Bipolar coagulation with small diameter forceps in animal models for in-utero cord obliteration.
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The objective of this study was to evaluate the feasibility, efficacy and safety of bipolar coagulation using endoscopic forceps of diameters of 2.7 mm and less in animal models mimicking in-utero conditions. Forceps of 2.2, 2.3, 2.5 and 2.7 mm were tested in rabbits (n = 36). Vessel pairs were dissected and coagulated in a fluid environment under endoscopic vision at 15 and 25 W. The main outcome measure was the perforation rate. In fetal lambs (n = 25), umbilical cords were coagulated under sono-endoscopic control with power settings from 10 to 35 W. Main outcome measures were the duration of coagulation, perforation rate, change in the temperature of the amniotic fluid and efficacy of vessel occlusion rate. At 20-25 W, all cords were coagulated successfully without any perforation using 2.3, 2.5 or 2.7 mm forceps. Coagulation with the 2. 2 mm forceps was associated with a high perforation rate, although the design rather than the diameter of the forceps may have influenced this outcome. Bipolar coagulation with forceps between 2. 3 and 2.7 mm and appropriate power settings achieves efficacious and safe coagulation in animal models for umbilical cord occlusion. (+info)
Prompt and durable engraftment in two older adult patients with high risk chronic myelogenous leukemia (CML) using ex vivo expanded and unmanipulated unrelated umbilical cord blood.
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Delayed engraftment, graft failure, and adverse transplant-related events have been observed in unrelated umbilical cord blood (UCB) recipients, particularly in those receiving a low leukocyte cell dose and in CML patients. We report the outcomes of two older adult patients with high risk CML who received a low leukocyte cell dose of unmanipulated UCB cells supplemented with ex vivo expanded (AastromReplicell System) UCB cells. Each engrafted promptly and neither patient experienced GVHD or life-threatening infection. Both remain engrafted with cells exclusively of donor origin and are in cytogenetic remission at 19 and 8 months follow-up. Ex vivo expanded UCB cells appear to facilitate hematopoietic recovery and therefore may increase the number of CML patients eligible for unrelated UCB transplant. (+info)
Apoptosis and regulation of Bax and Bcl-X proteins during human neonatal vascular remodeling.
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To verify that apoptosis is one of the possible mechanisms of neonatal vascular remodeling during the transition from fetal to neonatal circulation, we assayed for apoptosis and evaluated the expression of apoptosis-regulatory proteins in umbilical vessel versus ascending aorta, ductus arteriosus (DA) versus adjacent pulmonary artery and aorta, or aorta versus its branching arteries. Twenty-two umbilical cords (UCs), 6 DAs with adjacent aortas and pulmonary arteries, and 4 aortic arches with their branching great arteries were obtained from neonates. Smooth muscle cell (SMC) apoptosis in umbilical vessels was identified in all UCs. The expressions of Bax and Bcl-X were stronger in umbilical artery than in the neonatal aorta, but Bcl-2 was weak in both arteries in immunohistochemistry. In the immunoblot analysis of UCs, the expression of the proapoptotic short isoform of Bcl-X was stronger than in other tissue, and caspase-3 was selectively activated, whereas it was not in the other components of the cardiovascular system. In contrast, the expression patterns of the FasAg and Fas ligand were similar in umbilical artery and aorta. Regulation of Bcl-2 family proteins was also observed in other vascular sites at which SMCs undergo apoptosis on hemodynamic changes during birth, such as the DA and the branching points of the great arteries from the aortic arch. Apoptosis is involved in the regression of human umbilical vessels and the DA and in the remodeling of the branching great arteries during the neonatal period, when Bcl-2 family proteins are likely to play a key role. (+info)
Orosomucoid has a cAMP-dependent effect on human endothelial cells and inhibits the action of histamine.
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The plasma protein orosomucoid (alpha(1)-acid glycoprotein) has previously been shown to constitute a critical component of the capillary barrier. The protein has also been suggested to act as an anti-inflammatory mediator in a diversity of experimental situations. Recently we reported that orosomucoid is synthesized by the microvascular endothelial cells per se. In the present study, the effects of orosomucoid on primary cultures of human umbilical vein endothelial cells (HUVEC) were studied using the Cytosensor microphysiometer. We found that 1) orosomucoid (0.01 g/l) increased the metabolic activity of HUVEC as reflected by the increased acidification rate of +14 +/- 1%; 2) pretreatment with 0.5 mM 8-bromo-cAMP for 20 min markedly and reversibly inhibited the effect of orosomucoid, whereas 8-bromo-cGMP did not; 3) histamine elicited a dose-dependent response that was abolished by pretreatment with either cAMP or cGMP; and finally, 4) pretreatment of HUVEC for 6 min with orosomucoid (0.01 g/l) inhibited the action of histamine. In summary, this is the first report demonstrating that orosomucoid affects human endothelial cells and that it does so by using cAMP as a second messenger. This provides an explanation for previous findings of anti-inflammatory effects of the protein and shows that orosomucoid affects the endothelium during both normal and pathophysiological conditions. (+info)
The GPI-linked Ly-6 antigen E48 regulates expression levels of the FX enzyme and of E-selectin ligands on head and neck squamous carcinoma cells.
