Effect of liposomally encapsulated MTX-DMPE conjugates upon TNF alpha and PGE2 release by lipopolysaccharide stimulated rat peritoneal macrophages.

The ability of liposomally encapsulated preparations of methotrexate (MTX) and three of its lipophilic derivatives (MTX-gamma-DMPE, MTX-alpha-DMPE and MTX-alpha,gamma-diDMPE) to alter mediator release by lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages (PM theta) was investigated. The viability of these macrophages when incubated with approximately 6.0 nmol/10(5) cells of the respective liposomal preparations (MTX-LIPO, MTX-gamma-LIPO, MTX-alpha-LIPO and MTX-di-LIPO) for 20 h was greater than 80%. Treatment of macrophages, which had been incubated with MTX-alpha-LIPO (5.5 nmol/10(5) cells), MTX-gamma-LIPO (6.9 nmol/10(5) cells) and MTX-di-LIPO (4.5 nmol/10(5) cells) for 20 h, with antibody-coated sheep red blood cells resulted in 105 +/- 9.6%, 80.6 +/- 5.6% and 91 +/- 11.4% phagocytosis respectively (mean +/- S.E.M.). At similar concentrations of MTX-alpha-LIPO, MTX-gamma-LIPO and MTX-di-LIPO (6.5 nmol/10(5) cells), PGE2 release from LPS-stimulated rat peritoneal macrophages was inhibited by 85.3 +/- 3.7%, 68.7 +/- 0.6% and 88.8 +/- 2.2%, respectively (mean +/- S.E.M., n = 4). Incubation of these macrophages with 12, 10 and 9.4 nmol/10(5) cells of the respective liposomal preparations resulted in 89 +/- 3.3%, 62 +/- 5.5% and 85 +/- 3.9% inhibition of TNF alpha release (mean +/- S.E.M., n = 4). However, at this concentration MTX-di-LIPO was toxic. Neither MTX (20-2.5 nmol/10(5) cells) nor MTX-LIPO (5.6 nmol/10(5) cells) affected TNF alpha release from LPS-stimulated macrophages. Whilst free MTX was also ineffective at inhibiting PGE2 from these cells, incubation with MTX-LIPO at the above concentration resulted in 76.9 +/- 2.6% inhibition of the prostaglandins release.

Leukotriene B4 generation by human monocytes and neutrophils stimulated by uropathogenic strains of Escherichia coli.

The generation of the 5-lipoxygenase product, leukotriene B4 (LTB4) by human mononuclear phagocytes (monocytes) following incubation with 25 different uropathogenic strains of Escherichia coli correlated with the haemolytic activity of the strains (r = 0.572, P less than 0.01). LTB4 generation by human neutrophils (PMN), however, was unrelated to this haemolytic potential (r = 0.164). In contrast, both prelabelled monocytes and PMN were stimulated by haemolytic strains of E. coli and by haemolytic culture supernatants to release significant amounts of [3H]arachidonic acid. There was a significant correlation between haemolytic activity and [3H]arachidonic acid release generated by individual strains from monocytes (r = 0.804, P less than 0.001) and PMN (r = 0.888, P less than 0.001). In addition, nonhaemolytic strains but not their culture supernatants were capable of causing slow release of both [3H]arachidonic acid and LTB4 from PMN and mononuclear cells. These results suggest that both the possession of haemolytic activity, and the direct interaction of bacteria with the leukocyte surface are mechanisms by which uropathogenic strains of E. coli may cause the release and metabolism of arachidonic acid. In addition, there was synergistic augmentation by nonhaemolytic bacteria of the PMN LTB4 response to haemolytic culture supernatants or to low doses of the calcium ionophore A23187. These results support an ionophore-like mechanism for the activation of the cell by haemolysin. LTB4 generation by PMN incubated with haemolytic supernatants was also augmented by particulate zymosan in a manner dependent on the dose of zymosan, suggesting that the direct interaction of E. coli with PMN may involve an activation mechanism similar to that for zymosan. These results demonstrate differing responses of peripheral mononuclear cells and PMN from the same donors to identical strains of E. coli and suggest that the generation of the potent chemotactic agent LTB4 in response to E. coli infection in vivo need not depend solely on the elaboration of cytotoxic haemolysins by individual strains.

