Gut Microbiota Reconstruction Following Host Infection with Blood-stage Plasmodium berghei ANKA Strain in a Murine Model.

Malaria remains a global health problem. The relationship between Plasmodium spp. and the gut microbiota as well as the impact of Plasmodium spp. on the gut microbiota in vertebrate hosts is unclear. The aim of the current study was to evaluate the effect of blood-stage Plasmodium parasites on the gut microbiota of mice. The gut microbiota was analyzed by 16S rRNA sequencing and bioinformatic analyses at three stages. The gut microbiota changed during the three phases: the healthy stage, the infection stage, and the cure stage (on the 9th day after malarial elimination). Moreover, the gut microbiota of these infected animals did not recover after malaria infection. There were 254 operational taxonomic units (OTUs) across all three stages, and there were unique strains or OTUs at each stage of the experiment. The percentages of community abundance of 8 OTUs changed significantly (P<0.05). The dominant OTU in both the healthy mice and the mice with malaria was OTU265, while that in the cured mice was OTU234. In addition, the changes in OTU147 were the most noteworthy. Its percentage of community abundance varied greatly, with higher values during malaria than before malaria infection and after malaria elimination. These results indicated that the external environment influenced the gut microbiota after host C57BL/6 mice were infected with blood-stage P. berghei ANKA and that the same was true during and after elimination of blood-stage P. berghei ANKA. In addition, we could not isolate OTU147 for further study. This study identified gut microbiota components that were reconstructed after infection by and elimination of blood-stage P. berghei ANKA in host C57BL/6 mice, and this process was affected by P. berghei ANKA and the external environment of the host.

High mobility group [corrected] box 1 mediates neutrophil recruitment in myocardial ischemia-reperfusion injury through toll like receptor 4-related pathway.

This study aimed to explore the role of high mobility group [corrected] box 1 (HMGB1) and its receptor toll like receptor 4 (TLR4) on neutrophils in myocardial ischemia reperfusion (I/R) injury. We constructed TLR4-mutant (C3H/HeJ) and control (C3H/HeN) mouse models of myocardial I/R injury and subjected the mice to 30 min of ischemia and 6h of reperfusion. Light microscope was used to observe structural changes in the myocardium. HMGB1 levels were measured using quantitative real-time PCR and immunohistochemistry. Neutrophil accumulation, TNF-a expression and IL-8 levels were analyzed via myeloperoxidase (MPO) biochemical studies, quantitative real-time PCR and ELISA, respectively. The results demonstrated that fewer neutrophils infiltrated in the myocardium of TLR4-mutant mice after myocardial I/R and that TLR4 deficiency markedly decreased the ischemic injury caused by ischemia/reperfusion, and inhibited the expression of HMGB1, TNF-a, and IL-8, all of which were up-regulated by ischemia/reperfusion. These findings suggest that HMGB1 plays a central role in recruiting neutrophils during myocardial I/R leading to worsened myocardial I/R injury. This recruitment mechanism is possibly due to its inflammatory and chemokine functions based on the TLR4-dependent pathway.

Mitoxantrone exerts both cytotoxic and immunoregulatory effects on activated microglial cells.

Mitoxantrone (MX) is the most common immunosuppressive drug used in patients with rapidly worsening multiple sclerosis (MS), whose disease is not controlled by ß-interferon or glatiramer acetate. Although MX suppresses antigen-presenting cell (APC) and T-cell function in the periphery, its mechanism of action in the central nervous system (CNS) is not known. Given that MX can cross the disrupted blood-brain barrier, such as in MS patients, we in the present study have tested our hypothesis that MX in the CNS exerts cytotoxic and immunomodulatory effects on microglia, the major CNS-resident APCs that play a crucial role in MS pathogenesis. The cytotoxic effect of MX on microglial cells was determined by MTT and flow cytometry test, whereas the regulatory function was tested with enzyme-linked immunosorbent assay (ELISA) method. Indeed, we have found that MX induced microglial cell death in a dose-dependent manner, and the cell death was mainly from late apoptosis and necrosis. Further, MX induced significantly increased levels of interleukin (IL)-10 production of microglia, whereas IL-23p19 production/expression was significantly suppressed. Thus, our study for the first time demonstrates the immunosuppressive/regulatory effect of MX on microglia, which represents an important mechanism underlying the therapeutic effect of this drug on MS patients.

