Regulation of heme oxygenase 1 expression by miR-27b with stem cell therapy for liver regeneration in rats.

Adipose-derived mesenchymal stem cells (ASCs) have been considered to be attractive and readily available adult mesenchymal stem cells (MSCs) and are becoming increasingly popular for use in regenerating cell therapy. However, recent evidence attributed a fibrotic potential to MSCs which differentiated into myofibroblasts with highly increased α-smooth muscle actin (α-SMA) expression while transplanted into an injured/regenerating liver in mice. In this study, we studied the role of miR-27b in ASCs and their regenerative potential after partial liver resection in rats. ASCs transfected with control siRNA or miR-27b were intravenously injected into autologous rats undergoing 70% partial hepatectomy (PH). Our data showed that the regenerative capacities of ASCs with overexpressed miR-27b were significantly higher compared with control ASCs. However, the enhanced regeneration, hepatic differentiation, and suppressed liver inflammation, as well as fibrotic activity, were significantly reverted by ZnPP coadministration (heme oxygenase-1 [HO-1] inhibitor) indicating an important role of HO-1 in the regenerating and cytoprotective activities of miR-27b-transfected ASCs. We demonstrated that administration of autologous ASCs overexpressed with miR-27b enhances rapid and early liver regeneration and, importantly, preserves function after PH. The ASCs with miR-27b overexpression might offer a viable therapeutic option to facilitate rapid recovery after liver resection.

Elevation of C-reactive protein level and its correlation with psychiatric comorbidities in recipients after liver transplantation.

Orthotopic liver transplantation (OLT) is the gold standard procedure for treating end-stage liver disease resulting from a variety of causes. Psychiatric comorbidities are common problems among patients who have undergone OLT; however, the association between psychiatric comorbidity and biological factors in OLT has not been investigated. In our previous study, we found a positive correlation between serum C-reactive protein (CRP) level after OLT and the prevalence of psychiatric comorbidities in recipients. In this retrospective cohort analysis, we enrolled 279 recipients who underwent OLT during a 3-year period (from 2008 to 2011) at a single center. Our retrospective study showed that the recipients with a higher serum CRP level at 1 month after OLT had higher psychiatric comorbidities compared with the recipients with a normal serum CRP level. Additionally, an in vitro study demonstrated that the down-regulation of brain-derived neurotrophic factor (BDNF) gene expression by CRP treatment in retinoic acid (RA) differentiated SH-SY5Y neuroblastoma cells. These clinical and experimental results indicated that the overexpression of CRP may induce psychiatric comorbidities through suppressed expression of the BDNF, suggesting that OLT recipients who developed psychiatric comorbidities might be accompanied by an immunological or acute-phase protein response. In conclusion, an activation of systemic inflammatory processes may be one of the predictable signatures for psychiatric comorbidities in OLT recipients.

Significance of portosystemic shunt on graft survival in liver transplantation: a rat model.

AIMS: Distal splenorenal shunt effectively controls bleeding from esophageal and gastric varices but has a different effect on liver transplantation. This study sought to develop an animal model in rats to mimic the recipient with a portosystemic shunt and to investigate its hemodynamic consequences on liver transplantation. METHODS: We prepared 5 groups of allogeneic or syngeneic rat liver transplantation models with versus without portosystemic shunt, to investigate its effects on graft survival and portal flow. To explore the effects of excessive portal flow on graft survival in small-for-size liver transplantation, we transplanted partial liver grafts into syngeneic recipients. RESULTS: In allogeneic combinations, graft survival among the shunt group was shortened compared with their control counterparts. The graft survival of the large shunt group was significantly lower than that of a small shunt or without shunt group in a syngeneic liver transplantation model. Portal blood pressure of the large shunt group was significantly lower than that of the small shunt group. In contrast, excessive portal flow resulted in dysfunction of liver graft in small-for-size liver transplantation. CONCLUSIONS: These results suggested that reduction in portal flow by portosystemic shunt lead to an acceleration of acute rejection and subsequent liver graft dysfunction, but it may be applicable to regulate the excessive portal flow in small-for-size transplantations. This study showed a valuable model mimicking the recipient with a portosystemic shunt.