The assessment of relative surface hydrophobicity as a factor involved in the activation of human polymorphonuclear leukocytes by uropathogenic strains of Escherichia coli.

The initiation of the respiratory burst and the degranulation of human polymorphonuclear leukocytes (PMN) in response to stimulation by uropathogenic strains of Escherichia coli is dependent on the expression of Type 1 fimbriae by those strains. These PMN responses correlate with an increasing tendency of the interacting E. coli strain to be retained on hydrophobic columns. The present work assessed the measurement of relative surface hydrophobicity in relation to PMN activation. Type 1 fimbriate organisms bound most readily to Octyl-Sepharose columns and were strongly agglutinated in the salt aggregation test. In contrast, the same organisms partitioned to the dextran-rich (hydrophilic) phase of aqueous two-polymer phase systems. Electron microscopic observation of the organisms eluted from the Octyl-Sepharose columns and of the organisms recovered from both phases of the aqueous two-phase systems demonstrated, however, that both Type 1 and P-fimbriate organisms were retained on the columns and partitioned into the dextran-rich phase as a consequence of their being fimbriate and failed to identify this as a major factor in the activation of PMN. In addition electron microscopy demonstrated that each P-fimbriate population had fewer organisms expressing fimbriae than did Type 1 fimbriate populations, confirming the importance of phase variation as a factor affecting the physicochemical characteristics of a bacterial population.

TPA-induced differentiation and adhesion of U937 cells: changes in ultrastructure, cytoskeletal organization and expression of cell surface antigens.

Incubation of the human promonocytic cell line U937 with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h resulted in differentiation into immature macrophage-like cells and was accompanied by marked morphological and functional changes. U937 cells which normally grow in suspension and show a smooth surface, extended pseudopodia and became adherent to each other and to the surface of the culture vessel. Concomitant with the TPA-induced adherence U937 cells ceased to proliferate. Our results show that phorbol ester-treated U937 cells exhibited markedly increased levels of fibronectin and of the cytoskeletal proteins actin, myosin and vimentin including a reorganization of actin and vimentin filaments. The induction of both cellular adherence and growth inhibition were accompanied by a significantly reduced level of cells expressing transferrin receptors and changes in cell surface antigen expression. Here, the expression of the leukocytefunction antigens (LFA-1), including CD11 and CD18 was markedly enhanced during phorbol ester-induced differentiation. TPA-treatment, however, failed to enhance the small amount of U937 cells expressing the monocyte/macrophage-specific CD14 antigen or expressing MHC class-II antigens. A detailed analysis of the CD14 cluster by 7 differential antibodies resulted in an induction of TM1, UCHM1, MEM15, My4, and 3C10, whereas the epitopes recognized by TM2 and Mo2 remained unaltered. Neither indomethacin nor interferon-gamma were capable of inducing a marked expression of these antigen epitopes in TPA-treated cells. Although these data demonstrate that during phorbol ester-induced differentiation U937 cells acquire many properties typically associated with macrophages, the failure to express marked levels of macrophage-specific cell surface antigens suggests a transition of U937 cells from a promonocytic to an immature macrophage intermediate state rather than into mature macrophage-like cells.

In vivo exposure to bicarbonate/lactate- and bicarbonate-buffered peritoneal dialysis fluids improves ex vivo peritoneal macrophage function.

