Allogeneic Mesenchymal Stromal Cells (MSCs) are of Comparable Efficacy to Syngeneic MSCs for Therapeutic Revascularization in C57BKSdb/db Mice Despite the Induction of Alloantibody.

Intramuscular administration of mesenchymal stromal cells (MSCs) represents a therapeutic option for diabetic critical limb ischemia. Autologous or allogeneic approaches may be used but disease-induced cell dysfunction may limit therapeutic efficacy in the former. Our aim was to compare the efficacy of allogeneic and autologous MSC transplantation in a model of hindlimb ischemia in diabetes mellitus and to determine whether allogeneic transplantation would result in the activation of an immune response. MSCs were isolated from C57BL/6 (B6) and diabetic obese C57BKSdb/db mice. Phosphate-buffered saline (control group), and MSCs (1 × 106) from B6 (allogeneic group) or C57BKSdb/db (syngeneic group) were administered intramuscularly into the ischemic thigh of C57BKSdb/db mice following the induction of hindlimb ischemia. MSCs derived from both mouse strains secrete several angiogenic factors, suggesting that the potential therapeutic effect is due to paracrine signaling. Administration of allogeneic MSCs significantly improved blood perfusion as compared with the control group on week 2 and 3, post-operatively. In comparison with the control group, syngeneic MSCs significantly improved blood perfusion at week 2 only. There was no statistical difference in blood perfusion between allogeneic and syngeneic MSC groups at any stages. There was no statistical difference in ambulatory and necrosis score among the three groups. Amputation of toes was only observed in the control group (one out of seven animals). Alloantibody was detected in three out of the eight mice that received allogeneic MSCs but was not observed in the other groups. In summary, we demonstrated comparable efficacy after transplantation of autologous and allogeneic MSCs in a diabetic animal model despite generation of an immune response.

Analysis of clomiphene isomers in human plasma and detection of metabolites using reversed-phase chromatography and fluorescence detection.

A method is described for the quantitative clinical analysis of plasma concentrations of the E and Z isomers of clomiphene, which is used in the induction of ovulation. The isomers of clomiphene, in addition to metabolites, are extracted from plasma with tert-butyl methyl ether (MTB). The MTB layer is dried, reconstituted and an aliquot subjected to chromatography. The drug and metabolites are separated by reversed-phase high-performance liquid chromatography. The eluent is fed into a knitted or braided, cylindrical reaction coil made of Teflon, into which is inserted a low-energy mercury lamp. This results in a photoinduced stilbene-to-phenanthrene oxidation yielding highly fluorescent analytes; this provides excellent sensitivity for the quantitation of the intact drug isomers and the detection of presently uncharacterized metabolites. Use of the reversed-phase chromatographic mode results in elution of the polar metabolites prior to the intact drug isomers. A combination of reversed-phase chromatography and an in-line post-column reaction coil results in a sensitive method that is more reliable and rapid than those previously reported and is applicable to the routine analysis of clinical samples. The method has been applied to individual isomers of clomiphene in plasma at concentrations of 0.06-600 ng/ml.

High-performance liquid chromatographic determination of pilocarpine in aqueous humor: derivatization by quaternization of methylimidazole tertiary amine group.

A derivatization procedure and a high-performance liquid chromatographic (HPLC) method of analysis for pilocarpine are described. The method is based on the quaternization of the 3'-tertiary amino group of the methylimidazole ring of pilocarpine with p-nitrobenzyl bromide. The HPLC system employs and RP-ODS column with a methanol-water mobile phase containing octanesulfonate as an ion-pairing agent. The sensitivity of the method permits the analysis of pilocarpine in biological fluids such as aqueous humor. The method is selective for pilocarpine in the presence of isopilocarpine. Its applicability to the analysis of aromatic heterocyclic and alkyl tertiary amines is demonstrated.