High-mobility group box 1 protein activates hepatic stellate cells in vitro.

OBJECTIVES: Our previous study noticed remarkably elevated titers of anti-high-mobility group box 1 (HMGB1) antibodies in sera during the tolerance induction phase of a rat tolerogenic orthotopic liver transplantation (OLT) as well as in sera of clinically drug-free patients. We hypothesized that the release of nonhistone nuclear protein HMGB1 during rejection may play a pathogenic role in deteriorating post-OLT graft functions, such as inducing liver fibrosis. This study sought to investigate whether HMGB1 can directly activate hepatic stellate cells (HSCs) and drive them toward fibrogenesis. METHODS: The cultured HSCs were treated with recombinant HMGB1. RT-PCR and Western blotting analysis were used to measure alpha-smooth muscle actin (alpha-SMA) expression. Conditioned media were collected for gelatin zymography to monitor the activities of collagen-degrading matrix metalloproteinases (MMPs). RESULTS: HMGB1 at concentrations > 1 ng/mL significantly stimulated HSC growth as revealed by proliferation and BrdU assays. alpha-SMA gene and protein expression were significantly up-regulated by HMGB1, whereas the MMP-2, but not MMP-9, activity was suppressed by HMGB1 treatment. CONCLUSION: Our data suggested that HMGB1 protein, once released during the rejection phase of OLT, activated HSCs and exhibited profibrogenic effects on liver grafts either by increasing the HSC population and extracellular matrix content in liver grafts, or by transforming HSCs into myofibroblasts. Neutralization with anti-HMGB1 antibody was suggested to be a therapeutic modality applicable to prevent fibrogenesis in post-OLT liver grafts.

Induction of indoleamine 2,3-dioxygenase in livers following hepatectomy prolongs survival of allogeneic hepatocytes after transplantation.

OBJECTIVES: Indoleamine 2,3-dioxygenase (IDO), which catalyzes the breakdown of tryptophan into kyneurenine, has immunologic significance for the induction of maternal tolerance and liver allograft tolerance by inhibiting T-cell activation. In the present study, we compared survival of syngeneic or allogeneic hepatocytes in livers with or without hepatectomy. Subsequently, we investigated gene expression and localization of IDO in the recipient liver. METHODS: DA and Fisher 344 rats were used in the following experimental groups: group 1, DA hepatocytes transplanted into hepatectomized Fisher 344 rats; group 2, Fisher 344 hepatocytes transplanted into hepatectomized Fisher 344 rats; group 3, DA hepatocytes transplanted into nonhepatectomized Fisher 344 rats; and group 4, Fisher 344 hepatocytes transplanted into nonhepatectomized Fisher 344 rats. After transplantation, the surviving cells were evaluated on day 5. The IDO signal of the recipient liver was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: In the hepatectomized groups subjected to allogeneic or syngeneic hepatocyte transplantation, the number of surviving hepatocytes was greater than in the nonhepatectomized group after transplantation. The IDO signals (RT-PCR) in the hepatectomized groups were stronger than those in the nonhepatectomized groups. Immunohistochemistry demonstrated that the IDO signal is located in liver antigen-presenting cells, such as Kupffer cells or dendritic cells, and not expressed in hepatocytes. CONCLUSIONS: Our results demonstrated that IDO is induced in antigen-presenting cells of hepatectomized livers by which subsequently transplanted cells may be protected from rejection by inhibiting indirect or direct recognition of donor antigen and further T-cell activation.

The role of a nuclear protein, histone H1, on signalling pathways for the maturation of dendritic cells.