The impact on peritoneal macrophage (PMO) function of acidic lactate-buffered (Lac-PDF [PD4]; 40 mmol/L of lactate; pH 5.2) and neutral-pH, bicarbonate-buffered (TB; 38 mmol/L of bicarbonate; pH 7. 3) and bicarbonate/lactate-buffered (TBL; 25 mmol/L of bicarbonate/15 mmol/L of lactate; pH 7.3) peritoneal dialysis fluids (PDFs) was compared during a study of continuous therapy with PD4, TB, or TBL. During a run-in phase of 6 weeks when all patients (n = 15) were treated with their regular dialysis regimen with Lac-PDF, median PMO tumor necrosis factor alpha (TNFalpha) release values were 203.6, 89.9, and 115.5 pg TNFalpha/10(6) PMO in the patients subsequently randomized to the PD4, TB, and TBL treatment groups, respectively. Median stimulated TNFalpha values (serum-treated zymosan [STZ], 10 microgram/mL) were 1,894.6, 567.3, and 554.5 pg TNFalpha/10(6) PMO in the same groups, respectively. During the trial phase of 12 weeks, when the three groups of patients (n = 5 per group) were randomized to continuous treatment with PD4, TB, or TBL, median constitutive TNFalpha release values were 204.7, 131.4, and 155.4 pg TNFalpha/10(6) PMO, respectively. Stimulated TNFalpha values (STZ, 10 microgram/mL) were 1,911, 1,832, and 1,378 pg TNFalpha/10(6) PMO in the same groups, respectively. Repeated-measures analysis of variance comparing the run-in phase with the trial phase showed that PMO TNFalpha release was significantly elevated in patients treated with both TB (P = 0.040) and TBL (P = 0.014) but not in patients treated with Lac-PDF (P = 0. 795). These data suggest that patients continuously exposed to bicarbonate- and bicarbonate/lactate-buffered PDFs might have better preserved PMO function and thus improved host defense status.

Regulation of TNF-alpha, IL-1 and IL-6 synthesis in differentiating human monoblastoid leukemic U937 cells.

The human monoblastoid tumor cell line U937 was induced to differentiate along the monocyte/macrophage lineage by treatment with 5 x 10(-9) M 12-O-tetradecanoyl phorbol-13-acetate (TPA). Between 2 h and 4 h following TPA-treatment U937 cells started to release significant amounts of TNF-alpha which remained detectable until 8-10 days. A significant IL-1 beta release was measured 24 h-48 h post stimulation and increased levels of IL-1 beta persisted until 20-22 days of culture. In contrast no release of either IL-1 alpha or IL-6 could be detected with 5 x 10(-9) M TPA during the whole time course of the experiments. The sequential induction of TNF-alpha and IL-1 beta appeared to be independently regulated since TNF-alpha release was not required for the onset of IL-1 beta production. Northern-blot analysis confirmed the sequential induction and the long term expression of TNF-alpha and IL-1 beta mRNAs. Western-blot analysis predominantly showed a high molecular weight IL-1 beta protein of about 35 kD. Further investigations on the regulation of cytokine production and release by TPA-differentiated U937 cells revealed that TNF-alpha and IL-1 beta synthesis was not influenced by exogenously added rhTNF-alpha or PGE2, whereas rh gamma-IFN specifically enhanced the IL-1 beta production. Thus, the regulation and intracellular processing of cytokines generated by differentiating U937 cells shows some differences when compared to mature monocytes/macrophages which may be related to the tumorigenic origin of U937 cells or to an incomplete differentiation.

Macrophages and mesothelial cells in bacterial peritonitis.

