Gemifloxacin inhibits cytokine secretion by lipopolysaccharide stimulated human monocytes at the post-transcriptional level.

The fluroquinolone gemifloxacin was examined for its capacity to modulate secretion of cytokines by human monocytes stimulated with lipopolysaccharide (LPS). Monocytes from six male and two female healthy volunteers were stimulated with LPS, exposed to gemifloxacin and the amounts of secreted IL-1 alpha, IL-1 beta, IL-6, IL-10 and TNF-alpha measured at 3, 6 and 24 h. The results revealed that LPS alone increased secretion of each cytokine significantly. Treatment of the LPS-stimulated monocytes with gemifloxacin resulted in a significant inhibition (p < 0.01) of secretion of each of the cytokines from monocytes of the eight volunteers. Nuclear extracts of the human monocyte cell line, THP-1, were used in the electrophoretic mobility shift assay to determine whether gemifloxacin affects nuclear factor-kappa B (NF-kappa B) activation. In addition, RNA from THP-1 cells was used in Northern blots to determine whether inhibition of secretion of IL-1 beta and TNF-alpha by gemifloxacin occurred at the transcription or translation level. Whereas LPS induced a rapid increase in NF-kappa B activation, gemifloxacin alone did not. Gemifloxacin did not affect the kinetics or decrease the extent of activation. Northern blots indicated that the inhibitory activity of gemifloxacin occurred post-transcription. Thus, gemifloxacin may modulate the immune response by altering secretion of cytokines by human monocytes. Although the concentrations of gemifloxacin used were higher than those observed in the serum of human volunteers treated with the dose under clinical development, it should be taken into consideration that concentrations at tissue and intracellular levels may be considerably higher than serum concentrations.

Effect of moxifloxacin on secretion of cytokines by human monocytes stimulated with lipopolysaccharide.

OBJECTIVE: To determine the effect of moxifloxacin on secretion of cytokines by human monocytes stimulated with lipopolysaccharide (LPS) or Pansorbin. METHODS: Monocytes obtained from 10 healthy volunteer donors were stimulated with LPS or Pansorbin and exposed or not to different concentrations of the fluoroquinolone antibiotic moxifloxacin. At 3, 6 and 24 h, the amounts of interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-10, IL-12 (p70) and tumour necrosis factor-alpha (TNF-alpha) were measured in the supernatants of the monocyte cultures using enzyme-linked immunosorbent assay. RESULTS: Stimulation of human monocytes with either LPS or Pansorbin resulted in a significant increase in secretion of each of the cytokines examined. Treatment of LPS-stimulated monocytes with moxifloxacin significantly inhibited (P < 0.01) secretion of IL-1alpha by monocytes of each of 10 human donors; the secretion of TNF-alpha was significantly inhibited (P < 0.01) in monocytes from six of 10 donors. In general there was a trend towards inhibition of secretion of IL-6, IL-10 and IL-12 (p70), but the inhibitory effect was not statistically significant. Secretion of cytokines by Pansorbin-stimulated monocytes was not significantly inhibited by moxifloxacin. CONCLUSIONS: Moxifloxacin has immunomodulatory activity through its capacity to alter the secretion of IL-1alpha and TNF-alpha by human monocytes.

Activity of gatifloxacin alone or in combination with pyrimethamine or gamma interferon against Toxoplasma gondii.

