In vitro effect of cefodizime, imipenem/cilastatin and co-trimoxazole on dexamethasone and cyclosporin A depressed phagocytosis.

Phagocytosis is an important part of the host defense against infection. Antibiotics can influence phagocytic function. In the present study, leukocyte metabolic response to phagocytic challenge by latex was assessed in relation to in vitro addition of cotrimoxazole, imipenem/cilastatin, cefodizime, dexamethasone (DXM), and/or cyclosporin A (CsA). Using latex particles as phagocytic challenge, glucose-1-14C utilization and 14CO2 production were measured by liquid scintillation counting. The phagocytic response was impaired by in vitro addition of DXM or CsA and this setup was used as an experimental model of immunodepression. The addition of co-trimoxazole to control samples (without DXM or CsA) depressed the response to latex challenge, whereas imipenem and cefodizime had a neutral effect. In the presence of DXM, co-trimoxazole induced a further decrease. The depressive effect of DXM was partially neutralized in the presence of cefodizime. With CsA depression, co-trimoxazole also induced a further decrease, imipenem had a neutral effect, while cefodizime partially restored the CsA suppressed reaction. Co-trimoxazole depressed the phagocytic response, imipenem had a neutral effect, whereas cefodizime restored the experimentally induced immunosuppression.

An update on uremic toxins.

The author reviews trends and evolution in biochemical methodology, clinical symptomatology description, experimental models and definitions of uremic toxins. The assumption is made of one specific toxic effect for one specific toxin.

P-Cresol, a uremic compound, enhances the uptake of aluminum in hepatocytes.

In the end-stage renal disease patient, certain uremic compounds could influence the cellular accumulation of aluminum (Al). In this study, we examined the effect of 15 uremic ultrafiltrate fractions obtained by HPLC on the uptake and toxicity of Al in mouse hepatocytes (MH) in culture, a model system in which Al is taken up bound to transferrin (Tf). Uremic fractions 4 to 8, 12, 14, and 15 increased cellular Al uptake and aspartate aminotransferase release and decreased cell growth when Tf-Al, not Al citrate, was added to culture media. Compounds that have been extracted previously from these ultrafiltrate fractions (p-cresol, xanthine, tryptophan, hippuric acid, and o-hydroxyhippuric acid) were then tested for their effect on Al uptake and toxicity in MH at concentrations found in uremic serum. Significant Al uptake by MH was observed only when p-cresol was added together with Tf-Al. Time-response curves showed increased Al uptake and toxicity at p-cresol concentrations of 3 mg/dl in culture media. Dose-response curves confirmed that Al uptake and cell toxicity were proportional to p-cresol from 1.5 mg/dl to 3 mg/dl in culture media. p-Cresol was not toxic to MH in the absence of Tf-Al in media. p-Cresol increased Tf-associated Al uptake only because there was no effect on Al uptake when Al citrate was substituted, and studies with Tf-I125-Al in the presence of this compound showed increased Tf-I125 taken up by MH. p-Cresol did not increase Tf saturation with Al. p-Cresol also increased Tf-Al uptake in Friend erythroleukemia and neuroblastoma cells in culture. Our studies suggest that p-cresol and uremic fractions 4 to 8, 12, 14, and 15 increase the uptake and toxicity of Al in cultured MH. These compounds may play a role in the accumulation and toxicity of Al in the liver of end-stage renal disease patients and possibly in all cells that express Tf receptors.

Serum uremic toxins from patients with chronic renal failure displace the binding of L-tryptophan to human serum albumin.

The level of free tryptophan (Trp) and its metabolites in serum appears to be related to some pathologic states, such as chronic renal failure and neuropsychiatric disorders, so that a precise characterization of tryptophan binding to serum albumin is of interest. In the present paper, the binding of L-tryptophan to defatted human serum albumin at 37 degrees C and at pH 7.4 was studied by means of equilibrium dialysis. The competition between L-tryptophan and serum solutes extracted from uremic patients undergoing hemodialysis, before dialysis treatment, was also investigated. Solutes were extracted from uremic pools of sera using two different deproteinization methods: serum ultrafiltration and heat denaturation of serum proteins followed by ultrafiltration. We found 1.10 +/- 0.03 binding sites for Trp to defatted albumin with an association constant 11.37 +/- 1.03 x 10(3) M-1. The competition experiments suggested that the number of Trp binding sites were not significantly modified by the addition of solutes obtained with the method of ultrafiltration with respect to the binding of L-tryptophan to albumin in the absence of competitors, while their affinity constant was markedly reduced (2.66 +/- 0.18 x 10(3) M-1). Moreover, a significant reduction of the affinity constant was observed when competitors for Trp were obtained using heat deproteinization associated with ultrafiltration (1.91 +/- 0.15 x 10(3) M-1 vs. 2.66 +/- 0.18 x 10(3) M-1; P < 0.005). These results might be ascribed to the fact that the last procedure has a higher yield with a more complete liberation of uremic toxins from serum proteins, so that they became probably totally free thus competing at higher extent with L-tryptophan for albumin binding sites.

