevalent meningococcal B strains. Methodology: 9 individuals were inoculated with live N. lactamica, resulting in 5 successful colonisations. Colonisation was confirmed using nasopharyngeal swabs and gargles. Saliva and blood samples were taken at 0 weeks, 2, 4, 7, and 12 weeks. Findings: Results demonstrated a specific mucosal IgA and systemic IgG immune response to colonisation by N. lactamica. The specific mucosal IgA response from baseline to 12 weeks, comparing non colonised and colonised individuals was significant, p value Z0.0159. Baseline specific IgG and peak fold rise for each individual, using a Mann Whitney test, demonstrates a value of pZ0.063 in colonised individuals. Discussion: Most interestingly colonised individuals demonstrate a greater range of SBA responses to different meningococcal strains, than the non colonised group. Within the colonised individuals 2 volunteers had a significant (greater than 4 fold rise) response to 4 different meningococcal strains. Overall, all volunteers mounted
MATRIX METALLOPROTEINASE DRIVEN TISSUE DESTRUCTION IN CENTRAL NERVOUS SYSTEM TUBERCULOSIS IS REGULATED BY NFjB Justin Green, Shruti Dholakia, Rachel Moores, Lucinda Rand, Paul Ekington, Jon Friedland Imperial College, London, United Kingdom