An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT

A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.     

Rapid susceptibility testing of medically important zygomycetes by XTT assay

The XTT colorimetric assay quantifies fungal growth by measuring fungal metabolism and has been used successfully for susceptibility testing of Aspergillus species after 24 and 48 h of incubation. In the present study using 14 clinical isolates of Zygomycetes (Rhizopus oryzae [5 isolates], Cunninghamella spp. [3 isolates], Mucor spp. [3 isolates], and Absidia corymbifera [3 isolates]), significant metabolic activity was demonstrated before visual or spectrophotometric detection of fungal growth by performing the XTT assay as early as 6 h after inoculation. Testing of susceptibility to amphotericin B, posaconazole, and voriconazole was subsequently performed using the XTT method (100 microg/ml XTT, 25 microM menadione) at 6, 8, or 12 h after inoculation and the CLSI (formerly NCCLS) M38-A method with visual and spectrophotometric MIC determinations at 24 h after inoculation. Concentration-effect curves obtained with the use of the E(max) model (a sigmoid curve with variable slope) were comparable between the early XTT and spectrophotometric readings at 24 h. Complete inhibition of early metabolic activity with the azoles was delayed in comparison to that with amphotericin B. Using appropriate cutoff levels, agreement was demonstrated between the early XTT and 24-h spectrophotometric or visual readings. In particular, for MIC-0 (the lowest drug concentration showing absence of visual growth) of amphotericin B, overall agreement levels were 90 to 93% for the 6-h XTT assay and 100% for the 8- and 12-h time points. For MIC-0 of posaconazole, agreement levels were 86% for the 6-h XTT and 93 to 100% for the 8- and 12-h time points. The overall agreement levels for MIC-0 and MIC-2 (the lowest drug concentration showing prominent reduction of growth compared with the control well) of voriconazole (compared with 24-h spectrophotometric readings) were 93 to 98% for the 8- and 12-h XTT assays. These results support the use of the XTT method for rapid MIC determination for Zygomycetes.     

Susceptibility testing of Candida albicans and Aspergillus species by a simple microtiter menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay.

We describe a simple microtiter method for determining the susceptibility of Candida albicans and hyphal forms of Aspergillus fumigatus against antifungal agents. The assay measures mitochondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, a process that is enhanced in the presence of menadione. C. albicans or conidial suspensions of A. fumigatus are seeded into microtiter plates. Hyphal outgrowth of Aspergillus spp. was achieved by a 12 to 14-h culture at 30 degrees C. Antifungal agents (amphotericin B, fluconazole, itraconazole) were added to the cultures for 24 h. Thereafter, incubations were continued for 3 h in the presence of MTT plus 0.1 mM menadione. Formazan formation was quantified photometrically after extraction of the formazan with acid isopropanol. Well-defined dose-response curves reflecting impairment of mitochondrial function by the antifungal agents were obtained. With C. albicans, the results correlated excellently with the MIC determinations performed according to the standard macrodilution procedure. In confirmation of a recent report, it was found that fluconazole was unable to exert its fungistatic action on a sensitive C. albicans strain in the presence of serum. The presented method can easily be integrated in the standard repertoire of a diagnostic microbiology laboratory and should prove useful as a means to assess the antifungal action of various agents on yeasts and filamentous fungi in the presence and absence of serum proteins or body fluids.     

Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells.

