title: Characterisation of the B-lymphocyte response in delayed-type piperacillin hypersensitivity reactions.
Creators: name: Amali, Mohammed
Publisher: name: University of Liverpool Repository
Year: 2015

description: Adverse drug reactions remain a major health issue with delayed type hypersensitivity reactions developing in a high number of individuals. The cellular immunological processes that underlie drug-specific responses in hypersensitive patients have been previously described; however the involvement of the humoral immune system has not been studied in great detail. Consequently, this thesis explores the nature of the piperacillin-specific B cell response in hypersensitive patients and compares the cellular and humoral immune response that develops in patients with cystic fibrosis (CF) exposed to repeated courses of the drug. Initial studies involved characterization of B cell proliferation, B cell phenotype and the nature of total and drug-specific IgG antibody secretions using peripheral blood mononuclear cells (PBMC) from hypersensitive patients. For comparison PBMCs from 2 groups of individuals were assessed: piperacillin naïve healthy volunteers and piperacillin tolerant patients with CF. ELISA, and ELISpot were used to detect piperacillin-specific B cells responses and IgG secretion. T lymphocyte proliferation was assessed with the lymphocyte transformation test (LTT). T lymphocytes from hypersensitive patients, but not tolerant patients or naïve donors were stimulated to proliferate in the presence of the drug. The peak concentration for T cell activation was 1 mM. Phenotypic assessment of hypersensitive patients B-cells revealed an increase in CD19+CD27+ expression in response to piperacillin treatment in vitro. IgG secreting immortalized B-cell lines also expressed a pure CD19+CD27+ phenotype. Piperacillin stimulation of hypersensitive patient PBMC also led to an increase in the secretion of IgG. In contrast, IgG secretion was not detectable following piperacillin stimulation of PBMC from tolerant patients and healthy controls. Western blotting and mass spectrometric methods were applied to characterize -lactam-protein covalent binding. Bovine serum albumin (BSA) binding was time- and concentration-dependent with hapten densities (i.e., the extent of selective lysine residue modification) and anti-piperacillin antibody binding affinity increasing with increasing molar ratios. Lysine residues in BSA at positions 4, 12, 131, 132, 136, 211, 431, 524, and 537 were modified by piperacillin. Epitope profiles also showed similar lysine residues were modified with amoxicillin, benzylpenicillin and flucloxacillin though the extent of ionisation at each site of modification was drug-dependent. A hapten inhibition ELISA used to assess the specificity of the antidrug antibodies revealed the total antibody binding to aztreonam, amoxycillin, benzylpenicillin and penicillin V BSA adducts. This indicates a lack of cross-reactivity with piperacillin-specific IgG antibodies. Subsequently, LTT and ELISA were employed to screen the piperacillin-specific T cell response and IgG antibodies during piperacillin therapy. It was established that piperacillin-specific T cells were detectable on and following clinical diagnosis of hypersensitivity. Moreover, piperacillin-specific T cell responses were detected in a small number of patients currently classified as drug tolerant. A significant difference in piperacillin-specific IgG was observed when plasma form LTT positive and negative blood samples were compared. LTT positivity was associated with higher levels of piperacillin-specific IgG. Furthermore, a significant decrease in piperacillin-specific IgG was seen 24 h post-desensitisation (graded drug challenge). Piperacillin-specific T cell clones isolated from hypersensitive patients were used to explore the effect of plasma bearing anti-piperacillin IgG on the T cell response. Eleven piperacillin-specific CD4+ and CD8+ T-cell clones were generated from 2 hypersensitive patients. All clones were stimulated to proliferate with piperacillin in a concentration-dependent manner. IFN-γ and IL-5 secretion was seen to predominate following piperacillin stimulation. There were no differences in piperacillin-specific T-cell proliferation when piperacillin-specific antibody bearing plasma and plasma from naive volunteers were compared. However, attenuation in IFN-γ secretion was observed with plasma bearing anti-piperacillin antibodies alone. Collectively, the data presented in this thesis begins to describe the different components of the drug-specific humoral and cellular immune response that develops in piperacillin hypersensitive patients with CF.