The activity of gatifloxacin against Toxoplasma gondii, either alone or in combination with pyrimethamine or gamma interferon (IFN-gamma), was examined in vitro and in vivo. In vitro, gatifloxacin significantly inhibited intracellular replication of tachyzoites of the RH strain with a 50% inhibitory concentration of 0.21 microg/ml at 48 h after addition of the drug to the cultures. Toxicity for host cells was not observed at this concentration. A synergistic effect (combination indices < 0.5) was demonstrated in vitro following 48 h of treatment with the combination of gatifloxacin and pyrimethamine (1:1 ratio). Doses of gatifloxacin of 100 and 200 mg/kg of body weight/day administered orally to mice for 10 days resulted in significant (P values of 0.056 and <0.0001, respectively) prolongation in time to death following infection with a lethal inoculum of tachyzoites. A dose of 400 mg/kg resulted in 20% survival (P = 0.0001). Mortality was 100% in untreated control mice and in mice treated with 25 or 50 mg/kg/day. Treatment of infected mice with a combination of gatifloxacin at 200 mg/kg/day and pyrimethamine at 12.5 mg/kg/day resulted in 85% survival, whereas 100 and 80% of mice treated with gatifloxacin alone or pyrimethamine alone, respectively, died (P < 0.0001). Moreover, a gatifloxacin dose of 200 mg/kg/day administered orally for 10 days plus 2 microg of recombinant murine IFN-gamma/day administered intraperitoneally for 10 days resulted in significant survival compared with IFN-gamma alone (P < 0.0001) or gatifloxacin alone (P < 0.007).

Protection against lipopolysaccharide-induced death by fluoroquinolones.

Because fluoroquinolones have an immunomodulatory effect on cytokine production by lipopolysaccharide (LPS)-treated human monocytes, we examined the effect of fluoroquinolones on the survival of mice injected with a lethal dose of LPS. Trovafloxacin (100 mg/kg), ciprofloxacin (250 mg/kg), and tosufloxacin (100 mg/kg) protected 75% (P = 0.0001), 25% (P = 0.002), and 50% (P = 0.002), respectively, of mice against death. The fluoroquinolones significantly reduced serum levels of interleukin-6 and tumor necrosis factor alpha in LPS-treated mice. The protective effects of fluoroquinolones in LPS-induced shock in mice may also occur in humans.

Effect of quinupristin/dalfopristin on production of cytokines by human monocytes.

The effect of the novel streptogramin antibiotic quinupristin/dalfopristin (synercid) on cytokine production in vitro was examined in monocytes obtained from healthy human volunteers and stimulated with either lipopolysaccharide or heat-killed Staphylococcus aureus (Pansorbin). Synercid at concentrations that are achievable in humans (1, 5, and 10 microgram/mL) significantly suppressed production of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha in a concentration-dependent manner. Thus, synercid possesses significant immunomodulatory activity, in addition to its antimicrobial activity.

Quinupristin-dalfopristin is active against Toxoplasma gondii.

Synercid and each of its components (quinupristin and dalfopristin) were examined for their activities against Toxoplasma gondii. In vitro, intracellular replication of tachyzoites was inhibited by synercid and each of its two components. The 50% inhibitory concentrations of synercid, quinupristin, and dalfopristin were 1.6, 2.7, and 6.3 microg/ml, respectively. Thus, synercid was markedly more active than its components. Treatment of acutely infected mice with 100 or 200 mg of synercid per kg of body weight per day administered intraperitoneally for 10 days resulted in survival of 50% (P = 0.0002) and 100% (P < 0.0001) of infected mice, respectively, whereas all control mice died by day 18. In contrast, treatment with 200 mg of either quinupristin and dalfopristin per kg per day alone resulted in only 20% survival; treatment with 50 mg of either drug per kg per day resulted only in the prolongation of time to death. These results suggest that synercid may be useful for treatment of toxoplasmosis in humans.

Effect of clarithromycin and azithromycin on production of cytokines by human monocytes.