We have demonstrated previously that liver allograft tolerance is associated with the immunosuppressive activity of anti-histone H1 autoreactive antibodies induced in the serum of liver transplantation. Furthermore, we and others have shown that nuclear proteins such as histone H1 and high mobility group box 1 play an important role in maturation of dendritic cells (DCs), although the precise mechanisms are still unknown. In the present study, we focus upon the significance of histone H1 on DCs in terms of the intracellular signalling pathway of DCs. Our immunostaining and immunoblot studies demonstrated that histone H1 was detected in cytoplasm and culture supernatants upon the activation of DCs. Histone H1 blockage by anti-histone H1 antibody down-regulated the intracellular activation of mitogen-activated protein kinases (MAPKs) (p38) and IkappaBalpha of DCs, and inhibited DC activity in the proliferation of CD4+ T cells. On the other hand, the addition of histone H1 without endotoxin stimulation up-regulated major histocompatibility complex class II, the CD80 and CD86 surface markers of DCs and the activation of MAPKs (p38 and extracellular-regulated kinase 1/2) and IkappaBalpha. These results suggest that the translocation of histone H1 from nuclei to cytoplasm and the release of their own histone H1 are necessary for the maturation of DCs and the activation for T lymphocytes.

Histone H1 vaccine therapy for overcoming acute rejection in experimental organ transplantation.

OBJECTIVE: In a rat tolerogenic orthotopic liver transplantation (OLT) model, the recipient serum (post-OLT serum) shows strong immunosuppressive activity. In our previous reports, we suggested that autoreactive antibody (Ab) against histone H1 is a major immunosuppressive factor in this serum. The present study sought to determine whether up-regulation of anti-histone H1 Ab by histone H1 vaccination led to tolerance. MATERIALS AND METHODS: Using mixed lymphocyte reactions (MLR) and heterotopic heart transplantations (HHT), the alloreactive T-cell responses and allograft survivals of histone H1-immunized rats were compared with those of control rats. Cytokine and cellular profiles were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. RESULTS: The alloreactive T-cell response of histone H1-immunized rats was significantly lower than that of control rats, although there was no difference in nonspecific T-cell activation between the 2 groups. The allograft survival of histone H1-immunized rats was significantly prolonged after HHT. The major histocompatibility complex (MHC) class II and CD25 molecules of histone H1-immunized rats were significantly down-regulated compared with those of control rats. Moreover, the serum cytokine profile was modified by the immunization with histone H1. CONCLUSIONS: These results suggest that histone H1 vaccination of transplant recipients leads to the production of immunosuppressive factors and the modification of cytokine/cellular profiles.

Role of antinuclear antibodies in experimental and clinical liver transplantation.

OBJECTIVE: We recently reported that autoreactive antibodies (Abs) against nuclear histone H1 was transiently induced at an early phase after orthotopic liver transplantation (OLT) in a tolerogenic rat OLT model and possessed immunosuppressive activity. It was also reported that nuclear antigen, high-mobility group box 1 (HMGB1) protein was one of the initiators of the immune reaction. The present study sought to evaluate the role of antinuclear Abs in experimental and clinical liver transplantation. MATERIALS AND METHODS: We prepared 3 animal models: natural tolerance model (DA liver into PVG); acute rejection model (DA liver into LEW); and drug-induced tolerance model (acute rejection model + cyclosporine [CsA]). In addition, we examined clinical samples, including 1 drug-free patient, to measure the antihistone H1/HMGB1 titers at various times after OLT. RESULTS: In a natural tolerance model, antihistone H1 and HMGB1 Ab was induced during the rejection and the tolerance induction phases, respectively. Those Ab responses were also confirmed in a drug-induced tolerance model, whereas no such responses were shown in an acute rejection model. In our clinical drug-free patient, antihistone H1/HMGB1 titer was significantly higher after cessation of CsA than that in healthy volunteers. CONCLUSIONS: Antinuclear Ab is actively expressed in accordance with overcoming rejection episodes with subsequent tolerance induction in both a natural tolerance model and a drug-induced tolerance model. We also observed a similar tendency in our clinical drug-free patient. These results suggested that antinuclear Abs may be useful markers to determine the timing to withdraw immunosuppressants.

Characterization of immunosuppressive factors expressed in serum by rat tolerogenic liver transplantation.

