Sumuk Sundaram

Effect of two-chambered bicarbonate lactate-buffered peritoneal dialysis fluids on peripheral blood mononuclear cell and polymorphonuclear cell function in vitro☆☆☆

Low pH, high osmolality, increasing glucose concentration, and glucose degradation products (GDP) formed during heat sterilization of conventional peritoneal dialysis (PD) fluids have been shown to have a detrimental effect on cells involved in peritoneal host defense. The two-chambered PD fluid bag in which glucose at pH approximately 3 is separated from a bicarbonate (25 mmol/L)-lactate (15 mmol/L) buffer during heat sterilization permits PD fluids with lower GDP to be delivered to the patient at neutral pH. To establish the possible benefit of two-chambered bag PD fluids on peripheral blood mononuclear cell (PBMC) and polymorphonuclear (PMN) cell function, we compared conventional 1.5% Dianeal (1.5%D) with 1.5% two-chambered bag bicarbonate-lactate (1.5%D-B), and conventional 4.25% Dianeal (4.25%D) with 4.25% two-chambered bag bicarbonate-lactate (4.25%D-B). Furthermore, to study the effect of the sterilization process on PBMC and PMN function, we compared filter-sterilized 4.25%D (4.25%D-F) with 4.25%D and 4.25%D-B. PBMC were harvested by Ficoll-Hypaque separation, and 2.5 x 10(6) cells in RPMI were incubated with an equal volume of the test fluids for 4 hours, pelleted, and resuspended in RPMI containing 10 ng endotoxin for a further 20 hours. Tumor necrosis factor alpha (TNF-alpha) production by endotoxin-stimulated PBMC was not significantly different (P = 0.10) between 1.5%D-B and 1.5%D, but was significantly higher (P = 0.01) with 4.25%D-B compared with 4.25%D. PBMC exposed to filter-sterilized fluid (4.25%D-F) showed significantly higher endotoxin-stimulated TNF-alpha production compared with 4.25%D (P = 0.02), but was not significantly different from 4.25%D-B (P = 0.40). PMN were harvested by Ficoll-Hypaque separation and 10 x 10(6) cells incubated with test fluids for 30 minutes. After incubation, phagocytosis (phagocytosis index) was determined by the uptake of 14C-labeled Staphylococcus aureus, oxidative burst by reduction of ferricytochrome C to ferrocytochrome C on stimulation with PMA, and enzyme release by measurement of endotoxin-stimulated bactericidal/permeability increasing protein (BPI). Bicarbonate-lactate two-chambered fluids of similar osmolality and glucose concentration conferred a significant improvement in phagocytosis (P = 0.02 for 1.5%D-B and P < 0.001 for 4.25%D-B). Oxidative burst and BPI release were significantly higher in 4.25%D-B compared with 4.25%D (P < 0.001). Filter-sterilized 4.25%D-F conferred a significant improvement in phagocytosis and oxidative burst compared with 4.25%D (P < 0.001) or 4.25%D-B (P < 0.001). Furthermore, conventional 4.25%D was associated with significantly lower BPI release compared with 4.25%D-F (P = 0.01). GDPs acetaldehyde and 5-HMF were analyzed in 4.25%D-B, 4.25%D, and 4.25%D-F. Acetaldehyde was below the lower limit (0.79 ppm) of the standard curve in 4.25%D-B and 4.25%D-F fluids but was detected (3.76 to 5.12 ppm) in all of the 4.25%D fluids. Relative levels of 5-HMF in the 4.25%D-B (0.032 to 0.041 Abs @ 284 nm) and 4.25%D (0.031 to 0.036 Abs @ 284 nm) were similar. The lowest levels (0.001 Abs @ 284 nm) were observed in the filter-sterilized 4.25%D-F. The beneficial effects of two-chambered bicarbonate lactate-buffered PD fluids on PBMC and PMN function are probably related to reduction of GDP from heat sterilization of glucose in a separate chamber at a lower pH. This improvement in biocompatibility could have a beneficial affect on peritoneal defenses.

Shiga toxin induces superoxide production in polymorphonuclear cells with subsequent impairment of phagocytosis and responsiveness to phorbol esters

The role of inflammatory cells in the pathogenesis of hemolytic-uremic syndrome induced by Shiga toxin (Stx)-producing Escherichia coli remains unclear. The hypothesis that Stx has direct effects on polymorphonuclear cell (PMN) viability and function was examined by measuring apoptosis, necrosis, phagocytosis, and spontaneous and phorbol myristate acetate (PMA)-stimulated production of reactive oxygen intermediates. PMN from 6 healthy persons were exposed to medium, Stx1 (0.01-100 ng/mL), or heat-inactivated Stx1 or Stx1 B subunit (100 ng/mL). Stx1 induced a prominent dose-dependent respiratory burst from PMN at doses as low as 0.01 ng/mL; they were less responsive to PMA stimulation and had reduced ability for phagocytosis. This dysfunction was not due to cell death, as the magnitude of apoptosis and necrosis of PMN treated with Stx1 (100 ng/mL) for 20 h was identical to that of medium control. These results suggest that Stx has direct effects on PMN that could contribute to tissue injury early in the disease.