Research in recent years has examined the mechanisms underlying cellular host defence in the peritoneal cavity. These studies have established that the resident cells of the peritoneal cavity, the peritoneal macrophages (PM phi) and the mesothelial cells (HPMC) contribute to the initiation, amplification and resolution of peritoneal inflammation. Ex vivo measurements of intra-peritoneal inflammatory mediators during peritonitis has elucidated the time courses for the generation of proinflammatory, chemotactic and anti-inflammatory cytokines and have identified that their secretion occurs largely within the peritoneum. These studies provide evidence that both PM phi- and HPMC-derived mediators are directly involved in controlling inflammation. It has been widely accepted that resident PM phi form the first line of defence against peritoneal infection, a more contemporary view would suggest that the direct or indirect (via secreted pro-inflammatory cytokines) interaction between PM phi and HPMC is pivotal to the activation and subsequent amplification of the peritoneum's response to infection. Whilst the site of these interactions is unknown, considerable evidence suggests that it occurs on the surface of the mesothelium, where invading micro-organisms may colonize. In this respect Staphylococcal exoproducts can directly activate HPMC cytokine synthesis. Once the inflammatory response is initiated, recent evidence suggests, that mesothelial cells upon activation by PM phi-derived IL-1 beta and TNF-alpha, are capable of amplifying inflammation and generating signals (via the creation of a gradient of chemotactic cytokines, IL-8, MCP-1 and RANTES) for the recruitment of leukocytes into the peritoneum. This process is also facilitated via the cytokine driven up-regulation of adhesion molecule expression (ICAM-1 and VCAM-1) on HPMC. Much less is understood about the mechanisms by which inflammation is resolved, although the secretion of anti-inflammatory molecules (IL-6, IL-1ra and soluble TNF-p55/75) by receptors by PM phi and HPMC may be important in the process. The existence of a peritoneal cytokine network controlling inflammation is now well established, within this the interaction of PM phi and HPMC appears to play a pivotal role in the hosts response to peritoneal infection.

Prostaglandin and tumor necrosis factor secretion by peritoneal macrophages isolated from normal and arthritic rats treated with liposomal methotrexate.

The effect of a novel liposomal preparation containing a phospholipid conjugate of methotrexate (MTX-LIPO) upon macrophage mediator release was investigated in normal and arthritic rats ex vivo. Peritoneal macrophages isolated from MTX-LIPO-treated arthritic rats and stimulated with lipopolysaccharide produced significantly less tumor necrosis factor (TNF) and prostaglandin (PGE2) than did macrophages isolated from saline-treated controls. In the same experimental system, free methotrexate only inhibited prostaglandin release, but it was more potent than MTX-LIPO in this respect. Additional studies are presently underway to investigate the effect of MTX-LIPO and MTX treatment upon the lipopolysaccharide-induced rise in plasma levels of various proinflammatory mediators in vivo. Haematopoietic toxicity was demonstrated in blood isolated from rats treated with free MTX, and this was as characterized by a significant reduction in reticulocyte count compared with MTX-LIPO and saline-treated rats.

Differential biological activities of human interleukin-1 alpha and interleukin-1 beta.

We examined the biological effects induced by both human recombinant interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in five different cell types of human, rat and mouse origin. IL-1 alpha and beta preparations were standardized in terms of biological activity in the EL-4/CTLL bioassay and, in parallel, employed to stimulate PGE2 secretion in human fibroblasts, mesangial cells (MC), C57B1/6 mouse MC, DBA/2 mouse macrophages and Sprague Dawley rat MC. In addition, the co-mitogenic effects of IL-1 alpha and beta were determined in freshly prepared Sprague Dawley rat thymocytes. No significant differences in IL-1 alpha and beta concentration dependent PGE2 production were detectable in the different cell types (MC, fibroblasts and macrophages) of human or mouse origin. Incubation of Sprague Dawley rat MC with both IL-1 alpha and beta resulted in a concentration dependent production of PGE2. However, in contrast to mouse or human MC the potency of IL-1 beta to induce PGE2 in Sprague Dawley rat MC was 26-fold higher compared to IL-1 alpha. In addition, the potency of IL-1 beta to enhance co-stimulated proliferation of Sprague Dawley thymocytes was 200-fold higher than that of equal biological activities of IL-1 alpha. When we tested the additive effects on Sprague Dawley cells, increasing IL-1 beta concentrations added to a fixed IL-1 alpha concentration resulted in a cumulative rise in both, PGE2 secretion by MC and thymocyte proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Human glomerular mesangial cells inactivate leukotriene B4 by reduction into dihydro-leukotriene B4 metabolites.