High-mobility group box 1 exacerbates concanavalin A-induced hepatic injury in mice.

High-mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as an early endogenous alarmin of inflammation following injury and as a late mediator of lethality in sepsis. Although HMGB1 has been implicated in acute lung injury, rheumatoid arthritis, and allograft rejection, its role in T-cell mediated hepatitis remains obscure. Here, we investigated the role and the underlying mechanisms of HMGB1 in concanavalin A (Con A) induced hepatic injury. We demonstrate that high levels of HMGB1 were detected in the necrotic area and in the cytoplasm of hepatocytes after Con A treatment. Administration of exogenous recombinant HMGB1 enhanced Con A-induced hepatitis, while blockade of HMGB1 protected animals from T cell-mediated hepatitis as evidenced by decreased serum transaminase, associated with reduced hepatic necrosis and mortality. Blockade of HMGB1 by a neutralizing antibody inhibited proinflammatory cytokine production, NFκB activity, and the late stage of T/NKT cell activation. These finding thus suggest a pivotal factor of HMGB1 in Con A-induced hepatitis. Blockage of extracellular HMGB1 may represent a novel therapeutic strategy to prevent hepatic injury in T cell-mediated hepatitis.

IL-33 prolongs murine cardiac allograft survival through induction of TH2-type immune deviation.

BACKGROUND: In Th (T helper) 1/Th2 balance in response to signals given during donor antigen presentation, induction of allograft prolongation is more often related to Th2-type than with Th1-type immunity. Here, we examined the effect of interleukin (IL)-33, a novel member of the IL-1 family, on cardiac allograft survival in mice. METHODS: Mice heterotopic cardiac transplants were performed with sequential recipient sacrifice at anticipated time points to examine the immunoregulatory action of IL-33 in recipient mice. RESULTS: In vitro Th1-polarized CD4 T cells did not express ST2L; however, most CD4 T cells became ST2L on repeated stimulation under Th2-polarizing conditions. Similarly, we found that IL-33 was able to enhance the expression of Th2-associated cytokines (IL-5 and IL-13) but not interferon (IFN)-gamma. Treatment of recipient mice with IL-33 results in the improvement of allograft survival (more than 20 days) when compared with phosphate-buffered saline- or glutathione S-transferase-treated groups (all less than 9 days). Intracellular cytokine staining in CD4 splenocytes confirmed an increase in the percentage of IL-4 cells and a decrease in the percentage of IFN-gamma cells in IL-33 treated mice. In addition, IL-33 significantly enhanced the gene expression of Th2-type cytokines IL-4 and IL-5 but suppressed the Th1-type cytokine IFN-gamma mRNA levels in both allograft and recipient spleen. CONCLUSION: These data demonstrate that IL-33 serves as a potent inducer of Th2 immune response and can markedly contribute to the prolongation of cardiac allograft survival.

Ectopic B7-H4-Ig expression attenuates concanavalin A-induced hepatic injury.

Previous studies demonstrate that both membrane B7-H4 and B7-H4-Ig fusion protein could inhibit T-cell responses. In the present study, we explored the potential effect of B7-H4-Ig on liver injury in a hepatitis mouse model induced by concanavalin A (ConA). A B7-H4-Ig construct was introduced into animals by the hydrodynamic gene delivery approach. It was found that ectopic expression of B7-H4-Ig could inhibit ConA-induced elevation of serum levels of ALT and AST, suppress liver necrosis and even mortality of mice. Furthermore, we observed that pretreatment of B7-H4-Ig dramatically decreased serum levels and the expression of mRNA for IL-2, IFN-gamma and IL-4, but increased IL-10 in ConA-treated mice. Our results suggest that B7-H4-Ig may protect animals from liver injury induced by ConA, which could be associated with reduced serum levels for IL-2, IFN-gamma and IL-4 as well as enhanced IL-10 production.

CpG ODN pretreatment attenuates concanavalin A-induced hepatitis in mice.