Removal of cytokine inducing substances by polymyxin-B immobilized polystyrene-derivative fibers during in vitro hemoperfusion of 10% human plasma containing Staphylococcus aureus challenge.

Staphylococcus aureus (S. aureus) is frequently isolated from blood cultures in the hospital setting. The pathogenesis of S. aureus bacteremia probably replicates mechanisms implicated in gram negative bacterial infections. Cell wall components, such as peptidoglycans and lipoteichoic acids (LTA), can trigger cytokine production. Polymyxin-B (PMX-B) is a cationic peptide that binds endotoxin (ET) and inhibits its activity. Based on this principle, PMX-B was incorporated in polystyrene-derivative fibers, creating a hemoperfusion column (PMX-20R) that removes ET. The authors assessed whether S. aureus possesses PMX-B suppressible cytokine-inducing substances, and whether LTA, an anionic molecule, is one such substance. Heparinized blood was obtained from healthy volunteers, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque separation, and 10% human plasma prepared. PBMC were incubated with 1, 5, or 10 μg/ml of S. aureus LTA, with and without 10 μg/ml of PMX-B. Also, using PMX-20R, in vitro hemoperfusion (IVH) was performed with 10% human plasma containing a 1:1,000 dilution of S. aureus challenge at 100 ml/min for 2 hours at 37°C, and plasma obtained before and after IVH was incubated with PBMC. After a 24 hour incubation at 37°C, PBMC were subjected to three freeze-thaw cycles, and total TNFα was measured by radioimmunoassay. TNFα production by PBMC incubated with LTA was 164 ± 4 pg, 324 ± 54 pg, 657 ± 55 pg, and 1143 ± 215 pg in control, and LTA 1, 5, and 10 μg/ml, respectively. The addition of PMX-B resulted in a 40 ± 12% (p = 0.02), 61 ± 6% (p = 0.002), and 62 ± 14% (p = 0.02) decrease in TNFα production, respectively. Before IVH, TNFα production by PBMC incubated with 10% plasma containing S. aureus challenge was 1275 ± 70 pg. After 2 hours of IVH, the decrease in TNFα production was 20 ± 4% (p = 0.002). In conclusion, S. aureus LTA induces TNFα production that is significantly suppressed by PMX-B. Consequently, S. aureus cytokine-inducing substances are removed during IVH with PMX-20R, and this may be due to stoichiometric binding of LTA to PMX-B.

Biomaterials in cardiopulmonary bypass.

The improved utilization of biomaterials in cardiopulmonary bypass is dependent on polymer science and technology, procedures for blood compatibility assessment, optimization of biomaterial/antithrombotic agent combinations and the interpretation of clinical data.

Endotoxin Removal by Polymyxin-B Immobilized Polystyrene-Derivative Fibers During In Vitro Hemoperfusion of 10% Human Plasma

During gram-negative bacterial sepsis, lipid A, the biologically active moiety of endotoxin (ET), activates monocytes and induces the release of cytokines. PMX-B, a cationic peptide, binds to lipid A and inhibits its activity. Based on this principle, PMX-B was incorporated in polystyrene-derivative fibers, creating a hemoperfusion column (PMX-20R) that removes ET. After in vitro characterization of the cytokine inducing potency of three gram-negative bacterial challenges, the authors evaluated the in vitro efficacy of PMX-20R in a model using 10% human plasma. Cytokine production by peripheral blood mononuclear cells (PBMC) incubated with plasma before and after in vitro hemoperfusion (IVH) was used as the index of ET removal. One hundred forty milliliters of heparinized blood were obtained from healthy volunteers. Forty milliliters were used to harvest PBMC at baseline, and 10% plasma prepared from the rest, was challenged with: 1) 0.01, 1, or 100 ng/ml of purified Escherichia coli ET; or 2) 1:1,000 dilution of E. coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae. IVH was performed at 100 ml/min at 37°C for up to 6 hours. One half milliliter samples, drawn before and at designated time intervals after the start of IVH, were mixed with a 0.5 ml suspension of 5 × 106 PBMC/ml from the same donor, and incubated for 24 hours at 37°C. PBMC were subjected to three freeze-thaw cycles, and total tumor necrosis factor alpha (TNFα) was measured by radioimmunoassay. Before IVH, TNFα production by PBMC incubated with 10% plasma containing 0.01, 1, or 100 ng/ml of purified E. coli ET was 1905 ± 391 pg, 2076 ± 552 pg, and 5304 ± 1001 pg, respectively. After 2 hours of IVH, the respective decrease in TNFα production was 82 ± 5% (p = 0.005), 78 ± 10% (p = 0.01), and 95 ± 1% (p = 0.002). Before IVH, TNFα production by PBMC incubated with 10% plasma containing 1:1,000 dilution of E. coli, P. aeruginosa or K. pneumoniae was 2896 ± 273 pg, 1816 ± 122 pg, and 1131 ± 125 pg, respectively. After 2 hours of IVH, the respective decrease in TNFα production was 83 ± 4% (p < 0.001), 53 ± 4% (p < 0.001), and 70 ± 5% (p < 0.001). When IVH was extended to 6 hours, the further decrease in TNFα production was not statistically significant. These results suggest an impressive in vitro removal of ET by PMX-20R from 10% human plasma containing either purified E. coli ET or E. coli, P. aeruginosa, or K. pneumoniae. Further in vitro studies are required, using whole blood challenged with gram-negative bacteria.

