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O428 Aminoglycoside-induced apoptosis in cultured renal (LLC-PK1) and non-renal (J774 macrophages) cells: comparison between gentamicin and amikacin
Applied pharmacokinetics improve antimicrobial usage accumulation (range) of antibiotics (total drug) found was 26–106 (AZM), 4−19 (CXM), 2−13 (DCX), 21−36 (GEN), 2−25 (RIF). Conclusion: The concentrations found for CXM and DCX were higher than the concentrations found for AZM, GEN and RIF. This corresponds well with the higher dose injected and the relatively high antistaphylococcal activity observed in PD studies. The intracellular accumulation of AZM and RIF found here corresponds well with what has previously been reported. The intracellular accumulation for CXM, DCX and GEN found here, are surprisingly high, since b-lactams are notoriously considered not to accumulate intracellularly, and the 2−4 fold intracellular accumulation of aminoglycosides take several days, but the influx correlates well with the good effect seen in the in vivo model.
O427 Proinflammatory effects of erythromycin, clarithromycin and azithromycin on human endothelial cells in vitro
S89 Methods: We used non-confluent murine J774 macrophages and porcine LLC-PK1 renal cells grown to 80% of confluency. Electroporation was performed on trypsinised LLC-PK1 cells (8 square wave pulses; 800 v/cm; 1 ms) as previously described (Antimicrob Agents Chemother. 2006;50:1213−21). Cell viability was checked by measurement of LDH release (only cultures with <10% release were used for evaluation). Apoptotic cells were enumerated after DAPI staining by observers unaware of the experimental conditions, and expressed as percentage of all visible cells. Results: The Table shows the extent of apoptosis observed in controls (no aminoglycoside), in cells exposed to GEN concentrations known from previous studies to induce marked apoptosis or to AMK equimolar concentrations. For GEN, apoptosis developed on a concentrationdependent manner from an extracellular concentration of 1 mM for incubated cells and from 32 mM for electroporated cells. For AMK, no significant increase of apoptosis was seen at concentrations tested.
M. Millrose, I. Baumann-Wilschke, J. Webb, R. Stahlmann (Berlin, DE) An inflammatory reaction of the venous vessel is a common clinical problem that is observed after intravenous application of antibiotics and other drugs. The local irritation of the endothelium at the site of infusion leads to an inflammatory response with an increased expression of various cell surface antigens. Among these are CD 34, E-Selectin (CD 62E), ICAM-1 (CD 54) and VCAM-1 (CD 106). We studied the effects of three closely related antibiotics on human endothelial cells in vitro. We used the endothelial cell line EA.hy 926 and analysed the reaction by means of flow cytometry (FACScan, Becton Dickinson). Cells were incubated with clarithromycin or azithromycin at concentrations ranging from 100 mg/l to 800 mg/l and at concentrations ranging from 200 mg/l to 1400 mg/l for erythromycin. Such concentrations occur under therapeutic conditions at the site of infusion. Subsequently, they were stained with fluoresceinisothiocyanate- or phycoerythrin-conjugated monoclonal IgG mouse-antibodies for the four antigens mentioned above. Cells were incubated with the drugs for 2 h and analysis was carried out after an additional time period of 22 h. In control cells, we found positively stained cells at the following levels: CD 34 (2%), E-Selectin (4%), ICAM-1 (14%) and VCAM-1 (2%). The most pronounced changes were observed at 800 mg/l (erythromycin), 600 mg/l (azithroymcin), and 400 mg/l (clarithromycin). Erythromycin (800 mg/l) caused significantly increased expressions of all epitopes [CD 34 (+6%), E-Selectin (+5%), ICAM-1 (+15%) and VCAM-1 (+5%)]. At 600 mg/l the azalide azithromycin provokes a stronger upregulation of the proinflammatory antigens: CD 34 (+17%), E-Selectin (+16%), ICAM-1 (+27%) and VCAM-1 (+17%). Clarithromycin at a concentration of 400 mg/l causes a similar effect as erythromycin at twice this concentration [CD 34 (+6%), E-Selectin (+7%), ICAM-1 (+23%) and VCAM-1 (+4%)]. Analysis of the cell surface markers involved in cell-cell-interactions proved to be a useful approach to further study the mechanism of infusion phlebitis and to compare the proinflammatory effects of related compounds in vitro.
O428 Aminoglycoside-induced apoptosis in cultured renal (LLC-PK1) and non-renal (J774 macrophages) cells: comparison between gentamicin and amikacin S. Denamur, F. Van Bambeke, M.-P. Mingeot-Leclercq, P. Tulkens (Brussels, BE) Objectives: Apoptosis is now recognized as an early, and probably critical determinant in gentamicin (GEN)-induced nephrotoxicity in animals (Antimicrob Agents Chemother. 2000; 44: 665−75) as well as in renal cultured cells (Toxicol Sci. 2000; 56: 229−39). Models using electroporated cells also show that direct delivery of GEN in the cytosol of cultured renal cells enhances at least 30-fold its capacity to induce apoptosis (Antimicrob Agents Chemother. 2006; 50: 1213−21). Our aims were (i) to examine whether the capacity of GEN to induce apoptosis is restricted to renal cells; (ii) to compare amikacin (AMK) to GEN in this context, since AMK is generally considered to be less nephrotoxic than GEN (Antimicrob Agents Chemother. 1999; 43: 1003−12).
