Oxidative metabolism drives inflammation-induced platinum resistance in human ovarian cancer.

Tumour cells have long been considered defective in mitochondrial respiration and mostly dependent on glycolytic metabolism. However, this assumption is currently challenged by several lines of evidence in a growing number of tumours. Ovarian cancer (OC) is one of the most lethal cancers worldwide, but it continues to be a poorly understood disease and its metabolic features are far to be elucidated. In this context, we investigated the role of tumour necrosis factor receptor-associated protein 1 (TRAP1), which is found upregulated in several cancer types and is a key modulator of tumour cell metabolism. Surprisingly, we found that TRAP1 expression inversely correlated with grade, stage and lower survival in a large cohort of OC patients. Accordingly, TRAP1 silencing induced resistance to cisplatin, resistant cells showed increased oxidative metabolism compared with their sensitive counterpart, and the bioenergetics cellular index of higher grade tumours indicated increased mitochondrial respiration. Strikingly, cisplatin resistance was reversible upon pharmacological inhibition of mitochondrial oxidative phosphorylation by metformin/oligomycin. At molecular level, increased oxidative metabolism in low TRAP1-expressing OC cells and tissues enhanced production of inflammatory mediators such as interleukin (IL)-6 and IL-8. Mechanistically, we identified members of the multidrug resistance complex (MDR) as key mediators of such metabolism-driven, inflammation-induced process. Indeed, treatment of OC cell lines with TNFα and IL6 induced a selective increase in the expression of TAP1 and multidrug resistance protein 1, whereas TAP1 silencing sensitized cells to cisplatin-induced apoptosis. Our results unveil a novel role for TRAP1 and oxidative metabolism in cancer progression and suggest the targeting of mitochondrial bioenergetics to increase cisplatin efficacy in human OC.

In vitro activity and in vivo efficacy of tigecycline alone and in combination with daptomycin and rifampin against Gram-positive cocci isolated from surgical wound infection.

The aim of this work was to determine the in vitro activity of tigecycline and its bactericidal effect for a large number of Gram-positive cocci, as well as to investigate its in vitro interaction with six clinically used antibiotics. In vivo, a wound model was established through the panniculus carnosus of BALB/c mice, and then inoculated with 5 × 10(7) colony-forming units (CFU) of Staphylococcus aureus or Enterococcus faecalis. For each bacterial strain, the study included an infected or non-infected group that did not receive any treatment, three groups singly treated with tigecycline, rifampin, and daptomycin, and two groups that received tigecycline treatment plus rifampin or daptomycin. In the in vitro studies, tigecycline, daptomycin, and teicoplanin were active against all of the 48 Gram-positive isolates. The combination of tigecycline with rifampicin and daptomycin was synergistic against S. aureus and Enterococcus spp. In the in vivo studies, all groups treated with single drugs showed statistically significant results compared to the control group. The two groups treated with a combination of drugs showed the highest antimicrobial efficacy. In conclusion, our results suggested a strong activity of tigecycline alone and in combination with other antimicrobial agents against multi-resistant Gram-positive organisms isolated from wound infections.

In vitro activity of the lipopeptide PAL-Lys-Lys-NH2, alone and in combination with antifungal agents, against clinical isolates of Candida spp.

Candida albicans is known to be the organism most often associated with serious fungal infection, but other Candida spp. are emerging as clinical pathogens associated with opportunistic infections. Among antimycotic treatments, increasing attention is currently given to anti-infective drugs based upon naturally occurring peptides, such as the short lipopeptide palmitoyl PAL-Lys-Lys-NH2 (PAL). The aim of this study is to evaluate the activity of this peptide compared to the traditional antifungal agents Fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) on Candida spp. 24 clinical isolates of Candida spp. were tested against PAL, FLU, AMB and CAS using in vitro susceptibility tests, time killing and checkerboard assay. All of the drugs studied showed good activity against clinical isolates of candida; in particular CAS and AMB which have MICs value lower than PAL and FLU. Moreover we observed synergistic interactions for PAL/FLU (81.25%), PAL/AMB (75%) and particularly for PAL/CAS (87.5). We think that our results are interesting since synergy between PAL and CAS might be useful in clinic trails to treat invasive fungal infections.

In vitro activity of Tachyplesin III alone and in combination with terbinafine against clinical isolates of dermatophytes.