BACKGROUND: In a rat tolerogenic model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) possesses strong immunosuppressive activity. This study aimed to identify immunosuppressive factors present in early post-OLT serum. METHODS: Immunosuppressive activity was evaluated in vitro by inhibition of the mixed-lymphocyte reaction (MLR). Autoantigens recognized by MLR-inhibitory IgG were identified by the internal protein sequencing. RESULTS: Recipient post-OLT serum inhibited MLR, and OLT-inducible IgG was the major immunosuppressive factor. IgG from post-OLT sera (2 to 3 weeks) specifically reacted to 31; 34; and 73-kd autoantigens on spleen cells. The internal sequences of the 31- and 34-kd antigens coincided completely with those of histone H1 molecules. Immunodepletion of anti-histone H1 antibodies (Abs) from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal Ab-directed histone H1 not only significantly suppressed rat and human MLR but also prolonged survival of heart allografts. Flow-cytometric analysis revealed that some live PVG splenocytes were stained with antihistone H1 Abs, and that these positive cells increased on Con A stimulation. Western blot analysis indicated that several cross-reactive antigens against anti-histone H1 Abs were found in their membrane fraction. CONCLUSIONS: In this study we provide evidence that autoreactive Abs, against histone H1 are a major OLT-induced graft survival factor, and may play at least a part in overcoming the acute rejection phase to establish solid allograft tolerance.

Identification of two down-regulated genes in rat liver allografts by mRNA differential display.

Total RNA differential display (DD) using random primers was performed for rat orthotopic liver transplantation (OLT) models. DA (RT1a) donor livers were transplanted into DA, PVG (RT1c), and LEW (RT1l) recipients: (1) syngeneic OLT (DA-DA): no rejection occurs; (2) allogeneic OLT (DA-PVG): rejection occurs, but is naturally overcome without immunosuppression; (3) allogeneic OLT (DA-LEW): animals die of acute rejection within 14 days. cDNA was isolated from selected bands, re-amplified for sequencing, and confirmed by Northern blots. Two down-regulated genes were observed in day-7 allogeneic OLT livers (DA-PVG, DA-LEW), while they were consistently expressed in day-7 syngeneic OLT (DA-DA) livers. These two genes were identified as alpha-glutathione sulfotransferase (alpha-GST) Ya gene and estrogen sulfotransferase (EST), respectively. Northern blots confirmed that their expression was down-regulated in OLT (DA-PVG) livers on days 7-26 and gradually restored. The mRNA expression of GST and EST may be good markers to predict rejection or induction of tolerance.

Effect of different concentrations of recombinant leukemia inhibitory factor on different development stage of mouse embryo in vitro.

PURPOSE: To assess the influence of different concentrations of recombinant human leukemia inhibitory factor (LIF) on the in vitro development of mouse embryos. METHODS: The 2- to 4-cell embryos of CB6F1 mice were cultured in the human tubal fluid (HTF) media containing different concentrations of LIF. Mouse embryos were divided into seven groups: (1) HTF; (2) 1500 IU/ml LIF; (3) 1000 IU/ml LIF; (4) 750 IU/ml LIF; (5) 500 IU/ml LIF; (6) 250 IU/ml LIF; (7) 125 IU/ml LIF. The embryonic numbers of different stages including 5-8 cell, 9-16 cell, morula, blastocyst, and hatching blastocyst were recorded. RESULTS: The percentage of early embryo stage (2-cell embryos to 6- to 16-cell stages) in all groups were nonsignificantly different. There were higher formation rates of preimplantation embryos (morula to hatching blastocyst) in groups 2, 3, 4, and 5 than in groups 1, 6 and 7. CONCLUSIONS: LIF has positive effects on preimplantation embryo development and has nonsignificant influence on the early embryo development. The lowest concentration of LIF which could provide the optimal embryo development is 500 IU/ml.

Clusterin may be involved in rat liver allograft tolerance.

