A study of the sensitivity of erythrocytes to lysis by heterologous sera via the alternative complement pathway.

In order to get insight in the distribution of alternative complement pathway activities as detected by lysis of xenogeneic erythrocytes in the presence of magnesium and ethyleneglycol-bis-(2-aminoethyl)-tetra-acetic acid (EGTA) over the species, the 156 heterologous combinations of erythrocytes and sera out of thirteen animal species were tested. An order could be noticed in the species with respect to serum complement activity tending to negative correlation with the sensitivity of the corresponding erythrocytes to lysis by heterologous sera. So far, the most sensitive erythrocyte for each individual serum must be considered to be the target cell of choice for developing assays for alternative complement pathway activity in the serum involved. In this series of animals only for rabbit serum no sensitive target cell was found. The order observed, in connection with the failing lysis of erythrocytes by homologous sera, suggests further that in restriction of heterologous hemolysis in general one erythrocyte-associated, species-nonspecific regulatory principle may be involved, whereas in homologous restriction, most probably, also species-specific factors play a role.

Cytotoxic effects of commercial continuous ambulatory peritoneal dialysis (CAPD) fluids and of bacterial exoproducts on human mesothelial cells in vitro.

Cultured human mesothelial cells were exposed to peritoneal dialysis fluids, supernatants from cultures of Staphylococcus aureus and S. epidermidis, and antibiotics. Mesothelial cell monolayer cultures were derived from surgically removed omentum. The cytotoxicity of various agents for the cultured mesothelial cells was measured by a 51 Cr-release assay. All brands of fresh peritoneal dialysis fluids induced a more than 50% 51 Cr-release after 18 h. Morphological changes observed included retraction and shrinking of cells, pyknosis of the nuclei and, finally, detachment of cells over an 18-h period. Neutralization of the acid (pH 5.2-5.5) fluids to pH 7.3 did not abolish the cytotoxicity. In contrast, effluent dialysis fluids were not toxic for mesothelial cells; neither was acid (pH 5.5) culture medium nor culture medium with glucose up to 2%. However, higher glucose concentrations induced increasing 51 Cr-release. Furthermore, filter-sterilized supernatants of S. aureus were cytotoxic for mesothelial cell monolayers in 4/7 (57%) strains of S. aureus tested. In contrast, only 4/29 (14%) strains of S. epidermidis produced cytotoxic exoproducts (p = 0.03). Antibiotics were not found to be cytotoxic, with the possible exception of erythromycin. We conclude that currently available peritoneal dialysis fluids are cytotoxic for mesothelial cells in vitro and that during episodes of peritonitis exoproducts of some bacterial strains may further reduce mesothelial cell viability.

In vitro compatibility of a 1.1% amino acid containing peritoneal dialysis fluid with phagocyte function.

The effects of a recently introduced peritoneal dialysis fluid (PDF) containing amino acids (AA) were compared with those of a glucose-based PDF (G-PDF) on viability and function of donor granulocytes (PMNs) in vitro. After 30 min incubation in the PDF, viability, assessed by trypan blue exclusion, and phagocytosis capacity (PC), tested in two assays using a fluorescein and a 3H-labeled Staphylococcus epidermidis strain, were significantly better in AA-PDF than in G-PDF (p < 0.002 in the 3H-assay). Bactericidal activity was not different in the PDFs. If pH of G-PDF was adjusted from 5.2 to neutral, differences in PC disappeared. In AA-PDF, PMN chemiluminescence (CL) response was significantly higher than in G-PDF (p < 0.003). At neutral pH, however, PMNs showed a significant stronger CL-response in 1.36% G-PDF than in AA-PDF (p < 0.05). These data suggest that this AA-PDF has little detrimental effect on phagocyte viability and function. The improved compatibility over G-PDF in in vitro tests seems to be pH dependent. The reduced chemiluminescence response compared to 1.36% G-PDF with neutral pH is possibly due to quenching by (one of the) amino acids and osmolarity.

Antibacterial peritoneal defence in automated peritoneal dialysis: advantages of tidal over continuous cyclic peritoneal dialysis?

In tidal peritoneal dialysis (TPD) only a part of the infused dialysate is drained with each exchange, leaving a residual volume on top of which fresh fluid is cycled. As the persistent presence of a buffered intraperitoneal reserve volume might favour peritoneal macrophage (PMO) function, PMO obtained from eight patients during a 3-h continuous cyclic peritoneal dialysis (CCPD) or TPD session were studied in a randomized cross-over trial. PMO were studied for uptake of E. coli (complement-dependent) and S. epidermidis (antibody-dependent), as well as for their killing capacity and peak chemiluminescence response. In addition, dialysate was sampled during both treatment sessions and studied for pH, osmolality, and effect on the viability of donor phagocytes and mesothelial cells. TPD-derived PMO were significantly better able to phagocytose E. coli than CCPD-PMO (48 +/- 8 versus 33 +/- 6% uptake, P < 0.05), whereas the other tested functional capacities revealed no significant difference between TPD- and CCPD-PMO. During TPD dialysate pH ranged from 6 to 7 as compared to a pH range from 5 to 7 in CCPD. The presence of a residual dialysate volume resulted in less wash-out of cells and opsonins early in the treatment, and to some extent blunted the noxious effects of fresh dialysis solutions. Overall, however, tidal PD appeared to have no advantage over CCPD regarding preservation of peritoneal defences.