Due to its potent chemotactic properties leukotriene B4 is an important mediator of inflammatory reactions. Cultured human kidney mesangial cells converted exogenously added leukotriene B4 efficiently into three different more lipophilic metabolites, two of them probably representing dihydro-leukotriene B4 isomers. This represents an alternative metabolic pathway, in contrast to leukotriene B4 omega-oxidation found in human polymorphonuclear leukocytes. Both dihydro-leukotriene B4 isomers had nearly completely lost their ability to induce leukocyte chemotaxis as compared to leukotriene B4.

P1 blood group phenotype, secretor status in patients with urinary tract infections.

We have examined the distribution of P antigen, Lewis blood group phenotypes and secretor status of 65 patients with E. coli UTI (20 asymptomatic bacteriuria, 20 cystitis and normal radiology, 25 reflux nephropathy) and 45 controls who have never experienced a UTI episode. The distribution of Lewis blood group antigens was similar in all UTI groups and in the controls. The incidence of non-secretors in the reflux nephropathy group was similar to that in controls (24% versus 31%). The P1 phenotype was present in 100% of patients with asymptomatic bacteriuria, 80% with cystitis and controls and only 44% with reflux nephropathy. The combined P1/non-secretor phenotype was observed in 45% of patients with asymptomatic bacteriuria, 30% with cystitis, 12% with reflux nephropathy and in 22% of control healthy individuals. P2/secretor phenotype was demonstrated in 44% of patients with reflux nephropathy and in only 11% of controls. Our data suggest that having P2 blood group protects against asymptomatic colonization of the urinary tract, but is associated with the type of infection responsible for scarring in reflux nephropathy. It also appears that being a non-secretor does not predispose to renal scarring and that combined P2/secretor phenotype may be linked with susceptibility to reflux nephropathy.

Mucosal immunity in the urinary tract: changes in sIgA, FSC and total IgA with age and in urinary tract infection.

The incidence of primary urinary tract infection (UTI) is greatest in the first month of life and decreases with age throughout childhood. Secretory immunoglobulin A (sIgA) is an important component of mucosal immunity. The changes in secretory IgA, IgA and free secretory component (FSC) during the first year of life were examined in relation to age, sex and in infants, feeding practice. These constituents were further compared between healthy children and those with acute and recurrent UTI. Urine was collected from 41 healthy infants (16 female: 25 male) at intervals (mean age 1.4, 9.1, 44, 91, 210 and 412 days), 139 healthy children (75 female: 64 male), 29 children with histories of recurrent UTI (25 female: 4 male) and 10 with acute UTI (8 female: 2 male). sIgA, IgA and FSC were measured by enzyme linked immunoassay. In the majority of children sIgA and IgA were undetectable at birth. SIgA and IgA rose significantly during the first year then levelled off throughout childhood. FSC was detectable from birth (geometric mean [mean of logged values]-GOM at day 1.4, 362.2 ng/ml). No sex differences were apparent for any of the three constituents at any age. Breast feeding was associated with higher levels of sIgA and IgA than bottle feeding. This was highly significant at 9.1 days when sIgA and IgA levels of breast fed compared with bottle fed infants were 64.6 ng/ml vs 21.2 and 56.2 ng/ml vs 18.7 ng/ml respectively, giving a GOM ratio of 3.04 for sIgA and 3.0 for IgA (p < 0.001 for both). No significant difference in the three parameters were demonstrable when children with recurrent UTI-with normal or abnormal renal tracts-were compared with controls. Acute UTI resulted in raised sIgA, IgA and FSC compared with controls (GOM ratio of 4.9 [p < 0.002], 4.2 [p < 0.005] and 2.7 [p < 0.001] respectively). The proportion of total IgA present as sIgA (sIgA/total IgA) was not significantly different in the acute vs control groups. Urinary sIgA and IgA may be important for the observed variation with age in infant UTI and the reduced incidence in breast fed infants but does not appear to contribute to the sex associated difference in susceptibility to infection at any age.

Effect of three lipophilic methotrexate derivatives upon mediator release by lipopolysaccharide-stimulated rat peritoneal macrophages.