Little is known about the possible role of complement inhibitors on tolerance induced by liver allografts. Clusterin, which is a plasma glycoprotein, inhibits cytolytic membrane attack complex (MAC) of complement by binding to soluble C5b-7 complex. The role of clusterin in relation to the naturally achieved tolerance in a rat orthotopic liver transplantation (OLT) has not been investigated before. Here we determined the kinetics of clusterin expression at different post-transplantation time points in a tolerogenic model (DA-PVG) where rejection was naturally overcome without any immunosuppressive drugs in comparison with the syngenic OLT model (DA-DA). Peripheral blood and liver tissues were taken from OLT at various post-operative time points. A strong expression of soluble clusterin was observed on post-transplantation day 7, which occurred at the peak of the rejection in this tolerogenic OLT model. The expression of clusterin remained strong even after tolerance was achieved. The intensity of clusterin expression was much stronger when compared with the syngenic OLT (DA-DA) model after OLT. A strong expression of clusterin mRNA was also observed in the tolerogenic model on post-OLT day (POD) 7 and the expression persisted when compared with the syngenic model on post-OLT day 60. Our data have shown that the strongest levels of clusterin during the reaction phase in tolerogenic OLT may be involved in tolerance induction.

Prolonged culture of human cryopreserved embryos with recombinant human leukemia inhibitory factor.

PURPOSE: To evaluate the efficiency of recombinant human leukemia inhibitory factor (LIF) in the prolonged culture of human cryopreserved-thawing embryos. METHODS: After thawing, all embryos were divided into four groups: (1) Human tubal fluid (HTF), (2) HTF + LIF, (3) M3TH medium, and (4) M3TH medium plus LIF. Following prolonged culture, embryo development in each group was compared. RESULTS: In embryo development from about the 2- to 4-cell to 9- to 16-cell stage, there were nonsignificant differences between each group. There was lower morula formation rate in group 1 (6.9%) than those in other groups (23.2%, 19.7%, 23.1%). The lower blastocyst formation in group 1 and 3 (0%, 0%) than those in group 2 and 4 (11.0%, 12.8%) were noted. CONCLUSIONS: LIF is beneficial for preimplantation embryos. LIF does not influence the early embryo development. LIF-supplemented HTF provided a similar culture environment for thawing embryos as LIF-supplemented M3TH medium.

Recombinant human leukemia inhibitory factor enhances the development of preimplantation mouse embryo in vitro.

OBJECTIVE: To assess the effect of recombinant human leukemia inhibitory factor (rhLIF) on mouse embryos in vitro. DESIGN: Controlled prospective study. SETTING: Academic research environment. ANIMAL(S): Female CB6F1 mice between 6 and 8 weeks old. INTERVENTION(S): Mice were divided randomly into three groups, which included a control group in an in vivo study (group I) and two groups in an in vitro study (groups II and III). Mice were killed at 116-120 hours (group I) and 44-48 hours (groups II and III) after hCG injection. Two-cell embryos (groups II and III) and blastocysts (group I) were obtained. Embryos in group II were cocultured with human tubal fluid (HTF) + 0.5% human serum albumin and in group III with HTF + rhLIF (1,000 U/mL) under paraffin oil. MAIN OUTCOME MEASURE(S): The embryonic numbers in different stages were recorded and compared. RESULT(S): Similar early embryo development to the four- to eight-cell and morula stages was noted between groups II and III (87.3% versus 91.0% and 74.6% versus 87.1%, respectively). However, further embryo development to the blastocyst, expanded blastocyst, and hatching blastocyst in group II (48.1%, 31.7%, and 18.5%, respectively) was lower than that in group III (83.6%, 53.7%, and 37.8%). CONCLUSION(S): RhLIF does not provide obvious stimulation in the early mouse embryo. However, rhLIF has positive effects on preimplantation blastocyst growth, differentiation, and hatching.

Nutritional value for growing turkeys of corn of light test weight.

An experiment was conducted to determine the relationship between test weight of corn and growth, efficiency of feed utilization, nutrient retention, and carcass composition of young turkeys. The MEn values of corn were also measured. Two samples of corn differing substantially in test weight were fed to male turkey poults (Nicholas Large White) from 1 to 20 d of age. The test weights of the light-weight corn and the normal-weight corn were 55 kg/hL (43 lb/bu) and 70 kg/hL (54 lb/bu), respectively. The light-weight corn was lower in crude protein concentration (6.63%) than the normal-weight corn (8.53%). The determined MEn of the corn samples did not differ (3.22 vs 3.25 kcal/g). The light-weight corn diet had modest adverse effects on growth and feed efficiency of poults during the 1 to 7 d after hatching. Poult carcass composition and retention of nitrogen and energy were not affected by the test weight of corn.