Aim of our study was to investigate the in vitro effects of Tachyplesin III (TP), a potent disulfide-linked peptide, in dermatophytes infections, with respect to or in combination with terbinafine (TERB), against 20 clinical isolates of dermatophytes belonging to four species. A broth microdilution method following the CLSI recommendations (M38-A) was used for testing drugs alone and in combination. TERB MICs were significantly lower than those observed for TP (p<0.001). Testing for antifungal agents in combination was performed for TERB with TP for all the 20 isolates. TERB activity in combination with TP showed indifferent activity for 14 of the 20 isolates (70%); synergic activity for 6 of the 20 isolates (30%); no antagonistic activity was observed. Further experiments were conducted with Microsporum canis 133, Trichophyton rubrum 62 and Trichophyton mentagrophytes 91 for fungal biomass. TP and TERB did not show a significant growth reduction compared to the control against T. mentagrophytes and T. rubrum. A significant difference of growth reduction both for TP and TERB compared to controls was observed for M. canis (p<0.01). In conclusion our study demonstrated that Tachyplesin III has potential activity against dermatophytes. In addition, we observed that the in vitro activity of Tachyplesin III can be enhanced upon combination with terbinafine. Synergy could permit lower doses of the individual antifungal agents to be used more effectively and/or safely.

In vitro activity of the lipopeptide derivative (Pal-Lys-Lys-NH), alone and in combination with antifungal agents, against clinical isolates of dermatophytes.

BACKGROUND: An increasing number of antimycotics have become available for the treatment of dermatophytoses; however, there are reports suggesting recalcitrance to therapy or resistance of a dermatophyte against conventional treatment. Lipopeptides represent novel therapeutic drugs with a new mode of action. OBJECTIVES: The aim of this study was to investigate the in vitro effects of the lipopeptide Pal-Lys-Lys-NH(2) (PAL) alone and in combination with standard antifungal agents, such as fluconazole (FLU), itraconazole (ITRA) and terbinafine (TER) against 24 clinical isolates of dermatophytes belonging to four species. METHODS: A broth microdilution method following the Clinical and Laboratory Standards Institute recommendations (M38-A) was used for testing drugs alone and in combination. RESULTS: PAL minimum inhibitory concentrations (MICs) ranged from < or = 0.25 to > 16 microg mL(-1) and they were similar to those of FLU and higher than those of either ITRA or TER. Synergy, defined as a fractional inhibitory concentration (FIC) index of < or = 0.50, was observed in 67%, 52% and 15% of PAL/ITRA, PAL/TER and PAL/FLU interactions, respectively. None of these combinations yielded antagonistic interactions (FIC index > 4). When synergy was not achieved, there was still a decrease in the MIC of one or both drugs used in the combination. CONCLUSIONS: Our study demonstrates that PAL has potential activity against dermatophytes. In addition, the in vitro activity of PAL can be enhanced upon combination with standard drugs. This lipopeptide applied in the form of lacquer, spray or ointment, could represent an interesting new therapy, particularly when combined with conventional treatment in recalcitrant or resistant dermatophyte infections.

In-vitro activity of the synthetic protegrin IB-367 alone and in combination with antifungal agents against clinical isolates of Candida spp.

The in vitro activity of the peptide IB-367, alone or combined with either fluconazole (FLU) or amphotericin B (AMB), was investigated against 25 Candida isolates belonging to five species. IB-367 minimum inhibitory concentrations (MICs) ranged from 2.0 to 32 microg/ml and it was active against both FLU-susceptible and - resistant isolates. A rapid fungicidal activity was observed. Synergism was documented in 41.6% and 44% of IB-367/FLU and IB-367/AMB interactions, respectively. Antagonism was never observed. The broad antifungal activity and the positive interactions with AMB and FLU suggest that IB-367 represents a promising candidate against infections due to Candida spp.

Fluconazole susceptibility of Candida spp. isolates collected over nine years in a teaching hospital of Ancona, Italy.

The in vitro activity of fluconazole was investigated against 476 yeast isolates collected during a 9-year period (1997-2005) from patients hospitalised in a teaching hospital of Ancona. They included 373 isolates of Candida albicans, 53 of Candida glabrata and 50 of Candida parapsilosis. Minimum inhibitory concentrations (MICs) determined in accordance with the Clinical Laboratory Standards Institute methodology showed that 96% of the isolates were susceptible (MIC < or =8.0 microg/ml). The uncommon, resistant isolates (MIC > or =64 microg/ml) were randomly distributed over time. Our data show that resistance to fluconazole in this geographical area is a rare event and suggest that this triazole can still represent first-line therapy in our institution.

Epidemiology of onychomycosis and paronychia in the area of ANCONA (ITALY) over a period of 5 years.

We retrospectively evaluated the epidemiology of onychomycosis and/or paronychia in 172 patients attending the Clinic of Dermatology and Venereology over a 5 year period. Although yeast isolates, belonging to the Candida species, represented the most frequent etiologic agents of these infections, an increasing prevalence of fungal infections due to emerging fungal pathogens (EFP) was noted throughout this time period. In particular, EFP as causative agents of these infections increased from 0 to 28.4% from 1998 to 2002.