Low-calcium peritoneal dialysis fluid should not impact peritonitis rates in continuous ambulatory peritoneal dialysis.

It has been suggested that reducing the calcium content of peritoneal dialysis fluid (PDF) to 2.5 mEq/L decreases peritoneal macrophage (PMO) function and increases the incidence of peritonitis (especially Staphylococcus epidermidis peritonitis) in continuous ambulatory peritoneal dialysis patients. We studied the uptake and killing of S epidermidis and Escherichia coli by PMOs and peripheral blood leukocytes incubated in control buffer (Hank's balanced salt solution containing 0.1% gelatin [GHBSS]) and PDF containing varying concentrations of calcium (O to 3.5 mEq/L) and magnesium (O to 1.5 mEq/L) using ether diamine tetraacetic acid and ethylenediaminetetraacetic acid chelation, respectively. In addition, interleukin-1-beta-induced interleukin-6 production by human mesothelial cells was measured in the presence of concentrations of calcium increasing from 0 to 3.0 mmol/L. Fc receptor- mediated uptake of S epidermidis by PMO in the complete absence of Ca++ was comparable to that by PMO incubated in GHBSS with calcium. In contrast, the complement-dependent uptake of E coli was significantly lower in GHBSS devoid of Ca++ (46% +/- 5% v 24% +/- 3%; 0.05 < P < 0.02). No effect on intracellular killing of either microorganism by PMO was observed. The same held true for the phagocytic and killing capacity of polymorphonuclear granulocytes and monocytes obtained from healthy donors. Using Ca++ (2 to 3.5 mEq/L) and Mg++ (0.5 to 1.5 mEq/L) concentrations as applied in commercial PDFs, however, phagocytes performed as well as in control buffer. Interleukin-6 production by stimulated human mesothelial cells also required a small amount of Ca++ only, being normal above the 0.1 to 3 mmol/L Ca+ + range tested. Thus, complement- dependent uptake of bacteria by phagocytes is calcium dependent, whereas antibody-dependent uptake of S epidermidis is not. The concentrations of calcium in the current PDFs, however, will not compromise human mesothelial cells and leukocyte functions, and therefore should not impact the peritonitis rate.

Effects of granulocyte colony-stimulating factor (G-CSF) treatment on granulocyte function and receptor expression in patients with ventilator-dependent pneumonia.

Considerable experimental evidence in animals suggests that treatment with G-CSF may have a beneficial effect in the management of severe infections in non-neutropenic hosts. This beneficial effect is attributed to an enhancement of granulopoiesis and neutrophil function, the latter possibly involving up-regulation of receptors on neutrophils that are involved in antibody-mediated cytotoxicity and killing of microorganisms. We compared neutrophil function and phenotype in blood and bronchoalveolar lavage fluid (BALF) of 10 patients with severe ventilator-dependent pneumonia, at baseline and following initiation of G-CSF treatment as adjunct to standard therapy. G-CSF treatment was associated with three-fold increased blood neutrophil counts at day 3 of treatment compared with baseline counts. Mean serum G-CSF concentration increased from 313 to 2007 pg/ml. After correction for lavage dilution effects, BALF G-CSF levels did not differ significantly from baseline, nor did neutrophil receptor expression (FcgammaRI, FcgammaRII, FcgammaRIII, CR3, and L-selectin) or indicators of neutrophil function such as respiratory burst activity, phagocytosis and killing of Candida albicans in BALF or blood. The mortality in this group of patients was 30% and compared favourably to the APACHE II-derived predicted mortality of 60%. We conclude that the possible therapeutic benefit of G-CSF administration in the early phase of severe bacterial pneumonia is not readily explained by its effect on baseline indicators of neutrophil function or receptor expression.

Biocompatibility of a glucose-polymer-containing peritoneal dialysis fluid.

The currently available glucose-containing peritoneal dialysis fluids (PDF), which are all hyperosmolar, are toxic to the cells present in the peritoneal cavity. However, glucose-polymer solutions, being isosmolar, may have improved biocompatibility in this respect. We therefore compared in vitro the effects of PDF containing glucose-polymers with that of glucose solutions on the function of donor granulocytes and monocytes (MN), and on the viability of mesothelial cells. In addition, the function of peritoneal macrophages (PMO) of eight patients was studied in a randomized cross-over setting following intraperitoneal exposure to glucose-polymer-versus glucose-monomer-containing fluid of comparable ultrafiltration capacity. Donor granulocytes, as well as MN, showed significantly better phagocytosis of both Staphylococcus epidermidis and Escherichia coli after incubation in the glucose-polymer solution as compared with the 3.86% glucose-containing fluid. Their oxidative metabolism, as measured by chemiluminescence, also showed that the glucose-polymer solution was less inhibitory than fluids containing 2.27 or 3.86% glucose. Patient-derived PMO showed a significantly better phagocytic capacity for S epidermidis and E coli, a significantly higher killing of E coli, and a significantly higher chemiluminescence response after intraperitoneal exposure to the glucose-polymer solution as compared with the glucose-monomer-based fluid. Increasing the osmolality of the glucose-polymer solution to that of the respective glucose solutions blunted the favorable effect on phagocyte function, suggesting the beneficial effect to be osmolality-mediated. However, no major difference was observed between the glucose-polymer solution and the glucose-based fluid in their effects on mesothelial viability.(ABSTRACT TRUNCATED AT 250 WORDS)