Gerhard Lonnemann

The Effect of Dietary Supplementation with n—3 Polyunsaturated Fatty Acids on the Synthesis of Interleukin-1 and Tumor Necrosis Factor by Mononuclear Cells

We examined whether the synthesis of interleukin-1 or tumor necrosis factor, two cytokines with potent inflammatory activities, is influenced by dietary supplementation with n-3 fatty acids. Nine healthy volunteers added 18 g of fish-oil concentrate per day to their normal Western diet for six weeks. We used a radioimmunoassay to measure interleukin-1 (IL-1 beta and IL-1 alpha) and tumor necrosis factor produced in vitro by stimulated peripheral-blood mononuclear cells. With endotoxin as a stimulus, the synthesis of IL-1 beta was suppressed from 7.4 +/- 0.9 ng per milliliter at base line to 4.2 +/- 0.5 ng per milliliter after six weeks of supplementation (43 percent decrease; P = 0.048). Ten weeks after the end of n-3 supplementation, we observed a further decrease to 2.9 +/- 0.5 ng per milliliter (61 percent decrease; P = 0.005). The production of IL-1 alpha and tumor necrosis factor responded in a similar manner. Twenty weeks after the end of supplementation, the production of IL-1 beta, IL-1 alpha, and tumor necrosis factor had returned to the presupplement level. The decreased production of interleukin-1 and tumor necrosis factor was accompanied by a decreased ratio of arachidonic acid to eicosapentaenoic acid in the membrane phospholipids of mononuclear cells. We conclude that the synthesis of IL-1 beta, IL-1 alpha, and tumor necrosis factor can be suppressed by dietary supplementation with long-chain n-3 fatty acids. The reported antiinflammatory effect of these n-3 fatty acids may be mediated in part by their inhibitory effect on the production of interleukin-1 and tumor necrosis factor.

A pilot study of coupled plasma filtration with adsorption in septic shock

ObjectiveTo test the hypothesis that nonselective plasma adsorption by a hydrophobic resin (coupled plasmafiltration and adsorption) could improve hemodynamics and restore leukocyte responsiveness in patients with septic shock. DesignProspective, pilot, crossover clinical trial. SettingGeneral intensive care unit in a teaching hospital. SubjectsTen patients with hyperdynamic septic shock. InterventionsPatients were randomly allocated to 10 hrs of either coupled plasma filtration adsorption plus hemodialysis (treatment A) or continuous venovenous hemodiafiltration (treatment B) in random order. We measured the change in mean arterial pressure, norepinephrine requirements, and leukocyte tumor necrosis factor-&agr; (TNF-&agr;) production (both spontaneous and lipopolysaccharide-stimulated) after 10 hrs of each treatment. We also tested TNF-&agr; production from normal human adherent monocytes incubated with patients’ plasma obtained before and after the resin, both with or without incubation with an anti-interleukin-10 monoclonal antibody. ResultsMean arterial pressure increased after 10 hr by 11.8 mm Hg with treatment A and by 5.5 mm Hg with treatment B (p = .001). There was an average decrease of norepinephrine requirement of 0.08 &mgr;g/kg/min with treatment A and 0.0049 &mgr;g/kg/min with treatment B (p = .003). All patients but one survived. Spontaneous and lipopolysaccharide-induced TNF-&agr; production from patients’ whole blood increased over time with treatment A. This increase was more marked in blood drawn after the device (plasmafiltrate-sorbent plus hemodialyzer) (p = .009). Preresin plasma suppressed lipopolysaccharide-stimulated production of TNF-&agr; by 1 × 106 cultured adherent monocytes from healthy donors. This suppressive effect was significantly reduced after passage of plasma through the resin (p = .019) and after incubation with anti-interleukin-10 monoclonal antibodies (p = .028). ConclusionsIn patients with septic shock, coupled plasmafiltration-adsorption combined with hemodialysis was associated with improved hemodynamics compared with continuous venovenous hemodiafiltration. This result might be related to its ability to restore leukocyte responsiveness to lipopolysaccharide. These findings suggest a potential role for blood purification in the treatment of septic shock.

Concentrations of immunoreactive human tumor necrosis factor alpha produced by human mononuclear cells in vitro

The concentrations of tumor necrosis factor (TNF) produced by human peripheral blood mononuclear cells (MNC) were measured using a radioimmunoassay (RIA) for human TNF. This was developed using a rabbit antiserum against human recombinant TNF (Hu rTNF), and Hu rTNF labeled with Na1251 by a modification of the chloramine T method. This RIA does not detect human lymphotoxin, interieukin‐1 alpha or beta, interleukin 2, interleukin 6, interferon alpha or gamma, granulocyte‐macrophage‐colony stimulating factor, and C5a des arg. A good correlation (r = 0.89) was found between the RIA and the cytolytic bioassay for TNF. The sensitivity of the RIA is between 3 and 78 pg/ml (median 11 pg/ml).