POLYMYXIN-B STIMULATES TUMOR NECROSIS FACTOR-ALPHA PRODUCTION BY HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS

Gram-negative bacterial lipopolysaccharide (LPS) is a well known stimulus for cytokine production, particularly interleukin-1 (IL-1) and tumor necrosis tactor alpha (TNFα). Polymyxin B (PMX-B) is a cationic polypeptide that binds to LPS, neutralizing its biological effects. PMX-B also disrupts gram-negative bacterial cell membrane phospholipids but is highly toxic to mammalian cells, therefore is of limited use. PMX-B is used as additive to media, as a way to handle LPS contamination. To derive benefit from the ability of PMX-B to neutralize lipid A in vivo while avoiding its systemic toxicity, PMX-B was covalently bound to polystyrene-derivative fibers, creating a hemoperfusion column (PMX-F) for the selective removal of circulating ET In vitro PMX-F hemoperfusion studies have demonstrated effective ET removal, using either the Limulus amebocyte lysate assay or TNFα production by peripheral blood mononuclear cells (PBMC) as an index of ET removal. However, the question whether PMX-B itself could stimulate human PBMC to produce cytokines has not been adequately addressed. We examined the effect of increasing concentrations of PMX-B on cytokine production by PBMC in vitro. PBMC harvested from healthy volunteers were incubated for 24 hours at 37°C with control (tissue culture media RPMI), or 5 µg/ml, 10 µg/ml, 20 µg/ml or 100 µg/ml PMX-B. At the end of 24 hours, PBMC were subjected to three freeze-thaw cycles, and total TNFα production (pg/2.5x106 PBMC) was measured by radioimmunoassay. Total TNFα production by PBMC was 163 ± 3 pg, 171 ± 9 pg, 164 ± 4 pg, 323 ± 63 pg and 331 ± 58 pg, in the control, PMX-B 5 µg/ml, 10 µg/ml, 20 µg/ml and 100 µg/ml conditions, respectively. Compared to controls (RPMI), the percentage increase in TNFα production by PBMC was 5 ± 6% (P=0.23), 1 ± 3% (P=0.45), 99 ± 40% (P=0.03) and 103 ± 36% (P=0.02) in the presence of 5 µg/ml, 10 µg/ml, 20 µg/ml and 100 µg/ml of PMX-B, respectively. Furthermore, total TNFα production correlated significantly with increasing concentrations of PMX-B (R=0.53, P=0.007). We conclude that the use of PMX-B in in vitro studies as an LPS-neutralizing agent, or in the experimental treatment of endotoxic or septic shock can lead to erroneous interpretations of cytokine production by PBMC, and should be used cautiously in in vitro systems at high concentrations.

Cytokine production by human peripheral blood mononuclear cells stimulated by a Pseudomonas aeruginosa culture filtrate: Role of plasma and polymyxin B

The lack of consensus regarding the significance of transmembrane passage of bacterial products across hemodialysis membranes can be related to several methodological differences in the various studies, including the choice of circulating fluid in the blood compartment of the model, nature and concentration of the bacterial products employed to challenge the dialysate compartment and whether cytokine production by PMBC or the limulus amebocyte lysate (LAL) assay was used as the index of transfer and the cytokine used as the read-out. In this study, we examined the production of interleukin-1 alpha (IL-1α), interieukin-1 receptor antagonist (IL-1Ra) and interleukin-8 (IL-8) by peripheral blood mononuclear cells (PBMC) incubated with a Pseudomonas aeruginosa culture filtrate. Further, the effects of 10% autologous human plasma and Polymyxin B sulfate (PmB) on cytokine production by PBMC were also characterized. The results of our study indicate that the Ps. aeruginosa culture filtrate had both PmB suppressible and PmB non-suppressible components and that the addition of 10% human plasma significantly enhanced cytokine production by both PmB suppressible and PmB non-suppressible components. The enhancing effect of plasma was most evident at low concentrations of the filtrate. The inhibitory effect of PmB was most evident in samples cultured in the presence of 10% plasma. There was a direct correlation between the production of IL-1α and IL-1Ra suggesting that both pro-inflammatory cytokines and cytokine-specific inhibitory proteins are concurrently produced. There results have direct relevance to selection of study conditions for in vitro models used to study the transmembrane passage of bacterial products across hemodialysis membranes