Short Bacterial DNA Fragments

Abstract

Abstract. A number of bacterial cytokine-inducing substances (CIS) such as lipopolysaccharides (LPS) and exotoxins have been detected in dialysate and may contribute to inflammation in hemodialysis patients. Short DNA fragments, oligodeoxynucleotides (ODN) of 6 to 20 nucleotides, are able to bind to Toll-like receptors and are stimulatory on immune cells. ODN induce natural killer cell activity and induce IFN-␥, TNF-␣, and IL-6 from mononuclear cells. The presence of ODN in dialysate samples and bacterial cultures was investigated. ODN were extracted from fluids by adsorption to reverse-phase columns. ODN were detected in 18 of 20 investigated dialysate samples, in eight of 10 reverse-osmosis water samples, and in all cultures from various bacterial strains. The presence of bacterial DNA in dialysate was confirmed by PCR specific for bacterial tRNA gene sequences. Saline for intravenous use contained 0.02 Ϯ 0.01 g/ml DNA, dialysate samples contained 0.28 Ϯ 0.02 g/ml, and Pseudomonas cultures contained 1.0 Ϯ 0.03 g/ml DNA. ODN from bacterial cultures were only partially removed by ultrafiltration and were able to diffuse through regular high-flux dialyzer membranes. Synthetic cytosine-guanosine dinucleotide-containing ODN were able to induce IL-6 in human mononuclear cells. It is concluded that short bacterial-derived DNA fragments are present in clinically used fluids, e.g., dialysate. These fragments are of sufficient small size to pass through dialyzer membranes. Bacterial DNA fragments may be an overlooked factor contributing to inflammation in hemodialysis patients.

Dialysis Membrane Manipulation for Endotoxin Removal

“…As some studies have shown that typical endotoxin concentrations are higher in dialysis fluid than RO water entering the hemodialysis machine (von Sengbusch et al, 1993), the ultrapure criteria of the dialysis fluid needs to be created just prior to the patient receiving treatment. One method to produce and ensure the production of ultrapure dialysis fluid is to place an ultrafilter in the hydraulic path between the patient and the dialysis machine, as this filter will have the greatest impact to the final dialysis fluid solution prior to entrance into the dialyzer (Oliver et al, 1992;Schindler et al, 2004). Studies in the literature have revealed significant removal rates for ultrafilters in vitro, typically >99% for endotoxin (Oliver et al, 1992;Krautzig et al, 1996), with log reduction values >3 desirable (Tsuchida et al, 2009).…”

“…Going forward, future work may involve identifying new bacterial contaminants that cause adverse patient reactions, but may not be identified by the LAL assay (Glorieux et al, 2009). There are a number of smaller bacterial components released during cell lysis, with bacterial DNA fragments recently receiving considerable attention in research studies (Handelman et al, 2009;Schindler et al, 2004). Some of these studies have shown a correlation between bDNA fragments present in patient blood, and higher levels of CRP and IL-6 (Bossola et al, 2009).…”

The Evolution of Biocompatibility: From Microinflammation to Microvesiscles

“…A large number of studies have greatly contributed to increasing our knowledge in the mechanisms of endotoxin transfer across the membrane In fact, when using high permeability membranes, backfiltration and backdiffusion occur and have ebeen extensively described (Fiore & Ronco, 2007, Ronco, 2007. Thus, the transmembrane passage of endotoxins or other cytokine stimulating substances (CIS) occurs during HD (Schindler et al, 2004. The reduction of backfiltration of standard dialysate may reduce the plasma concentration of IL-1ra, a sensitive indicator of inflammation in HD patients (Dinarello personal communication, 2004), and IL-1 .…”

Did 20 Years of Technological Innovations in Hemodialysis Contribute to Better Patient Outcomes?

“…Traditional methods to detect microbial contamination overlook the majority of germs potentially present (88), whereas these difficult-to-detect germs are prone to generate proinflammatory agents other than intact endotoxin. Methods to measure these alternative reactants that may be present even in culture-negative and endotoxin-free dialysis water have been described (89,90). In contrast to the Limulus Amebocyte Lysate test, which detects only intact LPS, a new bioassay is sensitive to peptidoglycan, short bacterial DNA fragments, and LPS fragments Ͻ5 kD (89).…”