We examined the in vitro effect of clarithromycin and azithromycin on cytokine production by LPS and Pansorbin stimulated human monocytes. At concentrations that are physiologically achievable, both antibiotics affected in vitro production of IL-1alpha, IL-1beta, IL-6, IL-10, GM-CSF and TNF-alpha to varying degrees. Of those individuals in whom a significant increase or decrease in cytokine production was noted, clarithromycin treatment resulted in a significant suppression of production of each cytokine in 71% and a significant increase in 29% of the individuals. Similar results were noted with azithromycin. The results with IL-6 and TNF-alpha in the clarithromycin studies were most striking. A significant decrease was noted in 60% of individuals for IL-6 and 86% for TNF-alpha. For azithromycin, the most interesting results were for IL-1alpha (decrease in 100% of individuals) and for TNF-alpha (decrease in 100% of individuals). These results show that both clarithromycin and azithromycin alter cytokine production in human monocytes and thus possess immunomodulatory activity.

Effect of trovafloxacin on production of cytokines by human monocytes.

Antibiotics have previously been shown to have immunomodulatory effects. We examined the effect of the broad-spectrum fluoroquinoline antibiotic trovafloxacin on cytokine synthesis by monocytes obtained from healthy human volunteers and stimulated with either lipopolysaccharide or gram-positive cells (heat-killed Staphylococcus aureus [Pansorbin]). Trovafloxacin levels achievable in humans suppressed in vitro synthesis of each of the cytokines analyzed, viz., interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. This effect was not due to direct effects of the drug on cellular viability; at these concentrations, trovafloxacin did not have demonstrable cytotoxicity for the monocytes, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Although similar patterns of suppression of cytokine synthesis were observed in samples obtained from the same volunteers on different days, there were significant day-to-day variations. These results reveal that trovafloxacin possesses significant immunomodulatory activity in vitro and suggest that suppression of acute-phase inflammatory responses may occur in vivo, elicited through trovafloxacin's effect on cytokine synthesis by human monocytes.

The ketolide antibiotics HMR 3647 and HMR 3004 are active against Toxoplasma gondii in vitro and in murine models of infection.

Ketolides are a new class of macrolide antibiotics that have been shown to be active against a variety of bacteria including macrolide-resistant bacteria and mycobacteria. We examined two ketolides, HMR 3647 and HMR 3004, for their in vitro and in vivo activities against the protozoan parasite Toxoplasma gondii. In vitro, both ketolides at concentrations as low as 0.05 microg/ml markedly inhibited replication of tachyzoites of the RH strain within human foreskin fibroblasts. HMR 3004 demonstrated some toxicity for host cells after they were exposed to 5 microg of the drug per ml for 72 h. In contrast, HMR 3647 did not show any significant toxicity even at concentrations as high as 25 microg/ml. In vivo, both ketolides provided remarkable protection against death in mice lethally infected intraperitoneally with tachyzoites of the RH strain or orally with tissue cysts of the C56 strain of T. gondii. A dosage of 100 mg of HMR 3647 per kg of body weight per day administered for 10 days protected 50% of mice infected with tachyzoites. The same dosage of HMR 3004 protected 100% of the mice. In mice infected with cysts, a dosage of 30 mg of HMR 3647 per kg per day protected 100% of the mice, whereas a dosage of 40 mg of HMR 3004 per kg per day protected 75% of the mice. These results demonstrate that HMR 3647 and HMR 3004 possess excellent activities against two different strains of T. gondii and may be useful for the treatment of toxoplasmosis in humans.

Chronic infection with Toxoplasma gondii does not prevent acute disease or colonization of the brain with tissue cysts following reinfection with different strains of the parasite.

Two strains of Toxoplasma gondii with different capacities to induce disease and brain lesions in mice were used to study the effects of reinfection with the parasite on a previously infected host. In spite of marked antibody and cell-mediated immune responses, chronically infected mice developed disease and died of acute toxoplasmosis when reinfected with a strain different from the one causing the primary infection. Moreover, the marked antibody and cell-mediated immune responses of the chronically infected mice did not prevent invasion of their brains and formation of tissue cysts by the reinfecting strain. Tissue cysts of the reinfecting strain were demonstrated in the brains of the chronically infected and subsequently reinfected mice. These results highlight the importance of strain differences in the pathogenesis of toxoplasmosis.