Genotyping and antifungal susceptibility of human subgingival Candida albicans isolates.

Subgingival colonization by Candida albicans has been described in human immunodeficiency virus (HIV)-infected individuals, but subgingival isolates have scarcely been characterized, particularly with respect to genotype and antifungal susceptibility. A series of 29 subgingival strains of C. albicans isolated from nine HIV-infected individuals was typed by electrophoretic karyotyping and tested for susceptibility to fluconazole, itraconazole, the new investigational triazole posaconazole and amphotericin B. DNA typing showed genetic heterogeneity within subgingival isolates, as almost every individual harbored his/her own specific isolate. Genetic identity was usually demonstrated within oral and subgingival isolates simultaneously collected from the same individual, but a number of DNA types were found to be unique to subgingival strains. These findings suggest that colonization is not just the result of Candida spreading from oral surfaces, and that subgingivally adapted strains could be involved. All isolates were susceptible to all the triazole drugs tested and amphotericin B. Additional studies on subgingival Candida colonization and further characterization of subgingival isolates are now required to clarify the role of Candida as opportunistic periodontal pathogen.

In vitro activity of posaconazole against clinical isolates of dermatophytes.

A broth macrodilution method following the recommendations established by the National Committee for Clinical Laboratory Standards was used to compare the in vitro activity of posaconazole (PCZ) with that of itraconazole (ITC) against 30 clinical isolates of dermatophytes belonging to six different species. In terms of MICs, PCZ showed an activity equal to that of ITC. MICs of PCZ at which 50% (MIC(50)) and 90% (MIC(90)) of the isolates were inhibited were 0.5 and > 4.0 microg/ml, respectively. The MIC(50) and MIC(90) of ITC were 1.0 and > 4.0 microg/ml, respectively. However, PCZ showed a more potent fungicidal activity than that of ITC against isolates belonging to the genus Microsporum (P = 0.03). PCZ merits further investigation as a potentially useful agent for treatment of dermatophytosis.

Electrophoretic karyotyping and antifungal susceptibility patterns of Candida parapsilosis clinical isolates causing deep and superficial fungal infections.

Forty-six isolates of Candida parapsilosis, each from a single patient, were collected from July 1993 through March 1999 at the University of Ancona Hospitals and Clinics. Twenty-eight strains were isolated from superficial lesioned sites, including skin, nails and other sources while 18 strains were isolated from blood. The isolates were typed by electrophoretic karyotyping (EK) and tested for their susceptibility to fluconazole (FLC), itraconazole (ITC), flucytosine (5-FC), and amphotericin B (AMB). Our data confirmed that EK is a useful technique for DNA typing of isolates of Candida parapsilosis and showed that the source of isolation is not associated with a given DNA type. Although strains belonging to this species of Candida are susceptible to the most common antifungals, including the triazoles, the degree of ITC susceptibility was dose dependent (MIC ranging from 0.25-0.5 microgram/ml) for 98% of the isolates.

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 < or =0.50, was observed for 7% of the isolates in studies of the interactions of both FLC-AmB and ITC-AmB and for 33% of the isolates in studies of the SCH-AmB interactions; additivism (FICs, >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 < or =2.0) was observed for 26, 20, and 14% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively. Antagonism (FIC >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.

In vitro activities of the new antifungal triazole SCH 56592 against common and emerging yeast pathogens.

A broth microdilution method performed in accordance with the National Committee for Clinical Laboratory Standards guidelines was used to compare the in vitro activity of the new antifungal triazole SCH 56592 (SCH) to that of fluconazole (FLC), itraconazole (ITC), and ketoconazole (KETO) against 257 clinical yeast isolates. They included 220 isolates belonging to 12 different species of Candida, 15 isolates each of Cryptococcus neoformans and Saccharomyces cerevisiae, and seven isolates of Rhodotorula rubra. The MICs of SCH at which 50% (MIC(50)) and 90% (MIC(90)) of the isolates were inhibited were 0.06 and 2.0 microg/ml, respectively. In general, SCH was considerably more active than FLC (MIC(50) and MIC(90) of 1.0 and 64 microg/ml, respectively) and slightly more active than either ITC (MIC(50) and MIC(90) of 0.25 and 2.0 microg/ml, respectively) and KETO (MIC(50) and MIC(90) of 0.125 and 4.0 microg/ml, respectively). Our in vitro data suggest that SCH has significant potential for clinical development.

In-vitro interactions of itraconazole with flucytosine against clinical isolates of Cryptococcus neoformans.