% apoptotic cells
Gentamicin Amikacin
J774 macrophages
renal LLC-PK1 cells
incubateda
incubatedb
electroporatedc
none
3 mM
none
3 mM
none
128 mM
0.1 0.0
14.9 1.2
1.5 1.5
12.7 0.0
1.8 0.8
18.4 0.9
a 24 h incubation; b 48 h incubation; c 24 h incubation in drug-free medium after electroporation in the presence of the drug.
Conclusions: Apoptosis develops in both renal and non-renal cells upon incubation with GEN. The lack of apoptosis observed with AMK with both incubated (renal and non-renal) and electroporated (renal) cells support the concept that this aminoglycoside is intrinsically less toxic than GEN. O429 Inhibitors and activator of the P-glycoprotein (P gp) efflux pump modulate the accumulation of daptomycin (DAP) in THP-1 macrophages and its intracellular activity towards Staphylococcus aureus S. Lemaire, F. Van Bambeke, P. Tulkens (Brussels, BE) Objectives: The concentration of antibiotics in eukaryotic cells can be reduced by efflux pumps such as P-gp, which may impair their activity against intracellular bacteria (JAC 2003; 51: 1167–1173). DAP is a lipopeptide antibiotic with an amphiphilic character (calculated logD at pH 7 and 25ºC: 9.56). Since amphiphilicity is an important determinant for recognition and transport by P-gp, we have examined whether the activity of this transporter in macrophages could affect the handling and intracellular activity of DAP. Methods: Uninfected THP-1 macrophages were exposed to DAP (24 h) or to the fluorochrome dimethyloxadicarbocyanine iodide (DIOC2; a specific P-gp subtrate; 5 h) and their cell contents measured by fluorimetry (see JID 2005; 191: 2149–2152 for assay method of daptomycin). Intracellular activity of DAP was measured against phagocytised S. aureus (ATCC 25923; MIC in Ca2+ supplemented MH broth: 0.125 mg/L) after a 24 h exposure to an extracellular concentration of DAP yielding an apparent static effect for intracellular bacteria (1 mg/L; see AAC 2006; 50: 841–851 for description and validation of the model with other antibiotics). The activity of P-gp was inhibited by incubation with verapamil (100 mM) and GF120918 (0.25 mg/L), or stimulated with ouabain (1 mM). Gemfibrozil (an inhibitor of the MRP transporters) was used as control. Results: The data presented in the Table show that P-gp modulators affect the cell handling and the intracellular activity of DAP in the same directions (verapamil and GF120918 increase DAP accumulation and activity, while oubain decrease them both), whereas a MRP inhibitor is without effect.
O427 Proinflammatory effects of erythromycin, clarithromycin and azithromycin on human endothelial cells in vitro
S89 Methods: We used non-confluent murine J774 macrophages and porcine LLC-PK1 renal cells grown to 80% of confluency. Electroporation was performed on trypsinised LLC-PK1 cells (8 square wave pulses; 800 v/cm; 1 ms) as previously described (Antimicrob Agents Chemother. 2006;50:1213−21). Cell viability was checked by measurement of LDH release (only cultures with <10% release were used for evaluation). Apoptotic cells were enumerated after DAPI staining by observers unaware of the experimental conditions, and expressed as percentage of all visible cells. Results: The Table shows the extent of apoptosis observed in controls (no aminoglycoside), in cells exposed to GEN concentrations known from previous studies to induce marked apoptosis or to AMK equimolar concentrations. For GEN, apoptosis developed on a concentrationdependent manner from an extracellular concentration of 1 mM for incubated cells and from 32 mM for electroporated cells. For AMK, no significant increase of apoptosis was seen at concentrations tested.