Pharmacokinetics of recombinant hirudin in hemodialyzed end-stage renal failure patients.

Recently, hirudin was used for the first time as an anticoagulant during hemodialysis in men. Pharmacokinetic data of this compound in end-stage renal failure are however not available. In this study, the pharmacokinetics of recombinant hirudin (HBW 023) was evaluated in hemodialysis-treated end-stage renal failure patients. HBW 023 was administered as a bolus at the start of a single dialysis (0.02 to 0.08 mg/kg) in 20 patients, and plasma hirudin levels were followed during this and the 5 following dialyses, without additional hirudin administration. The initial dialysis (HD1) was performed with a low flux polysulfone dialyzer, the following dialyses (up to HD6) with a high flux polysulfone dialyzer and regular heparin. Hirudin levels averaged 504.0 +/- 214.0 and 527.7 +/- 217.1 ng/ml in the middle and at the end of HD1, and then gradually decreased to 15.2 +/- 15.2 ng/ml at the end of HD6. Pharmacokinetic data were compared to those obtained in healthy controls (n = 5), receiving the same dose, and reaching the same peak hirudin level. Hirudin half-life was > 30 times longer in hemodialysis patients (51.8 +/- 15.6 vs. 1.7 +/- 1.5 h, p < 0.001), whereas area under the curve was > 60 times higher (34,669 +/- 14,898 vs. 545 +/- 205 ng/ml x h, p < 0.001). Distribution volume was lower in hemodialysis patients (11.0 +/- 3.1 vs. 14.1 +/- 2.01, p < 0.05). Hirudin disappearance rate was the same during high flux polysulfone dialysis as during interdialytic periods. Hirudin removal was markedly higher in those patients still maintaining some residual renal function and parameters of hirudin removal were significantly correlated to residual creatinine clearance. It is concluded that hirudin removal from the body is markedly depressed in hemodialyzed end-stage renal failure patients and that even minor residual renal function may increase this removal rate.

HPLC fractions of human uremic plasma inhibit the RBC membrane calcium pump.

We have reported that uremic plasma filtrates (UF) inhibit the red blood cell (RBC) membrane calcium pump. The inhibitor was dialyzable, smaller than 3,000 molecular weight, heat-stable, and protease-resistant. In the present study, we used reverse-phase preparative HPLC, analytical HPLC, and Sephadex G-25 elution to identify inhibitory fractions. Inhibition was confirmed in three different bioassays: (1) Sr2+ efflux in intact RBC, the primary bio-assay; (2) 45Ca efflux in intact RBC; and (3) calcium ATPase activity in isolated RBC membranes. Active fractions were analyzed by mass spectrometry, capillary electrophoresis, enzymatic analysis, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy. These demonstrated a number of compounds, including: sugars, polyols, osmolytes like betaine and myoinositol, amino acids, and other metabolites, such as 3-D-hydroxybutyrate, dimethylglycine, trimethylamine-N-oxide, guanidinoacetic acid and glycine. Many individual compounds were then tested for an effect on the calcium pump. Thus, HPLC was able to separate a substantial number of compounds in inhibitory fractions. Efforts are under way for precise identification of the inhibitor, to advance our understanding of uremic toxicity and/or hypertension in CRF.

Estimation of parameters in a two-pool urea kinetic model for hemodialysis.

A two-pool, variable volume urea kinetic model for estimation of solute removal in hemodialysis is solved analytically, and closed form expressions are presented for urea concentration in both compartments, both during dialysis and between dialyses. This approach also includes an estimation of the extent of the post dialysis rebound phenomenon of urea concentration. A method is presented to estimate values for the urea generation rate G, the distribution volume V and its partition in two compartments with volumes alpha 1V and alpha 2V (alpha 1-alpha 2-1), the total clearance K, and intercompartmental transfer coefficient X. To apply this analysis, several measurements are needed as input; the urea concentration at the end of a dialysis, the evolution of this concentration during the next dialysis, with at least four measurements including the initial and the final concentration, the volume of the dialysate, and its urea concentration. The main results are: the magnitude of the rebound is approximately proportional to alpha 2(2) K/X; the accuracy of the parameter estimation does not improve much further by taking more than six measurements during dialysis.

Binding of indole-3-acetic acid to human serum albumin and competition with L-tryptophan.