The ability of methotrexate and three lipophilic derivatives (methotrexate-gamma-dimyristoylphosphatidylethanolamine (M gamma D), methotrexate-alpha-dimyristoylphosphatidylethanolamine (M alpha D) and methotrexate-alpha-gamma-di-dimyristoylphosphatidylethanolamine (M alpha gamma D) to modulate mediator release by lipopolysaccharide-stimulated rat peritoneal macrophages was investigated. At nontoxic concentrations, approximately 10 nmol/10(5) cells, M alpha D and M gamma D produced 11.06 +/- 1.0 and 75.6 +/- 5.2%, respectively, inhibition of tumour necrosis factor (TNF) release (mean +/- s.e.m., n = 4). At this same dose M alpha gamma D resulted in 68.8 +/- 2.1% inhibition of TNF but cellular ATP levels were reduced by 80%. The inhibitory activity of all three derivatives was dose-dependent. Non-derivatized methotrexate at a concentration of 25 nmol/10(5) cells had no inhibitory effect upon TNF release (14.7 +/- 0.8%, n = 3). Determination of prostaglandin E2 (PGE2) levels in the same samples demonstrated that all three conjugates were powerful inhibitors of prostaglandin release. At a quarter of the conjugate concentrations described above the monoamides M alpha (3.1 nmol/10(5) cells) and M gamma D (2.5 nmol/10(5) cells) maintained their effects on PGE2 production with 73 +/- 2.3 and 71 +/- 2.0% (n = 4) inhibition, respectively. At this lower concentration, however, the diamide M alpha gamma D (3.1 nmol/10(5) cells) was less effective in reducing the amount of PGE2 released from the macrophages (29 +/- 18%, n = 4). Maximal PGE2 inhibition by each of the conjugates was attained at approximately 5 nmol/10(5) cells. Unconjugated methotrexate (range of 2.5-20 nmol/10(5) cells) did not inhibit the release of PGE2 from lipopolysaccharide-stimulated macrophages.

In vitro biocompatibility of bicarbonate-based peritoneal dialysis solutions.

OBJECTIVE: To review the current literature on the in vitro biocompatibility of peritoneal dialysis solutions buffered with bicarbonate and compare them to existing conventional lactate-buffered solutions. RESULTS: Conventional lactate-buffered solutions are known to inhibit or modulate cell functions related to host defense mechanisms; these effects are mediated by their low pH in combination with lactate concentration, as well as their glucose content, associated osmolality, and the presence of glucose breakdown products. CONCLUSIONS: Bicarbonate-buffered solutions appear to present a viable alternative for use in CAPD based on their significantly improved biocompatibility profile. The results of on-going clinical trials will clarify whether these in vitro data will impact significantly on peritoneal host defence, infection rate and the long term function of the peritoneal membrane in vivo.

Biocompatibility studies on peritoneal cells.

This review outlines the problems involved in assessing the biocompatibility of PD fluids. It has summarized the data available from conventional in vitro studies and highlights many of the inadequacies of this approach. In vivo data are lacking both on host defense and on the clinical effect of changing conventional PD fluids for a more "ideal" formulation. The best parameters for assessing biocompatibility need to be defined. Alternative formulation of fluids must be aimed towards (1) a system that interferes minimally with host defense, and (2) a system that maintains the integrity of the peritoneal membrane for ultrafiltration and clearance. Cell culture studies should be designed to model the in vivo situation. Ex vivo studies (cells exposed within the peritoneal cavity) should be used to support in vivo findings. Finally, in vitro results must be related to clinical significance, and changes in fluid composition should be followed by improvements in clinical outcome.

Impact of peritoneal dialysis solutions on peritoneal immune defense.

In summary, conventional CAPD fluids may interfere with most pathways of the intraperitoneal network of cytokines (Figure 4), potentially resulting in an impairment of important host defense functions. Thus alternative approaches are needed in order to improve dialysis fluid biocompatibility. Initial results indicate that pH neutralization and a restriction to moderate osmolality might constitute important determinants of modern dialysis fluid technology; however, clinical studies will have to prove that these fluids indeed improve the clinical outcome of the patients on long-term CAPD therapy.