M. Millrose, I. Baumann-Wilschke, J. Webb, R. Stahlmann (Berlin, DE) An inflammatory reaction of the venous vessel is a common clinical problem that is observed after intravenous application of antibiotics and other drugs. The local irritation of the endothelium at the site of infusion leads to an inflammatory response with an increased expression of various cell surface antigens. Among these are CD 34, E-Selectin (CD 62E), ICAM-1 (CD 54) and VCAM-1 (CD 106). We studied the effects of three closely related antibiotics on human endothelial cells in vitro. We used the endothelial cell line EA.hy 926 and analysed the reaction by means of flow cytometry (FACScan, Becton Dickinson). Cells were incubated with clarithromycin or azithromycin at concentrations ranging from 100 mg/l to 800 mg/l and at concentrations ranging from 200 mg/l to 1400 mg/l for erythromycin. Such concentrations occur under therapeutic conditions at the site of infusion. Subsequently, they were stained with fluoresceinisothiocyanate- or phycoerythrin-conjugated monoclonal IgG mouse-antibodies for the four antigens mentioned above. Cells were incubated with the drugs for 2 h and analysis was carried out after an additional time period of 22 h. In control cells, we found positively stained cells at the following levels: CD 34 (2%), E-Selectin (4%), ICAM-1 (14%) and VCAM-1 (2%). The most pronounced changes were observed at 800 mg/l (erythromycin), 600 mg/l (azithroymcin), and 400 mg/l (clarithromycin). Erythromycin (800 mg/l) caused significantly increased expressions of all epitopes [CD 34 (+6%), E-Selectin (+5%), ICAM-1 (+15%) and VCAM-1 (+5%)]. At 600 mg/l the azalide azithromycin provokes a stronger upregulation of the proinflammatory antigens: CD 34 (+17%), E-Selectin (+16%), ICAM-1 (+27%) and VCAM-1 (+17%). Clarithromycin at a concentration of 400 mg/l causes a similar effect as erythromycin at twice this concentration [CD 34 (+6%), E-Selectin (+7%), ICAM-1 (+23%) and VCAM-1 (+4%)]. Analysis of the cell surface markers involved in cell-cell-interactions proved to be a useful approach to further study the mechanism of infusion phlebitis and to compare the proinflammatory effects of related compounds in vitro.
O428 Aminoglycoside-induced apoptosis in cultured renal (LLC-PK1) and non-renal (J774 macrophages) cells: comparison between gentamicin and amikacin S. Denamur, F. Van Bambeke, M.-P. Mingeot-Leclercq, P. Tulkens (Brussels, BE) Objectives: Apoptosis is now recognized as an early, and probably critical determinant in gentamicin (GEN)-induced nephrotoxicity in animals (Antimicrob Agents Chemother. 2000; 44: 665−75) as well as in renal cultured cells (Toxicol Sci. 2000; 56: 229−39). Models using electroporated cells also show that direct delivery of GEN in the cytosol of cultured renal cells enhances at least 30-fold its capacity to induce apoptosis (Antimicrob Agents Chemother. 2006; 50: 1213−21). Our aims were (i) to examine whether the capacity of GEN to induce apoptosis is restricted to renal cells; (ii) to compare amikacin (AMK) to GEN in this context, since AMK is generally considered to be less nephrotoxic than GEN (Antimicrob Agents Chemother. 1999; 43: 1003−12).
% apoptotic cells
Gentamicin Amikacin
J774 macrophages
renal LLC-PK1 cells
incubateda
incubatedb
electroporatedc
none
3 mM
none
3 mM
none
128 mM
0.1 0.0
14.9 1.2
1.5 1.5
12.7 0.0
1.8 0.8
18.4 0.9
a 24 h incubation; b 48 h incubation; c 24 h incubation in drug-free medium after electroporation in the presence of the drug.
Conclusions: Apoptosis develops in both renal and non-renal cells upon incubation with GEN. The lack of apoptosis observed with AMK with both incubated (renal and non-renal) and electroporated (renal) cells support the concept that this aminoglycoside is intrinsically less toxic than GEN. O429 Inhibitors and activator of the P-glycoprotein (P gp) efflux pump modulate the accumulation of daptomycin (DAP) in THP-1 macrophages and its intracellular activity towards Staphylococcus aureus S. Lemaire, F. Van Bambeke, P. Tulkens (Brussels, BE) Objectives: The concentration of antibiotics in eukaryotic cells can be reduced by efflux pumps such as P-gp, which may impair their activity against intracellular bacteria (JAC 2003; 51: 1167–1173). DAP is a lipopeptide antibiotic with an amphiphilic character (calculated logD at pH 7 and 25ºC: 9.56). Since amphiphilicity is an important determinant for recognition and transport by P-gp, we have examined whether the activity of this transporter in macrophages could affect the handling and intracellular activity of DAP. Methods: Uninfected THP-1 macrophages were exposed to DAP (24 h) or to the fluorochrome dimethyloxadicarbocyanine iodide (DIOC2; a specific P-gp subtrate; 5 h) and their cell contents measured by fluorimetry (see JID 2005; 191: 2149–2152 for assay method of daptomycin). Intracellular activity of DAP was measured against phagocytised S. aureus (ATCC 25923; MIC in Ca2+ supplemented MH broth: 0.125 mg/L) after a 24 h exposure to an extracellular concentration of DAP yielding an apparent static effect for intracellular bacteria (1 mg/L; see AAC 2006; 50: 841–851 for description and validation of the model with other antibiotics). The activity of P-gp was inhibited by incubation with verapamil (100 mM) and GF120918 (0.25 mg/L), or stimulated with ouabain (1 mM). Gemfibrozil (an inhibitor of the MRP transporters) was used as control. Results: The data presented in the Table show that P-gp modulators affect the cell handling and the intracellular activity of DAP in the same directions (verapamil and GF120918 increase DAP accumulation and activity, while oubain decrease them both), whereas a MRP inhibitor is without effect.