Measurement of immunoreactive interleukin-1β from human mononuclear cells: optimization of recovery, intrasubject consistency, and comparison with interleukin-1α and tumor necrosis factor

Numerous studies have reported altered levels of in vitro production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) from blood leukocytes in various human disease states. Most of these studies have used bioassays which are vulnerable to inhibitors produced by these cells. Furthermore in vitro cytokine production is often assessed on a single occasion. The present study was designed to standardize stimulation conditions for in vitro IL-1 beta production and to employ a competitive radioimmunoassay (RIA) to demonstrate reproducibility and long-term variation of in vitro cytokine production in a cohort of healthy human subjects. We also examined relative amounts of immunoreactive IL-1 beta, IL-1 alpha, and TNF induced by the stimuli endotoxin, phytohemagglutinin, or Staphylococcus epidermidis. We show that the RIA can reliably detect IL-1 beta produced from mononuclear cells in concentrations as low as 115 pg/ml. Lysing cells by repeated freeze-thawing yields maximal recovery of total (i.e., secreted plus cell-associated) immunoreactive IL-1 beta, when compared to extraction with the detergent CHAPS or addition of protease inhibitors. Repeated measurement of in vitro cytokine production on different days within 1 week shows good reproducibility for a given individual and a given stimulus (variation coefficient 20 to 30%). Over a long time period (6 months) in vitro cytokine production is stable in some individuals but changes considerably in others. The soluble stimulus endotoxin induces twofold more IL-1 alpha than IL-1 beta or TNF; in contrast the phagocytic stimulus heat-killed S. epidermidis induces fourfold more IL-1 beta and TNF than IL-1 alpha. This distinct pattern of cytokine response indicates differential stimulation of the mononuclear cells by different stimuli. The results form the basis for studying in vitro cytokine production in different human disease states.

Enhancement of in-vitro human interleukin-1 production by sodium acetate.

Human blood monocytes were incubated in vitro in the presence of various concentrations of sodium acetate or sodium chloride or with medium alone. Intracellular and extracellular levels of interleukin-1 (IL-1) were measured. The production of intracellular IL-1 and the release of extracellular IL-1 were higher in the presence of acetate than in the presence of chloride or in medium alone. The concentrations of acetate used were comparable to those encountered by blood monocytes on the surface of haemodialysis membranes. Since complications of peritoneal dialysis, such as loss of ultrafiltration and progressive fibrosis of the peritoneum, have been associated with the use of sodium acetate as the exchange-fluid buffer, these results suggest that the widespread use of sodium acetate as a buffer during haemodialysis may be contraindicated.

Clinical, hematologic, and immunologic effects of interleukin-10 in humans.

We conducted a double-blind, placebo-controlled study to investigate the safety, pharmacokinetics, and immunological properties of interleukin-10 (IL-10) administration in healthy humans. Volunteers received a single intravenous bolus injection of recombinant human IL-10 (1, 10, or 25μg/kg) or placebo. Cytokine production in whole blood and peripheral blood mononuclear cells (PBMC) was assessed before and 3, 6, 24, and 48 hr after the injection. Peak serum concentrations of IL-10 (15±1.1, 208±20.1, and 505±22.3 ng/ml) occurred after 2–5 min for 1, 10, and 25μg/kg IL-10, respectively. The terminal-phase half-life was 3.18 hr. A transient leukocytosis (24–63% above baseline) was observed 6 hr after injection, which coincided with a dose-dependent decrease (12–24%) in neutrophil superoxide generation. There was a marked inhibition (60–95%) of endotoxin-induced IL-6 production from whole blood in each group receiving IL-10. Production of IL-8 in endotoxin-stimulated blood was reduced in the 10μg/kg group. In PBMC stimulated with phytohemagglutinin and phorbol ester, there was a decrease (72–87%) in interferon-γ (IFNγ) production 6 hr after IL-10 with a return to pre-IL-10 levels after 24 hr. This reduction was only partially associated with a decrease in the number of CD2-bearing cells. We conclude that IL-10 administration into humans is without significant side effects, and a single injection reducesex vivo production of IL-6, IL-8, and IFNγ.

