Los profesorados en la UNGS 1 Estructura curricular

Piperacillin is a piprazine ampicillin derivative (Oh et al., 2009). By the end of 1970s, the drug was launched following thorough research on its antimicrobial efficacy compared with other available antibiotics. Piperacillin is effective against resistant microbes such as Pseudomonas aeruginosa and Klebsiella pneumonia

(Jones et al., 1977; Ueo et al., 1977; Wise, 1977; Dickinson et al., 1978; Verbist, 1978). In vitro experiments on bacterial cultures revealed a synergistic effect

between aminoglycosides and piperacillin against Pseudomonas aeruginosa.

Moreover,  piperacillin  activity  against  β-lactamase producing resistant bacteria (e.g.

Escherichia coli, Haemophilus influenza, staphylococci) can be improved by

combining piperacillin with tazobactam (Speich et al., 1998; Sweetman, 2009).

Our investigation focussed on patients with a history of allergic reactions to piperacillin, aztreonam and meropenem. Piperacillin is a member of the pencillin class of antibiotics, which contains β-lactam and thiazolidine ring structures [Figure 1.6]. Meropenem, a carbapenem, contains similar ring structures but the sulfur in the thiazolidine ring has been replaced with a carbon atom, and unsaturation has been introduced. Finally, aztreonam, a monobactam, contains an unfused β-lactam ring.

Figure 1.6 - Chemical structures of piperacillin, meropenem, and aztreonam.

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Piperacillin is administered as a sodium salt by intravenous or intramuscular injection as it is poorly absorbed from the intestine. The usual piperacillin dosage is 3-4g every 4 or 6 hours, while the maximum daily dose for uncomplicated or mild infections is at 100 to 125 mg/kg. However, adults can be given piperacillin at 200 to 300 mg/kg in divided doses daily intravenously if they are suffering from complicated or serious type of infections (Sweetman, 2009). Approximately 20% of piperacillin in the blood circulation is attached to plasma proteins. Only 20% of piperacillin is excreted unchanged in bile, whereas 60% to 80% of a dose is excreted unchanged in the urine within 24 hours (Tjandramaga et al., 1978; Sweetman, 2009).

In vivo duodenal samples and in vitro studies utilising microsomes of human liver revealed that a small piperacillin fraction is metabolised in the liver in to desethyl- piperacillin and a glucuronide conjugate of the desethyl metabolite. Both desethyl- piperacillin and desethyl-piperacillin glucuronide are detected in human bile and urine (Minami et al., 1991; Komuro et al., 1997; Ghibellini et al., 2006; Ghibellini et al., 2007). Moreover, piperacillin and desethyl-piperacillin are believed to form adducts with proteins, which may act as a functional antigens leading to piperacillin hypersensitivity reactions (Whitaker et al., 2011a).

Multiple reports were published in the mid-1980s describing the high incidence of piperacillin reactions, particularly fever in patients with CF (Brock and Roach, 1984;

Moss et al., 1984). However, the manufacturers’  suggested  that  mostly  patients  with  

CF are liable to hypersensitivity reactions and to support their view they cited reactions with other semisynthetic penicillins including carbenicillin (Sweetman, 2009). Therefore, throughout this thesis piperacillin clones were tested against other

structurally  related  β-lactams [Figure 1.7].

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Figure 1.7 -Structurally related drugs to piperacillin.

Piperacillin Penicillin G Ampicillin _Amoxicillin Carbenicillin Cefoperazone Cefalexin 7-Amino-desacetoxycephalosporanic acid D-penicillamine

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To investigate drugs of our interest, cloning method was used throughout this thesis to generate enough numbers of T cell clones for further studies. T lymphocytes are a pool of cells with exceedingly different characteristics. Firstly, each cell that drives from a specific precursor has reordered its genes for the T cell receptor (TCR) in a different manner from other cells. Therefore, there is a great heterogeneity of T cells in peripheral blood resulted from different TCR expression by the cells that distinguish different epitopes. Additionally, T cells can be further divided in isolated subsets, which are differentiated by different molecules expression, such as CD8, CD4, CD45R and CD25. Such molecules are variably related to the function that a single T cell is intended to accomplish or to the differentiation stage in which at a certain time that cell belongs. Furthermore, the pool of peripheral cells also involves T lymphocytes with variable capacity to release function-associated cytokines. By analysing peripheral blood mononuclear cells (PBMC) many studies on T cell functions can be performed, however since PBMCs cells including a heterogenous population, in many experiments it might be difficult to attain a clear-cut data and to attribute a define function to a defined T cell population. The accessibility of T lymphocytes population that originate from the same progenitor (clone) with a distinctive function and phenotype may be of a great benefit. Oppositely, limitation on the results interpretation should be defined also when a clone is utilised. A clone will comprise cells that share similar phenotypic and functional characteristics, but they may not be demonstrative of the whole T lymphocytes population in in vivo settings (Mariotti and Nisini, 2009).

One of the important sources for T cell clones is isolation of PBMC from patients with or shortly recovered from a specific disease, which is related to an antigen- specific T cell expansion (Mariotti and Nisini, 2009). The next two figures illustrate the methods used throughout this thesis to study T-cell responses to antigen using

both  volunteers  and  patients’  blood  samples [Figure 1.8; Figure 1.9].

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Figure 1.8- A schematicillustration to the methods used to study T-cell responses to antigen  using  patients’  blood  samples.    

Figure 1.9 - A schematic illustration to the methods used to study T-cell responses to antigen using volunteers’ blood samples.

49 1.9 Aims of the thesis

β-lactam hypersensitivity remains one of the most common causes of immune- mediated drug hypersensitivity.   Furthermore,   β-lactam antibiotics can induce both skin and liver injury. The incidence of hypersensitivity reactions to β-lactams is three times (28%) greater in CF patients in comparison to patients without CF (8%). The incidence of piperacillin reactions is the highest amongst all β-lactam antibiotics (30- 50%) (Parmar and Nasser, 2005; Burrows et al., 2007).

Flucloxacillin   is   a   β-lactam antibiotic used mainly for the treatment of staphylococcal infections. Even though well tolerated flucloxacillin use is accompanied by the appearance of cholestatic liver injury at an estimated rate of 8.5 in 100 000 new users. GWAS in 2009 found that 84% of patients with flucloxacillin- induced liver injury expressed the HLA-B*57:01 allele. To date, there are no studies characterising the drug-specific immune response underlying both piperacillin and flucloxacillin hypersensitivity.

The primary objective of this thesis was to study the role of T lymphocytes in patients  with  β-lactam mediated skin and liver injury. Secondary objectives were (1) to investigate the mechanisms of drug antigenicity and immunogenicity and (2) to determine whether the HLA associations linked to drug- induced liver injury relate to the HLA-restricted presentation of the drug-derived antigen.

The two drugs piperacillin and flucloxacillin were selected as (1) their use in humans is associated with a high incidence of skin and liver injury, respectively and (2) large number of patients to promote the research.

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