Activity of trovafloxacin in combination with other drugs for treatment of acute murine toxoplasmosis.

Current therapy for toxoplasmosis with a synergistic combination of pyrimethamine plus sulfadiazine or pyrimethamine plus clindamycin is not always efficacious and is frequently discontinued due to intolerable toxic effects in immunocompromised individuals, particularly those with AIDS. Trovafloxacin, a new fluoroquinolone with potent activity against Toxoplasma gondii, was examined for potential synergistic activity when combined with other drugs used for treatment of human toxoplasmosis. Combinations of trovafloxacin with clarithromycin, pyrimethamine, or sulfadiazine demonstrated significantly enhanced activities compared to those observed with each drug alone. Our results suggest that combinations of trovafloxacin and other anti-toxoplasma drugs should be further explored for treatment of toxoplasmosis in humans.

IL-10 is required to prevent immune hyperactivity during infection with Trypanosoma cruzi.

Previous studies have associated the production of IL-10 with suppression of the protective cell-mediated immune response to Trypanosoma cruzi. To further understand the role of IL-10 in the resistance to and pathogenesis of Chagas' disease, we infected C57BL/6 wild-type (IL-10 +/+) or C57BL/6 IL-10 knockout (IL-10 -/-) mice with the virulent Tulahuen strain of T. cruzi. IL-10 -/- mice had a lower parasite burden and higher levels of serum TNF-alpha, IL-12, and IFN-gamma compared with infected IL-10 +/+ mice. However, infection resulted in earlier mortality of IL-10 -/- mice compared with IL-10 +/+ controls. The earlier mortality of IL-10 -/- mice could be reversed by administering rIL-10 or a neutralizing Ab specific for IL-12. A role for T cells in the early mortality of IL-10 -/- mice was suggested by experiments in which SCID IL-10 -/- mice infected with T. cruzi had a delay in time to death and significantly lower serum levels of IFN-gamma compared with IL-10 -/- mice. Furthermore, treatment of infected IL-10 -/- mice with a mAb specific for CD4 resulted in reduced serum levels of IFN-gamma and a delay in time to death. Altogether, our results demonstrate for the first time that during infection with T. cruzi there is a critical requirement for IL-10 to prevent the development of a pathologic immune response associated with CD4+ T cells and overproduction of IL-12.

Trovafloxacin is active against Toxoplasma gondii.

Drugs currently used for treatment of toxoplasmosis in pregnant women, congenital infections, immunocompromised patients, and patients with the ocular disease are not always effective or may be dangerous to use; therefore, there is a need for more-effective and less-toxic drugs. Recently, we examined a group of fluoroquinolones for in vitro and in vivo activities against Toxoplasma gondii. Among those examined in vitro (ciprofloxacin, fleroxacin, ofloxacin, temafloxacin, and trovafloxacin), only trovafloxacin significantly inhibited intracellular replication of T. gondii without significant toxicity for host cells. In a murine model of acute toxoplasmosis, 100 or 200 mg of trovafloxacin per kg of body weight per day for 10 days protected 100% of infected mice against death. A dose of 50 mg/kg/day protected 90% of the mice, and a dose of 25 mg/kg/day effected prolongation of time to death. The other fluoroquinolones did not have such in vivo activities. These results indicate that trovafloxacin may be useful for treatment of toxoplasmosis in humans.

Interleukin-12-mediated resistance to Trypanosoma cruzi is dependent on tumor necrosis factor alpha and gamma interferon.

