Inhibitory effects of transcription factor Ikaros on the expression of liver cancer stem cell marker CD133 in hepatocellular carcinoma.

CD133 is a cellular surface glycoprotein that has been reported as a marker for the enrichment of cancer stem cells (CSCs). However, the regulatory mechanism of CD133 remains unknown. CSCs have been proposed to contribute to radioresistance and multi-drug resistance. The elucidation of key regulators of CD133 and CSCs is critical for the development of CSC-targeted therapy. In this study, we showed that Ikarosinhibited the expression of CD133 via direct binding to the CD133 P1 promoter and repressed the tumorigenic and self-renewal capacity of CD133(+) cancer stem-like cells in hepatocellular carcinoma (HCC). We found that Ikaros interacted with CtBP as a transcription repressor complex, which inhibited CD133 expression in HCC. We also demonstrated that Ikaros expression was up-regulated by ETS1 which activity was regulated by MAPKs pathway. Furthermore, decreased expression of Ikaroswas significantly associated with poor survival in HCC patients. Overall, our study identifies that Ikaros plays a role as a transcription repressor in HCC and is a new reactivated therapeutic target for the treatment of HCC. Meanwhile, our findings provide evidence that Ikaros could be an attractive inhibitor of the target gene CD133, which reactivates anticancer mechanisms in targeted CSC therapy.

A new method for evaluating tricuspid valve displacement in children with Ebstein's anomaly: using the annulus and coronary sinus as a reference point.

This study aimed to investigate the reference point for the downward displacement of the posterior and anterior leaflets of the tricuspid valve using echocardiography in children with Ebstein's anomaly. This study enrolled 25 patients with Ebstein's anomaly. The extent of downward displacement of the posterior and anterior leaflets of the tricuspid valve was evaluated by echocardiography using the tricuspid annulus and the coronary sinus as reference points. These results were compared with the surgical findings. The findings showed displacement of the simple septal leaflet in 1 patient, displacement of both the septal and posterior leaflets in 22 patients, displacement of both the anterior and posterior leaflets in 1 patient, and displacement of all the leaflets in 1 patient. Because the septal and posterior leaflets were close to the apex or because the posterior leaflet was nearly absent, the displacement distance of the septal and posterior leaflets could not be measured accurately in two patients. The displacement distance of the septal and posterior leaflets in the remaining 22 patients were 2.08 ± 1.15 and 2.58 ± 1.06 cm, respectively. The displacement distances of the anterior leaflet in two patients were respectively 1.0 and 2.2 cm. These results were similar to those measured during surgery. The direction of the valvular regurgitation flow was anterolateral in the apical four-chamber and apical right heart two-chamber views in patients with the downward displacement of the anterior leaflet. The tricuspid valve annulus and the coronary sinus are ideal reference points for evaluating the downward displacement of the posterior and anterior leaflets of the tricuspid valve. It is critical to evaluate the downward displacement of the anterior leaflet that the direction of the tricuspid regurgitation flow is changed.

[Echocardiographic diagnosis of partial anomalous pulmonary venous connection].

OBJECTIVE: To investigate partial anomalous pulmonary venous connection (PAPVC) with echocardiography. METHODS: The right ventricular volume overload was detected by routine echocardiography in 37 child patients, who underwent further echocardiography to find the abnormal locations of pulmonary vein opening at superior, inferior vena cava and right atrium. The ultrasound results were compared with surgical findings. RESULTS: In 30 patients the ultrasound diagnosis was consistent with surgery results, 7 were misdiagnosed by ultrasound with a detective rate of 81.1 %. All 37 PAPVC patients presented varying degrees of right heart enlargement; PAPVC combined with atrial septal defect (ASD) was found in 34 cases. CONCLUSION: The possibility of PAPVC should be considered when unexplained right heart volume overload was detected by echocardiography. Superior, inferior vena cava and right atrium should be inspected when the pulmonary veins were not seen in echocardiography.

Th17 promotes acute rejection following liver transplantation in rats.

