Unusual grafts for living-donor liver transplantation.

PURPOSE: Unusual grafts, including extended left liver plus caudate lobe, right anterior section, and right posterior section grafts, are alternatives to left and right lobe grafts for living-donor liver transplantation. This study aimed to investigate unusual grafts from the perspectives of recipients and donors. METHODS: From 2016 to 2021, 497 patients received living-donor liver transplantation at Severance Hospital. Among them, 10 patients received unusual grafts. Three patients received extended left liver plus caudate lobe grafts, two patients received right anterior section grafts, and five patients received right posterior section grafts. Liver volumetrics and anatomy were analyzed for all recipients and donors. We collected data on laboratory examinations (alanine aminotransferase, total bilirubin, international normalized ratio), imaging studies, graft survival, and complications. A 1:2 ratio propensity-score matching method was used to reduce selection bias and balance variables between the unusual and conventional graft groups. RESULTS: The median of Model for End-stage Liver Disease score of unusual graft recipients was 13.5 (interquartile range 11.5-19.3) and that of graft-recipient weight ratio was 0.767 (0.7-0.9). ABO incompatibility was observed in four cases. The alanine aminotransferase level, total bilirubin level, and international normalized ratio decreased in both recipients and donors. Unusual and conventional grafts had similar survival rates (p = 0.492). The right and left subgroups did not differ from each counter-conventional subgroup (p = 0.339 and p = 0.695, respectively). The incidence of major complications was not significantly different between unusual and conventional graft recipients (p = 0.513). Wound seromas were reported by unusual graft donors; the complication ratio was similar to that in conventional graft donors (p = 0.169). CONCLUSION: Although unusual grafts require a complex indication, they may show feasible surgical outcomes for recipients with an acceptable donor complication.

Chitosan-TPP nanoparticle as a release system of antisense oligonucleotide in the oral environment.

Antisense oligonucleotide loaded chitosan nanoparticles were prepared and the release of oligonucleotide from chitosan-TPP/oligonucleotide nanoparticles was investigated. Morphological property, zeta potential and particle size of the prepared chitosan/oligonucleotide nanoparticles were investigated using Field Emission-Scanning Electron Microscope (FE-SEM) and particle size analyzer. The interaction between chitosan and oligonucleotide was confirmed by using capillary zone electrophoresis (CZE), and the released oligonucleotides were determined by spectrophotometric method. Oligonucleotides formed the complexes with chitosan with a unique morphological property. The release of oligonucleotides from nanoparticles was dependent on loading methods and pH conditions. Chitosan/oligomer-TPP nanoparticles, which was prepared by adding TPP after the formation of chitosan/oligonucleotide complex, showed the lowest release percent of oligonucleotides with 41.3% at pH 7.0 among the loading methods. The percent release of oligonucleotide from oligonucleotide loaded chitosan nanoparticle at pH 10 was higher than the one in acidic condition (pH 5.0). The released oligonucleotides from chitosan/oligonucleotide nanoparticles were stable enough for 12 h under the 20% saliva solution. Our results suggest that the sustained release of oligonucleotide from chitosan nanoparticles may be suitable for the local therapeutic application in periodontal diseases.

A facile synthesis and physical properties of nano-sized dendritic alpha,epsilon-poly(L-lysine)s for the delivery of nucleic acids.

A series of nano-sized dendritic alpha,epsilon-poly(L-lysine)s (DPL) were synthesized by the solid-phase peptide synthesis method, using a core epsilon-peptide structure consisting of eight lysine residues. Surface amines of dendritic alpha,epsilon-poly(L-lysine) were characterized by comparing the retention times of a reverse phase HPLC with the electrophoretic mobilities of capillary zone electrophoresis (CZE) and non-denatured polyacrylamide gel electrophoresis (PAGE). The elution times of alpha,epsilon-poly(Llysine) in HPLC were well correlated with the electrophoretic mobilities of CZE and PAGE. The separation was dependent on size, shape of molecule and the number of surface amine. The alpha,epsilon-poly(L-lysine) formed a complex with nucleic acids at various charge ratios and the degree of complex formation was size- and structure-dependent. Atomic force microscopy of the complex visualized the size and morphology of alpha,epsilon-poly(L-lysine)/DNA complex as a nano-sized spherical shape. The small size in complex formation provides a promise for in vivo therapeutic application of dendritic alpha,epsilon-poly(L-lysine)s or their derivatives in the delivery of gene or oligonucleotide.