The aim of this study was to determine if interleukin-12 (IL-12) has a role in the immune response to Trypanosoma cruzi. Infection of BALB/c mice with the virulent Tulahuen strain of T. cruzi is characterized by a high-level parasitemia, pathology in the heart associated with the presence of amastigotes, and death during the acute phase of the disease. Administration of IL-12 to BALB/c mice infected with T. cruzi resulted in a reduced parasitemia and a significant delay in the time to death compared with those for infected controls. This protective effect was correlated with increased levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in serum. To determine if these cytokines were involved in the protective effects of IL-12, we treated infected mice with IL-12 alone or in combination with monoclonal antibodies specific for IFN-gamma or TNF-alpha. These antibodies antagonized the protective effect of exogenous IL-12. Treatment of infected mice with a polygonal antibody specific for IL-12 resulted in a significant increase in parasitemia but did not affect the time to death. These latter studies demonstrate a role for endogenous IL-12 in resistance to T. cruzi. Together, our data identify an IL-12-mediated mechanism of resistance to T. cruzi, which is dependent on IFN-gamma and TNF-alpha.

Nonionic block copolymers potentiate activities of drugs for treatment of infections with Toxoplasma gondii.

We studied the interaction between drugs and the nonionic block copolymers CRL 8131 and CRL 8142 in the treatment of toxoplasmosis in murine models of the disease. Treatment of acute toxoplasmosis with copolymers alone caused slight prolongation of time to death but not survival. In contrast, significant survival occurred when mice were treated with either copolymer combined with doses of sulfadiazine, pyrimethamine, clindamycin, or atovaquone, which did not prevent mortality when used alone. Treatment with CRL 8131 plus sulfadiazine or pyrimethamine resulted in 50 or 40% survival, respectively. Treatment with the same copolymer plus a dose of clindamycin that protected 40% of the mice when used alone resulted in 100% survival. Treatment of toxoplasmic encephalitis with CRL 8131 plus an ineffective dose of atovaquone reduced the inflammation and numbers of Toxoplasma gondii cysts in the brain. Studies to investigate the drug-enhancing activity of CRL 8131 revealed that mice immunized with toxoplasma lysate plus copolymer had lymphocyte proliferation responses to T. gondii antigens significantly higher than those in mice immunized with lysate alone. Challenge of immunized mice with a lethal inoculum of T. gondii resulted in significant survival. Administration of CRL 8131 alone appeared to cause a down-regulation in the production of gamma interferon and up-regulation in the production of interleukin-2. No differences were noted in the production of tumor necrosis factor alpha between mice treated with CRL 8131 and controls.

Rifabutin is active in murine models of toxoplasmosis.

Rifabutin, a semisynthetic derivative of rifamycin S, was examined alone and in combination with other drugs for activity in treatment of systemic toxoplasmosis and toxoplasmic encephalitis in murine models. One hundred percent of the mice infected with a lethal inoculum of tachyzoites or cysts of Toxoplasma gondii were protected against death by treatment with doses of 400 or 300 mg of rifabutin per kg administered alone for 10 days. Doses of 200 mg/kg protected at least 80% of the mice, and doses of 100 mg/kg protected 10 to 40% of the infected mice against death. Doses of 50 mg/kg were not protective but caused a delay in time to death. Combination of nonprotective (50-mg/kg) or slightly protective (100-mg/kg) doses of rifabutin with doses of sulfadiazine, pyrimethamine, clindamycin, or atovaquone that did not confer any protection against death from toxoplasmosis when administered alone resulted in remarkable enhancement of the in vivo activities of all of these drugs. Seventy-five percent of the infected mice survived when treated with 100 mg of rifabutin per kg per day combined with the ineffective dose of 10 mg of pyrimethamine per kg. A dose of 50 mg of rifabutin per kg in combination with the ineffective dosages of clindamycin (25 mg/kg/day), atovoquone (5 mg/kg/day), and sulfadiazine (80 mg per liter of drinking water) protected at least 80, 60, and 60% of the mice against death, respectively. The inflammatory responses in the brains of mice treated for 30 days with 200 mg of rifabutin per kg per day were significantly reduced compared with those in the brains of untreated controls. These observations suggest that clinical trials with rifabutin for treatment and prevention of human toxoplasmosis may be justified, particularly when the drug is used in combination with other drugs with activity against T. gondii.