T help cell 17 (Th17), recently identified as a new subset of CD4(+) T cells, has been implicated in autoimmune diseases, tumor immunity, and transplant rejection. To investigate the role of Th17 in acute hepatic rejection, a rat model of allogeneic liver transplantation (Dark Agouti (DA) to Brown Norway (BN)) was established and isogeneic liver transplantation (BN to BN) was used as controls in the study. The expression of Th17-related cytokines in the liver and peripheral blood was determined by immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), or real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Strong expression of interleukin-17A (IL-17A), IL-6, transforming growth factor-ß (TGF-ß), IL-8, and myeloperoxidase (MPO) was observed in liver allografts. The ratios of Th17 to CD4(+) lymphocytes in the liver and peripheral blood were dramatically increased in the allograft group compared with the control (P<0.01). Secreted IL-17 and IL-6 in liver homogenate and serum were significantly elevated in the allograft group, while secreted TGF-ß was increased in liver homogenate and decreased in serum compared with the control (P<0.01). The messenger RNA (mRNA) levels of IL-17, IL-21, and IL-23 were enhanced in the allografts compared with the control (P<0.01). Correlation analysis showed significant correlations between IL-17 and IL-6 and TGF-ß and between IL-17 and IL-21 and IL-23. The present study demonstrates that Th17 plays a role in promoting rat liver allograft rejection.

[Role of B7-H1 in pancreatic carcinoma immune evasion].

OBJECTIVE: To investigate the role of B7-H1 expression in IL-10 production, the B7-H1 and IL-10 expression levels in pancreatic carcinoma tissues and to analyze the correlation between B7-H1 expression and IL-10 level. METHODS: The mRNA and protein levels expressions of B7-H1 and IL-10 in 35 cases of pancreatic cancer and corresponding paracarcinoma tissues and 5 cases of normal pancreas tissues were detected by RT-PCR, Western blot and immunohistochemistry respectively. RESULTS: The findings for the first time provided the evidences that there was a clear trend for B7-H1 and IL-10 expressions to be most highly expressed in carcinoma tissue, intermediately expressed in paracarcinoma tissue, and expressed at the lowest level in normal pancreatic tissue at mRNA and protein levels. Moreover, there were statistically significant differences in B7-H1 and IL-10 expression between pancreatic carcinoma tissues, corresponding paracarcinoma tissues and normal pancreatic tissues at mRNA and protein levels (P < 0.05). Furthermore, the immunohistochemistry indicated that there were high expression levels of B7-H1 (60.5% +/- 12.7%) and IL-10 (65.3% +/- 16.2%) in pancreatic carcinoma tissues while there were no significant expressions in normal pancreatic tissues. Meanwhile, correlation analysis revealed that B7-H1 expression was significant associated with IL-10 level in tumor tissues at mRNA (P = 0.008, r = 0.841) and protein levels (P = 0.007, r = 0.838). CONCLUSIONS: Over-expression of B7-H1 may be responsible for the increasing IL-10 production in pancreatic cancer, which caused reduced immune response to tumor cells and contributed to pancreatic carcinoma escape from immune attack.

Protective effect of raloxifene on lipopolysaccharide and acid- induced acute lung injury in rats.

AIM: To evaluate the protective effect of oral raloxifene on acute lung injury. METHODS: Thirty adult, male Sprague-Dawley rats each weighing 180-210 g were used and divided into 3 groups: the raloxifene-lipopolysaccharide (LPS)-HCl group (n=10), the LPS-raloxifene-HCl group (n=10), and the placebo group (n=10). All the rats were injected intraperitoneally (ip) with 5 mg/kg LPS, and raloxifene (30 mg/kg) was orally administered 1 h before and 14 h after LPS injection into the raloxifene-LPS-HCl and the LPS-raloxifene-HCl groups, respectively; the placebo group received nothing. Sixteen hours after LPS injection, all the animals were anesthetized and the femoral artery was cannulated. All the rats received a direct intratracheal (IT) injection of HCl (pH 1.2; 0.5 mL/kg). The mean arterial pressure (MAP) and blood gas concentrations were measured. Fifteen rats (5 in each group, respectively) underwent a micro positron emission tomography (microPET) scan of the thorax 4 h after HCl instillation. The wet/dry (W/D) weight ratio determination and histopathological examination were also performed. RESULTS: The rats in the LPS-raloxifene-HCl group had a lower [18F]fluorodeoxyglucose uptake compared with the rats in the placebo group (4.67+/-1.33 vs 9.01+/-1.58, respectively, P<0.01). The rats in the LPS-raloxifene-HCl group also had a lower histological lung injury score (8.20+/-1.23 vs 12.6+/-0.97, respectively, P<0.01) and W/D weight ratio (5.335+/-0.198 vs 5.886+/-0.257, respectively, P<0.01) compared to the placebo group. The rats in this group also showed better pulmonary gas exchange and more stable mean arterial pressure (MAP) compared to the placebo group. CONCLUSION: Raloxifene provides a significant protective effect on acute lung injury in rats induced first by LPS ip injection and then by HCl IT instillation.

