In vitro susceptibility of filamentous fungi from mycotic keratitis to azole drugs.

OBJECTIVE: The in vitro antifungal activities of azole drugs viz., itraconazole, voriconazole, ketoconazole, econazole and clotrimazole were investigated in order to evaluate their efficacy against filamentous fungi isolated from mycotic keratitis. METHODS: The specimen collection was carried out from fungal keratitis patients attending Aravind eye hospital and Post-graduate institute of ophthalmology, Coimbatore, India and was subsequently processed for the isolation of fungi. The dilutions of antifungal drugs were prepared in RPMI 1640 medium. Minimum inhibitory concentrations (MICs) were determined and MIC50 and MIC90 were calculated for each drug tested. RESULTS: A total of 60 fungal isolates were identified as Fusarium spp. (n=30), non-sporulating moulds (n=9), Aspergillus flavus (n=6), Bipolaris spp. (n=6), Exserohilum spp. (n=4), Curvularia spp. (n=3), Alternaria spp. (n=1) and Exophiala spp. (n=1). The MICs of ketoconazole, clotrimazole, voriconazole, econazole and itraconazole for all the fungal isolates ranged between 16 µg/mL and 0.03 µg/mL, 4 µg/mL and 0.015 µg/mL, 8 µg/mL and 0.015 µg/mL, 8 µg/mL and 0.015 µg/mL and 32 µg/mL and 0.06 µg/mL respectively. From the MIC50 and MIC90 values, it could be deciphered that in the present study, clotrimazole was more active against the test isolates at lower concentrations (0.12-5 µg/mL) when compared to other drugs tested. CONCLUSION: The results suggest that amongst the tested azole drugs, clotrimazole followed by voriconazole and econazole had lower MICs against moulds isolated from mycotic keratitis.

In vitro effects of fetal rat cerebrospinal fluid on viability and neuronal differentiation of PC12 cells.

BACKGROUND: Fetal cerebrospinal fluid (CSF) contains many neurotrophic and growth factors and has been shown to be capable of supporting viability, proliferation and differentiation of primary cortical progenitor cells. Rat pheochromocytoma PC12 cells have been widely used as an in vitro model of neuronal differentiation since they differentiate into sympathetic neuron-like cells in response to growth factors. This study aimed to establish whether PC12 cells were responsive to fetal CSF and therefore whether they might be used to investigate CSF physiology in a stable cell line lacking the time-specific response patterns of primary cells previously described. METHODS: In vitro assays of viability, proliferation and differentiation were carried out after incubation of PC12 cells in media with and without addition of fetal rat CSF. An MTT tetrazolium assay was used to assess cell viability and/or cell proliferation. Expression of neural differentiation markers (MAP-2 and ß-III tubulin) was determined by immunocytochemistry. Formation and growth of neurites was measured by image analysis. RESULTS: PC12 cells differentiate into neuronal cell types when exposed to bFGF. Viability and cell proliferation of PC12 cells cultured in CSF-supplemented medium from E18 rat fetuses were significantly elevated relative to the control group. Neuronal-like outgrowths from cells appeared following the application of bFGF or CSF from E17 and E19 fetuses but not E18 or E20 CSF. Beta-III tubulin was expressed in PC12 cells cultured in any media except that supplemented with E18 CSF. MAP-2 expression was found in control cultures and in those with E17 and E19 CSF. MAP2 was located in neurites except in E17 CSF when the whole cell was positive. CONCLUSIONS: Fetal rat CSF supports viability and stimulates proliferation and neurogenic differentiation of PC12 cells in an age-dependent way, suggesting that CSF composition changes with age. This feature may be important in vivo for the promotion of normal brain development. There were significant differences in the effects on PC12 cells compared to primary cortical cells. This suggests there is an interaction in vivo between developmental stage of cells and the composition of CSF. The data presented here support an important, perhaps driving role for CSF composition, specifically neurotrophic factors, in neuronal survival, proliferation and differentiation. The effects of CSF on PC12 cells can thus be used to further investigate the role of CSF in driving development without the confounding issues of using primary cells.

The effects of blood glucose testing versus urine sugar testing on the metabolic control of insulin-dependent diabetic children.

Glycosylated hemoglobin A1c was measured in 43 insulin-dependent diabetic children before and after training in blood glucose testing. These measurements were compared with those obtained from a group of matched controls who tested urine sugars for the same period. Mean HbA1c measurements for the blood glucose testing group were significantly improved at 6 mo after training in blood glucose testing, and were significantly lower than those measured in the urine sugar testing group. However, HbA1c levels in the blood glucose testing group were well above levels for nondiabetic persons, and were generally higher than those reported for adult insulin-dependent diabetic persons following training in blood glucose testing. Explanations for this difference were considered. It was noted that 81% of the trained children accepted and continued to use blood glucose testing for at least 9 mo after training.