A tetrazolium compound, XTT, bioreducible to a water-soluble formazan was used to develop a simplified cellular cytotoxicity assay. Most (13/15 melanoma and 2/3 colon carcinoma cell lines tested metabolized XTT greater than 50 times more efficiently than the lymphoid effector cells, and thus the test could be performed without separation of the effector from the target cells. The XTT assay (XTT-A) was compared to the standard 51chromium-release assay (51CrA) in terms of sensitivity as well as intra- and interassay variability using low effector to target cell (E:T) ratios and both short and long incubation periods. The correlation coefficient (r) for percent specific lysis (%SL: 35.0 +/- 15.0 versus 30.2 +/- 15.8) or lytic units (LU20/10(7) effector cells: 405 +/- 208 versus 357 +/- 227) between XTT-A and 51CrA was 0.86 for 4 h XTT-A and 51CrA (n = 37). Due to a poor performance of the 51CrA after 24 h incubation of effector and target cells, the correlation coefficient for 24 h assays was reduced to 0.79 (n = 44,%SL = 63.3 +/- 23.9 versus 55.5 +/- 26.6, and LU = 1267 +/- 982 versus 1017 +/- 691). Inter- and intra-assay variability of XTT-A were significantly lower than those for 51CrA. The total background values for XTT-A and 51CrA were similar in 4 h cytotoxicity assay and lower for XTT-A in assays with 24 h incubation. The sensitivity, in terms of discrimination between effector cells with different lytic capacity and targets with different susceptibility, was identical. The XTT-A was simpler, cheaper, and safer to perform than the 51CrA. Furthermore, the XTT-A was suitable for long-term assays and allowed experiments without requiring trypsinization of tumor cells grown in 96-well plates prior to testing.     

Evaluation of MTS, XTT, MTT and3HTdR incorporation for assessing hepatocyte density, viability and proliferation

The new 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric assays for assessing hepatocyte density, viability and proliferation were evaluated and compared with 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and tritiated thymidine (³HTdR) incorporation. OD values of MTS or XTT, which are metabolically reduced in viable cells to a watersoluble formazan product and can be read directly, had a good correlation coefficient with hepatocyte densities in a range of 2.5–40×10⁴ cells/ml (MTSr=0.952; XTTr=0.902) and with hepatocyte viability (MTSr=0.974; XTTr=0.975). At 0, 20, 40 and 60 hr cultures, the correlation coefficients with³HTdR for assessing hepatocyte viability and proliferation (MTSr=0.942–0.981; XTTr=0.953–0.992) were excellent. In contrast with MTS and XTT, MTT OD values had a poor correlation coefficient with hepatocyte densities (r=0.672), viability (r=0.622) and³HTdR incorporation (r=0.701–0.818 at 0, 20 and 40 hour cultures). This study shows that the MTS or XTT colorimetric assays for assessing hepatocyte density, viability and proliferation are more accurate, reliable and simpler than the widely used MTT assay. They avoid use of radioactive material as required for³HTdR incorporation. Of the two, the MTS colorimetric assay is more sensitive than the XTT.     

应用四唑鎓盐法快速检测卡介苗活菌含量

目的通过四唑鎓盐（XTT）方法快速检测卡介苗样品中的活菌含量,结果与传统培养法菌落形成单位计数结果进行相关性分析,探讨应用XTT方法快速检测卡介苗（BCG）制品中的活菌含量的可行性。 方法活菌含量是BCG疫苗重要的质控指标,它决定疫苗的效力。传统活菌含量的检测采用固体培养法计数菌落形成单位,但耗时长,重复性差。因此快速检测卡介苗活菌含量的方法急需建立和应用。四唑鎓盐（XTT）由于其水溶性的显色反应能够反映细胞的活力,同样也能应用于细菌的活力检测。其原理是细菌在代谢过程中,通过氧化一还原反应将XTT还原为可溶性的高色产物甲臜（formazan）,甲腊含量反应了细菌的活菌数量。将该方法应用于卡介苗活菌含量的快速检测中,通过已知活菌含量BCG参考品制备活菌含量和XTT有色产物吸光度值的参比曲线,根据未知样品的吸光度值,在此参比曲线上读出未知样品的活菌含量。同时将该快速检测方法得到的结果和传统培养法活菌含量结果进行相关性分析。 结果XTT法能反应BCG制品中活菌的含量差异,在一定的浓度范围内,活菌含量和有色产物吸光度值间有线性关系,相关系数R^2达到0．99以上,应用该方法对10批BCG样品活菌含量进行快速检测,结果和培养法菌落形成单位（CFU）结果的相关系数r=0．834,实验可在24h左右完成。 结论XTT法能够快速检测BCG制品中的活菌含量,结果与培养法菌落形成单位计数结果有较好的相关性,实验耗时由4周减少到24h,该法可以用于BCG活菌含量快速检测。     