T cell-mediated hepatic damage plays a key role in the pathogenesis of liver diseases such as autoimmune hepatitis, viral hepatitis and acute liver failure. CpG-containing oligodeoxynucleotides (CpG ODN), a ligand for toll-like receptor (TLR) 9, is widely used as an immunological adjuvant. In the present study, we investigated the effect of CpG ODN on T cell-mediated liver injury in a murine model of concanavalin A (Con A)-induced hepatitis. We found that the aminotransferase level was significantly decreased in CpG ODN pretreated mice and the survival of the mice was markedly prolonged. CpG ODN pretreatment inhibited NF-kappaB DNA binding activity. As a result, the systemic/liver levels of TNF-alpha and IFN-gamma were significantly suppressed. Furthermore, the activation of inflammatory cells was diminished by CpG ODN pretreatment. These results suggest that CpG ODN pretreatment protects the mice from Con A-induced liver injury via inhibiting hepatocyte apoptosis, inflammation and activation of lymphocytes.

B7-H4 transfection prolongs beta-cell graft survival.

B7-H4, a recently discovered member of B7 family, can negatively regulate T cell responses. However, it is not clear whether B7-H4 negatively function in cell transplantation. In this study we investigated the immunosuppressive effect of B7-H4 on beta-cell transplantation. An insulinoma cell line, NIT-1, transfected with B7-H4 (B7-H4-NIT) was established, and transplanted to diabetic C57BL/6 mice by intraperitoneal injection. Proliferation assay of splenocytes in vitro showed that B7-H4-NIT suppressed alloreactive T cell activation. The proportion of IFN-gamma-producing cells in recipient spleen was significantly reduced and the number of Treg cells was upregulated in B7-H4-NIT group compared to the control, EGFP-NIT. The expression of mRNA coding IFN-gamma was lower but that of IL-4 was higher in B7-H4-NIT transplanted recipients than in the control animals. The results of ELISA also revealed the same trends. Diabetic mice reached normalglycemic quickly and gained weight after transplantation of B7-H4-NIT. More importantly, the survival time for recipients transplanted with B7-H4-NIT cells was significantly longer than that with EGFP-NIT cells. These results indicate that B7-H4 transfection prolongs beta-cell graft survival.

Fructose-1,6-diphosphate attenuates acute lung injury induced by lipopolysaccharide in mice.

Fructose-1,6-diphosphate (FDP), a high-energy glycolytic pathway intermediate, is reported to have a salutary effect in endotoxic shock and sepsis, but its underlying mechanism of action in inflammation is incompletely understood. In this study, our aim was to examine the function of FDP on acute lung injury (ALI) induced by lipopolysaccharide (LPS). We found that in vitro pretreatment with FDP remarkably repressed the production of TNF-alpha and IL-6 in murine alveolar macrophages MH-S exposed to LPS. In the mouse model of LPS-induced inflammatory lung injury, intravenous precondition of a single 400 mg/kg dose of FDP resulted in a significant reduction in LPS-mediated extravasation of Evans blue dye albumin, bronchoalveolar lavage leucocyte content, and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Furthermore, histopathologic examination indicated that alveolitis with inflammatory cells infiltration and alveolar hemorrhage in the alveolar space was less severe in the FDP-treated mice than in the mice treated by LPS alone at 24 h. Additionally, pretreatment with FDP markedly decreased the transcription of TNF-alpha, IL-6 and inducible NO synthase (iNOS), and suppressed the nuclear translocation of NF-kappaB in lung tissues in response to LPS challenge. These results thus suggested that FDP plays an anti-inflammatory role in LPS-mediated acute lung injury, possibly through abrogation of NF-kappaB activation.

Heme oxygenase-1 upregulation significantly inhibits TNF-alpha and Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury in mice.

