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Posaconazole prophylaxis has proven highly effective in preventing invasive fungal infections, despite relatively low serum concentrations. However, high tissue levels of this agent have been reported in treated patients. We therefore hypothesized that the intracellular levels of antifungal agents are an important factor in determining the success of fungal prophylaxis. To examine the effect of host cell-associated antifungals on the growth of medically important molds, we exposed cells to antifungal agents and removed the extracellular drug prior to infection. Epithelial cells loaded with posaconazole and its parent molecule itraconazole, but not other antifungals, were able to inhibit fungal growth for at least 48 h and were protected from damage caused by infection. Cell-associated posaconazole levels were 40-to 50-fold higher than extracellular levels, and the drug was predominantly detected in cellular membranes. Fungistatic levels of posaconazole persisted within epithelial cells for up to 48 h. Therefore, the concentration of posaconazole in mammalian host cell membranes mediates its efficacy in prophylactic regimens and likely explains the observed discrepancy between serum antifungal levels and efficacy.

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Microencapsulation of islets of Langerhans has been proposed as a means of preventing their immune destruction following transplantation. Microcapsules of diameters <350 microm made with an electrostatic pulse system present many advantages relative to standard microcapsules (700-1500 microm), including smaller total implant volume, better insulin kinetics, better cell oxygenation, and accessibility to new implantation sites. To evaluate their biocompatibility, 200, 1000, 1120, 1340, or 3000 of these smaller microcapsules (<350 microm) or 20 standard microcapsules (1247+/-120 microm) were implanted into rat epididymal fat pads, retrieved after 2 weeks, and evaluated histologically. The average pericapsular reaction increased with the number of small microcapsules implanted (p<0.05; 3000 vs. 200, 3000 vs. 1000, and 1000 vs. 200 microcapsules). At equal volume and alginate content, standard microcapsules caused a more intense fibrosis reaction than smaller microcapsules (p<0.05). In addition, 20 standard microcapsules elicited a stronger pericapsular reaction than 200 and 1000 smaller microcapsules (p<0.05) although the latter represented a 3.4-fold larger total implant surface exposed. We conclude that microcapsules of diameters <350 microm made with an electrostatic pulse system are more biocompatible than standard microcapsules.

IGF-II has been reported to decrease neonatal islet cell apoptosis and in vitro adult islet cell necrosis and apoptosis, but the usefulness of IGF-II in a transplantation setting is unknown. We evaluated the effect of in vitro IGF-II incubations on microencapsulated rat islet survival both in vitro and in minimal mass transplantations into diabetic mice. After 6 d in culture, fresh examinations, histology, fluorescence microscopy, sodium 3'-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)-benzene sulfonic acid hydrate assay, and apoptosis studies all indicated that IGF-II significantly improves islet cell viability in a dose-dependent fashion. IGF-II 100 ng/ml and 500 ng/ml induced a 51% and 83% increase of viable islets (P = 0.052, P < 0.01). A 20%, 29%, and 33% reduction of the apoptotic index was observed with 50, 100, and 500 ng/ml incubations respectively (P < 0.05; P < 0.005; P < 0.001). Ten weeks after transplantation of 150 encapsulated rat islet equivalents incubated with IGF-II 500 ng/ml, 80% of diabetic mice were normoglycemic. Without IGF-II preincubation, only 8% of the recipients remained normoglycemic with the transplantation of 150 islets and 42% with 300 islets (P < 0.05). In conclusion, IGF-II promotes islet cell survival, and allows successful transplantation using a smaller number of islets.

A competitive binding assay based on localized surface plasmon resonance (LSPR) of folic acid-functionalized gold nanoparticles (FA-AuNPs) and human dihydrofolate reductase enzyme (hDHFR) was developed to detect nanomolar to micromolar concentrations of the widely applied anti-cancer drug, methotrexate (MTX). By the nature of the competitive assay for MTX, the LSPR shift from specific binding between FA-AuNPs and the free enzyme was inversely proportional to the concentration of MTX. In addition, the dynamic range for MTX was tuned from 10(-11) to 10(-6) M by varying the concentration of hDHFR from 1 to 100 nM. Inter-day reproducibility and recovery of MTX spiked in phosphate buffer saline (PBS) were excellent. Potential interferents such as FA, trimethoprim (TMP) and 4-amino-4-deoxy-N-methylpteroic acid (DAMPA) did not occur in the concentration range of interest for MTX. Clinical samples of human serum from patients undergoing MTX chemotherapy were analyzed following a simple solid-phase extraction step to isolate MTX from the serum matrix, with a limit of detection of 155 nM. Validation of the LSPR method was carried out in comparison to Fluorescence Polarization Immunoassay (FPIA), a commonly used method in clinical settings, and LC-MS/MS, a reference technique. The results of the LSPR competitive assay compared well to FPIA and LC-MS/MS, with a slope of 2.4 and 1.1, respectively, for the correlation plots. The method established herein is intended for therapeutic drug monitoring (TDM) of MTX levels in patients undergoing chemotherapy to ensure safety and efficacy of the treatment.