Abstract
Silkworm larvae plasma (SLP) reagent is activated by peptidoglycan (PG), a fragment of both the gram-positive and the gram-negative bacterial cell wall, as well as β-glucan (BG), a component of fungi. It is possible to measure contamination by PG quantitatively by combining the limulus amebocyte lysate (LAL) and SLP measurement methods. Therefore, a more accurate analysis of a dialysate can be made by using both SLP and LAL methods to detect endotoxin (ET) and/or PG contamination. We studied the effects of contaminated dialysates on human peripheral blood mononuclear cells (PBMC) by measuring production of various cytokines in vitro. Muramyl dipeptide (MDP) was used as the biologically active minimum constituent of PG. A total of 54 dialysate samples were obtained in sterile conditions from 4 sites, 1) a reverse osmosis-treated water system, 2) a central dialysate proportioning system, 3) a multiple patient station, 4) a personal patient station, at 9 dialysis facilities. To detect bacterial contamination, the samples were measured by the LAL (C), LAL (G) and SLP methods. PBMC were collected from 10 healthy controls and from 10 hemodialysis patients and cultured for 24 hours with lipopolysaccharide (LPS), MDP, LPS+MDP and a contaminated dialysate. Levels of IL-1 Ra, IL-1 β and TNF-α in the culture medium supernatants were measured by ELISA. PG was not detected in dialysates from site 1) or site 2). However, dialysates from the inlets of the dialyzer at a bedside monitor of the central patient stations and the personal patient stations showed 4.1±6.1ng/ml at site 3) (in 3 out of 18 samples 17%) and 3.3±4.6ng/ml at site 4) (in 7 out of 18 samples 39%). Not only contamination by PG alone but also combined contamination by PG and ET were detected. Furthermore, IL-1 Ra, IL-1 β and TNF-α production by PBMC increased in accordance with the concentrations of MDP. Cytokine production was 5 to 10 times higher where MDP and ET coexisted than where either MDP or ET existed alone, showing a synergic effect of MDP and ET. On the basis of these results, there is a high possibility that PG may also be a pyrogen, although ET has been considered the only pyrogen in dialysates. Therefore, it is essential to recognize the presence of both ET and PG in investigating dialysate contamination.