[Assessment of the characteristics of echocardiography in pediatric patients with total anomalous pulmonary venous connection].

OBJECTIVE: To assess the accuracy of echocardiography in diagnosis of total anomalous pulmonary venous connection (TAPVC). METHODS: A combination of suprasternal, parasternal, subcostal and apical views were employed to diagnose TAPVC and to trace the course of the anomalous pulmonary venous connection, the direction of the inter-atrial shunt, enlargement of right atrium (RA) and right ventricle (RV), superior and inferior vena cava. All pediatric patients underwent surgical repair. The results of echocardiography were compared with surgical findings. RESULT: A total of 28 consecutive pediatric patients with suspected TAPVC were included in this study. The TAPVC diagnosis was confirmed in 26 cases after surgery, partial anomalous pulmonary venous connection (PAPVC) in one case, and Cor Triatriatum and possible TAPVC in another. The diagnostic accuracy of TAPVC by echocardiography in the study was 92.86%. There were 17 supracardiac TAPVC, 11 intracardiac TAPVC. In all patients, enlargement of the RA and RV, inter-atrial right-to-left shunt via atrial septal defects were documented in parasternal and subcostal views. Common pulmonary vein or four pulmonary vein direct to RA or via coronary sinus to RA were the draining sites of intracardiac TAPVC. The enlargement of left innominate vein-right superior vena cava draining to RA was seen in supracardiac TAPVC. CONCLUSION: A combination of suprasternal and subcostal multi-views in echocardiography can increase the diagnostic accuracy of TAPVC in pediatric patients.

Adenovirus mediated CTLA4Ig gene inhibits infiltration of immune cells and cell apoptosis in rats after liver transplantation.

AIM: To investigate the role of adenovirus-mediated CTLA4Ig gene therapy in inhibiting the infiltration of macrophages and CD8(+) T cells and cell apoptosis after liver transplantation. METHODS: The rat orthotopic liver transplantation model was applied. The rats were divided into three groups: group I: rejection control (SD-to-Wistar); group II: acute rejection treated with intramuscular injection of CsA 3.0 mg/(kg x d) for 12 d (SD-to-Wistar+CsA); groupIII: injection of 1 x 10(9) PFU adenovirus-mediated CTLA4Ig gene liquor in dorsal vein of penis 7 d before liver transplantation (SD-to-Wistar+CTLA4Ig). Immunohistochemistry and transferase-mediated dUTP nick-end labeling (TUNEL) were used to analyze the expression of CTLA4Ig gene in liver, infiltration of macrophages and CD8(+) T cells, cell apoptosis in grafts at different time-points after liver transplantation. Histopathological examination was done. RESULTS: CTLA4Ig gene expression was positive in liver on d 7 after administering adenovirus-mediated CTLA4Ig gene via vein, and remained positive until day 60 after liver transplantation. Infiltration of macrophages and CD8(+) T cells in CTLA4Ig-treated group was less than in rejection control group and CsA-treated group. The apoptotic index of rejection group on d 3, 5, and 7 were significantly higher than that of CTLA4Ig-treated group. A good correlation was found between severity of rejection reaction and infiltration of immune activator cells or cell apoptotic index in grafts. CONCLUSION: CTLA4Ig gene is constantly expressed in liver and plays an important role in inducing immune tolerance.

[Antitumor effect of immunizations with fusions of dendritic and hepatocellular carcinoma cells in mice].

