Impact of Early Reoperation following Living-Donor Liver Transplantation on Graft Survival

Background

The reoperation rate remains high after liver transplantation and the impact of reoperation on graft and recipient outcome is unclear. The aim of our study is to evaluate the impact of early reoperation following living-donor liver transplantation (LDLT) on graft and recipient survival.

Methods

Recipients that underwent LDLT (n = 111) at the University of Tokyo Hospital between January 2007 and December 2012 were divided into two groups, a reoperation group (n = 27) and a non-reoperation group (n = 84), and case-control study was conducted.

Results

Early reoperation was performed in 27 recipients (24.3%). Mean time [standard deviation] from LDLT to reoperation was 10 [9.4] days. Female sex, Child-Pugh class C, Non-HCV etiology, fulminant hepatitis, and the amount of intraoperative fresh frozen plasma administered were identified as possibly predictive variables, among which females and the amount of FFP were identified as independent risk factors for early reoperation by multivariable analysis. The 3-, and 6- month graft survival rates were 88.9% (95%confidential intervals [CI], 70.7–96.4), and 85.2% (95%CI, 66.5–94.3), respectively, in the reoperation group (n = 27), and 95.2% (95%CI, 88.0–98.2), and 92.9% (95%CI, 85.0–96.8), respectively, in the non-reoperation group (n = 84) (the log-rank test, p = 0.31). The 12- and 36- month overall survival rates were 96.3% (95%CI, 77.9–99.5), and 88.3% (95%CI, 69.3–96.2), respectively, in the reoperation group, and 89.3% (95%CI, 80.7–94.3) and 88.0% (95%CI, 79.2–93.4), respectively, in the non-reoperation group (the log-rank test, p = 0.59).

Conclusions

Observed graft survival for the recipients who underwent reoperation was lower compared to those who did not undergo reoperation, though the result was not significantly different. Recipient overall survival with reoperation was comparable to that without reoperation. The present findings enhance the importance of vigilant surveillance for postoperative complication and surgical rescue at an early postoperative stage in the LDLT setting.     

The role of endogenous glucocorticoids in lymphocyte development in melanocortin receptor 2-deficient mice

Glucocorticoids are extensively used in anti-inflammatory therapy and are thought to contribute to the steady-state regulation of hematopoiesis and lymphopoiesis. We have previously established MC2R(-/-) mice, a model of familial glucocorticoid deficiency, that show several similarities to patients with this disease, including undetectable levels of corticosterone, despite high levels of ACTH and unresponsiveness to ACTH. In this study, we analyzed the possible roles of endogenous glucocorticoids in hematopoiesis and lymphopoiesis in MC2R(-/-) and CRH(-/-) mice as models of chronic adrenal insufficiency. Our analysis of total peripheral blood cell counts revealed that the number of lymphocytes was increased and the number of erythrocytes was slightly, but significantly, decreased in MC2R(-/-) mice. Numbers of immature double negative (CD4(-) CD8(-)) thymocytes, transitional type 1 B cells in the spleen, and pre-B cells in the bone marrow, were significantly increased in MC2R(-/-) mice, suggesting that endogenous glucocorticoids contribute to steady-state regulation of lymphopoiesis. Oral glucocorticoid supplementation reversed peripheral blood cell counts and reduced numbers of T and B cells in the thymus and the spleen. T cells in the thymus and B cells in the spleen were also increased in CRH(-/-) mice, another animal model of chronic adrenal insufficiency. MC2R(-/-) mice were sensitive to age-related thymic involution, but they were resistant to fasting-associated thymic involution. Our data support the idea that endogenous glucocorticoids contribute to stress-induced as well as steady-state regulation of hematopoiesis and lymphopoiesis.     

Sugar-inducible expression of the nucleolin-1 gene of Arabidopsis thaliana and its role in ribosome synthesis, growth and development

Animal and yeast nucleolin function as global regulators of ribosome synthesis, and their expression is tightly linked to cell proliferation. Although Arabidopsis contains two genes for nucleolin, AtNuc-L1 is the predominant if not only form of the protein found in most tissues, and GFP-AtNuc-L1 fusion proteins were targeted to the nucleolus. Expression of AtNuc-L1 was strongly induced by sucrose or glucose but not by non-metabolizable mannitol or 2-deoxyglucose. Sucrose also caused enhanced expression of genes for subunits of C/D and H/ACA small nucleolar ribonucleoproteins, as well as a large number of genes for ribosomal proteins (RPs), suggesting that carbohydrate availability regulates de novo ribosome synthesis. In sugar-starved cells, induction of AtNuc-L1 occurred with 10 mM glucose, which seemed to be a prerequisite for resumption of growth. Disruption of AtNuc-L1 caused an increased steady-state level of pre-rRNA relative to mature 25S rRNA, and resulted in various phenotypes that overlap those reported for several RP gene mutants, including a reduced growth rate, prolonged lifetime, bushy growth, pointed leaf, and defective vascular patterns and pod development. These results suggest that the rate of ribosome synthesis in the meristem has a strong impact not only on the growth but also the structure of plants. The AtNuc-L1 disruptant exhibited significantly reduced sugar-induced expression of RP genes, suggesting that AtNuc-L1 is involved in the sugar-inducible expression of RP genes.     

