This research was undertaken to: 1) Develop techniques for the recovery of DNA from fixed and paraffin wax embedded tissues. 2) Assess the potential for use of this DNA for the diagnosis of lymphoma. 3) Contribute to the understanding of the effects of fixation on DNA. Initial investigations used pure DNA and lymphocytes to determine the effect of five fixatives on the integrity, recovery and restriction endonuclease digestion of the nucleic acid. Two fixatives, Bouin and formol sublimate, proved unsuitable for further analysis. DNA recovery and Southern analysis was then attempted using Carnoy, formol saline and neutral buffered formalin fixed and paraffin wax embedded tissue. From this an optimised method featuring prolonged incubation of tissue with Proteinase K and SDS at 37°C followed by purification was developed. Within limits imposed by fixation this DNA was suitable for Southern analysis and amplification by PCR. A simplified DNA recovery method involving overnight incubation of paraffin wax sections in Proteinase K at 55°C without purification was also evaluated. This gave satisfactory results by PCR but the maximum product size obtained was 500 bp compared with 1250 bp using the optimised method. DNA was better preserved after Carnoy than following formalin fixation and this was reflected in consistently superior Southern and PCR results. Formalin fixation induced degradation of DNA, which increased as fixation time was extended. This made Southern analysis for the identification of B and T cell rearrangements in lymphomas unreliable. However, the t(14:18) translocation was demonstrated successfully by PCR in formalin fixed follicular lymphomas. The results of these investigations show the importance of the pH of fixatives on the preservation of DNA. They also suggest that chemical interactions with DNA and associated proteins occur using fixatives containing formalin and mercury.

Streptokinase (SK) is a naturally occurring bacterial protein. It is used to lyse blood clots and restore blood flow to the heart during acute myocardial infarction (AMI) by initiating the conversion of plasminogen to plasmin. SK is substantially cheaper than most alternative thrombolytic drugs but is immunogenic in humans. Natural antibodies specific for SK are present at low levels in most people due to streptococcal infections. Following SK treatment, anti-SK antibody levels increase in a manner consistent with a secondary immune response. These anti-SK antibodies may neutralise a subsequent dose of SK before activation of plasmin can occur (SK resistance). Individuals who have received SK treatment are treated for a second infarct with a more expensive, and possibly less safe thrombolytic drug. In this thesis, in vitro responsiveness to SK of lymphocytes isolated from SK-treated AMI patients before and for several years after treatment was determined. Secondly, a system for the production of recombinant SK fragments, the detection of their fibrinolytic activity and evaluation of their immunogenicity was optimised. A fibrinolytically active recombinant SK fusion protein and nine non-overlapping recombinant peptide fragments of SK were produced. Two peptides were preferentially recognised in vitro by lymphocytes isolated from 5 out of 7 volunteers tested. The in vitro peripheral T cell response to SK was shown to increase significantly in the first week after SK treatment, but return to control levels within 12 months. Significantly elevated total anti-SK IgG levels were detected in the serum of SK-treated AMI patients for up to 93 months after SK treatment. These data provide support for the possibility of effective T cell epitope modification to produce an immunologically silent form of SK. In addition, they suggest that the acquired immune response to SK may persist for up to 93 months after SK treatment for AMI.

The objective of this study was to determine whether the haptenation of protein changes qualitatively the peptides produced by antigen processing. This hypothesis suggests that T cells will have no self-tolerance to neo-epitopes exposed by haptenation. Any T cell recognising such an epitope may therefore be involved in the pathogenesis of hapten-induced autoimmunity. The model protein used was the house dust mite antigen, Der p II. To minimise the complexity of processed peptides visualised following antigen processing, the Der p II cDNA was mutated by PCR extension overlap mutagenesis, to replace three tyrosine residues with alanines. The single tyrosine residue of the mutated Der p II fusion protein (Der p II-3Y) was iodinated and Der p II-3Y was haptenated with trinitrophenol (TNP). Iodinated Der p II-3Y +/- TNP was incubated with EBV-transformed B cells. To overcome low antigen uptake by non-specific EBV-transformed B cells, a system developed by C. Watts and A. Lanzavecchia using EBV-transformed B cell clones specific for the C-fragment of tetanus toxin was employed. The C-fragment protein was iodinated throughout and the haptenation studies repeated. The peptide fragmentation patterns seen were unaltered +/- TNP. T cell proliferation offered a more sensitive readout than autoradiography to visualise possible neo-epitope production. Haptenation of Der p II-3Y and C-fragment led to the loss of a proliferation response of specific T cell clones and T cell lines, respectfully. A lower level of C-fragment haptenation caused increased T cell proliferation. To prolong the life of T cells in culture, the caspase inhibitor z-VAD-FMK was investigated for its ability to prevent activation induced cell death (AICD), z-VAD-FMK reduced the level of AICD in PMA-PHA-P and anti-Fas-stimulated Jurkat and C-fragment-specific T cell lines. Der p II-3Y-specific T cell clones appeared to proliferate normally following prior treatment with z-VAD-FMK. Further studies, not only on the effects of haptenation on antigen processing, but also on the uptake, trafficking and efficient presentation of antigen by antigen presenting cells will help in our understanding of the possible role of haptenation in the induction of autoimmune disease.

The three pathways of complement activation lead to the proteolysis of the same component, C3, that will then carry on the enzymatic cascade of the downstream components and the MAC proteins. In the context of the alternative pathway, the enzyme responsible for the proteolysis of C3 relies upon properdin, a six thrombospondin repeat domains protein, for its stability and activity. Thus properdin has a key role in ensuring the full activity of the alternative pathway. Cases studies of properdin deficiency show that the patients have a higher risk to suffer recurrent bacterial infections, in particular of meningococcal nature, and that the ensuing level of mortality is also higher than non-deficient patients. The work described in this thesis is the characterisation of the murine gene for properdin. High homology between humans and mice of the complement activation, regulation and components, makes these animals a privileged laboratory model. The mouse properdin gene was isolated by screening a mouse genomic library of the SVJ129 strain and sequence analysis of phage clones determined to represent the genomic sequence for properdin. The properdin gene was determined to contain 9 exons encoding for the leader peptide, N-terminus, the six thrombospondin repeats and the 3' untranslated region. Each thrombospondin repeat was shown to be encoded by a single exon with the exception of the sixth repeat which is encoded by two separate exons interrupted by an intronic sequence. The sequencing of the gene confirmed the homology to human properdin at the genomic level, thus validating further the value of the mouse model to study properdin deficiency. The characterisation of the mouse gene for properdin will allow the generation of a mouse strain deficient for the properdin gene to study the phenotype of these transgenic animals.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.