Expression and functional characterization of the system L amino acid transporter in KB human oral epidermoid carcinoma cells.

We have examined the expression and function of system l amino acid transporter in KB human oral epidermoid carcinoma cells. The KB cells express l-type amino acid transporter 1 (LAT1) in plasma membrane, but not l-type amino acid transporter 2 (LAT2). The [14C]l-leucine uptake by KB cells is inhibited by system l selective inhibitor BCH. The majority of [14C]l-leucine uptake is, therefore, mediated by LAT1. These results suggest that the transport of neutral amino acids including several essential amino acids into the KB cells mediated by LAT1 and the specific inhibition of LAT1 in oral cancer cells will be a new rationale for anti-cancer therapy.

Expression of inducible nitric oxide synthase in the lower esophageal sphincter of the endotoxemic opossum.

BACKGROUND: Endotoxin modulates esophageal motor function by increasing nitric oxide (NO) production. The aims of this study were to examine inducible nitric oxide synthase (iNOS) induction in the lower esophageal sphincter (LES) of endotoxemic opossums and to investigate the effects of aminoguanidine (AG), a selective inhibitor of iNOS, on plasma nitrite/nitrate levels and on iNOS protein and mRNA expression after exposure to lipopolysaccharide (LPS). METHODS: Before and 12 h after the intravenous administration of LPS and/or AG, plasma nitrite/nitrate levels were determined. The iNOS protein and mRNA expression were investigated in the tissues taken from the LES by Western blot and reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: Plasma nitrite/nitrate levels were significantly increased by LPS. The increase in plasma nitrite/nitrate produced by LPS was significantly decreased by AG. Western blot and RT-PCR demonstrated that iNOS expression was markedly increased by LPS, and attenuated slightly by AG. CONCLUSIONS: These studies support the hypothesis that endotoxin increases NO production by the induction of iNOS protein and mRNA.

Dendritic alpha,epsilon-poly(L-lysine)s as delivery agents for antisense oligonucleotides.

PURPOSE: To evaluate the potential use of dendritic alpha,epsilon-poly(L-lysine)s (DPL) for the efficient cellular delivery of antisense oligonucleotides. METHODS: A series of dendritic alpha,epsilon-polylysines of various generations were prepared. Their physical properties and the ability to form complex with oligonucleotide were investigated by polyacrylamide gel electrophoresis, capillary zone electrophoresis (CZE), agarose gel electrophoresis, fluorescence titration and atomic force microscopy (AFM). The efficiency to deliver oligonucleotide to HeLa cells, stably transfected with plasmid pLuc/705, was evaluated by using antisense splicing correction assay and confocal microscopy. RESULTS: DPLs formed the complexes with antisense oligonucleotide with modest cytotoxicity. The charge ratio of oligonucleotide to DPL and the size (generation) of DPLs were all critical variables for the antisense effect. Compared to low generation DPLs, high generation DPLs were more effective in delivering oligonucleotide into cells. CONCLUSIONS: High generation DPL-oligonucleotide complexes were moderately effective for delivery antisense oligonucleotide. The complex formation provides a promise for in vivo therapeutic application of DPLs or their derivatives in the delivery of gene or oligonucleotide.

Roles of mitogen-activated protein kinase and phosphoinositide 3'-kinase in ErbB2/ErbB3 coreceptor-mediated heregulin signaling.

