HPTLC method for direct determination of gemifloxacin mesylate in human plasma.

Novel, simple and sensitive high performance thin-layer chromatography (HPTLC) with fluorescence detection has been successfully developed and validated for determination of gemifloxacin mesylate (GFX) in plasma samples without prior pretreatment. Montelukast (MK) was used as internal standard. GFX and MK in plasma samples were separated using a mobile phase consisting of a mixture of ethyl acetate:methanol:25% ammonia, (8:4.5:3, v/v/v). The emission intensity was measured using optical filter K400 after excitation at 342 nm. The Rf values for GFX and MK were 0.45±0.03 and 0.79±0.02, respectively. Under the optimum conditions, a linear relationship with good correlation coefficient (r=0.9965, n=6) was obtained in concentration range of 3-180 ng/band. The LOD and LOQ of the proposed method were 0.45 and 1.5 ng/band, respectively. The accuracy of the method was proved as the recovery % of GFX from spiked human plasma was 94.21-101.85%. The efficiency of the proposed method was confirmed by in-vivo application on human plasma in real patient samples. Moreover, the stability of GFX in plasma was carefully tested at different conditions and compared to others in aqueous solution.

Human preformed IgG combining with membrane-bound porcine serotransferrin lyse porcine endothelial cells through antibody-dependent cellular cytotoxicity.

Preformed antibodies are involved in xenograft rejection. The purpose of this work was to characterize porcine xenoantigens recognized by human preformed IgG (hpIgG), and to investigate the role of hpIgG in xenogeneic rejection. IgG eluted from porcine livers perfused with human plasma, human sera and total human IgG were immunoblotted on porcine aortic endothelial cell extracts. The amino acid sequence of a 76-kDa antigen constantly revealed was 100% homologous with porcine serotransferrin (psTf). hpIgG from human sera, human IgG1 and IgG2 and F(ab')2gamma specifically bound to psTf. Neutralization by psTf abolished that binding. Although alpha1,3-linked galactose residues (Gal(alpha)1,3Gal) is the dominant epitope recognized by preformed antibodies in the swine-to-human combination, the analysis of carbohydrate composition of psTf showed that the molecule was devoid of Gal(alpha)1,3Gal moieties and that preformed anti-psTf IgG bound to epitopes localized on the peptide core of the molecule. Purified human anti-psTf IgG antibodies were able to bind to psTf linked to its receptor on porcine endothelial cells, and to kill those cells through antibody-dependent cellular cytotoxicity.

A newly established porcine aortic endothelial cell line: characterization and application to the study of human-to-swine graft rejection.

The establishment of cell lines allows reproductible in vitro studies that would be far more difficult to perform using primary cells that rapidly undergo phenotypical alterations in culture. The purpose of this work was to establish an endothelial cell line appropriate for in vitro study of endothelial cell activation during xenograft rejection. Porcine aortic endothelial cells were transfected with the early region of SV40 and selected on the basis of morphological, phenotypical, and functional features. By light and electron microscopy, the porcine aortic endothelial cell line (PAEC11) and primary cells were similar except that PAEC11 was slightly smaller. PAEC11 displayed endothelial cell characteristics since it endocytosed acetylated low density lipoproteins, produced von Willebrand factor, and expressed E-selectin. Human natural antibodies bound to the same xenoantigens on PAEC11 and primary cells. That binding was followed by human complement activation and cell lysis. In addition, PAEC11 was found appropriate for genetic engineering since it could be transfected with a plasmid encoding a foreign gene. Therefore, this cell line should be a useful model for in vitro study of endothelial cell function in general and human-to-swine xenograft rejection in particular.

Comparison between aortic and sinusoidal liver endothelial cells as targets of hyperacute xenogeneic rejection in the pig to human combination.

Endothelial cells of aortic origin are usually used in vitro as targets of hyperacute xenogeneic rejection, although endothelial cells from organs may have different properties. The sensitivities of aortic and liver endothelial cells to hyperacute xenogeneic rejection were compared in the pig to human combination. Sinusoidal liver endothelial cells were isolated and purified by collagenase perfusion of pig livers, sedimentation on a percoll gradient and selective adherence. Purity and viability of isolated liver endothelial cells after adherence were 85+/-6% and >95%, respectively. Endothelial cells from pig aortae (purity and viability >95%) were isolated by scraping. Immunoblotting analysis of xenoantigens on liver and aortic endothelial cell membranes preparations showed identical patterns. The strongest bands revealed by human IgM were located between 110 and 135 kD, while human IgG detected two major bands at 115 and 75kD. The membrane expression of xenoantigens recognized by human sera, analyzed by flow cytometry, was significantly lower on liver than on aortic endothelial cells (IgM: P=0.0006; IgG: P=0.0009). However, the complement-dependent cytotoxic activity of human sera was the same whether liver (54.5+/-1.4%) or aortic endothelial cells (50.0+/-4.2%) were used as targets. Taken together, those results allow the use of aortic instead of sinusoidal liver endothelial cells in the characterization of pig antigens recognized by human natural antibodies.