Colorimetric assay fop cellular activity in microcapsules

Cellular activity in microcapsules was determined by a simple colorimetric assay, based on the cellular transformation of a tetrazolium salt, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, into an insoluble formazan which was quantified in a spectrophotometer. The results showed that when encapsulated Chinese hamster ovary fibroblasts were exposed to the tetrazolium salt containing tissue culture medium, the formazan crystals were formed inside the poly(hydroxyethyl methacrylate-methyl methacrylate) microcapsules; capsules containing no cells or dead cells formed no formazan. A detectable amount of formazan was readily obtained even from single capsules. Formazan production was dependent on the incubation time, but not on the amount of added reagent. Capsules from a high cell-density encapsulation (4 × 10⁶ cells/ml) formed more formazan than capsules from a low cell-density (4 × 10⁵ cells/ml) encapsulation, suggesting a positive correlation between the cell density and tetrazolium transformation in microcapsules. The tetrazolium assay indicated the maintenance of cellular activity but slow, if any, proliferation in microcapsules over a 2 wk testing period.     

Comparison between the standardized clinical and laboratory standards institute M38-A2 method and a 2,3-Bis(2-Methoxy-4-Nitro-5-[(Sulphenylamino)Carbonyl]-2H-tetrazolium hydroxide- based method for testing antifungal susceptibility of dermatophytes

In this study, we determined the utility of a 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based assay for determining antifungal susceptibilities of dermatophytes to terbinafine, ciclopirox, and voriconazole in comparison to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method. Forty-eight dermatophyte isolates, including Trichophyton rubrum (n = 15), Trichophyton mentagrophytes (n = 7), Trichophyton tonsurans (n = 11), and Epidermophyton floccosum (n = 13), and two quality control strains, were tested. In the XTT-based method, MICs were determined spectrophotometrically at 490 nm after addition of XTT and menadione. For the CLSI method, the MICs were determined visually. With T. rubrum, the XTT assay revealed MIC ranges of 0.004 to >64 μg/ml, 0.125 to 0.25 μg/ml, and 0.008 to 0.025 μg/ml for terbinafine, ciclopirox, and voriconazole, respectively. Similar MIC ranges were obtained against T. rubrum by using the CLSI method. Additionally, when tested with T. mentagrophytes, T. tonsurans, and E. floccosum isolates, the XTT and CLSI methods resulted in comparable MIC ranges. Both methods revealed similar lowest drug concentrations that inhibited 90% of the isolates for the majority of tested drug-dermatophyte combinations. The levels of agreement within 1 dilution between both methods were as follows: 100% with terbinafine, 97.8% with ciclopirox, and 89.1% with voriconazole. However, the agreement within 2 dilutions between these two methods was 100% for all tested drugs. Our results revealed that the XTT assay can be a useful tool for antifungal susceptibility testing of dermatophytes.     

Comparison of Two Rapid Colorimetric Methods for Determining Resistance of Mycobacterium tuberculosis to Rifampin, Isoniazid, and Streptomycin in Liquid Medium