OBJECTIVE: To investigate the effects of immunization with fusions of dendritic cells and H22 cells on tumor-bearing mice and their possible mechanisms. METHODS: Fusion cells of DC and H22 cells were prepared with polyethylene glycol (PEG). Expression of MHC and costimulatory molecules by dendritomas were determined by FACs. To study the antitumor immune preventative and therapeutic effects, fusions were subcutaneously injected into tumor-bearing mice. The cytotoxic T lymphocyte (CTL) activity was determined by LDH method, the expression of TNF-a and IFN-g in tumors were assayed by RT-PCR. RESULTS: The data showed that the hybridomas of DC and H22 cells acquired both DC and H22 cell phenotypes. Immunization of BALB/C mice with DC/H22 fusions induced potent CTL activity (mean CTL activity=0.624+/-0.024, compared with DC + H22, DC, H22 groups, F = 65.46) and a protective immunity against a high dose of H22 tumor challenge. After treatment with hybridomas, the survival time of tumor-bearing mice was greatly extended (x2=18.45). The expression levels of TNF-a and IFN-g mRNA were remarkably increased (TNF-a, F = 47.84; IFN-g, F = 37.23). CONCLUSIONS: The hybridomas of DC and H22 cells could induce effective antitumor immune responses and may have a useful potential in prevention and management of the recurrences and metastases of HCC.

Lymphotactin enhances the in-vitro immune efficacy of dendritoma formed by dendritic cells and mouse hepatocellular carcinoma cells.

OBJECTIVE: To investigate the in-vitro antitumor immune responses of dendritoma formed by mouse hepatocellular carcinoma (HCC) cells and lymphotactin (Lptn) gene modified dendritic cells (DCs). METHOD: DCs prepared from mouse bone marrow were genetically modified by lymphotactin adenovirus, and fused with H22 cells by polyethylene glycol (PEG). RT-PCR and ELISA were employed to identify lymphotactin expression at mRNA and protein level. Cell phenotypes and fusion efficiency was detected by FACS. The stimulatory effect of DC on T cells was detected by mixed lymphocyte reaction. The cytotoxicity activity against H22 cells was assayed by LDH method. RESULTS: Lymphotactin could be efficiently expressed by DCLptn/H22 hybridoma. DCLptn/H22 cells could induce potent T cell proliferation effect and generate strong cytotoxic T lymphocyte (CTL) reaction against allogenic H22 cells. CONCLUSION: Lymphotactin genetic modification could enhance the in vitro immune activity of the dendritoma.

Antitumor effect of immunization with fusion of dendritic cells and hepatocellular carcinoma cells in mice.

BACKGROUND: Important advances have made within the past few years in the treatment of hepatocellular carcinoma (HCC), however, the long-term prognosis after resection of HCC remains unsatisfactory as a result of a high incidence of recurrence. This study was to investigate immunization with fusions of DCs and HCC cells against HCC tumors transplanted to mice. METHODS: Fusion cells of dendritic cells (DCs) and H22 cells were prepared with polyethylene glycol. Expression of MHC and costimulatory molecules by dendritomas were determined by FACs. To study the antitumor immune preventitive and therapeutic effects, fusions were subcutaneously injected into naive or tumor-bearing mice; the CTL activity was assumed by the LDH method, and the expression of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma(IFN-gamma) in tumors were assayed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The hybridomas of DCs and H22 cells acquired both DCs and H22 cells phenotypes. Immunization of BALB/C mice with DC/H22 fusions induced protective immunity against a high dose of H22 tumor challenge. After treatment with hybridomas, the survival time of tumor-bearing mice was extended. The expression level of TNF-alpha and IFN-gamma mRNA was markedly increased. CONCLUSION: The hybridomas of DCs and H22 cells could induce effective antitumor immune responses and may be potentially used in prevention and management of recurrence and metastasis of HCC.

[Influence of combined cyclosporine A and tacrolimus with 5-fluorouracil on hepatocellular carcinoma rats].

