Expression of small heat shock proteins by the third-stage larva of Brugia pahangi.

Changes in proteins synthesised by the infective third-stage larvae (L3) of the filarial nematode Brugia pahangi were examined with respect to the temperature shift encountered by the parasite as it migrates from insect to mammal, and the presence of serum in the culture medium. While the synthesis of a number of polypeptides is regulated by the temperature shift of the L3 from 28 degrees C to 37 degrees C in vitro, there is no evidence that serum has any significant effect on protein synthesis. Two complexes of small acidic polypeptides (22-24 kDa and 18 kDa) are synthesised for a limited period only by L3 transferred to 37 degrees C. One component of each complex appears to be constitutively expressed at 28 degrees C, but its synthesis is up-regulated at 37 degrees C, while the remaining members of each complex are synthesised only at 37 degrees C. Subjection of L3 and post-infective (p.i.) L3 to heat shock (41 degrees C) also induces synthesis of both complexes, indicating that these heat-inducible polypeptides are related to the family of small heat shock proteins. The possible role of the heat shock-related proteins in this important environmental transition is considered.

The expression of small heat shock proteins in the microfilaria of Brugia pahangi and their possible role in development.

Development of the microfilariae of Brugia pahangi in the mammalian host is blocked until uptake by a mosquito vector when the developmental cycle is re-initiated. Comparison of the profile of polypeptides labelled in microfilariae cultured at mammalian temperature (37 degrees C) or mosquito temperature (28 degrees C) revealed a complex of low-molecular-weight proteins (18 kDa and 22-24 kDa) synthesized only in microfilariae at 37 degrees C. The synthesis of these proteins was also induced by transfer of microfilariae to 41 degrees C (i.e., heat shock conditions), suggesting that these are heat shock proteins. The expression of the small heat shock proteins in the Brugia life cycle is developmentally regulated, as they are not observed in the mature adult female. Their synthesis is strictly temperature dependent and is repressed upon transfer of the microfilariae to 28 degrees C.

Commissioning health services research: an iterative method.

The standard linear method of commissioning research involves many stages, some lengthy. While assessment criteria are usually explicit, their weighting and interaction are not. Output is assessed on completion. This method is suitable where the research question is clear-cut. However, it has drawbacks when the research question and the form and scope of the research are not clear at the outset, as is often the case with research on the delivery and organisation of services. Also, it does not encourage potential users of the research to develop a sense of ownership. An alternative method is proposed by which the scope, form and content of research are not specified in advance but are developed iteratively. A programme director, advised by a group of potential users and research commissioners, has devolved authority to commit funding for the stages of the work as it unfolds, predicated on evolving need. There are foreseeable but avoidable risks of the group over-identifying with the researchers, of research management becoming cumbersome, and of unproductive friction between research groups when they are required to work together. The iterative method, being new and untried, is itself an organisational change requiring evaluation. However, from our local experience, it provides for productive dialogue between research commissioners, researchers and potential users.

The expression of the Mr 30,000 antigen in the third stage larvae of Brugia pahangi.

The expression of the Mr 30,000 surface antigen in the third stage larvae (L3) of Brugia pahangi has been investigated. The antigen could be detected only with great difficulty in the mosquito derived L3 externally labelled with 125I but was more easily labelled in 24 and 48 h post-infective larvae harvested from the vertebrate host. Labelling of a detergent extract of mosquito derived L3 with 125I demonstrated that the Mr 30,000 antigen was indeed present in this life cycle stage, presumably in an internal localization. It seems likely that the Mr 30,000 antigen is not fully expressed in the parasite cuticle until after infection of the vertebrate host. The data presented also suggest that there are major differences in the surface properties of the mosquito derived L3 compared to the p.i. L3 harvested from the vertebrate host.

Identification and characterization of a DR4-restricted T cell epitope within chlamydia heat shock protein 60.

An epitope within the 60 kD Chlamydia trachomatis heat shock protein (hsp) 60, recognized by a HLA-DRB1*0401-restricted T cell clone from a reactive arthritis patient, has been characterized. Stimulatory peptides contained a nine amino acid sequence (residues 38-46) predicted by algorithm to confer strong binding to DRB1*0401, with valine in the P1 position. The overall length of the peptide was critical for efficient recognition; peptides with at least one residue N-terminal to the putative P1 position were markedly more stimulatory than a peptide whose N-terminal is the P1 valine. Optimal responses were seen with 14mer peptides having two to three amino acids N- and C-terminal to the core 9mer. The sequence of the defined epitope is identical in hsp60 from both C. trachomatis and C. pneumoniae. Since the latter is a common respiratory pathogen, patients infected with C. trachomatis may already be primed for responses to hsp60 by prior infection with C. pneumoniae. Such secondary responses are important in the pathogenesis of chlamydia-induced inflammatory diseases such as trachoma. Priming by infection with enteric organisms was considered because of the similarity of the epitope sequence in Escherichia coli hsp60. However, although an E. coli-related peptide was recognized, intact E. coli hsp60 was not, suggesting that the epitope is cryptic in E. coli hsp60. Human hsp60 has six amino acid differences from chlamydial hsp60 in the epitope sequence and was not recognized. Thus cross-reactive recognition of self hsp60 could not be implicated in the pathogenesis of chlamydia-induced reactive arthritis in this patient.

Identification of 2 Chlamydia trachomatis antigens recognized by synovial fluid T cells from patients with Chlamydia induced reactive arthritis.

OBJECTIVE: To identify antigens of Chlamydia trachomatis recognized by synovial T cell clones from patients with reactive arthritis (ReA). METHODS: C. trachomatis specific T cell clones were isolated from synovial fluid of 2 patients with chlamydia induced ReA. The particular antigens/epitopes recognized by these clones were identified using T cell immunoblotting and testing recombinant chlamydial proteins and synthetic peptides. RESULTS: Two sets of clones were shown by immunoblotting to recognize antigens of roughly 60 and 18 kilodaltons (kDa) respectively. By testing recombinant chlamydial proteins these antigens were identified as the 57 kDa heat shock protein and the 18 kDa histone-like protein, Hc1. Mapping the epitope in Hc1 using synthetic peptides identified a peptide containing a sequence motif compatible with binding to HLA-DR1, the restricting antigen for the Hc1 specific clones. CONCLUSION: These are the first 2 chlamydial antigens to be identified as targets of the synovial T cell response in chlamydia induced ReA. Both have properties that are shared with target antigens identified in ReA induced by enteric infection and relevant to the pathogenesis of joint inflammation.