424 Abstracts

cytochrome C translocation was observed in D39 and DR6,but not SC infected cells. These data were corroborated bynuclear morphology, which showed significantly higherlevels of apoptosis in D39 and DR6 than in SC infected or MITHP-1s or MDMs. Infection with D39 resulted in the trans-location and activation of the lysosomal protease, cathepsinD. Treatment of cells with the acid protease inhibitorpepstatin A reduced MMP and cytochrome C translocation.These data imply that the pore forming cytolytic activity ofPLY is not essential for the induction of macrophageapoptosis, and that protein-protein interactions may be ofmore relevance, in particular, any PLY interactions withlysosomal factors which may serve to drive apoptosis throughnovel pathways such as cathepsin activation. Identificationof these early events is essential before therapeutic manip-ulation of host-mediated apoptosis can be carried out.

PILOT STUDY OF HUMAN EXPERIMENTALCHALLENGE WITH NEISSERIA LACTAMICA

C. Evans 1, A. Gorringe 2, M. Matheson 2, C. Pratt 2,R. Borrow 3, J. Findlow 3, R.C. Read 1

1University of Sheffield, Sheffield, United Kingdom2Health Protection Agency, Porton Down, UnitedKingdom3Health Protection Agency, Manchester, UnitedKingdom

AbstractIntroduction: Neisseria lactamica is a non pathogenic

commensal bacteria commonly found in the upper respira-tory tract of infants. Its carriage dynamics have beenextensively studied in young children and a clear correla-tion has been documented between colonisation by N. lac-tamica and protection from meningococcal disease. This isthought to be due to common antigens resulting in a crossprotective immune response.

Objectives: The aim of this study was to inoculate Neisse-ria negative healthy adult volunteers, with live N. lactamicaand monitor their specific mucosal and systemic immuneresponse, then analyse their serum bactericidal antibodyresponse against 6 UK prevalent meningococcal B strains.

Methodology: 9 individuals were inoculated with liveN. lactamica, resulting in 5 successful colonisations. Coloni-sation was confirmed using nasopharyngeal swabs andgargles. Saliva and blood samples were taken at 0 weeks,2, 4, 7, and 12 weeks.

Findings: Results demonstrated a specific mucosal IgA andsystemic IgG immune response to colonisation by N. lactamica.The specific mucosal IgA response from baseline to 12 weeks,comparing non colonised and colonised individuals wassignificant, p value Z0.0159. Baseline specific IgG and peakfold rise for each individual, using a Mann Whitney test,demonstrates a value of pZ0.063 in colonised individuals.

Discussion: Most interestingly colonised individualsdemonstrate a greater range of SBA responses to differentmeningococcal strains, than the non colonised group.Within the colonised individuals 2 volunteers had a signifi-cant (greater than 4 fold rise) response to 4 differentmeningococcal strains. Overall, all volunteers mounted

a response to at least one strain of meningococci, it is likelythat individual exposure to N. lactamica is resulting ina booster response of one or more specific antigens whichare found on the outer membrane of these different strainsof meningococci. These results support the hypothesis thatcolonisation by Neisseria lactamica does result in crossprotection against meningococci.

MATRIX METALLOPROTEINASE DRIVEN TISSUEDESTRUCTION IN CENTRAL NERVOUS SYSTEMTUBERCULOSIS IS REGULATED BY NFjB

Justin Green, Shruti Dholakia, Rachel Moores,Lucinda Rand, Paul Ekington, Jon FriedlandImperial College, London, United Kingdom

AbstractIntroduction: Involvement of the central nervous system

(CNS) is the most serious manifestation of extra pulmonarytuberculosis (TB). Matrix metalloproteinases (MMPs) are up-regulated in CNS TB and are implicated in the breakdown ofthe blood brain barrier. The inflammatory phenotype ofmicroglia, resident CNS macrophages, is distinct from thatof infiltrating monocytes/macrophages. The transcriptionfactor NFjB has a central role in the up-regulation ofmany inflammatory genes, thus we hypothesised thatNFjB differentially controls secretion of MMPs and theirspecific tissue inhibitors (TIMPs).

Methods: In a cellular model of CNS TB human microglialcells were stimulated with conditioned media from Myco-bacterium tuberculosis (M. tb)-infected human monocytes(CoMTB). Cells were pre-incubated with NFjB blockingchemicals and secretion of MMP-1 was measured by caseinzymography and western blotting. MMP-3 and TIMP-1 weremeasured by ELISA. Gene expression and regulation werestudied by real time RT-PCR and dual luciferase promoterreporter assays. NFjB subunit kinetics were studied usingoligonucleotide based transcription factor ELISAs and bywestern blotting.

Results: CoMTB increased MMP-1 secretion 13-fold andMMP-3 secretion 5-fold compared to controls (P<0.05).Blockade of the NFjB p65 subunit by helenalin and upstreamIKK-2 inhibition by SC-514 demonstrated dose-dependentdecreases 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 mRNAaccumulation. Dual luciferase promotor reporter assays,using specific constructs with a mutated NFjB binding site,further established the critical importance of the consensusNFjB site in the MMP-1 promoter. CoMTB rapidly up-regulated p65-p50 heterodimer recruitment to the nucleusat 30 minutes, with concomittant IjB-a degradation.However, the p65 signal only returned to baseline at 24hours despite resynthesis of IjB-a by 60 minutes.

Discussion: M. tb driven monocyte networks up-regulateMMP-1 and 3 secretion in human microglia. Early and sus-tained NFjB activity appears to be a key early step in thisprocess. Better understanding of the mechanisms involvedin CNS TB tissue degradation could identify novel thera-peutic targets for this devastating disease.