Expression Levels of a Kinesin-13 Microtubule Depolymerase Modulates the Effectiveness of Anti-Microtubule Agents

Chemotherapeutic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs. In this report, we test the hypothesis that the cellular levels of microtubule depolymerases, in this case kinesin-13 s, modulate the effectiveness of the microtubule disrupting drug colchicine.We used a combination of RNA interference (RNAi), high-throughput microscopy, and time-lapse video microscopy in Drosophila S2 cells to identify a specific MAP, kinesin-like protein 10A (KLP10A), that contributes to the efficacy of the anti-microtubule drug colchicine. KLP10A is an essential microtubule depolymerase throughout the cell cycle. We find that depletion of KLP10A in S2 cells confers resistance to colchicine-induced microtubule depolymerization to a much greater extent than depletion of several other destabilizing MAPs. Using image-based assays, we determined that control cells retained 58% (+/-2%SEM) of microtubule polymer when after treatment with 2 microM colchicine for 1 hour, while cells depleted of KLP10A by RNAi retained 74% (+/-1%SEM). Likewise, overexpression of KLP10A-GFP results in increased susceptibility to microtubule depolymerization by colchicine.Our results demonstrate that the efficacy of microtubule destabilization by a pharmacological agent is dependent upon the cellular expression of a microtubule depolymerase. These findings suggest that expression levels of Kif2A, the human kinesin-13 family member, may be an attractive biomarker to assess the effectiveness of anti-microtubule chemotherapies. Knowledge of how MAP expression levels affect the action of anti-microtubule drugs may prove useful for evaluating possible modes of cancer treatment

Epidemiology and microbiology of surgical wound infections.

This study included 676 surgery patients with signs and symptoms indicative of wound infections, who presented over the course of 6 years. Bacterial pathogens were isolated from 614 individuals. A single etiologic agent was identified in 271 patients, multiple agents were found in 343, and no agent was identified in 62. A high preponderance of aerobic bacteria was observed. Among the common pathogens were Staphylococcus aureus (191 patients, 28.2%), Pseudomonas aeruginosa (170 patients, 25.2%), Escherichia coli (53 patients, 7.8%), Staphylococcus epidermidis (48 patients, 7.1%), and Enterococcus faecalis (38 patients, 5.6%)

ACTIVITY OF THE NEW ANTIFUNGAL TRIAZOLE, POSACONAZOLE, AGAINST CRYPTOCOCCUS NEOFORMANS

The new antifungal derivative posaconazole was tested against three clinical isolates of Cryptococcus neoformans var. neoformans using a broth microdilution procedure performed according to the guidelines established by the NCCLS. Posaconazole MICs were 0.125, 0.25 and 1.0 mg/L for isolates 491, 2337 and 486, respectively. To investigate the in vivo activity of this new compound, we established an experimental model of systemic cryptococcosis in CD1 mice by iv injection of cells of each strain of C. neoformans. Low (3 mg/kg/day) and high (10 mg/kg/day) doses of posaconazole were compared with amphotericin B given at 0.3 mg/kg/day for 10 consecutive days. Survival studies showed that all treatment regimens were effective in prolonging the survival of mice infected with C. neoformans 486 (P < 0.001). Only posaconazole at 10 mg/kg and amphotericin B were effective in prolonging the survival in mice infected with C. neoformans 2337 (P from <0.01 to <0.001), while neither agent was effective in mice infected with C. neoformans 491. Tissue burden experiments performed 24 h after the end of therapy revealed that posaconazole at 10 mg/kg was effective at reducing the fungal burden in both lung and brain tissues of all three strains of C. neoformans. In particular, for C. neoformans 491 and 2337 posaconazole was superior to amphotericin B at reducing the fungal burden in the brain (P < 0.05). The efficacy of posaconazole was also confirmed by determining the capsular antigen serum levels of treated mice versus untreated mice. Our study underlines the excellent activity of posaconazole against this pathogenic yeast

Interactions of Posaconazole and Flucytosine against Cryptococcus neoformans

A checkerboard methodology, based on standardized methods proposed by the National Committee for Clinical Laboratory Standards for broth microdilution antifungal susceptibility testing, was applied to study the in vitro interactions of flucytosine (FC) and posaconazole (SCH 56592) (FC-SCH) against 15 isolates of Cryptococcus neoformans. Synergy, defined as a fractional inhibitory concentration (FIC) index of <0.50, was observed for 33% of the isolates tested. When synergy was not achieved, there was still a decrease in the MIC of one or both drugs when they were used in combination. Antagonism, defined as a FIC of >4.0, was not observed. The in vitro efficacy of combined therapy was confirmed by quantitative determination of the CFU of C. neoformans 486, an isolate against which the FC-SCH association yielded a synergistic interaction. To investigate the potential beneficial effects of this combination therapy in vivo, we established two experimental murine models of cryptococcosis by intracranial or intravenous injection of cells of C. neoformans 486. At 1 day postinfection, the mice were randomized into different treatment groups. One group each received each drug alone, and one group received the drugs in combination. While combination therapy was not found to be significantly more effective than each single drug in terms of survival, tissue burden experiments confirmed the potentiation of antifungal activity with the combination. Our study demonstrates that SCH and FC combined are significantly more active than either drug alone against C. neoformans in vitro as well in vivo. These findings suggest that this therapeutic approach could be useful in the treatment of cryptococcal infections

Interactions between triazoles and amphotericin B against Cryptococcus neoformans.

The interaction of amphotericin B (AmB) and azole antifungal agents in the treatment of fungal infections is still a controversial issue. A checkerboard titration broth microdilution-based method that adhered to the recommendations of the National Committee for Clinical Laboratory Standards was applied to study the in vitro interactions of AmB with fluconazole (FLC), itraconazole (ITC), and the new investigational triazole SCH 56592 (SCH) against 15 clinical isolates of Cryptococcus neoformans. Synergy, defined as a fractional inhibitory concentration (FIC) index of 0.50 to 1.0) was observed for 67, 73, and 53% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively; indifference (FICs, >1.0 to 2.0) was not observed. When synergy was not achieved, there was still a decrease, although not as dramatic, in the MIC of one or both drugs when they were used in combination. To investigate the effects of FLC-AmB combination therapy in vivo, we established an experimental model of systemic cryptococcosis in BALB/c mice by intravenous injection of cells of C. neoformans 2337, a clinical isolate belonging to serotype D against which the combination of FLC and AmB yielded an additive interaction in vitro. Both survival and tissue burden studies showed that combination therapy was more effective than FLC alone and that combination therapy was at least as effective as AmB given as a single drug. On the other hand, when cells of C. neoformans 2337 were grown in FLC-containing medium, a pronounced increase in resistance to subsequent exposures to AmB was observed. In particular, killing experiments conducted with nonreplicating cells showed that preexposure to FLC abolished the fungicidal activity of the polyene. However, this apparent antagonism was not observed in vivo. Rather, when the two drugs were used sequentially for the treatment of systemic murine cryptococcosis, a reciprocal potentiation was often observed. Our study shows that (i) the combination of triazoles and AmB is significantly more active than either drug alone against C. neoformans in vitro and (ii) the concomitant or sequential use of FLC and AmB for the treatment of systemic murine cryptococcosis results in a positive interaction