Indole-3-acetic acid (IAA) is a product of tryptophan (Trp) metabolism and is found to be markedly increased in uremic sera. IAA binding to defatted human serum albumin at 37 degrees C and pH 5, 7.4, and 8.5 was studied by equilibrium dialysis, and data were analyzed assuming two independent high affinity binding sites plus a class of low affinity sites. The estimated values of the association constant of dominant site were: 7.96 x 10(3) M-1 at pH 5, 11.57 x 10(3) M-1 at pH 7.4, and 6.30 x 10(3) M-1 at pH 8.5. The competition between IAA and Trp for albumin binding at pH 7.4 was investigated. The results suggest that one specific albumin site is common for IAA and Trp, but the data were not adequately predicted by a purely competitive scheme. A better prediction was achieved assuming that the binding of IAA to a site different from the common site inhibits Trp binding.

Disturbed host defense in peritoneal cavity during CAPD: characterization of responsible factors in dwell fluid.

In this study, the factors in overnight dwell fluid (8 to 10 hr dwell) depressing granulocyte (GC) NAD(P)H-oxidase dependent radical species production are characterized. At present, most studies have essentially focused on fresh, unspent dialysate and on peritoneal macrophages. The response to Staphylococcus aureus (Staph A) was dose-dependently depressed for both GC CO2 production (from 91.3 +/- 8.4 to 9.0 +/- 1.5 dpm/10(3) GC, P < 0.01) and chemiluminescence (CL) (peak from 7.3 +/- 0.8 to 1.6 +/- 0.8 cps x 10(3)/GC, P < 0.01). Stimulation with formyl-methionine-leucine-phenylalanine (f-MLP), phorbol myristic acid (PMA), Staphylococcus epidermidis (Staph Epi), E. coli, latex and zymosan revealed a parallel depression, pointing to an intrinsic metabolic defect, rather than failure of particle ingestion. The addition of glucose to the normal cell medium to obtain the same concentration as in the CAPD effluent (2.9 +/- 0.3 mg/dl) depressed function but not to the same extent as the genuine PD effluent. Opsonization of Staph A and E. coli induced a partial correction. No effect of pH or osmolality was observed. HPLC fractionation of CAPD effluent on a polarity based gradient revealed an elution of depressive factors in hydrophobic fractions with a nadir in F7 and F12. Analysis of the elution pattern of various uremic solutes revealed elution in F12 of p-cresol, a solute with known inhibitory effect on GC function. These events may be related to recent peritonitis (CL in response to Staph A 0.3 +/- 0.1 in effluent of 6 patients with recent peritonitis versus 2.6 +/- 0.8 cps x 10(3)/GC in 12 patients without recent peritonitis (P < 0.01). We conclude that the GC response is depressed in the presence of CAPD effluent due to excess glucose, lack of opsonization, and uremic solutes of which p-cresol is one of the responsible compounds.

Immunomodulating effects of antibiotics: literature review.

Antibiotics can interact directly with the immune system. This is a review of the immunomodulating effects of antibiotics. The Medline database on CD-ROM was searched for the years 1987 to 1994 using the following search string: "thesaurus explode antibiotics/all AND (thesaurus explode immune-system/drug effects OR thesaurus immune-tolerance/drug effects)." Aspects of the immune system studied were aspects of phagocyte functions: phagocytosis and killing, and chemotaxis and aspects of lymphocyte functions: lymphocyte proliferation, cytokine production, antibody production, delayed hypersensitivity and natural killer-cell activity. In order to quantify and to compare immunomodulatory properties of antibiotics we calculated an "immune index," defined as: number of positive statements--number of negative statements/total number of statements. Concerning phagocytosis, positive effects were observed for cefodizime, imipenem, cefoxitin, amphotericin B and clindamycin and negative effects for erythromycin, roxithromycin, cefotaxime, tetracycline, ampicillin and gentamicin. Clindamycin, cefoxition and imipenem induce enhancement of chemotaxis, whereas cefotazime, rifampicin and teicoplanin decrease chemotaxis. Regarding lymphocyte proliferation, cefodizime has the strongest stimulating effect, whereas tetracycline has the strongest negative effect. Except for erythromycin and amphotericin B the number of statements reported is too small to be conclusive for the interpretation of effects on cytokine production. Erythromycin and amphotericin B appear to stimulate cytokine production. As to antibody production, cefodizime has the strongest positive effect, whereas josamycin, rifampicin and tetracycline have marked negative effects. For delayed hypersensitivity and the natural killer-cell activity the number of statements is too small for any single antibiotic to be conclusive. There are three markedly immuno-enhancing antibiotics (imipenem, cefodizime and clindamycin) and eight markedly immuno-depressing antibiotics (erythromycin, roxithromycin, cefotaxime, tetracycline, rifampicin, gentamicin, teicoplanin and ampicillin).

Two-pool versus single-pool models in the determination of urea kinetic parameters.