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By differential display we demonstrated that antibody-mediated ligation of the GPI-linked protein product of E48, a newly discovered human Ly-6 gene, up-regulates the expression of the FX enzyme in 3 lines of head and neck squamous carcinoma cells. FX is responsible for the last step in the synthesis of GDP-L-fucose. The up-regulation of FX was E48 ligand-specific. 22AWT head and neck squamous carcinoma cells expressing high levels of E48 expressed significantly higher levels of FX than the E48 antisense transfected 22AWT cells (8-3 cells). The former cells also expressed higher levels of two major fucosylated glycans (the selectin ligand, Sialyl Lewis a, and VIM-2) than the E48 antisense transfectants. Conversely, transfection of cells from the 14CWT line expressing very low levels of E48 with E48 cDNA caused an up-regulated expression of FX and of the two fucosylated glycans in the 14C-CMV16 transfectants. Moreover, the expression levels of Sialyl Lewis a was significantly up-regulated on HNSCC upon ligation of E48 by anti-E48 antibodies. The functional significance of the E48-mediated up-regulation of Sialyl Lewis a was demonstrated in rolling experiments on E-selectin bearing surfaces under physiological conditions of shear flow and on tumor necrosis factor alpha-activated human umbilical venous endothelial cells. Only high E48/FX/Sialyl Lewis a expressing 14C-CMV16 cells could roll on purified E-selectin or establish E-selectin dependent rolling on the activated human umbilical venous endothelial cells. Low E48/FX/Sialyl Lewis a expressing 14CWT cells did not roll. These results show that E48 controls the expression of the FX enzyme and of certain fucosylated E-selectin ligands by HNSCC. E48 may thus function as a key regulator of the adhesiveness of these tumor cells to inflamed vessel walls expressing E-selectin. (+info)
Pairing of oligosaccharides in the Fc region of immunoglobulin G.
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The Fc portion of immunoglobulin G (IgG) expresses paired oligosaccharides with microheterogeneities, which are associated with efficiencies of effector functions and with pathological states. A comparison of electrospray ionization mass spectrometry data obtained using a variety of Fc fragments derived from human and mouse IgG that do and do not retain the inter-chain disulfide bridge(s) revealed that (1) the Fc portion can be asymmetric as well as symmetric with respect to glycosylation and (2) the ratios of the individual glycoforms are different from what is expected from the random pairing. (+info)
Differential display identification of 40 genes with altered expression in activated human smooth muscle cells. Local expression in atherosclerotic lesions of smags, smooth muscle activation-specific genes.
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Detailed knowledge on the molecular and cellular mechanisms that control (re)-differentiation of vascular smooth muscle cells (SMCs) is critical to understanding the pathological processes underlying atherogenesis. We identified by differential display/reverse transcriptase-polymerase chain reaction 40 genes with altered expression in cultured SMCs upon stimulation with the conditioned medium of activated macrophages. This set of genes comprises 10 known genes and 30 novel genes, which we call "smags" (for smooth muscle activation-specific genes). To determine the in vivo significance of these (novel) genes in atherogenesis, we performed in situ hybridization experiments on vascular tissue. Specifically, FLICE (Fas-associated death domain-like interleukin-1beta-converting enzyme)-like inhibitory protein (FLIP) is expressed in neointimal SMCs as well as in lesion macrophages and endothelial cells, whereas the expression of the novel genes smag-63, smag-64, and smag-84 is restricted to neointimal SMCs. Characterization of full-length smag-64 cDNA revealed that it encodes a novel protein of 66 amino acids. smag-82 cDNA comprises the complete, unknown, 3'-untranslated region of fibroblast growth factor-5. Collectively, our results illustrate the complex changes of SMC gene expression that occur in response to stimulation with cytokines and growth factors secreted by activated macrophages. Moreover, we identified interesting candidate genes that may play a role in the differentiation of SMCs during atherogenesis. (+info)
Ceramide mimics tumour necrosis factor-alpha in the induction of cell cycle arrest in endothelial cells. Induction of the tumour suppressor p53 with decrease in retinoblastoma/protein levels.
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Tumour necrosis factor (TNF)-alpha induces a transient increase in N-octanoylsphingosine (C8-ceramide) which has been postulated as an intracellular mediator in TNF-alpha signalling. We tested the ability of C8-ceramide to reproduce the TNF-alpha-mediated interference with endothelial cell proliferation and DNA synthesis. TNF-alpha (10 ng.mL-1) and C8-ceramide (20 microM) inhibited the incorporation of [3H]thymidine into DNA and led to an accumulation of cells in the G1 phase of the cell cycle. When the responses of the tumour suppressors p53 and RB were analysed, it was found that TNF-alpha and C8-ceramide induced increased expression of p53. Treatment with TNF-alpha or C8-ceramide lead to a significant decrease in total retinoblastoma protein (RB) content that correlated with high levels of p53. These results suggest that p53 and RB may complement each other in their contribution to cell cycle arrest. TNF-alpha prevented RB phosphorylation whereas C8-ceramide did not interfere with this process, suggesting that it follows a ceramide-independent pathway. (+info)