The biological characteristics of cytokine-induced killer cells

AIM:To investigate the biological characteristics of cytokine-induced killer (CIK) cellsin vitro.METHODS:The non-adhere peripheral blood monoclear cells from healthy donors were induced into CIK cells in the presence of IFN-γ, IL-1β, IL-2 and anti-CD3 antibody. LAK (lymphokine activated killer) cells were prepared as a control. The cellular phenotype were detected by FCM and immunocytochemistry and the cytotoxicity was measured by LDH release assay.RESULTS:After 2 weeks of induction, the proliferation rate of CIK cells reached a peak and the proportion of CD3⁺ population was above 95%, and then the cells growth entered to plateau phase at week 3. The proportion of CD3⁺CD56⁺ NKT subset cells was 16.5% on day 15 and it had no obvious variety between 2 and 4 weeks. Correspondingly, LAK cells grew slowly and had lower proliferation rate compared with the CIK cells (P<0.01). CIK cells showed higher specific lysis rates to BeL-7402 hepatoma cells than those of LAK cells at different effector to target ratio (P<0.01). Immunocytochemical staining showed the CIK cells highly co-expressed HLA-DR and CD54 antigens. The NKT cells were slightly bigger than CD3⁺CD56^- cells and a large quantity of pseudopodia were observed on their surface.CONCLUSION:The CIK cells have higher proliferation potency and stronger cytotoxicity to lyse tumor cells than LAK cellsin vitro.Within the span of time from 14 to 21 days, the proliferation rate and the proportion of CD3⁺CD56⁺ subset of CIK cells all reach peaks. Therefore, CIK cells in this period are suitable for clinical application.     

Effect of SC58125 on cell proliferation and apoptosis in HepG-2 cells

AIM: To clarify the effect of SC58125 on cell proliferation and apoptosis in HepG-2 cells and explore the molecular mechanisms. METHODS: Cell culture, MTT, TUNEL, DNA ladder, flow cytometry and Western blot analysis were employed in the present study. RESULTS: SC58125 inhibited the growth of HepG-2 cells and induced the apoptosis. Furthermore, it arrested G₀/G₁ phase and inhibited S phase in HepG-2 cells. Depressed expression of P33^cdk2, P34^cdc2, cyclin B₁, cyclin E, Mpm-2, Rb, PCNA proteins were found in HepG-2 cells treated with SC58125. CONCLUSION: SC58125 inhibits cell proliferation and induces apoptosis, which may be related to the altered low protein levels of P33^cdk2, P34^cdc2, cyclin B₁, cyclin E, Mpm-2, Rb, PCNA.     

Effect of heat shock protein on dendritic cell maturation

AIM: To study the effect of heat shock protein (HSP) on the maturation of dendritic cells (DCs) and observe the morphological changes of DCs dynamically. METHODS: The HSP (gp96)-peptide complex was purified from the tissues of hepatocellular carcinoma. Hepatoma cells were treated by heat shock for preparation of the HSP expressed on cell surface and then marked with DiI fluorescence. Immature DCs from peripheral blood mononuclear cells were cocultured with two types of HSPs. The morphological changes of DCs were observed dynamically and the effects of HSPs on the maturation of DCs analyzed by Flowcytometer. RESULTS: The morphological changes and the processes of antigen capture of DCs cocultured with DiI marked tumor cells were well showed. Four ways to capture antigens of DCs were observed, including direct contact, besieging, forming bubbles and extending pseudopodia with bubbles on the terminals. Results also indicated that both types of HSP could promote the maturation of DCs. CONCLUSION: DiI is a good fluorescent dye suitable for the morphological studies of DCs. Four ways for DCs to capture antigens were indicated in this paper. Different types of HSP, purifed from tumor tissues or expressed on tumor cells surface, promote DC maturation, and the purified HSP is more effective.     

Changes of serum levels of TXA2 and PGl2 in cirrhosis patients during liver transplantation