Population structure of the invasive forest pathogen Hymenoscyphus pseudoalbidus

Understanding the genetic diversity and structure of invasive pathogens in source and in introduced areas is crucial to the revelation of hidden biological features of an organism, to the reconstruction of the course of invasions and to the establishment of effective control measures. Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) is an invasive and highly destructive fungal pathogen found on common ash Fraxinus excelsior in Europe and is native to East Asia. To gain insights into its dispersal mechanisms and history of invasion, we used microsatellite markers and characterized the genetic structure and diversity of H. pseudoalbidus populations at three spatial levels: (i) between Europe and Japan, (ii) in Europe and (iii) at the epidemic's front in Switzerland. Phylogenetic and network analysis demonstrated that individuals from both regions are conspecific. However, populations from Japan harboured a higher genetic diversity and were genetically differentiated from European ones. No evident population structure was found among the 1208 European strains using Bayesian and multivariate clustering analysis. Only the distribution of genetic diversity in space, pairwise population differentiation (G_ST) and the spatial analysis of principal components revealed a faint geographical pattern around Europe. A significant allele deficiency in most European populations pointed to a recent genetic bottleneck, whereas no pattern of isolation by distance was found. Our data suggest that H. pseudoalbidus was introduced just once by at least two individuals. The potential source region of H. pseudoalbidus is vast, and further investigations are required for a more accurate localization of the source population.     

Fbxw7 acts as an E3 ubiquitin ligase that targets c-Myb for nemo-like kinase (NLK)-induced degradation

The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via a pathway involving TAK1 (transforming growth factor-beta-activated kinase 1), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK directly binds to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, and induces its ubiquitination and proteasome-dependent degradation. Here, we report that Fbxw7, the F-box protein of an SCF complex, targets c-Myb for degradation in a Wnt-1- and NLK-dependent manner. Fbxw7alpha directly binds to c-Myb via its C-terminal WD40 domain and induces the ubiquitination of c-Myb in the presence of NLK in vivo and in vitro. The c-Myb phosphorylation site mutant failed to interact with Fbxw7alpha, suggesting that the c-Myb/Fbxw7alpha interaction is enhanced by NLK phosphorylation of c-Myb. Treatment of M1 cells with Fbxw7 small interfering RNA (siRNA) rescued the Wnt-induced c-Myb degradation and also the Wnt-induced inhibition of cell proliferation. NLK bound to Cul1, a component of the SCF complex, while HIPK2 interacted with both Fbxw7alpha and Cul1, suggesting that both kinases enhance the c-Myb/SCF interaction. In contrast to c-Myb, the v-myb gene product (v-Myb) encoded by the avian myeloblastosis virus was resistant to NLK/Fbxw7alpha-induced degradation. Thus, Fbxw7 is an E3 ubiquitin ligase of c-Myb, and the increased c-Myb levels may contribute, at least partly, to transformation induced by mutation of Fbxw7.     

Dendritic Cells from Spleen, Mesenteric Lymph Node and Peyer's Patch Can Induce the Production of Both IL-4 and IFN-γ from Primary Cultures of Naive CD4+ T Cells in a Dose-Dependent Manner

Dendritic cells (DCs) as antigen presenting cells can stimulate naive CD4⁺ T cells and initiate the primary immune response which controls Th1/Th2 development. It has been suggested that DCs derived from different tissues have distinct properties. We investigated whether DCs from mesenteric lymph nodes (MLN), Peyer's patches (PP) and spleen (SPL) could induce different responses of naive CD4⁺ T cells to varying doses of antigen by using a co-culture system of DCs and T cells. DCs from each tissue induced IL-4 secretion from naive CD4⁺T cells in the presence of low dose antigenic peptide, and induced IFN-γ production at high doses of antigen. When purified CD11c⁺/B220^? DCs were used, MLN-derived DCs induced a higher amount of IFN-γ secretion from naive CD4⁺ T cells, compared with SPL-derived DCs. We could not detect large differences in the expressions of costimulatory molecules on the surface of these two populations of DCs. On the other hand, we found that large amounts of IL-12 were secreted from MLN DCs in an antigen dose-dependent fashion. In conclusion, DCs from SPL, MLN and PP can induce the production of both IL-4 and IFN-γ from naive CD4⁺ T cells, depending on antigen dose. MLN-derived CD11c⁺/B220^? DCs induce higher IFN-γ production from naive CD4⁺ T cells than SPL-derived DCs, through efficient IL-12 secretion.     

Selection and structural analysis of de novo proteins from an α3β3 genetic library

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.     

Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H-303]+) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of approximately 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress). These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.     

Augmentation of the generation of cell-mediated cytotoxicity in culture by mitomycin C

Summary The effects of mitomycin C (MMC) on the generation of cell-mediated cytotoxicity in primary stimulation culture of human peripheral blood mononuclear cells (PBM) with the B lymphoblastoid Raji cell line were assessed. The cell-mediated cytotoxicity induced in culture was significantly augmented when MMC was added to cultures on day –1 to day 3 for 24 h at concentrations of 2.5×10^–2 g/ml and 2.5×10^–3 g/ml. To identify the cell populations affected by MMC, PBM were separated by adherence to plastic after treatment with MMC for 24 h (day –1). The two populations were recombined with untreated separated cells and stimulated with antigen. The ability to develop an augmented cell-mediated cytotoxicity was associated with the adherent cell fraction of MMC-treated PBM. Therefore, the ability of MMC-treated adherent cells to produce interleukin 1 (IL 1) was examined. Significantly higher levels of IL 1 were produced by treated cells as compared to untreated adherent cells. The results appear to indicate that the selective effects of MMC on the adherent cell fraction, especially the modification of IL 1 production, may be involved in the mechanisms of MMC-induced augmented cell-mediated cytotoxicity.     

Comparison of Japanese isolates of Coxiella burnetii by PCR-RFLP and sequence analysis

The genetic variation of Japanese isolates of Coxiella burnetii, the agent of Q fever, was found for the first time. Forty-nine out of 72 isolates had the chronic pattern of the isocitrate hydrogenase gene. Sequence analysis revealed that the isolates have a specific nucleotide sequence. The putative amino acid sequence was the same as that of chronic reference strains. These results suggest the variation of C. burnetii isolates in Japan.