GC-MS characterisation and biological activity of essential oils from different vegetative organs of Plectranthus barbatus and Plectranthus caninus cultivated in north Italy.

Essential oils (EOs) from the roots, stems and leaves of Plectranthus barbatus (A) and Plectranthus caninus (B), cultivated in north Italy, were obtained by steam distillation and chemically characterised by gas chromatography-mass spectrometry. The highest yields were obtained from roots (268.15 and 673.60 mg/kg from A and B), followed by leaves (64.34 and 26.65 mg/kg) and stems (19.76 and 18.63 mg/kg). A total of 128 structures were identified in A and 121 in B. Fe(++) chelating and antiradical activities (DPPH and ABTS) were evaluated: root and stem EOs showed the strongest activities, while EOs from leaves did not show relevant activities. All EOs were tested for their in vitro antimicrobial activity, showing optimal growth-inhibition in antibiogram (∅>35 mm) and MIC tests (32-64 µg/mL) against Candida albicans, while EOs from leaves of both species showed a good activity (25 < ∅ < 34 mm, MIC 64-128 µg/mL) against Escherichia coli.

Xenogeneic serum promotes leukocyte-endothelium interaction under flow through two temporally distinct pathways: role of complement and nuclear factor-kappaB.

Endothelial cell activation and mononuclear cell infiltration are consistent features of discordant xenograft rejection. This study evaluated whether xenogeneic serum--as a source of xenoreactive natural antibodies and complement--induced endothelial activation with consequent leukocyte adhesion and transmigration under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 30 min, 1 h 30 min, or 5 h with 10% human serum or 10% porcine serum and then perfused with human leukocytes in a parallel plate flow chamber under flow (1.5 dynes/cm2). Adherent and transmigrated cells were counted by digital image analysis. Results showed that human serum significantly (P < 0.01) increased over time the number of adherent leukocytes compared with porcine serum. Stimulation of PAEC with human serum also promoted a progressive increase in leukocyte transmigration that reached statistical significance (P < 0.01) at 1 h 30 min and at 5 h compared with porcine serum. Studying the role of complement in leukocyte-endothelium interaction in xenogeneic conditions, a marked complement C3 deposition on PAEC exposed to human serum was shown by immunofluorescence, whereas cells incubated with porcine serum were negative. Next, it was documented that human serum decomplemented by heating and C3-deficient human serum failed to promote both leukocyte adhesion and transmigration, results that were comparable to porcine serum. To elucidate the intracellular mediators involved in endothelial cell activation by xenogeneic serum, this study focused on transcriptional factor nuclear factor-kappaB (NF-kappaB), a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. Positive nuclear staining of NF-kappaB (p65 subunit) found by confocal fluorescence microscopy of PAEC exposed to human serum was taken to reflect NF-kappaB activation. NF-kappaB was instead strictly localized in the cell cytoplasm in PAEC incubated with the homologous serum. Heat-inactivated human serum failed to activate NF-kappaB. Electrophoretic mobility shift assay of nuclear extracts from PAEC exposed to human serum revealed an intense NF-kappaB activation that was inhibited by the NF-kappaB inhibitor pyrrolidinedithiocarbamate. The NF-kappaB inhibitors pyrrolidinedithiocarbamate and tosyl-phe-chloromethylketone did not affect the number of adherent and transmigrated leukocytes in PAEC exposed to human serum for 30 min and 1 h 30 min. Both inhibitors instead significantly reduced leukocyte adhesion and transmigration induced by human serum at 5 h. Confocal fluorescence microscopy studies showed that human serum induced an increase in the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. Functional blocking of these adhesive molecules with the corresponding antibodies significantly inhibited xenogeneic serum-induced leukocyte adhesion. These data suggest that leukocyte adhesion and transmigration are directly dependent on complement deposited on PAEC in the early phase of cell activation (30 min and 1 h 30 min) induced by xenogeneic serum, whereas leukocyte adhesive events observed after 5 h of incubation of endothelial cells with xenogeneic serum are possibly regulated by transcription of NF-kappaB-dependent genes. The finding that xenogeneic serum promotes leukocyte-endothelial interaction depending on NF-kappaB activation might be relevant for designing future therapeutic strategies intended to prolong xenograft survival.