Treatment failures can occur in AIDS patients infected with Cryptococcus neoformans, despite aggressive antifungal therapy. Combination regimens with additive or synergic drugs could provide additional options for treating cryptococcosis. We studied the effects of itraconazole combined with flucytosine against 16 strains of C. neoformans var. neoformans. Combination therapy revealed different results for the various strains, including synergy (fractional inhibitory concentration (FIC) index 0.5, 63% of the interactions), addition (FIC >0.5 to 1.0, 31% of the interactions) and indifference (FIC >1.0 to <2.0, 6% of the interactions). Antagonism (FIC >2.0) was not observed. The efficacy of combination therapy was confirmed by quantitative cfu and killing curve assays. In particular, killing curves conducted in replicating cells showed that the addition of itraconazole prevented the development of flucytosine-resistant mutants of C. neoformans. These data show that the combination of itraconazole and flucytosine is significantly more active than either drug alone against C. neoformans in vitro.

Infection due to Absidia corymbifera in a patient with a massive crush trauma of the foot.

We report the case of a patient with a massive crushing trauma of the right foot who developed a local infection due to Absidia corymbifera. Systemic and local antifungal therapy with ketoconazole associated with hyperbaric oxygen therapy (HBO) yielded a rapid clinical and microbiological resolution. Controlled clinical studies are warranted to further elucidate the potential utility of HBO/antifungal combination therapy.

Electrophoretic karyotyping and triazole susceptibility of Candida glabrata clinical isolates.

A series of 35 strains of Candida glabrata isolated from 29 subjects (5 AIDS patients and 24 HIV-seronegative individuals) were typed by electrophoretic karyotyping and tested for their susceptibilities to both fluconazole and itraconazole. Almost every individual harboured his/her own specific isolate (DNA type). Neither the source of isolation nor the patient's HIV status was associated with a given DNA type. Recurrences were generally due to the persistence of the same DNA type over time. Only 9% of the isolates showed reduced susceptibility to fluconazole (MIC > or = 8.0 microg/ml), while 43% of the isolates showed reduced susceptibility to itraconazole (MIC > or = 0.25 microg/ml) (P = 0.02). These data show that electrophoretic karyotyping is a useful technique for DNA typing of isolates of Candida glabrata. Care must be taken prior to initiation of antifungal therapy with either of these drugs.

In vitro activity of terbinafine against clinical isolates of dermatophytes.

A broth macrodilution method following the recommendations established by the National Committee for Clinical Laboratory Standards was employed for testing terbinafine against 20 clinical isolates of dermatophytes belonging to seven species. The minimal inhibitory concentrations resulting in either 80% or 100% inhibition of growth, compared to growth in drug-free control tubes, ranged from

Tinea capitis due to Microsporum gypseum in an adult.

A case of tinea capitis due to Microsporum gypseum in an adult is described. An otherwise healthy 69-year-old woman presented two large patches of slightly erythematous scaling alopecia localized on the vertex and on the left parietal region of the scalp. The only subjective sign was itching. A mycological culture was positive for Microsporum gypseum. Tinea capitis is uncommon in adults; furthermore, isolation of Microsporum gypseum from this type of lesion is rare.

Comparison of three methods for testing azole susceptibilities of Candida albicans strains isolated sequentially from oral cavities of AIDS patients.

Three susceptibility testing procedures were compared to determine fluconazole, itraconazole, and ketoconazole MICs against 47 Candida albicans strains isolated sequentially from the oral cavities of five AIDS patients undergoing azole therapy. They included the broth microdilution method (BM), performed according to the National Committee for Clinical Laboratory Standards' tentative standard, the agar dilution method (AD), and the Etest; the latter two tests were performed both in Casitone agar (AD-Cas and Etest-Cas) and in RPMI (AD-RPMI and Etest-RPMI). Twenty-four- and 48-h MICs obtained by AD and Etest were compared with 48-h MICs obtained by BM. The MICs of all the azoles determined by BM were usually lower than those obtained by the other methods, mainly due to different reading criteria. In order to assess the most appropriate way of evaluating the agreement of MICs obtained by different methods with those produced by the proposed reference method (BM), we used the mean differences calculated according to Bland and Altman's method. Comparison of fluconazole MICs obtained by BM and AD-Cas yielded a mean difference of 3, and the percentages of agreement within +/-2 dilutions were 98 and 100% at 24 and 48 h, respectively. For ketoconazole and itraconazole MICs, lower mean differences were noted, and agreement ranged from 96 to 100%. Agreement between the AD-RPMI and BM results was poor for all azoles, and an increase in MICs was always observed between the 1st- and 2nd-day readings. Similarly, Etest-Cas gave better agreement with BM than did Etest-RPMI for all the azoles. BM, AD-Cas, and Etest-Cas each demonstrated a progressive increase in fluconazole MICs against strains isolated sequentially from a given patient, in accordance with the decreased clinical response to fluconazole.