Chloramphenicol in children: dose, plasma levels and clinical effects.

Despite concerns about adverse effects, chloramphenicol (CMC) continues to be used in certain situations and, due to its low therapeutic index and variable pharmacokinetics, therapeutic drug monitoring (TDM) is often recommended. At our centre, CMC finds applications in typhoid and meningitis and TDM is routinely performed. Elsewhere in Malaysia, however, CMC is used without TDM. We therefore decided to evaluate our TDM for CMC in relation to its roles in CMC therapy in children, who constitute most of our patients. Our objective was also to develop strategies to improve our TDM for CMC use. Data were collected from 168 children given CMC for various indications and monitored by the TDM service. Plasma CMC was determined by HPLC and used to adjust doses to maintain concentrations within a range of 10-25 micrograms/ml. Outcomes measured included daily temperatures and haematological indices. Daily doses and plasma CMC varied greatly. Doses averaged 40.5 mg/kg for neonates and 75.5 for older children. Average peak concentrations were therapeutic in 60% and trough in 42%. Average duration of fever was 6.3 days and it was unaffected by plasma CMC. Typhoid was eradicated in 97% but nine children with other diagnoses died. Side-effects were confined to mild reversible haematological abnormalities which developed in 11% of children at plasma concentrations which tended to be high. We conclude that CMC remains useful in children with typhoid. Its use for other indications, however, should be reviewed. Routine TDM for CMC is probably not warranted, at least until a clearer role is defined by well designed prospective studies.

Alternative splicing of human inducible nitric-oxide synthase mRNA. tissue-specific regulation and induction by cytokines.

Human inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide synthesis in response to inflammatory mediators. The human iNOS gene, containing 26 exons, encodes a protein of 131 kDa. This study was aimed at investigating the presence of alternative splicing of human iNOS mRNA. Total RNA from human alveolar macrophages, nasal and bronchial epithelial cells, and several human tissues was transcribed to cDNA and analyzed using polymerase chain reaction with specific primers for segmental analysis of the iNOS gene. Four sites of alternative splicing were identified by sequence analysis; these included deletion of: (i) exon 5; (ii) exons 8 and 9; (iii) exons 9, 10, and 11; and (iv) exons 15 and 16. The deduced amino acid sequences of the novel iNOS cDNAs predict one truncated protein (resulting from exon 5 deletion) and three iNOS proteins with in-frame deletions. Southern analyses of polymerase chain reaction products were consistent with tissue-specific regulation of alternative splicing. In cultured cells, iNOS induction by cytokines and lipopolysaccharide was associated with an increase in alternatively spliced mRNA transcripts. Because iNOS is active as a dimer, the novel forms of alternatively spliced iNOS may be involved in regulation of nitric oxide synthesis.

Cloning and characterization of human inducible nitric oxide synthase splice variants: a domain, encoded by exons 8 and 9, is critical for dimerization.

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242-335 of the oxygenase domain. In this study, iNOS8(-)9(-) and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8(-)9(-) in 293 cell line resulted in generation of iNOS8(-)9(-) mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8(-)9(-) did not form dimers. Similar to iNOSFL, iNOS8(-)9(-) exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two "control" iNOS deletion mutants were synthesized, lacking either residues 45-52 of the oxygenase domain or residues 1131-1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.