Identification of two elements involved in regulating expression of the murine leukaemia inhibitory factor gene.

Mouse leukaemia inhibitory factor (LIF) is a polyfunctional cytokine which exhibits multiple functions in vitro and in vivo. Two forms of LIF cDNA, differing at their 5' ends, have been described encoding either diffusible (D-LIF) or matrix-associated (M-LIF) forms of the protein [Rathjen, Toth, Willis, Heath and Smith (1990) (Cell 62, 1105-1114]. The present report describes the DNA sequence and functional characterization of the murine LIF gene and its surrounding transcriptional regulatory elements. Transient transfection of constructs containing the LIF gene and various amounts of 5'-non-coding sequence failed to give detectable levels of expression, suggesting the presence of inhibitory sequences within the LIF gene. Stable cell lines were produced by transfection of experimental constructs containing various lengths of 5'-non-coding sequence of the LIF gene, or the heterologous phosphoglycerate kinase promoter, linked to an LIF/neomycin-resistance-hybrid-coding sequence. The frequency of recovery of stable clones indicated that sequences located in the first intron between the transcriptional start sites for D-LIF and M-LIF act to suppress expression of the gene in most genomic locations. This region is rich in GC residues and has been shown to be hypomethylated in vitro [Kaspar, Dvorak and Bartunek (1993) FEBS Lett. 319, 159-162]. Analysis of the LIF/neomycin-resistance transgene expression in these stable cell clones demonstrated that transcripts containing the M-LIF or D-LIF exons required the presence of sequences located between -1200 and -3200 in the LIF gene. In the absence of these sequences, transcription is initiated elsewhere within the first intron. These sequences can be replaced by the heterologous phosphoglycerate kinase promoter. Deletion of the GC-rich region between the D-LIF and M-LIF transcriptional start sites results in the appearance of transcripts that do not splice out the first intron of the LIF gene. These may result from gene or promoter trapping of the LIF gene. Sequence analysis of the region between -1200 and -3200 revealed a number of minimal steroid-response elements, regions of similarity to DNAase I-hypersensitive sites in the uteroglobin gene and a region of alternating purine/pyrimidine sequence. This study therefore defines two important regulatory regions in the LIF gene: a GC-rich region in the first intron and a distal 'enhancer' located between -3200 and -1200.

Growth and differentiation factors of pluripotential stem cells.

The mammalian embryo develops as a quasi-stem cell system whose differentiation and pluripotentiality in vitro is controlled by a single regulatory factor, Differentiation Inhibiting Activity/Leukemia Inhibitory Factor (DIA/LIF). DIA/LIF is expressed in two distinct functional forms, derived from the use of alternate transcriptional start sites, one of which is freely diffusible and the other tightly associated with the extracellular matrix. The dissemination of the DIA/LIF signal is therefore under specific molecular control. The expression of DIA/LIF in vitro is both developmentally programmed and controlled by the action of other growth factors, the most notable of which are members of the fibroblast growth factor family expressed by the stem cells themselves. This indicates that differentiation and proliferation in early development of the mouse are controlled, at least in part, by an interactive network of specific growth and differentiation regulatory factors.

Response to and concentration of interleukin-2 in patients with nasopharyngeal carcinoma.

To seek beneficial immunomodulation with interleukin-2 (IL-2) for patients with nasopharyngeal carcinoma (NPC), a study was conducted of the response to and concentration of IL-2 by mononuclear cells (MNC) from 45 NPC patients who had been pathologically verified, but pre-treatment, at the time they were studied. Thirty-eight normal healthy controls were studied simultaneously. Lymphocyte subsets did not change much after IL-2 stimulation in either NPC patients or the controls. A decreased IL-2 concentration was found in NPC, and a trend of inverse correlation to clinical staging was detected; the more advanced stage, the less concentration of IL-2. It was concluded that there was a normal response to and decreased concentration of, IL-2 in NPC patients. Whether decreased concentration of IL-2 in NPC patients resulted from decreased production or increased consumption is not clear. A further research of IL-2 immunomodulation in NPC patients should be done before its clinical application.