Effects of anidulafungin and voriconazole, singly and in combination, on cytokine/chemokine production by human monocyte-derived macrophages infected with Candida glabrata or activated by lipopolysaccharide.

BACKGROUND: Candida glabrata causes infections associated with severe sepsis, production of high concentrations of cytokines/chemokines, and high mortality. This study describes the effects of anidulafungin (ANF) and voriconazole (VRC), singly and in combination, on the production of eight cytokines/chemokines by human monocyte-derived macrophages (MDM) infected with C. glabrata or activated by lipopolysaccharide (LPS). METHODS: MDM monolayers were established, infected with C. glabrata or activated with LPS, and then treated with high or low concentrations of ANF, VRC, or both. Cytokine/chemokine levels in MDM supernatants were determined. RESULTS: Levels of cytokines/chemokines were significantly elevated in supernatants of infected or LPS-activated MDM. Except for interleukin-10, all significant decreases in cytokine/chemokine concentrations (p < 0.01) occurred in supernatants of infected MDM treated with high concentrations of ANF or ANF + VRC. CONCLUSIONS: Decreases in cytokine/chemokine levels in supernatants of infected MDM treated with high concentrations of ANF or ANF + VRC suggest that similar treatment could improve survival in patients with severe, invasive C. glabrata infections and markedly elevated levels of serum cytokines/chemokines.

IFN-gamma enhances killing of methicillin-resistant Staphylococcus aureus by human monocytes more effectively than GM-CSF in the presence of daptomycin and other antibiotics.

Because cytokines have been utilized in treatment of sepsis in neonates, we studied the effects of interferon-gamma (IFN-gamma) and GM-CSF on killing of intracellular methicilin-resistant Staphylococcus aureus (MRSA) by human monocyte derived macrophages (MDM) in the presence of daptomycin (Dap), rifampin (Rif), gentamicin (Gen), and combinations of these drugs. MDM infected with MRSA were treated with Dap (1 x MIC), Gen (0.5 x MIC), or Rif (1 x MIC), singly or in combination, with or without cytokines. MDM were lysed and viable bacteria counted. With antibiotics, MDM activated by IFN-gamma had a more rapid and prolonged bacterial killing effect than MDM activated by GM-CSF. This effect was most obvious with the triple-drug combination. In contrast, GM-CSF reduced intracellular killing under most experimental conditions compared to the effect of antibiotics alone. Dap alone and two- and three-drug combinations demonstrated significant killing effect for the 48 h of the assay. IFN-gamma enhanced rapid intracellular killing of MRSA in the presence of triple-drug treatment or Dap alone. GM-CSF in combination with the antibiotics reduced killing under most conditions studied. Further studies to confirm these observations with IFN-gamma-activated MDM and other MRSA strains are needed to support clinical trials for difficult-to-treat MRSA infections.

Anticandidal effects of voriconazole and caspofungin, singly and in combination, against Candida glabrata, extracellularly and intracellularly in granulocyte-macrophage colony stimulating factor (GM-CSF)-activated human monocytes.

OBJECTIVES: The antifungal effects of voriconazole and caspofungin, singly and in combination, were determined against Candida glabrata in time-kill curves in broth, in human monocyte-derived macrophages (MDMs) and in MDMs activated by granulocyte-macrophage colony-stimulating factor (GM-CSF). METHODS: Three strains of fluconazole-resistant C. glabrata were evaluated. For intracellular studies, MDM monolayers, with or without GM-CSF activation, were infected with C. glabrata and treated with voriconazole and caspofungin at 2.5x and 5x MIC, respectively, or at 1x MIC. Extracellular studies in broth were performed using drug concentrations from 0.1 to 10x MIC. Viable yeast were enumerated at 0, 24 and 48 h. RESULTS: Significantly greater killing of C. glabrata occurred with the drug combination than with either single drug, both intracellularly and extracellularly (P < 0.01). For voriconazole, the antifungal activity in MDM activated by GM-CSF was greater than that in unactivated MDM, regardless of antibiotic concentration or time of exposure. However, for caspofungin and for the two-drug combination, enhanced activity in GM-CSF-activated MDM depended on the drug concentration and time of exposure. CONCLUSIONS: Our data suggest that combinations of voriconazole and caspofungin may be efficacious for the treatment of serious C. glabrata infections. With single-drug therapy, especially voriconazole, GM-CSF activation of monocytes could be considered.