 The usefulness of two colorimetric methods for the determination of the susceptibility or resistance of Mycobacterium tuberculosis to rifampin, streptomycin, and isoniazid in liquid medium based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was investigated. The agar proportion method was used as the reference method. Results obtained indicate that the sensitivity of the XTT reduction assay for the detection of rifampin resistance was comparable to that observed, and previously described, for the MTT assay. However, the reduction of XTT yields a water-soluble formazan that can be easily quantified without performing additional steps such as addition of lysing buffer and solubilization. Furthermore, the colorimetric assays, based on the reduction of XTT and MTT for the detection of isoniazid and streptomycin resistance in Mycobacterium tuberculosis, were standardized. The inhibition of MTT and XTT reduction after treatment with rifampin, streptomycin, or isoniazid was directly proportional to the reduction in the number of viable bacteria, and a strain of Mycobacterium tuberculosis could be reported as susceptible or resistant to rifampin, streptomycin, or isoniazid after 3, 6, or 8 days, respectively. The XTT and MTT reduction assays are rapid, reliable, and affordable and do not require the use of radioisotopes. Moreover, they can be performed with common laboratory equipment.     

Comparison of Spectrophotometric and Visual Readings of NCCLS Method and Evaluation of a Colorimetric Method Based on Reduction of a Soluble Tetrazolium Salt, 2,3-Bis {2-Methoxy-4-Nitro-5-[(Sulfenylamino) Carbonyl]-2H- Tetrazolium-Hydroxide}, for Antifungal Susceptibility Testing of Aspergillus Species

The susceptibilities of 25 clinical isolates of various Aspergillus species (Aspergillus fumigatus, A. flavus, A. terreus, A. ustus, and A. nidulans) to itraconazole (ITC) and amphotericin B (AMB) were determined using the standard proposed by NCCLS for antifungal susceptibility testing of[filamentous fungi, a modification of this method using spectrophotometric readings, and a colorimetric method using the tetrazolium salt 2,3-bis [2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide] (XTT). Five MIC end points for ITC (MIC-0, no visible growth or 93%. Between visual and spectrophotometric readings, high levels of agreement were found for AMB (approximately 97%) and MIC-1 (approximately 92%) and MIC-2 (approximately 88%) of ITC. Poor agreement was found for MIC-0 of ITC (51% after 24 h), since the spectrophotometric readings resulted in higher MIC-0 values than the visual readings. The agreement was increased to 98% by shifting the threshold level of MIC-0 from 5 to 10% relative optical density and by establishing an optical density of greater than 0.1 for the GC as the validation criterion. No statistically significant differences were found between the NCCLS method and the XTT method, with the levels of agreement exceeding 97% for MIC-0 of AMB and 83% for MIC-0, MIC-1, and MIC-2 of ITC. The XTT method and spectrophotometric readings can increase the sensitivity and the precision, respectively, of in vitro susceptibility testing of Aspergillus species.     

Microplate Alamar blue assay for susceptibility testing of Candida albicans biofilms.

Although biofilm-based fungal infections are an important cause of morbidity and mortality in patients, there is no standardized method for the in vitro evaluation of the drug susceptibility of biofilms. We investigated a high-throughput method for determining the susceptibility of Candida albicans biofilms that uses the oxidation reduction indicator Alamar blue (AB). Biofilms from the tested Candida albicans strains were markedly resistant to amphotericin B (AMB), nystatin (NYT), fluconazole (FLC) and 5-fluorouracil (5FC), but susceptible to Conflikt disinfectant. The latter was used in comparative studies of AB reduction with two other methods for assessing in vitro drug susceptibility i.e., 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and enumeration of viable colony counts (CFU/ml). AB results correlated well with XTT (r=0.88-0.93) and CFU/ml (r=0.93-0.99) for all four C. albicans test strains. This simple, reproducible method for determining in vitro drug susceptibility should facilitate discovery of antifungals active against Candida biofilms.     