Encapsulating peritoneal sclerosis: definition, etiology, diagnosis, and treatment. International Society for Peritoneal Dialysis Ad Hoc Committee on Ultrafiltration Management in Peritoneal Dialysis.

Current definitions of encapsulating peritoneal sclerosis are practical and clinically relevant. It is important to adhere to a more uniform use of the proper terminology, and it is the recommendation of the authors that EPS be adopted as the more appropriate term. The best literal definition of EPS is based on clinical-pathologic criteria. Differentiation of EPS from the general category of ultrafiltration failure is required. Further, better appreciation of the diverse pathways that can lead to the same final common clinical-pathologic picture should not be overshadowed by the requirement of uniform terminology. Incidence and prevalence of the syndrome have been defined in some large populations and a few single-center experiences. The former show an incidence of less than 1%, while higher percentages are reported in the latter. The reported increased incidence with duration on therapy requires validation. The epidemiology of the syndrome offers limited insight into its pathogenesis. A list of factors, both dialysis-related and non dialysis-related. has been accumulated. Except in a few categories where agents are clearly related to the development of EPS, the majority of the listed factors for dialysis-related BPS remain, at best, associations and at worst, simple conjecture. The same limitations that plague the issue of etiology apply in the area of pathogenesis. More basic, focused work is required. The diagnosis of EPS remains based on clinical suspicion confirmed with, primarily, radiologic findings. Pathologic confirmation is obtained in cases that come to surgery for management or for catheter removal. Radiologic studies are precise enough for confirmation, but none have been evaluated for early diagnosis for possible early intervention or prevention. Studies based on transport characteristics or effluent dialysate constituents are not useful for EPS. At present, there are no reliable predictive tests for BPS that can be used in individual patients. Therapy of BPS is based on anecdotal evidence. The possible variable etiologies and probable distinct pathways leading to the syndrome may make a uniform therapeutic approach unlikely. Further, the limited number of cases and the sporadic pattern of occurrences make therapeutic trials not readily feasible. This is distinct from the case of ultrafiltration failure, where significant advances in mechanism elucidation and rationale-based interventions have been made.

Insulin stimulates the activity of Na+/K(+)-ATPase in human peritoneal mesothelial cells.

OBJECTIVE: To assess the effect of insulin on the Na+/K(+)-ATPase expression and activity in human peritoneal mesothelial cells (HPMC). METHODS: HPMC were isolated from the omental tissue of non-uremic patients, grown to confluence and rendered quiescent by serum deprivation for 24 hours. The activity of Na+/K(+)-ATPase was determined by measuring the ouabain-sensitive 86Rb uptake. To assess whether the effect of insulin was related to changes in [Na+]i the sodium influx was measured with 22Na and the activity of Na+/K(+)-ATPase was assessed in the presence of amiloride. Expression of Na+/K(+)-ATPase alpha 1,alpha 2 and beta 1-subunit mRNAs was determined by RT/PCR. RESULTS: Exposure of HPMC to insulin resulted in a time- and dose-dependent increase in the Na+/K(+)-ATPase activity. After 60 minutes the ouabain-sensitive 86Rb uptake (cpm/10(4) cells) was increased from 6650 +/- 796 in control cells to 9763 +/- 1212 in HPMC exposed to 100 mU/mL insulin (1.5-fold increase; n = 4, P < 0.05). In addition, incubation of HPMC with 100 mU/mL insulin resulted in a time-dependent increase in the 22Na influx. Pre-exposure of HPMC to 1mM amiloride reduced the activity of Na+/K(+)-ATPase but did not block the stimulatory effect of insulin. RT/PCR analysis revealed that HPMC constitutively expressed alpha 1- and beta 1-subunit mRNAs while the alpha 2-subunit mRNA was barely detectable. Exposure of HPMC to insulin for up to 24 hours was not associated with any changes in the expression of either alpha 1, alpha 2 or beta 1-subunit. CONCLUSION: Insulin stimulates the Na+/K(+)-ATPase activity in HPMC in a time- and dose-dependent manner. This effect appears to mediated by an increase in [Na+]i and is not related to alterations in Na+/K(+)-ATPase subunit mRNAs expression.

