Learning to safely play with explosives. Or, does pathology recapitulate phylogeny?
Humoral (antibody) immune responses to infection or vaccination is the one instance where the cell will intentionally mutate the genome (specifically of the antibody encoding immunoglobulin genes by an enzyme called AID or Aicda). This process, if properly conducted, greatly improves our ability to combat infectious diseases. However, if anything goes wrong in this highly orchestrated cellular and molecular dance the outcome can be cancer or autoimmune diseases. This process is referred to as antibody affinity maturation and it normally occurs in secondary lymphoid organs in a transient process that leads to the generation of germinal centres - the site where cell genomes are mutated and the mutated cells undergo checks and balances to ensure that only 'good' infection fighting cells emerge.
My interests are in understanding the checks and balances that prevent inadvertent damage to the host. I'm also interested in how such a complex and potentially damaging process could have evolved.
Our lab uses a variety of technologies from zebrafish transgenics & CRISPR genome editing to in vitro cell manipulations for a variety of projects, that include:
Somatic Hypermutation & Class Switching in catfish B-cells
Catfish B-cell lines are being used to determine if fish have all or part of the proteins necessary to drive somatic hypermutation and class switch recombination. We previously cloned the gene for the fish AID mutator and established that it could drive hypermutation and class switch recombination in AID knockout mice. The ability to drive class switching is of particular interest because fish B-cells do not undergo class switch recombination. The catfish AID gene, under control of an inducible promoter, has been integrated into the genome of catfish B-cell lines. These cells are being used to characterize the ability of this 'primordial' AID protein to drive hypermutation and class switch recombination in fish cells.
Evolution of cell type specific transcriptional enhancers
The mouse IgH transcriptional enhancer was perhaps the earliest and most thoroughly studied cell-type-specific enhancer. It was determined that to achieve B-cell specific transcriptional activity there needed to be precise spacing and organization of transcription factor binding motifs 10,11 . However we subsequently determined that there was considerable flexibility in how B-cell specific transcriptional activity could be achieved 12,13 . We are presently examining the IgH and AID enhancer function and structure for a number of different species to determine how much plasticity there has been in the organization of these cell type specific enhancers over time. We have also found evidence that the position of the enhancer within the IgH locus has changed over time and that this relocation of the enhancer influenced the evolutionary course of the immunoglobulin gene and antibodies in vertebrates 14 .
Organization of 'Germinal Centres' in fish
In Situ Hybridization (ISH) and Immunocytochemistry techniques are being used to identify the organization of T- and B-cells (including those expressing the AID mutator) in the lymphoid tissues of catfish and zebrafish. These studies complement those being done with transgenic zebrafish.
Transgenic zebrafish to track humoral immune responses
Using transcriptional enhancers and promoters that are specific for fish immunoglobulin and AID genes, we have constructed reporter transgenes. These genes are integrated into the genomes of zebrafish embryo's and can be used to track those cells expressing immunoglobulin and the immunoglobulin mutator AID. These studies explore where, and under what conditions, zebrafish develop humoral immune responses.
Shark monoclonal antibodies
Sharks produce an antibody form that is comprised of only immunoglobulin heavy chain (i.e. there is no light chain). This give the antibody distinct binding characteristics and also means that the entire antibody is encoded by a single gene. This latter trait means that shark antibodies can be easily expressed in a bacteriophage screening system. We are testing different vaccination strategies aimed at developing strong humoral immune responses in nurse sharks. Antibody cDNA's from these sharks are to be used for development of monoclonal antibodies for research purposes.
Fish as a model of human autoimmune disease
In some types of human autoimmune disease there is formation of ectopic germinal centers outside of the secondary lymphoid organs, e.g. in the synovium of arthritic joints. These aberrant germinal centers lack the organization and structure of conventional germinal centers and have been implicated in the propagation and exacerbation of autoimmunity 6,7 . A question we are trying to resolve is whether the poorly structured ‘normal' germinal centers of fishes have similar organization to the ectopic germinal centers of autoimmune diseases. If so, then fish may provide an excellent model for the study of certain human autoimmune diseases. To develop this model system we have studies to determine how prone fish are to developing autoimmune diseases.