Reimbursement of Dialysis: A Comparison of Seven Countries

Reimbursement for chronic dialysis consumes a substantial portion of healthcare costs for a relatively small proportion of the total population. Each country has a unique reimbursement system that attempts to control rising costs. Thus, comparing the reimbursement systems between countries might be helpful to find solutions to minimize costs to society without jeopardizing quality of treatment and outcomes. We conducted a survey of seven countries to compare crude reimbursement for various dialysis modalities and evaluated additional factors, such as inclusion of drugs or physician payments in the reimbursement package, adjustment in rates for specific patient subgroups, and pay for performance therapeutic thresholds. The comparison examines the United States, the province of Ontario in Canada, and five European countries (Belgium, France, Germany, The Netherlands, and the United Kingdom). Important differences between countries exist, resulting in as much as a 3.3-fold difference between highest and lowest reimbursement rates for chronic hemodialysis. Differences persist even when our data were adjusted for per capita gross domestic product. Reimbursement for peritoneal dialysis is lower in most countries except Germany and the United States. The United Kingdom is the only country that has implemented an incentive if patients use an arteriovenous fistula. Although home hemodialysis (prolonged or daily dialysis) allows greater flexibility and better patient outcomes, reimbursement is only incentivized in The Netherlands. Unfortunately, it is not yet clear that such differences save money or improve quality of care. Future research should focus on directly testing both outcomes.

The Effect of Ultrafiltered Dialysate on the Cellular Content of lnterleukin-1 Receptor Antagonist in Patients on Chronic Hemodialysis

We investigated the effect of dialysate ultrafiltration on the content of IL-1 receptor antagonist (IL-1Ra) in mononuclear cells (PBMC) as a marker of the inflammatory response. 11 patients on Cuprophan dialyzers were randomly assigned to treatment with standard bicarbonate dialysate first and then to ultrafiltered dialysate or the reverse order in a crossover design. In each treatment period (at least 4 weeks) weekly separations of PBMC were performed before the start of dialysis. Cellular content of IL-1Ra was determined in PBMC that were frozen immediately after separation; all values of IL-1Ra in each treatment period were averaged. The dialysate contained a median of 148 (range, 61-400) colony-forming units without dialysate filter; no bacterial growth was detected in ultrafiltered dialysate. The median endotoxin content was 80 pg/ml in nonfiltered dialysate; endotoxin was below 5 pg/ml in all ultrafiltered dialysate samples. Cellular content of IL-1Ra decreased in all but 1 patient with the use of ultrafiltered dialysate (mean +/- SEM: 1,467 +/- 113 pg/ml without dialysate filter vs. 1,166 +/- 104 pg/ml with filter, p = 0.016). The present study demonstrates that the bacterial contamination of the dialysate induces a systemic inflammatory response in hemodialysis patients.

Chronic Inflammation in Hemodialysis: The Role of Contaminated Dialysate

Routine sodium bicarbonate-buffered dialysate is contaminated with predominantly gram-negative micro-organisms. These bacteria release pyrogenic substances such as endotoxins, peptidoglycans, exotoxins and fragments thereof. Pyrogens derived from contaminated dialysate either alone or in costimulation with activated complement components are the most important activators of circulating mononuclear cells in patients on chronic intermittent hemodialysis. Activated mononuclear cells release proinflammatory cytokines which are key mediators in acute and chronic inflammatory diseases associated with long-term hemodialysis therapy. Recent experimental and clinical data suggest that the use of pyrogen-free dialysate prevents activation of mononuclear cells and improves the state of chronic inflammation, as indicated by decreased plasma levels of C-reactive protein in chronic hemodialysis patients. Future clinical studies have to prove whether the use of pyrogen-free dialysate in combination with biocompatible dialyzer membranes and tubings reduces the incidence and severity of chronic inflammatory diseases (β2-microglobulin amyloidosis, muscle protein wasting, atherosclerosis) in long-term hemodialysis patients.

Plasma Interleukin-1 Activity during Hemodialysis: The Influence of Dialysis Membranes

Plasma interleukin-1 (IL-1) activity was measured in 7 stable ESRD patients on regular hemodialysis for no less than 5 months. Predialysis levels were significantly raised compared to 8 normal control subjects. During hemodialysis with four different membranes, plasma IL-1 activity rose with Cuprophan and Hemophan and was unchanged or reduced with Gambrane and Polysulfon. In spite of these differences, body temperature rose in all forms of hemodialysis. Factors responsible for the predialysis elevation included the absence of renal function and/or the repeated stimulus of human blood monocytes by hemodialysis. In view of the uniform increase of body temperature during hemodialysis, the differences in changes of plasma IL-1 activity observed with the various membranes may not be caused by a variable stimulation of monocytes but rather by the presence or absence of the membranes ability to remove and/or absorb IL-1. Thus, the consequences of monocyte hemodialysis stimulation may be obtained locally, even in the presence of unchanged or reduced plasma IL-1 activity.