The mathematics used for urea kinetic modeling are currently based on a single-pool distribution of urea throughout the body. In this study, we evaluated which one of a single- or a two-pool model would be more appropriate for the prediction of directly measured urea decay during hemodialysis. A numerical method was used which minimizes the relative root mean square (RMS) error between a calculated single- or two-pool urea decay curve and the measured intradialysis decay in 13 equilibrated dialysis patients. Using a two-pool model, the RMS error was markedly lower (1.27 +/- 0.72%) than the values obtained with a single-pool model, either based on multiple urea concentrations (RMS error 3.14 +/- 1.36%; p < 0.01 vs. two-pool model) or only on pre- and postdialysis urea (RMS error 5.00 +/- 2.38%; p < 0.001). This resulted for the single-pool model in an overall underestimation of urea generation, distribution volume (V) and protein catabolic rate and in an overestimation of Kt/V versus the two-pool model. In individual cases, the difference reached up to 18.7%. Comparison of V calculated from the two-pool model versus V values determined from anthropometric formulae (Watson) resulted in similar mean values (34.05 +/- 4.87 vs. 33.09 +/- 4.19 liters; p = NS), with a weak correlation (n = 13, r = 0.75, p = 0.003). Individual values, however, again differed by up to more than 20%. In conclusion, the use of single-pool kinetic models, as well as of anthropometric estimations of V, should be regarded with care, especially when individual patients are considered instead of groups. The two-pool model follows the directly measured urea decay more exactly which results in substantial differences in calculated kinetic parameters.

Leukocyte CD14 and CD45 expression during hemodialysis: polysulfone versus cuprophane.

The expression of CD14 on monocytes and CD45 on monocytes and granulocytes was evaluated during hemodialysis by flow cytometric analysis in the 'resting state' and after in vitro stimulation with phorbol myristate acetate (PMA). A comparison of complement activating cuprophane (CU) versus less complement activating polysulfone (PS) was undertaken. 'Resting state' CD45 expression on granulocytes increased markedly during CU dialysis compared to time 0, whereas this rise was only moderate with PS (CU vs. PS, p < 0.01). When considering the increase in expression upon PMA stimulation, a lower value was obtained during CU dialysis for both CD14 (monocytes at 60 and 240 min) and for CD45 (monocytes and granulocytes at 15 min). In conclusion, granulocytes in the 'resting state' expressed more CD45 on their cell membranes during CU dialysis, whereas CD14 and CD45 upregulation after ex vivo addition of PMA was blunted during CU dialysis.

Novel frameshift mutation in a heterozygous woman with Fabry disease and end-stage renal failure.

UNLABELLED: It is generally accepted that Fabry disease (angiokeratoma corporis diffusum) is an X-linked disorder resulting from the deficient activity of the lysosomal enzyme alpha-galactosidase. In males, the enzymatic defect leads to accumulation of glycosphingolipids, particularly in the kidney which causes end-stage renal disease. We report here a woman who presented in 1987 with focal and segmental glomerulosclerosis and required hemodialysis 4 years later when her son was evaluated for proteinuria. In these patients morphologic, biochemical, and genetic investigations were performed to explore the possibility of a hereditary renal disorder. Ultrastructural examination of the son's renal biopsy specimen revealed lamellated osmiophilic inclusions in the glomeruli, typical of Fabry disease. Four months after kidney transplantation in the mother, a graft biopsy specimen also revealed dense lamellated inclusions on electron microscopy. The leukocyte alpha-galactosidase activity was 0.008 mumol/min.10(9) cells in the son and 0.070 in the mother (range 0.100-0.500 mumol/min.10(9) cells). The diagnosis of Fabry disease was confirmed in both patients by the identification by DNA sequencing of a novel mutation in the alpha-galactosidase gene: one single base pair deletion in exon 3 (7317delA). IN CONCLUSION: (1) end-stage renal disease may occur in heterozygous women with Fabry disease; (2) morphologic lesions due to glycosphingolipid accumulation may be observed in the renal allograft after transplantation, and (3) DNA analysis confirmed the diagnosis by demonstrating a frameshift mutation, which has as yet not been reported.

Middle molecules: toxicity and removal by hemodialysis and related strategies.

Renal failure results in the retention of metabolites which may arbitrarily be grouped according to their molecular weight: low (< 300 daltons molecular weight), middle (300-15,000 daltons), and high (> 15,000 daltons). Opinion in respect to the relative importance of these groups varies. Initially it was thought that small molecules were important. In the mid-1970s, investigators identified the possible pathophysiological role of middle molecules. However, since positive identification of such molecules was difficult, opinion has shifted back in favor of small molecules, and little attention, with the exception of beta 2 microglobulin, has been paid to middle molecules and their removal by hemodialysis and related therapies. In this review current knowledge regarding middle molecules identified as uremic toxins and their removal by hemodialysis and associated therapies are discussed.