Xenogeneic human serum promotes leukocyte adhesion to porcine endothelium under flow conditions, possibly through the activation of the transcription factor NF-kappa B.

Endothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P < 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-kappa B activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-kappa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidinedithiocarbamate (PDTC, 25 microM) and tosyl-phechloromethylketone (TPCK, 25 microM) significantly (P < 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival.

Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion.

We addressed the role of hyperglycemia in leukocyte-endothelium interaction under flow conditions by exposing human umbilical vein endothelial cells for 24 h to normal (5 mM), high concentration of glucose (30 mM), advanced glycosylation end product-albumin (100 microg/ml), or hyperglycemic (174-316 mg/dl) sera from patients with diabetes and abnormal hemoglobin A1c (8.1+/-1.4%). At the end of incubation endothelial cells were perfused with total leukocyte suspension in a parallel plate flow chamber under laminar flow (1.5 dyn/cm2). Rolling and adherent cells were evaluated by digital image processing. Results showed that 30 mM glucose significantly (P < 0. 01) increased the number of adherent leukocytes to endothelial cells in respect to control (5 mM glucose; 151+/-19 versus 33+/-8 cells/mm2). A similar response was induced by endothelial stimulation with IL-1beta, here used as positive control (195+/-20 cells/mm2). The number of rolling cells on endothelial surface was not affected by high glucose level. Stable adhesion of leukocytes to glucose-treated as well as to IL-1beta-stimulated endothelial cells was preceded by short interaction of leukocytes with the endothelial surface. The distance travelled by leukocytes before arrest on 30 mM glucose, or on IL-1beta-treated endothelial cells, was significantly (P < 0.01) higher than that observed for leukocytes adhering on control endothelium (30 mM glucose: 76.7+/-3.5; IL1beta: 69.7+/-4 versus 5 mM glucose: 21.5+/-5 microm). Functional blocking of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 on endothelial cells with the corresponding mouse mAb significantly inhibited glucose-induced increase in leukocyte adhesion (67+/-16, 83+/-12, 62+/-8 versus 144+/-21 cells/ mm2). Confocal fluorescence microscopy studies showed that 30 mM glucose induced an increase in endothelial surface expression of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Electrophoretic mobility shift assay of nuclear extracts of human umbilical vein endothelial cells (HUVEC) exposed for 1 h to 30 mM glucose revealed an intense NF-kB activation. Treatment of HUVEC exposed to high glucose with the NF-kB inhibitors pyrrolidinedithiocarbamate (100 microM) and tosyl-phe-chloromethylketone (25 microM) significantly reduced (P < 0.05) leukocyte adhesion in respect to HUVEC treated with glucose alone. A significant (P < 0.01) inhibitory effect on glucose-induced leukocyte adhesion was observed after blocking protein kinase C activity with staurosporine (5 nM). When HUVEC were treated with specific antisense oligodesoxynucleotides against PKCalpha and PKCepsilon isoforms before the addition of 30 mM glucose, a significant (P < 0.05) reduction in the adhesion was also seen. Advanced glycosylation end product-albumin significantly increased the number of adhering leukocytes in respect to native albumin used as control (110+/-16 versus 66+/-7, P < 0.01). Sera from diabetic patients significantly (P < 0.01) enhanced leukocyte adhesion as compared with controls, despite normal levels of IL-1beta and TNFalpha in these sera. These data indicate that high glucose concentration and hyperglycemia promote leukocyte adhesion to the endothelium through upregulation of cell surface expression of adhesive proteins, possibly depending on NF-kB activation.