It was imaginative to have a special oral session devoted to non-adaptive immune responses. Alison Quayle (California) gave an excellent overview on defensins in the genital tract. Among the six alpha defensins, four (HMP1-4) are found in neutrophil granules and two, HD5 & 6, are associated with epithelial cells. HD5 is present at the cervix and endometrium at high levels, with expression highest late in the menstrual cycle. Glandular epithelium was particularly associated with HD5 production.
Unusually, the male genital tract was not neglected! Alison was able to demonstrate strong expression of HD5 in the penile urethra at autopsy. The HD5 is secreted in a propeptide form but cleaved, probably by neutophil proteases. Both HD5 and HMP1 were elevated in gonococcal and chlamydial infection [Fichorova et al., 2002]. Processed HD5 was mildly anti-chlamydial. Thus, when 5 micrograms per ml of activated HD5 was incubated with _ C. trachomatis_ serovar F elementary bodies there was an approximately 50% reduction in inclusion formation on HeLa cells. When HeLa cells were transfected with HD5, the peptide was synthesized but not fully processed and the transfected HeLa cells were less susceptible to chlamydial infection. [See also Wiesenfeld et al., 2002].
*Robin Ingalls* (Boston) gave a fascinating presentation on Toll receptors. These receptors, which are thought to be a relatively ancient immune system, play a central role in the recognition of microbial molecular patterns and they are expressed at the appropriate sites to interact with these molecules [ For a review see Imler & Hoffmann, 2001]. The extracellular domain of the toll receptors includes a leucine rich repeat region while the intracellular region has an interleukin-1 receptor domain. On activation, toll receptors induce cytokines and ArchiveDocsBiologyImunolProtectCostim. Human TLR2 dimerises with TLR6 and recognizes bacterial peptidoglycans, lipoproteins, lipopolysacchardes etc. TLR9 recognizes CpG motifs in bacterial DNA; TLR5 flagellin; TLR3 double stranded RNA; TLR7/8 various antiviral compounds. Interestingly endocervical cells lack TLR4 or MD2 and are unable to respond to bacterial lipopolysaccharide (LPS) via Toll receptors. Chlamydial LPS interacts with TLR2 transfected cells to make IL-8, one of the dominant epithelial cytokine responses to chlamydial infection [See: * ArchiveDocsBiologyImunolProtectCkines*]. In macrophages TLR2 is recruited to the phagosome and seems to be involved in sifting phagosome contents for microbial components. TLR2 is present at high amounts at the cell membrane and localises to the Golgi area. In cells infected with C. trachomatis, TLR2 is enriched in the cytoplasm around the inclusion membrane and may form part of an intracellular signalling system. Robin suggested that _ C. trachomatis_ probably activates epithelial cells primarily via TLR2. This is an MyD88 dependent process and the activation requires replicating bacteria. Chlamydia also have TLR4 ligands, but do not activate TLR4 well. Key questions are what is the chlamydial TLR2 ligand and what is the TLR2-dependent signalling mechanism in response to chlamydiae? [See also Fichorova et al., 2002].
In the oral session on adapative immune responses to chlamydial infection, Kathleen Kelly (Arkansas) reported that the homing of CD4+ cells to the genital tract of C. muridarum infected p-selectin knock out mice was impaired. Antibody to p-selectin ligand 1 also blocked T cell migration to the genital tract of wild type infected mice.
Cap1 is an important, inclusion membrane-associated antigen for generating protective CD8+ T cell responses [See: * Cap 1*]. Lisa Steel (Harvard) infected bone-marrow derived macrophages or Immunopathogenesis.AntigenProcessing with chlamydiae and tested their ability to process Cap1 peptide into association with MHC Class 1 antigen to serve as a target for Cap1 specific H-2d-restricted CD8+ cytotoxic T cells. It was concluded that Cap1 processing occurs by both the Bafilomycin A1-sensitive exogenous pathway and the lactacystin-sensitive endogenous pathway. Wendy Loomis (also Harvard) reported that a further CD8+ cytotoxic T cell antigen, Cap2, had been identified. This was an H-2b-restricted antigen identified in C57/Bl6 mice. Recombinant vaccinia vector expressing Cap2 for presentation via the endogenous pathway resulted in excellent Cap2-specific memory cell responses which generated equivalent protection to live chlamydiae in an intravenous challenge model. Cap2 is a T cell antigen of the crpA gene, CT442, the 15KDa Antigens_Proteomics.CysteineRichProteins protein.
*Natasha Telyatnikova* (Cambridge, UK) updated the meeting on the antigenic specificities of CD4+ T cell clones isolated in the presence of GMCSF from patients with reactive arthritis. Initially characterized against expression libraries, these clones were further identified using overlapping peptides. Epitopes identified included: Omp2 (OmpCB) amino acids 439 - 546 with amino acids 450 - 462 almost identical in _ C. pneumoniae_; peptide 626 - 716 of ArchiveDocsBiologyEnvPmps.htm; and peptide 88 - 108 of _ C. trachomatis_ Enolase, which was non cross-reactive with _ E. coli_ enolase. Tina Mygind (Denmark) reported that PmpC was recognized by murine CD4+ T cells during experimental _ C. pneumoniae_ infection.
In the session on experimental animal models and chlamydial vaccines, Lisa Tormakangas (Oulu, Finland) reported that repeated C. pneumoniae infection increased lipid accumulation in the aortic sinuses of C57/Bl6 mice. High cholesterol diet and genetic factors played a role. However hypercholesterolaemia was not necessary for _ C. pneumoniae_-induced changes. Benjamin Wizel (Tyler, Texas) in an interesting study identified 12 C. pneumoniae proteins serving as targets for H-2b-restricted CD8+ T cells in mice. 18 H-2b binding peptides capable of sensitizing target cells for MHC Class 1 T cells were also identified. The CD8+ T cells identified had a Tc1 cytokine secretion pattern, secreting interferon gamma and lysing chlamydial-infected macrophages. A mini-gene CpnCTL7 construct was created consisting of Omp5, Omp85, DnaK, OmpB etc and the Padre T helper epitope. This was used to transfect EL-4 (H-2b) cells. In experimental animals the construct showed no sign of immunopathology and caused a 4 log units reduction in pulmonary bacterial load from a respiratory challenge of 106 ifu compared with mock immunized animals. Simultaneous priming was achieved against all epitopes. Francis Eko (Morehouse and CDC) expressed outer membrane proteins Omp1, 2 and 3 in a _ Vibrio cholerae_ ghost construct known to have strong mucosal adjuvant properties [See: * Immunopathogenesis.MucosalImmunolPix*]. This was used to immunize mice and was found to induce Th1 responses and to be partially protective.
Regretfully I had almost no time to get round the poster presentations, largely due to all the informal discussions which occurred in the various breaks.
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*Fichorova, R. N., Cronin, A. O., Lien, E., Anderson, D. J. & Ingalls, R. R. (2002). Response to Neisseria gonorrhoeae by cervicovaginal epithelial cells occurs in the absence of toll-like receptor 4-mediated signaling. Journal of Immunology *168, 2424 - 2432. * Full article*
*Imler, J. L. & Hoffmann, J. A. (2001). Toll receptors in innate immunity. Trends in Cell Biology *11, 304 - 311.
*Wiesenfeld, H. C,, Heine, R. P., Krohn, M. A., Hillier, S. L., Amortegui, A. A., Nicolazzo, M. & Sweet, R. L. *(2002). Association between elevated neutrophil defensin levels and endometritis. Journal of Infectious Diseases 186, 792 - 797.