Antifungal effect of voriconazole on intracellular Candida glabrata, Candida krusei and Candida parapsilosis in human monocyte-derived macrophages.

Infections caused by Candida species other than Candida albicans are increasingly common, and decreased susceptibility to azoles has made them more difficult to treat. Since phagocytic killing is important in elimination of Candida infections, intracellular killing of fluconazole-resistant Candida glabrata, Candida krusei and Candida parapsilosis (four strains each) by voriconazole was investigated in human monocyte-derived macrophages (MDMs). MDMs were infected with Candida, and voriconazole was then added. MDMs were lysed at 0, 24 or 48 h after infection, and viable Candida in the lysates enumerated. Compared to the starting inoculum, the number of viable intracellular C. parapsilosis and C. glabrata in untreated MDMs increased to 28,121 and 351 %, respectively, in 48 h. In contrast, the number of C. krusei decreased to 42 %. In MDMs treated with voriconazole, the decrease in viable count was dependent upon drug concentration. At 48 h, C. glabrata was killed only at 5x MIC (P < 0.05), C. krusei was killed at all voriconazole concentrations, while C. parapsilosis was inhibited at 0.5 and 1x MIC and killed at > or = 2.5x MIC (P < 0.05). The data show that intracellular growth and survival of these Candida species in the absence or presence of voriconazole vary markedly. The activity of voriconazole depends on the concentration of the drug and the time of exposure. For the 12 Candida strains studied, regression curves show that the maximum intracellular anticandidal activity of voriconazole was reached at 3.5-5x MIC.

Effects of voriconazole, granulocyte-macrophage colony-stimulating factor, and interferon gamma on intracellular fluconazole-resistant Candida glabrata and Candida krusei in human monocyte-derived macrophages.

Infections caused by fluconazole-resistant Candida glabrata and Candida krusei are increasingly common causes of morbidity and mortality. We investigated the intracellular killing of fluconazole-resistant C. glabrata and C. krusei by cytokine-activated human monocyte-derived macrophages (MDM) in the presence and absence of voriconazole. For C. glabrata, MDM were activated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interferon gamma (IFN-gamma) before infection, after infection, or both before and after infection, whereas for C. krusei MDM were activated with cytokines both before and after infection. Activated MDM were infected, treated with voriconazole, and then lysed, and viable yeast in the lysates enumerated at 0, 24, or 48 h after infection. In the presence of voriconazole (2.5 x MIC), the best activity against C. glabrata occurred when MDM were activated with GM-CSF for 24 h before infection as well as after infection or when they were activated for 24 h before infection alone. A lesser effect was observed when MDM were activated for at least 1 h before infection or when they were treated with cytokines only after infection. IFN-gamma activation had a significant but lesser effect than GM-CSF. Activity against C. krusei in the presence of voriconazole was greatest when MDM were activated with IFN-gamma rather than GM-CSF. Our results suggest that cytokines increase the intracellular anticandidal effect of voriconazole and may be useful as therapeutic adjuvants to voriconazole for treatment of infections caused by fluconazole-resistant C. glabrata and C. krusei.

Effect of levofloxacin on the viability of intracellular Chlamydia pneumoniae and modulation of proinflammatory cytokine production by human monocytes.