OBJECTIVE: To investigate the effect of combined CsA and FK506 with 5-FU on hepatocellular carcinoma rats. METHODS: A syngeneic rat model of hepatocellular carcinoma was used. Control group (A) underwent 4 ml 5% GS. Treatment group was divided into 3 groups namely, group B: only 5-FU and 5% GS; group C: 5-FU, CsA and 5% GS; group D: 5-FU, FK506 and 5%GS. Cell cycle, apoptosis, necrosis and mitochondrial transmembrane potential were measured by flow cytometry, laser scanning confocal microscopy, and electron transmission microscopy. Statistical analysis was performed by SPSS 10.0 for Windows software. Statistical comparisons were made with ANOVA followed by Dunnett's T3 or LSD test. RESULTS: Compared to the control group, the percentage of apoptotic cells including trifle necrotic cells was significantly higher, and among the treatment group, group D was the highest, and group C was higher than group B. In the treatment group, cell cycle of hepatoma cells was mainly arrested at S phase, but in group D, G0/G1 phase cells were significantly decreased and S phase cells significantly increased. Compared to the control group, mitochondrial transmembrane potential was significantly decreased in the treatment group, among with, group B was the lowest, group C was higher than group D. Morphological changes demonstrated by electron microscopy included dispersed nuclear chromatin, loss of nucleoli, membrane bleeding, cell shrinkage, typical apoptotic bodies and marked swelling of mitochondria in the treatment group. In the control group, however, they were characterized by normal cell ultrastructure. CONCLUSIONS: The present study reveals that 5-FU combined with CsA or FK506 demonstrated a synergistic effect on hepatocellular carcinoma rats. For FK506, the powerful mutual effect is related to the increase of tumor cell's quantity in S phase. Both CsA and FK506 can provide protection on mitochondrial transmembrane potential reduction against hepatoma cells damage from 5-FU.

[Early expression of pro-inflammatory cytokines in the lung of rat with small-for-size liver transplantation and the significance].

OBJECTIVE: To investigate the expression of tumor necrosis factor-alpha(TNF-alpha), interleukin-1 beta (IL-1beta), and intercellular adhesion molecule-1 (ICAM-1) in the lung of rat with small-for-size liver transplantation and the significance of the expression of these cytokine in lung injury after liver transplantation. METHODS: 150 SD rats were randomly divided into 4 groups: sham operation group (n = 6), whole graft liver transplantation group (n = 48), 50% size graft liver transplantation group (n = 48), and 30% size liver transplantation group (n = 48). Six rats from each group were killed 0.5, 2, 6, and 24 hours after operation. Blood samples from subhepatic inferior vena cava were obtained to examine the plasma TNF-alpha by ELISA. Specimens of lung were obtained to be examined pathologically. RT-PCR was used to examine the expression of TNF-alpha mRNA, IL-1beta mRNA, and ICAM-1 mRNA in lung, and chromatometry was performed to detect the activity of myeloperoxidase (MPO). RESULTS: (1) The plasma TNF-alpha at any time point was higher in the 3 transplantation groups than in the sham operation group. The plasma TNF-alpha 2 hours after operation in the whole graft group was significantly higher than that in the 30% size group (P < 0.05). (2) The expression levels of TNF-alpha mRNA 2 and 6 hours after operation in the whole graft group and in the 50% graft group were significantly higher than those in the 30% graft group (all P < 0.01). The expression levels of TNF-alpha mRNA remained significantly higher than those in the sham operation group since the second hour after operation (all P < 0.01). (3) IL-1beta mRNA was expressed in the 3 liver transplantation groups without significant differences between any levels at all the time points and was not expressed in the sham operation group. The expression of ICAM-1 mRNA was higher at all the time points in the liver transplantation groups than in the sham operation group (P < 0.01 or P < 0.05) without significant difference between the values of any transplantation group at any time point (all P > 0.05). The MPO activity was stronger in the 3 liver transplantation groups at any time point than in the sham operation group (all P < 0.01). The peak occurred 2 hours after operation in the whole graft group and 50% size group and occurred 6 hours after operation in the 30% graft group. The plasma TNF-alpha level was positively correlated to the MPO activity in lung tissue with a correlation coefficient of 0.422 (P < 0.05). (4) The morphology of lung was normal in the sham operation group. Obvious interstitial hyperemia and hemorrhage, neutrophil infiltration, and pulmonary septal thickening were found in the 3 transplantation groups, in particular being severe at the 2-hour time point. CONCLUSION: Increase of plasma TNF-alpha is one of the causes of lung injury after small-for-size liver transplantation. Upregulation of TNF-alpha mRNA, IL-1beta mRNA, and ICAM-1 mRNA expression may be also responsible for the lung injury and their expression may be correlated to the size of graft.