Comparison of NCCLS and 3-(4,5-dimethyl-2-Thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) methods of in vitro susceptibility testing of filamentous fungi and development of a new simplified method

The susceptibility of 30 clinical isolates belonging to six different species of filamentous fungi (Aspergillus fumigatus, Aspergillus flavus, Scedosporium prolificans, Scedosporium apiospermum, Fusarium solani, and Fusarium oxysporum) was tested against six antifungal drugs (miconazole, voriconazole, itraconazole, UR9825, terbinafine, and amphotericin B) with the microdilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (M38-P). The MICs were compared with the MICs obtained by a colorimetric method measuring the reduction of the dye 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan by viable fungi. The levels of agreement between the two methods were 96 and 92% for MIC-0 (clear wells) and MIC-1 (75% growth reduction), respectively. The levels of agreement were always higher for Aspergillus spp. (97% +/- 2.5%), followed by Scedosporium spp. (87% +/- 10.3%) and Fusarium spp. (78% +/- 7.8%). The NCCLS method was more reproducible than the MTT method: 98 versus 95% for MIC-0 and 97 versus 90% for MIC-1. However, the percentage of hyphal growth as determined visually by the NCCLS method showed several discrepancies when they were compared with the percentages of MTT reduction. A new simplified assay that incorporates the dye MTT with the initial inoculum and in which the fungi are incubated with the dye for 48 h or more was developed, showing comparable levels of agreement and reproducibility with the other two methods. Furthermore, the new assay was easier to perform and more sensitive than the MTT method.     

Effects of the insecticide amitraz, an alpha2-adrenergic receptor agonist, on human luteinized granulosa cells

BACKGROUND: Amitraz, an insecticide used to prevent tick and mite infestation of cattle, crops and dogs, is an alpha2-adrenergic receptor agonist that inhibits GnRH release and the ovulatory LH surge in rats. Noradrenalin, the physiological ligand for adrenergic receptors, inhibits progesterone production by IVF-derived granulosa cells, but the effects of amitraz are unknown. METHODS: Luteinized granulosa cells obtained from women undergoing ovarian stimulation were exposed to amitraz (1, 10, 50, 100 microg/ml) for 2-72 h, and to amitraz (50 microg/ml) +/- hCG or the specific alpha2-adrenergic receptor antagonist yohimbine, for 6 h. Cell numbers were determined by 3-(4, 5-dimethylthiazol-(2)-yl)-2, 5-diphenyl tetrazolium bromide(MTT) assay and hormone production by radioimmunoassay. RESULTS: Amitraz 10 microg/ml did not affect cell numbers or estrogen production, but reduced progesterone production to 58 +/- 8% (p < 0.01, 24 h, n = 6) of control values. Amitraz (100 microg/ml) was cytotoxic and caused a corresponding reduction in hormone production. Amitraz 50 microg/ml did not affect cell numbers or estrogen production, but reduced progesterone per cell production to 82 +/- 6% of control values after 6 h. This was prevented by 0.2 mmol/l yohimbine. Exposure to amitraz 50 microg/ml for 6 h exposure abolished hCG-stimulated progesterone production but not estrogen production. CONCLUSIONS: Amitraz inhibited basal and hCG-stimulated progesterone but not estrogen production. The inhibitory action of amitraz and its antagonism by yohimbine suggest that alpha2-adrenergic receptors are expressed by luteinized human granulosa cells.     

Interleukin-12 Enhances Antifungal Activity of Human Mononuclear Phagocytes against Aspergillus fumigatus: Implications for a Gamma Interferon-Independent Pathway

The potential of recombinant human interleukin-12 (IL-12) to enhance the capacity of human monocytes (MNC) to elicit an oxidative burst and damage hyphae of Aspergillus fumigatus was investigated. Incubation of peripheral blood mononuclear cells (PBMC) from healthy adults with 10 to 100 ng of IL-12/ml at 37 degrees C for 2 to 3 days enhanced the production of superoxide anion (O2-) in response to phorbol myristate acetate (PMA) (P = 0.04) and unopsonized A. fumigatus hyphae (P = 0.03) and further enhanced hyphal damage (P = 0.009). Anti-gamma interferon (anti-IFN-gamma) blocked secretion of IFN-gamma by IL-12-treated PBMC but did not inhibit IL-12-induced O2- production by these cells in response to PMA. In addition, IL-12-treated elutriated MNC secreted no IFN-gamma or tumor necrosis factor alpha but exhibited enhanced O2- production compared to controls (P = 0.013). These findings demonstrate that IL-12 augments oxidative antifungal activities of MNC via an IFN-gamma-independent route, suggesting a novel pathway of IL-12 action in antifungal defense.     