Although antibiotics are known to affect the intracellular growth of Chlamydia pneumoniae in acute infections, their efficacy in therapy for chronic infections, including atherosclerosis, remains debatable. Human monocyte-derived macrophages (MDM) obtained from monocytes of healthy donors were infected with C. pneumoniae AR-39 and treated with levofloxacin (8 microg/mL) immediately after infection (0 hours) or 24 hours after infection. Levofloxacin treatment at 24 hours, but not at 0 hours, resulted in a significant decrease in the number of C. pneumoniae inclusions within the MDM (p < 0.05). Also decreased were concentrations of proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, and IL-8 in the extracellular medium (p < 0.01). Viable counts in titrations remained similar to those in untreated controls. In summary, levofloxacin administered to MDM at serum-attainable levels 24 hours after C. pneumoniae infection significantly decreased inclusion counts and proinflammatory cytokine production, but did not eliminate the C. pneumoniae infection.

Effects of echinocandins on cytokine/chemokine production by human monocytes activated by infection with Candida glabrata or by lipopolysaccharide.

Serious Candida glabrata infections, which can be difficult to treat, are often treated with echinocandins. We compared in vitro the effects of high and low concentrations of 3 echinocandins (micafungin [MCF], caspofungin [CAS], and anidulafungin [ANF]), voriconazole (VRC), and amphotericin B (AmB), singly and VRC in combination with MCF, CAS, and ANF, on the production of cytokines/chemokines by human monocyte-derived macrophages (MDM). MDM were activated by infection with C. glabrata or lipopolysaccharide (LPS). Luminex multi-analyte microsphere technology was used for cytokine/chemokine analysis. Concentrations of cytokines/chemokines were significantly elevated following activation by infection or LPS. Treatment with high concentrations of echinocandins, singly or in combination with VRC, was most effective in lowering the elevated cytokine/chemokine levels. This effect occurred only with MDM activated by infection with C. glabrata and not with LPS. Treatment with VRC or AmB alone had little or no effect on cytokine/chemokine levels. In severe C. glabrata infection associated with very high concentrations of dysregulated cytokines/chemokines, echinocandins, singly or in combination with VRC, may decrease cytokine/chemokine concentrations and thus may improve host survival.

Time-kill studies with micafungin and voriconazole against Candida glabrata intracellularly in human monocyte-derived macrophages and extracellularly in broth.

The purpose of this study was to determine by time-kill methodology the anticandidal effectiveness and durability of micafungin (MCF) and voriconazole (VRC), singly and in combination, against Candida glabrata (Cgl), intracellularly in human monocyte-derived macrophages and extracellularly in RPMI-MOPS broth with and without fetal calf serum (FCS) or pooled human serum (PHS). The anticandidal activity of MCF was concentration-dependent and durable. Combinations of MCF + VRC both intra- and extracellularly were more effective than single drugs. However, in extracellular experiments, FCS, and even more PHS, significantly decreased the anticandidal activity of MCF and VRC. Our in vitro studies suggest that MCF alone may be adequate where protein concentration is low (intracellular or extravascular sites), while MCF + VRC combinations may be preferred where protein concentration is high such as in the intravascular space.

Activities of tigecycline and comparators against Legionella pneumophila and Legionella micdadei extracellularly and in human monocyte-derived macrophages.

The activity of tigecycline against Legionellae, which are intracellular pathogens, was evaluated intracellularly in human phagocytes and extracellularly, and compared to the activities of erythromycin and levofloxacin. Clinical isolates of L. pneumophila serogroups 1, 5, and 6 and L. micdadei were tested in time-kill experiments. Extracellular experiments were done using buffered yeast extract broth. For intracellular assays, monolayers of human monocyte-derived macrophages (MDM) were infected with L. pneumophila or L. micdadei. Antibiotics (0.05-2.5 × MIC) were then added. MDM were lysed at 0, 24, 48, and 72 h and viable bacteria in the lysates were enumerated. Based on multiples of the MICs, tigecycline was less active extracellularly than levofloxacin or erythromycin. However, intracellular killing of both L. pneumophila and L. micdadei by tigecycline at 72 h was greater than for erythromycin or levofloxacin. Currently, evidence does not support the use of tigecycline as a first-line drug for treatment of Legionella infections. However, since Legionellae are intracellular pathogens, these results suggest that tigecycline should be effective for treatment of infections caused by these bacteria.