AIM：To study the changes of serum levels of thromboxane A₂ (TXA₂) and prostacyclin (PGI₂) in cirrhosis patients during liver transplantation.METHODS：Samples were obtained from 24 cirrhosis patients in end at five time points during liver transplantation.TXA₂ and PGI₂ level were measured by radioimmunoassay.Arterial and mixed venous blood samples used for blood gas analysis were taken at the same time.Intrapulmonary shunt (Qs/Qt) was calculated according to the standard formula.The hemodynamics parameters including continuous cardiac output index (CI),HR,mean artery blood pressure (MABP),MPAP,CVP,PAWP,SVRI,PVRI were measured during liver transplantation.RESULTS：(1) MABP decreased significantly in the early stage of anhepatic period and neohepatic period.(2) CVP,MPAP and PAWP decreased significantly during anhepatic period.They increased significantly after graft reperfusion and remain the high level.(3) CI declined significantly during anhepatic period and increased at 10 min postreperfusion of new liver.(4) SVRI and PVRI increased during anhepatic period and were higher than baseline level at 15 min after reperfusion.SVRI was lower than baseline level at 30 min after reperfusion.(5) Compared with the baseline level,6-keto-PGF1α and TXB₂ increased significantly.Compared with the level before vascular cross-clamping,6-keto-PGF1α decreased during neohepatic period and it had significant difference in statistics at the end of operation.CONCLUSION：Serum levels of TXA₂ and PGI₂ significantly change during liver transplantation and may affect the system and pulmonary circulation to some extent.     

Nile tilapia (Oreochromis niloticus), liver morphology, CYP1A activity and thyroid hormones after Endosulfan dietary exposure

Endosulfan is a worldwide used insecticide suspected to be highly toxic to aquatic organisms, including fish. Most of the available studies have focused in water exposures, although this pollutant can be transferred through food chain. Therefore, in the present study, the effects of Endosulfan on tilapia (Oreochromis niloticus), when administered through the diet. Fish were fed 21 days with diets containing 1 and 0.5 μg g⁻¹ of Endosulfan, after which qualitative histological liver analysis showed that Endosulfan induced hepatocyte destruction, vessel endothelium rupture and increased melanomacrophages aggregates. To test lower environmentally relevant doses of Endosulfan could induce hepatic damage, as well as other negative effects, such as altered phase I metabolism and plasma thyroid hormone levels. Hence, tilapia were orally exposed to 0.1 and 0.001 μg g⁻¹ for 35 days. Low environmentally realistic doses of Endosulfan were still able to induce liver histopathological damage such as increased hepatocyte vacuolization and increased eosinophil granular cell aggregates. Liver cytochrome P450 1A activity, evaluated through ethoxyresorufin-o-deethylase (EROD), was enhanced in tilapia exposed to 0.001 μg g⁻¹, whereas the highest dose had no measurable effects in this enzyme activity. Fish exposed to 0.1 μg g⁻¹ of Endosulfan had depressed T4 plasma levels. Overall, the results of the present study further demonstrate the toxic effects of Endosulfan in tilapia when administered in the diet at environmentally relevant concentrations, which indicates that in the field food chain transfer may also be an importance source of this pollutant.     

Comparison of the inhibition effect of different anticoagulants on vitamin K epoxide reductase activity from warfarin-susceptible and resistant rat

Anti-vitamin K drugs are widely used as anticoagulant in human thromboembolic diseases. Similar compounds have also been used as rodenticides to control rodent population since 1950s. Massive use of first generation anticoagulants, especially warfarin, has lead to the development of genetic resistances in rodents. Similar resistances have been reported in human. In both cases, polymorphisms in VKORC1 (Vitamin K epoxide reductase subunit 1), the subunit 1 of the VKOR (Vitamin K epoxide reductase) complex, were involved. In rats (Rattus norvegicus), the Y139F mutation confers a high degree of resistance to warfarin. Little is known about the in vitro consequences of Y139F mutation on inhibitory effect of different anticoagulants available. A warfarin-susceptible and a warfarin-resistant Y139F strain of wild rats (Rattus norvegicus) are maintained in enclosures of the Lyon College of Veterinary Medicine (France). Using liver microsomes from susceptible or resistant rats, we studied inhibition parameters by warfarin (K_i = 0.72 ± 0.1 μM; 29 ± 4.1 μM), chlorophacinone (K_i = 0.08 ± 0.01 μM; 1.6 ± 0.1 μM), diphacinone (K_i = 0.07 ± 0.01 μM; 5.0 ± 0.8 μM), coumachlor (K_i = 0.12 ± 0.02 μM; 1.9 ± 0.2 μM), coumatetralyl (K_i = 0.13 ± 0.02 μM; 3.1 ± 0.4 μM), difenacoum (K_i = 0.07 ± 0.01 μM; 0.26 ± 0.02 μM), bromadiolone (K_i = 0.13 ± 0.02 μM; 0.91 ± 0.07 μM), and brodifacoum (K_i = 0.04 ± 0.01 μM; 0.09 ± 0.01 μM) on VKOR activity. Analysis of the results leads us to highlight different anticoagulant structural elements, which influence inhibition parameters in both susceptible and Y139F resistant rats.     