Interleukin-6 levels decrease in effluent from patients dialyzed with bicarbonate/lactate-based peritoneal dialysis solutions.

OBJECTIVE: Conventional lactate-buffered peritoneal dialysis (PD) solutions have several bioincompatible characteristics, including acidic pH, lactate buffer, and the presence of glucose degradation products (GDPs). These characteristics, along with inflammation, are believed to contribute to membrane dysfunction in peritoneal dialysis patients. A new PD solution containing a bicarbonate/lactate buffer system with physiologic pH and low GDPs has shown improved biocompatibility in both in vitro and ex vivo studies. In the present study, the concentrations of cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), and vascular endothelial growth factor (VEGF), were measured in timed overnight effluents from PD patients continuously dialyzed with either lactate-based control solution (C) or bicarbonate/lactate-based solution (B/L) for 6 months. METHODS: Effluents from 92 continuous ambulatory peritoneal dialysis (CAPD) patients were collected when the patients were entered into the study (baseline, all patients on C for more than 3 months), and at 3 and 6 months following randomization to C (n = 31) or to B/L (n = 61). Effluent samples were filtered, stored frozen, and then assayed for IL-6, TNFalpha, and VEGF by ELISA. RESULTS: A significant decrease in effluent IL-6 was seen at 3 months and at 6 months in the B/L-treated patients. Levels of VEGF were significantly reduced at 3 months. No changes in the levels of IL-6 or VEGF were seen in the C-treated patients, and no changes in TNFalpha were seen in either group over time. CONCLUSIONS: Treatment with B/L is associated with decreased IL-6 synthesis and decreased VEGF secretion. The data suggest that the use of B/L solution is associated with reduced intraperitoneal inflammation and potential for angiogenesis. The use of B/L solution may, over time, help to restore peritoneal homeostasis and therefore preserve the function of the membrane in peritoneal dialysis.

Cell function and viability in glucose polymer peritoneal dialysis fluids.

OBJECTIVE: To investigate the biocompatibility profile of a new peritoneal dialysis fluid containing glucose polymer (GPF). DESIGN: Viability and function of peripheral neutrophils (PMN) from healthy donors and cultured human peritoneal mesothelial cells were assessed in vitro after exposure to dialysis fluids. Phagocytosis, leukotriene B4 synthesis, and respiratory burst activation were measured following stimulation with serum-treated zymosan (STZ) or opsonized Staphylococcus epidermidis (S. epidermidis). Bacterial growth in the fluids was also investigated. In vivo pH equilibration of GPF and subsequent respiratory burst activation following incubation in spent dialysate were studied. RESULTS: For all the host defense parameters measured, commercial dialysis fluids (Dianeal; 1.36% and 3.86% glucose) and GPF (pH 5.2) were significantly more inhibitory than the control buffer (pH 7.3). Mesothelial cell viability was reduced by all the fluids tested irrespective of pH. Glucose polymer fluid was significantly more inhibitory than Dianeal 1.36% for STZ phagocytosis and respiratory burst activation. In contrast, it was less suppressive than Dianeal 3.86% for LTB4 synthesis. For all parameters tested, except LTB4 generation, there was a marked effect of pH, with GPF being significantly more inhibitory at pH 5.2 than at pH 7.3. None of the fluids tested supported the growth of S. epidermidis, although the viable counts in GFP were significantly higher than in Dianeal. Fluid inhibition of PMN respiratory burst activation and cytotoxicity were reduced in a time-dependent manner following increasing dwell time in vivo. CONCLUSIONS: GPF does not appear to be significantly different from Dianeal as far as host defense parameters are concerned. However, the cell viability and bacterial survival data suggest some possibly negative aspects of this fluid formation.