Antimicrobial activities of daptomycin, vancomycin, and oxacillin in human monocytes and of daptomycin in combination with gentamicin and/or rifampin in human monocytes and in broth against Staphylococcus aureus.

We investigated the antistaphylococcal activity of daptomycin, vancomycin, oxacillin, gentamicin, and rifampin in human monocyte-derived macrophages. Compared with vancomycin and oxacillin, daptomycin had the most rapid and greatest antibacterial activity, but that of oxacillin was most sustained. The combination of daptomycin, gentamicin, and rifampin was most effective intracellularly, while daptomycin plus gentamicin and the three-drug combination were most effective extracellularly, completely eliminating viable Staphylococcus aureus.

Effect of recombinant human activated protein C on the bactericidal activity of human monocytes and modulation of pro-inflammatory cytokines in the presence of antimicrobial agents.

OBJECTIVES: To determine the effects of recombinant human activated protein C (rhAPC) on the antimicrobial activity and cytokine production of normal human monocyte-derived macrophages (MDMs) in the presence and absence of Escherichia coli infection, with and without treatment with levofloxacin or ampicillin. METHODS: MDM monolayers were infected with E. coli ATCC 25922 and treated with levofloxacin or ampicillin in the presence or absence of rhAPC. Antimicrobial activity and cytokine (TNF-alpha, IL-1beta, IL-6 and IL-8) concentrations in the supernatants were measured. RESULTS: When low concentrations of levofloxacin were used, a therapeutic concentration of rhAPC enhanced intracellular antibacterial activity at all time points. With ampicillin, antibacterial activity increased, was unaffected or diminished depending upon the drug concentration and assay time. Without antibiotics, rhAPC had no antibacterial effect. E. coli caused cytokine production to increase many fold. This increase was significantly greater with antibiotics (P < 0.01). Without antibiotics, rhAPC decreased production of TNF-alpha, IL-1beta and IL-6, but not IL-8. At high levofloxacin concentrations, rhAPC was associated with further increases in the concentrations of these cytokines. Cytokine concentrations at 24 h were unaffected by rhAPC in the presence of ampicillin and E. coli. CONCLUSIONS: rhAPC can affect the bactericidal activity and cytokine production of human MDM in the presence of infection and antibiotic therapy. Importantly, factors such as type and concentration of antibiotics, presence of bacteria and timing must be taken into consideration when evaluating cytokine data from septic patients.

Antibacterial activities of gemifloxacin, levofloxacin, gatifloxacin, moxifloxacin and erythromycin against intracellular Legionella pneumophila and Legionella micdadei in human monocytes.

OBJECTIVES: The antibacterial activity of a new fluoroquinolone, gemifloxacin, was tested against intracellular Legionella pneumophila and Legionella micdadei and was compared with the activities of levofloxacin, gatifloxacin, moxifloxacin and erythromycin. METHODS: For intracellular assays, bacteria were used to infect human monocyte-derived macrophages prepared from heparinized blood of healthy volunteers. Antibiotics were added following phagocytosis. Numbers of viable bacteria were determined at 0, 24, 48, 72 and 96 h. RESULTS: The intracellular antibacterial activity of gemifloxacin was concentration- and time-dependent. All of the quinolones had similar activities against L. pneumophila and L. micdadei at 10 x MIC, but there were minor differences: at 24 h moxifloxacin was significantly more active than the other quinolones against L. pneumophila, while gemifloxacin was more active against L. micdadei (P < 0.01). All of the quinolones were markedly more active than erythromycin (P < 0.01). The antibacterial effect of gemifloxacin against L. pneumophila following drug removal at 24 h persisted for 72 h at 20 x MIC but not at 10 x MIC, while for L. micdadei the antibacterial effect persisted for 24 h at 10 x MIC. CONCLUSIONS: All of the quinolones had similar activities against intracellular L. pneumophila and L. micdadei and were markedly more effective than erythromycin.