Analysis of the influence of Tween concentration, inoculum size, assay medium, and reading time on susceptibility testing of Aspergillus spp

The influence of several test variables on susceptibility testing of Aspergillus spp. was assessed. A collection of 28 clinical isolates was tested against amphotericin B, itraconazole, voriconazole, and terbinafine. Inoculum size (10(4) CFU/ml versus 10(5) CFU/ml) and glucose supplementation (0.2% versus 2%) did not have significant effects on antifungal susceptibility testing results and higher inoculum size and glucose concentration did not falsely elevate MICs. In addition, antifungal susceptibility testing procedure with an inoculum size of 10(5) CFU/ml distinctly differentiated amphotericin B or itraconazole-resistant Aspergillus strains in vivo from the susceptible ones. Time of incubation significantly affected the final values of MICs, showing major increases (two to six twofold dilutions, P < 0.01 by analysis of variance) between MIC readings at 24 and 48 h, but no differences were observed between antifungal susceptibility testing results obtained at 48 h and at 72 h. Significantly higher MICs were uniformly associated with higher concentrations of Tween (P < 0.01), used as a dispersing agent in the preparation of inoculum suspensions. The geometric mean MICs showed increases of between 1.5- and 10-fold when the Tween concentration varied from 0.1% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.29, 0.69, 1.06, and 0.64 mug/ml, respectively) to 5% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.97, 5.79, 1.60, and 4.66 mug/ml, respectively). The inhibitory effect of Tween was clearly increased with inoculum sizes of 10(5) CFU/ml and was particularly dramatic for itraconazole, terbinafine, and Aspergillus terreus. The inoculum effect was not observed when the Tween concentration was below 0.5% (P > 0.01).     

Menadione biphasically controls JNK-linked cell death in leukemia Jurkat T cells

Signals for cell-death induction by menadione were studied in Jurkat T cells. Low concentrations of menadione (10-20 microM) and H(2)O(2) (10-50 microM) induced cell death accompanying low (menadione: <5%) or moderate (H(2)O(2): 10-15%) levels of DNA fragmentation in Jurkat cells. These concentrations of menadione (10 microM) and H(2)O(2) also caused membrane (necrotic) cell death at unproportionally high (80%) and proportional (10-30%) levels, respectively. Higher concentrations (100-5,000 microM) of H(2)O(2) exclusively induced membrane cell death. Unexpectedly, 30-300 microM menadione induced ever-decreasing levels of necrotic cell death in a concentration-dependent manner. An in vitro kinase assay showed that 20-50 microM, but not >100 microM, menadione induced activation of c-Jun NH(2)-terminal kinase (JNK), whereas a striking activation of JNK was induced by 500-5,000 microM H(2)O(2). Induction of cell death by a low concentration of menadione was partially inhibited in dominant negative JNK gene-transfected Jurkat/VPF cells. A high concentration (300 microM) of menadione was found to inhibit cell-death induction by high concentrations (200-5,000 microM) of H(2)O(2). The JNK inhibitory activity of menadione was also demonstrated in a cell-free system. However, menadione did not activate JNK in vitro. These results suggest that JNK is required for induction of not only apoptotic cell death, but also necrotic cell death in Jurkat T cells and that menadione biphasically controls this JNK-linked signal for inducing cell death.     

