Effect of pro-inflammatory cytokines on the toxicity of the arylhydroxylamine metabolites of sulphamethoxazole and dapsone in normal human keratinocytes.

Sulphonamides, such as sulphamethoxazole (SMX) and the related sulphone dapsone (DDS), show a higher incidence of cutaneous drug reactions (CDRs) in patients with the acquired immunodeficiency syndrome (AIDS) compared with human immunodeficiency virus (HIV) negative patients. During HIV infection, pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are increased. We hypothesized that this increase in pro-inflammatory cytokines may increase the toxicity of the arylhydroxylamine metabolites of SMX (S-NOH) and DDS (D-NOH) in keratinocytes through a reduction in glutathione (GSH) content. We evaluated the effect of TNF-alpha on GSH levels in normal human epidermal keratinocytes (NHEK) and found a significant decrease in GSH after 24h. Pre-treatment with TNF-alpha also resulted in an increase in the recovery of D-NOH, but failed to alter drug-protein covalent adduct formation in NHEK. We also evaluated the effect of TNF-alpha, IL-1 beta, interferon-gamma (IFN-gamma), lipopolysaccharide (LPS) and conditioned media (obtained from monocytes stimulated with LPS) on the cytotoxicity of pre-formed arylhydroxylamine metabolites in NHEK. Priming cells with cytokines did not significantly alter the cytotoxicity of the metabolites. The effect of pre-treatment with TNF-alpha on reactive oxygen species (ROS) generation in NHEK was also determined. While ROS formation in NHEK was increased in the presence of D-NOH, TNF-alpha did not alter the level of ROS generation. Our data suggest that the level of GSH reduction induced by pro-inflammatory cytokines does not predispose NHEK to cellular toxicity from either S-NOH or D-NOH.

Inadequacy of manual cleaning for reprocessing single-use, triple-lumen sphinctertomes: simulated-use testing comparing manual with automated cleaning methods.

BACKGROUND AND AIMS: Despite widespread reuse of single-use sphinctertomes, publications regarding the adequacy of reprocessing are conflicting and the cautery wire channel is seldom evaluated. Our objective was to use thickened artificial test soil containing microorganisms to perform simulated-use tests combined with in-situ and destructive testing to evaluate cleaning efficacy and ethylene oxide sterilization of single-use triple lumen sphinctertomes. METHODS: New triple-lumen sphinctertomes were soiled with thickened artificial test soil containing 6 log(10) per milliliter of Enterococcus faecalis and Bacillus stearothermophilus by inoculation through the distal end and dried for 1 hour, 24 hours, or 7 days before cleaning. The efficacy of manual cleaning was compared with that of automated cleaning with the Medisafe SI-Auto narrow-lumen cleaner. After cleaning, Bradford's reagent was injected into the channels as a direct method of detecting residual protein. Destructive testing was done to determine the levels of residual protein, carbohydrate, hemoglobin, endotoxin, and viable bacteria in the cleaned device. Destructive sterility testing of the devices also was performed after ethylene oxide sterilization. RESULTS: Both in-situ and destructive testing demonstrated that manual cleaning and automated washers connected via the luer ports did not remove soil or organisms from the cautery wire channel. Only retro-flushing in the SI-Auto provided adequate cleaning of all 3 channels. If reprocessing was delayed for more than 24 hours, retro-flush cleaning was no longer effective. Ethylene oxide sterilization failure was detected only for devices held for 7 days before cleaning and sterilization. In-use testing showed that patient secretions gained access to the cautery wire channel. CONCLUSIONS: Only retro-flushing done within 24 hours of use provided adequate cleaning for multi-lumen, single-use sphinctertomes. Our data validate the efficacy of reprocessing sphinctertomes once with SI-Auto retro-flush cleaning followed by 2 hours of ethylene oxide sterilization.