Use of the microbial growth curve in postantibiotic effect studies of Legionella pneumophila.

Using the standard Craig and Gudmundsson method (W. A. Craig and S. Gudmundsson, p. 296-329, in V. Lorian, ed., Antibiotics in Laboratory Medicine, 1996) as a guideline for determination of postantibiotic effects (PAE), we studied a large series of growth curves for two strains of Legionella pneumophila. We found that the intensity of the PAE was best determined by using a statistically fitted line over hours 3 to 9 following antibiotic removal. We further determined the PAE duration by using a series of observations of the assay interval from hours 3 to 24. We determined that inoculum reduction was not necessarily the only predictor of the PAE but that the PAE was subject to the type and dose of the drug used in the study. In addition, there was a variation between strains. Only levofloxacin at five and ten times the minimum inhibitory concentration (MIC) resulted in a PAE duration of 4 to 10 h for both strains of L. pneumophila tested. Ciprofloxacin at five and ten times the MIC and azithromycin at ten times the MIC caused a PAE for one strain only. No PAE could be demonstrated for either strain with erythromycin or doxycycline. Using the presently described method of measuring PAE for L. pneumophila, we were able to detect differences in PAE which were dependent upon the L. pneumophila strain, the antibiotic tested, and the antibiotic concentration. We suggest the use of mathematically fitted curves for comparison of bacterial growth in order to measure PAE for L. pneumophila.

Levofloxacin kills Chlamydia pneumoniae and modulates interleukin 6 production by HEp-2 cells.

BACKGROUND: Chlamydia pneumoniae is known to cause acute respiratory infection and more recently it has been studied as a pathogen causing inflammatory changes in chronic diseases such as atherosclerosis. This study addresses the antichlamydial effect of levofloxacin and its role in modulation of a proinflammatory cytokine IL-6 production by uninfected and infected HEp-2 cells. METHODS: HEp-2 cell monolayers were infected with previously prepared and frozen aliquots of C.pneumoniae [1 x 10(3) inclusion-forming units (IFU)/ml] by centrifugation for 30 min and incubation at 37 degrees C for 1 h. Infected monolayers were treated with levofloxacin (3 or 8 microg/ml) immediately after infection (0 h) or 24 h after infection. Monolayers were examined daily for 96 h after infection by counting inclusions with fluorescently labeled antichlamydial monoclonal antibody. Aliquots of disrupted monolayers were titrated to determine the numbers of viable C. pneumoniae IFU/ml. IL-6 concentrations in cell supernatants were determined by ELISA assays. RESULTS: Infected HEp-2 cells produced IL-6. Noninfected HEp-2 cells demonstrated modulation of IL-6 production by levofloxacin. No viable C. Pneumoniae were detected in infected HEp-2 cells when the monolayer was treated with levofloxacin immediately after infection (0 h). In contrast, when cells were treated 24 h after infection, a gradual decline in the number of viable C. pneumoniae occurred; by 96 h into the assay >or=98% of C. pneumoniae were killed. IL-6 concentrations were similar in the supernatants of levofloxacin-treated and nontreated HEp-2 cells. CONCLUSIONS: (1). Levofloxacin is effective in eliminating C. pneumoniae from infected HEp-2 cells; (2). although levofloxacin modulates the production of IL-6 in untreated HEp-2 cells, no evidence for such modulation was observed in HEp-2 cells infected with C. pneumoniae. (3). Presence of viable C. pneumoniae may not be necessary for IL-6 production by infected and treated HEp-2 cells.