Jetty G. Veenstra

Molecular characterisation of β-tubulin genes present in benzimidazole-resistant populations of Haemonchus contortus

A beta-tubulin isotype 1 gene, gru-1, from a benzimidazole (BZ)-resistant population of the nematode parasite, Haemonchus contortus, was cloned and sequenced. The predicted gene organisation showed 10 exons and 9 introns, one of which was H. contortus specific. Using probes and restriction sites selected from this sequence, restriction maps were constructed from and around beta-tubulin genes of 3 BZ-susceptible and 7 BZ-resistant populations. There was a reduction in beta-tubulin isotype 1 genes to usually one, in BZ-resistant populations. So, our previously reported reduction of beta-tubulin probe-reactive RFLP fragments in resistant populations correlated with the reduction of beta-tubulin isotype 1 genes. The beta-tubulin isotype 1 gene present on the apparently selected fragment and was not always the same, and the geographical origin of the resistant populations indicated independent development rather than geographical spread of the resistant populations. The beta-tubulin genes on the apparently selected fragments were transcribed and processed to mRNA using the nematode-specific trans-spliced leader (SL1). Comparison of the derived amino acid sequence of gru-1, with known sequences from a susceptible population, identified 3 mutations that could be involved in BZ resistance.

Molecular aspects of drug resistance in parasitic helminths

Parasitic helminths (worms) cause serious infectious diseases in humans and animals. As control of these infections relies mostly on chemotherapeutics, the anthelmintics, resistance has developed against most of these drugs in several parasite species. These resistant parasites are being used to elucidate the molecular mechanisms of drug action.

Effector and regulatory T cells derived from the same T cell clone differ in MHC class II-peptide multimer binding.

MHC class II‐peptide multimers are a valuable tool for antigen‐specific detection of CD4+ T cells. However, it has been proposed that T cells in a hypo‐responsive state can have diminished binding of such multimers. In the present study, we investigated this phenomenon at the clonal level. We found that anergic CD4+ T cells had a reduced capacity to bind MHC class II‐peptide multimers compared to their non‐anergic counterparts. Increasing the incubation temperature, time, or MHC‐peptide valency could not equalize multimer binding by anergic and non‐anergic T cells. Neither anergic T cells nor non‐anergic T cells internalized the MHC class II‐peptide dimers efficiently, and in both cases the dimers bound to the plasma membrane at locations containing a low amount of raft‐associated lipids. Disruption of lipid rafts, however, led to decreased dimer binding by non‐anergic T cells and to a lesser extent by anergic T cells. Finally, we show that the depth of the anergic state of the T cell, which determines its ability to regulate other T cell responses, correlates with the reduced dimer binding. We here demonstrate for the first time differential MHC class II‐peptide multimer binding by regulatory (anergic) and effector T cells with identical TCR.

Induction of experimental autoimmune arthritis by a public epitope of the T cell receptor variable α domain of an arthritogenic T cell clone

T cell receptor (TCR) peptide immunizations have been demonstrated to protect against experimental autoimmune diseases. These findings have led to clinical trials employing TCR peptides in multiple sclerosis and rheumatoid arthritis patients. Previously, we identified a strongly immunogenic region of the TCR α chain of an arthritogenic T cell clone (AV11 66‐80). In this report, we show that rats immunized with AV11 66‐80 developed arthritis with clinical symptoms and histology similar to adjuvant arthritis (AA). Transfer of this disease into naive rats using AV11 66‐80‐specific T cells proved the T cell‐mediated character of the disease. The AV11 66‐80 arthritic rats developed resistance to Mycobacterium tuberculosis‐induced AA, indicating that both forms of arthritis depended on similar regulatory mechanisms. This first demonstration of TCR peptide‐induced arthritis, together with an earlier report on a polymorphism in this very same AV11 66‐80 region involved in arthritis resistance in mice, suggests a central role of the public epitope AV11 66‐80 in the control of autoimmune arthritis. Although TCR peptide immunizations can be exploited to prevent experimental autoimmunity, caution should be taken in the induction of TCR peptide‐specific T cells for immunotherapy to avoid adverse effects as shown here.