ABSTRACT
Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.
Subject(s)
Acanthamoeba Keratitis/diagnosis , Acanthamoeba/immunology , Antibodies, Protozoan/analysis , Carboxylesterase/immunology , Culture Media, Conditioned/metabolism , Epithelium, Corneal/cytology , Acanthamoeba/classification , Acanthamoeba/growth & development , Acanthamoeba/isolation & purification , Animals , Antibodies, Protozoan/blood , Antibody Specificity , Carboxylesterase/administration & dosage , Carboxylesterase/genetics , Cell Line , Cells, Cultured , Contact Lenses/parasitology , Early Diagnosis , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/parasitology , Epithelium, Corneal/metabolism , Epithelium, Corneal/parasitology , Humans , Immunization , Male , Mice , Protozoan Proteins/administration & dosage , Protozoan Proteins/genetics , Protozoan Proteins/immunologyABSTRACT
Prediction of intestinal availability (FaFg) of carboxylesterase (CES) substrates is of critical importance in designing oral prodrugs with optimal properties, projecting human pharmacokinetics and dose, and estimating drug-drug interaction potentials. A set of ester prodrugs were evaluated using in vitro permeability (parallel artificial membrane permeability assay and Madin-Darby canine kidney cell line-low efflux) and intestinal stability (intestine S9) assays, as well as in vivo portal vein-cannulated cynomolgus monkey. In vitro-in vivo extrapolation (IVIVE) of FaFg was developed with a number of modeling approaches, including a full physiologically based pharmacokinetic (PBPK) model as well as a simplified competitive-rate analytical solution. Both methods converged as in the PBPK simulations enterocyte blood flow behaved as a sink, a key assumption in the competitive-rate analysis. For this specific compound set, the straightforward analytical solution therefore can be used to generate in vivo predictions. Strong IVIVE of FaFg was observed for cynomolgus monkey with R2 of 0.71-0.93. The results suggested in vitro assays can be used to predict in vivo FaFg for CES substrates with high confidence.
Subject(s)
Carboxylesterase/administration & dosage , Carboxylesterase/blood , Intestinal Absorption/drug effects , Intestinal Absorption/physiology , Portal Vein/drug effects , Portal Vein/metabolism , Administration, Oral , Animals , Catheterization/methods , Dogs , Female , Macaca fascicularis , Madin Darby Canine Kidney Cells , Male , Substrate Specificity/physiologyABSTRACT
Human mesenchymal stem cells (hMSCs) have emerged as attractive cellular vehicles for gene therapy against brain malignancy because of their targeted tropism for cancer and the intrinsic attribute of autologous transplantation. We evaluated the characteristics and therapeutic potential of human adipose tissue-derived MSCs (hAT-MSCs) and prodrug gene therapy against diffuse pontine gliomas. The hAT-MSCs were isolated from human adipose tissue and characterised for morphology, surface markers and potential to differentiate into mesenchymal and neuronal lineages. We genetically modified hAT-MSCs to express rabbit carboxylesterase (rCE) enzyme, which can efficiently convert the prodrug CPT-11 (irinotecan-7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin), into the active drug SN-38 (7-ethyl-10-hydroxycamptothecin). The migratory capacity of hAT-MSCs expressing rCE (hAT-MSC.rCE), their ability to convert CPT-11 to SN-38 and cytotoxic effect on F98 cells were evaluated in vitro. The therapeutic potential of hAT-MSC.rCE was confirmed using a rat brainstem glioma model. The hAT-MSCs showed fibroblast-like morphology and expressed hMSC-specific markers including CD73, CD90 and CD105. The hAT-MSCs could differentiate into a mesenchymal lineage and transdifferentiate into a neuronal lineage under optimum culture conditions. The hAT-MSC.rCE converted CPT-11 to SN-38 and preserved the tumour tropism of hAT-MSCs. Brainstem glioma-bearing rats treated with hAT-MSC.rCE and CPT-11 survived 5d more than rats treated with CPT-11 only (p=0.0018). Our study demonstrates that hAT-MSCs can be easily prepared and genetically modified as cellular vehicles for prodrug gene therapy and that they have therapeutic potential against brainstem gliomas.