ErbB2/HER2 and ErbB3/HER3, two members of the ErbB/HER family, together constitute a heregulin coreceptor complex that elicits a potent mitogenic and transforming signal. Among known intracellular effectors of the ErbB2/ErbB3 heregulin coreceptor are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. Activation of the distinct MAPK and PI 3-kinase signaling pathways by the ErbB2/ErbB3 coreceptor in response to heregulin and their relative contributions to the mitogenic and transformation potentials of the activated coreceptor were investigated here. To this end, cDNAs encoding the wild-type ErbB3 protein (ErbB3-WT) and ErbB3 proteins with amino acid substitutions in either the Shc-binding site (ErbB3-Y1325F), the six putative PI 3-kinase-binding sites (ErbB3-6F), or both (ErbB3-7F) were generated and expressed in NIH-3T3 cells to form functional ErbB2/ErbB3 heregulin coreceptors. While the coreceptor incorporating ErbB3-WT activated both the MAPK and the PI 3-kinase signaling pathways, those incorporating ErbB3-Y1325F or ErbB3-6F activated either PI 3-kinase or MAPK, respectively. The ErbB2/ErbB3-7F coreceptor activated neither. Elimination of either signaling pathway lowered basal and eliminated heregulin-dependent expression of cyclin D1, which was in each case accompanied by an attenuated mitogenic response. Selective elimination of the PI 3-kinase pathway severely impaired the ability of heregulin to transform cells expressing the coreceptor, whereas attenuation of the MAPK pathway had a lesser effect. Thus, while both pathways contributed in a roughly additive manner to the mitogenic response elicited by the activated ErbB2/ErbB3 coreceptor, the PI 3-kinase pathway predominated in the induction of cellular transformation.

The effect of high local concentrations of antibiotics on demineralized bone induction in rats.

PURPOSE: This study evaluated the effect of high local concentrations of antibiotics on bone repair induced by demineralized bone in rats. MATERIALS AND METHODS: Seventy-two 3-week-old rats, weighing 200 to 300 g, were used for the experiment. Rats were divided into 4 groups: control group (group 1), saline-impregnation group (group 2), gentamicin-impregnation group (group 3), and tetracycline-impregnation group (group 4). The rats were killed at 3, 8, and 12 weeks after surgery. Samples were stained with hematoxylin-eosin and subjected to the histomorphometric analyses. RESULTS: There were significant differences in new bone formation among groups at all time periods. Comparing differences between groups at each time point, significantly more new bone formation was present in group 2 than in group 1 at 3 weeks, more in group 2 than in group 1 and more in group 2 than in group 4 at 8 weeks, and the most in group 2 among all groups at 12 weeks. In terms of the time period, significantly more new bone formation was observed at 8 weeks during the time period between 3 and 8 weeks, and at 12 weeks during the time period between 3 and 12 weeks. CONCLUSIONS: Our result suggests that the bone graft material is most effective when mixed with saline for the regeneration of osseous defects.

Stabilization of hypoxia-inducible factor-1alpha is involved in the hypoxic stimuli-induced expression of vascular endothelial growth factor in osteoblastic cells.

It has been suggested that blood vessel formation is an important event coupled to bone formation. The expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been shown to be greatly stimulated in osteoblasts by hypoxic stimuli such as deprivation of oxygen and treatment with cobalt. In other cell types, hypoxia-inducible factor-1 (HIF-1) that binds hypoxia-response element (HRE) has been shown to mediate gene expression induced by hypoxic stimuli. In this study, we investigated the effects of hypoxic stimuli on HIF-1, HRE, and VEGF in osteoblastic cell lines. Exposure of these cells to hypoxia or cobalt resulted in a great increase in the protein level of HIF-1alpha and the gene expression of VEGF. Transforming growth factor-beta1, prostaglandin E2, dexamethasone, and 1,25-dihydroxyvitamin D3 that have been shown to regulate VEGF gene expression in osteoblasts had no effect on HIF-1alpha induction. Blocking the enzymatic activity of phosphatidylinositol 3-kinase, p38, MEK-1 did not have any effect on the cobalt-stimulated increase of HIF-1alpha in these cells. In contrast, N-acetylcysteine (NAC), a scavenger of reactive oxygen species, abolished the cobalt induction of HIF-1alpha and that of the VEGF and a HRE-driven reporter genes. However, the hypoxia responses were not affected by NAC. These findings suggest that hypoxia and cobalt can induce VEGF gene expression in osteoblasts by increasing the level of HIF-1alpha protein through different mechanisms.