Investigation on the ability of different strains and doses of exogenous Bifidobacteria, to translocate in the liver of weaning pigs

Our aim was to evaluate the ability of different strains of Bifidobacteria to cross the gut wall and reach the liver of weaning pigs, and to study the influence of different doses of a probiotic on this parameter. The ability of bacteria to pass from the intestinal lumen to mesenteric lymph nodes and other tissues is called translocation. Several factors promote bacterial translocation, including intestinal bacterial overgrowth, deficiencies in host immune response, and gut barrier damage. Translocation can also be used as a parameter to evaluate a candidate probiotic. Few data are available on the probiotic translocation in weaning pig. We analysed the liver samples from two trials performed with 32 and 64 pigs, respectively, reared from 21 to 35 days of age, fed seven different diets: control diet (C), or C supplemented with one of six different Bifidobacteria strains (10¹⁰/CFU/d) (trial 1); or supplemented with four different levels of B. animalis (0, 10⁷, 10⁹, 10¹¹) crossed with 0% or 2% sugar beet fructo-oligosaccharides (trial 2). We found Bifidobacterium spp. genus-specific DNA in liver of subjects from all dietary treatments (control fed pigs included), and no relationship between oral dose and strain appeared. On average 53.1% (trial 1) and 48.4% (trial 2) of the pigs showed the presence of Bifidobacterium spp. genus-specific DNA. We conclude that both endogen and exogenous bacteria can translocate to the liver.     

Expressions of VEGF,ANG-1,ANG-2 and TSP-1 in cholangiocellular carcinoma and relationship with tumor angiogenesis,differentiation,invasion and metastasis

AIM: To investigate the angiogenesis status,the expression of vascular endothelial growth factor (VEGF),angiopoietin-1 (ANG-1),angiopoietin-2 (ANG-2),thrombospondin-1 (TSP-1) in cholangiocellular carcinoma (CCC) and relationship with tumor angiogenesis,differentiation,invasion and metastasis.METHODS: 33 specimen of surgically resected CCC were investigated.Immunohistochemical staining of CD34,VEGF,ANG-1,ANG-2 and TSP-1 was carried out.RESULTS: The mean MVD was (87.2±52.6)/mm2.VEGF positive expression was found in 75.6% cases;ANG-1 positive expression was observed in 36% cases;ANG-2 positive was detected in 57.6% cases and 45.5% cases exhibited positive TSP-1 expression.VEGF and ANG-2 expressions were found to be associated with significant higher level of MVD (P<0.01 and P<0.05,respectively).TSP-1 expression was found to be associated with significant low level of MVD (P<0.01).Positive TSP-1 expression was also found to be associated with higher level of intrahepatic metastasis (46.7% vs 5.6%,P<0.05).CONCLUSION: Considerable angiogenesis compared to other solid tumors can be observed in CCC.VEGF and ANG-2 might play a proangiogenic role and TSP-1 may play an inhibitory role.Although TSP-1 may increase the intrahepatic metastasis of CCC,neither MVD levels nor the expression of VEGF,ANG-1,or ANG-2 is associated with tumor differentiation,invasion and metastasis.     

Changes of mitochondrial peripheral-type benzodiazepine receptor during rat live regeneration

AIM: To investigate the expression profile of peripheral-type benzodiazepine receptor (PBR) involved in mitochondrial permeability transition (PT) regulation, and to observe the binding dynamic of the mitochondrial PBR with specificity ligand during rat live regeneration. METHODS: Liver regeneration model was produced by 70% partial hepatectomy (PH) performed in male SD rats. The animals of sham groups underwent the same surgical operations as PH groups did, but the liver lobes were not resected. The animals in the PH groups and corresponding sham groups were sacrificed at 3, 6, 12, 24, 48, 72, 120 and 168 hours after the operation. The livers were removed, weighted and processed for isolation of mitochondria. Semi-quantitative RT-PCR was performed to examine the expression level of PBR in 70% hepatectomized rat livers during the whole regeneration process and compared to that in the sham and normal groups. Compared with healthy rats, the kinetic parameters of PBR was evaluated by using a specific radioligand ［3H］-PK11195. RESULTS: Compared with healthy rats, the expression of PBR was unchanged. Meanwhile, the results obtained in the present experiments by scatchard analysis, Bmax of PK11195 for PBR significantly decreased, returned to normal level in 168 h after PH. Kd of PK11195 for PBR significantly decreased at 72 h and 168 h after PH of rat liver regeneration (P<0.01). CONCLUSION: The mRNA expression and evaluation of kinetic parameters of PBR may be related to the time-phase change of mitochondrial PT during rat liver regeneration after partial hepatectomy.