Utility of 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5-[(Phenyl-Amino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT) and Minimum Effective Concentration Assays in the Determination of Antifungal Susceptibility of Aspergillus fumigatus to the Lipopeptide Class of Compounds

The susceptibility of Aspergillus fumigatus to mulundocandin, an echinocandin-like compound, and other antifungal agents was assessed by the National Committee for Clinical Laboratory Standards (NCCLS) M38-P method, a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based colorimetric assay, and determination of morphologic alterations by microscopy. In contrast to the NCCLS M38-P method, which does not predict the activity in vivo, the XTT-based assay showed that A. fumigatus is susceptible to mulundocandin. Thus, the XTT-based assay might be useful for determination of the susceptibilities of molds to echinocandins. Further evaluation is warranted.     

Biosynthesis of tannase and gallic acid from tannin rich substrates by Rhizopus oryzae and Aspergillus foetidus

Modified solid-state fermentation (MSSF) of tannin-rich substrates for production of tannase and gallic acid was carried out using two fungal cultures, Rhizopus oryzae (RO IIT RB-13, NRRL 21498) and Aspergillus foetidus (GMRB013 MTCC 3557). The tannin rich substrates included powdered fruits of Terminalia chebula and Caesalpinia digyna pod cover powder. The different environmental parameters for the maximum production of tannase and gallic acid were optimized through media engineering. The highest yield of tannase and gallic acid was obtained after 60 h in case of Rhizopus oryzae and after 72 h by Aspergillus foetidus with 3 ml of induced inoculum. The optimum initial pH of the fermentation was found to be 4.5 in case of Rhizopus oryzae and 5.0 for Aspergillus foetidus. MSSF was carried out at the optimum conditions of 30 degrees C and 80% relative humidity. Collectively, the data reveal the potential of the modified solid-state fermentation process for the production of tannase and gallic acid from tannin-rich substrates with R. oryzae and A. foetidus.     

Inteferon gamma and granulocyte-macrophage colony-stimulating factor augment the antifungal activity of human polymorphonuclear leukocytes against Scedosporium spp.: comparison with Aspergillus spp.

While Aspergillus spp. have been the most frequent filamentous fungi causing infections in immunocompromised patients, Scedosporium spp. are emerging as life-threatening pathogens. We studied the effects of interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or combined on the antifungal activities of human polymorphonuclear leukocytes (PMN) against Scedosporium apiospermum and Scedosporium prolificans. We paralleled these activities to those against Aspergillus fumigatus and Aspergillus flavus. Incubation of PMN with IFN-gamma and GM-CSF for 22 h enhanced PMN-induced hyphal damage of both Aspergillus spp. and S. prolificans (p < 0.05) but not of S. apiospermum. However, hyphae of S. apiospermum were damaged significantly more after incubation with PMN that had been treated with IFN-gamma and GM-CSF for 2 h. In addition, incubation of PMN with GM-CSF for 2 h enhanced PMN oxidative burst measured as superoxide anion (O2-) production in response to nonopsonized hyphae of A. flavus and Scedosporium spp. (p < 0.05). In contrast, after 2 h, IFN-gamma and GM-CSF alone did not enhance PMN O2- in response to opsonized hyphae of A. flavus and Scedosporium spp.; however, the combination of IFN-gamma and GM-CSF showed significant enhancement against these species. Thus, IFN-gamma and GM-CSF, particularly in combination, demonstrate a species- and time-dependent augmentation of PMN responses to Scedosporium spp.     

Antifungal potential of disulfiram.

Disulfiram, an alcohol antagonistic drug has been on the market since 1949 with 80% bioavailability and an established safety profile. Recently it has been reported as a P-glycoprotein efflux pump modulator. Herein we report its antifungal potential. The MIC50 and MIC90 of disulfiram for yeast isolates is 4 and 8 microg/ml, respectively, and the MIC range is 1-16 micro g/ml for both fluconazole sensitive and resistant strains. Interestingly, disulfiram also showed fungicidal activity on Aspergillus spp. with MIC50 and MIC90 of 2 and 8 microg/ml, respectively.