The present study was designed to investigate whether administration of CoPPIX, an HO-1 inducer, could significantly inhibit TNF-alpha and Hmgb1 expression and thus attenuate the acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. Acute lung injury was induced successfully by intratracheal administration of LPS (0.5 mg/kg) in male BALB/c mice. CoPPIX or ZnPPIX (an HO-1 inhibitor) was administered to mice 24 h prior to LPS exposure. It was found that CoPPIX (5, 10 mg/kg, i.p.) caused a significant reduction in the total cells and neutrophils in BALF, a significant reduction in the W/D ratio and EBA leakage at 24 h after LPS challenge. Furthermore, the histopathologic findings indicated that alveolitis with leukocyte infiltration in the alveolar space was less severe in the CoPPIX-treated mice than in the mice treated with LPS alone. In addition, CoPPIX was also believed to have down-regulated the expression of LPS-induced proinflammatory cytokines, including early proinflammatory cytokine TNF-a, and late proinflammatory cytokine Hmgb1. In contrast, no obvious difference was observed between the ZnPPIX group and the LPS group. These findings demonstrate the significant protection of CoPPIX against LPS-induced ALI, and the effect mechanism of CoPPIX was associated with decreasing the expression of TNF-a and Hmgb1.

[Common linear B-cell epitopes in human hepatitis B virus core protein and woodchuck hepatitis virus core protein].

OBJECTIVE: To search for and verify some common B cell epitopes in the core proteins of woodchuck hepatitis virus and human hepatitis B virus. METHODS: Monoclonal antibodies against both core proteins of woodchuck hepatitis virus (WHV) and human hepatitis B virus (HBV) were prepared by inoculating Balb/c mice with denatured recombination WHV and HBV core proteins. ELISA and immunoblotting assays for WHcAg and HBcAg were carried out by using these antibodies. Immunohistochemistry was carried out with liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients. The epitopes were mapped with the mouse mAbs (6D1 and 1H4) by using a panel of 24 16mer overlapping peptides covering the entire WHcAg. The amino acid sequences of WHcAg and HBcAg were compared. RESULTS: Cross-reactions were observed between mAbs (6D1 and 1H4) and WHcAg and between Mabs and HBcAg/HBcAg in ELISA and immunoblotting assay. Liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients could be stained specifically by mAbs. The epitopes were mapped at aa1-8 (6D1) and aa125-140 (1H4) of the core proteins of both WHV and HBV by using ELISA assay. WHcAg and HBcAg share similar amino acids sequences at aa1-8 and aa125-140 respectively. CONCLUSION: The core proteins of woodchuck hepatitis virus and human hepatitis B virus share common linear B cell epitopes which span aa1-8 and aa125-140 respectively.

A limited course of soluble CD83 delays acute cellular rejection of MHC-mismatched mouse skin allografts.

CD83 is a surface marker expressed on matured dendritic cells (DCs). It plays a pivotal role in the mediation of DC/T cell interaction and induction of T-cell activation. Previous studies have suggested that a soluble form of CD83 could suppress DC maturation and inhibit T-cell activation and, as a result, it can prevent paralysis associated with experimental autoimmune encephalomyelitis. Here, we explored its potential effect on allograft rejection in a fully major histocompatibility complex-mismatched murine skin transplantation model. A form of mouse soluble CD83 (CD83-Ig) fused the extracellular domain of murine CD83 with human IgG1alpha Fc tail was purified from transfected COS-7 cell. It was found that the treatment of recipient mice with CD83-Ig significantly delayed allograft rejection. Especially, when T cells originated from recipients treated with CD83-Ig re-stimulated with donor-specific splenocytes, they showed a significant reduced responding capability as compared with that of originated from control recipients. In line with these results, a reduction for serum IFN-gamma and IL-2 and a decreased mRNA expression of IFN-gamma and IL-2 in allograft infiltrated immune cells were also observed. Our results suggest that CD83-Ig could be useful for the treatment of allograft rejection in combination with other therapeutic strategies.

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication.

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

Pretreatment with soluble ST2 reduces warm hepatic ischemia/reperfusion injury.

The interleukin-1 receptor-like protein ST2 exists in both membrane-bound (ST2L) and soluble form (sST2). ST2L has been found to play an important regulatory role in Th2-type immune response, but the function of soluble form of ST2 remains to be elucidated. In this study, we report the protective effect of soluble ST2 on warm hepatic ischemia/reperfusion injury. We constructed a eukaryotic expression plasmid, psST2-Fc, which expresses functional murine soluble ST2-human IgG1 Fc (sST2-Fc) fusion protein. The liver damage after ischemia/reperfusion was significantly attenuated by the expression of this plasmid in vivo. sST2-Fc remarkably inhibited the activation of Kupffer cells and the production of proinflammatory mediators TNF-alpha and IL-6. Furthermore, the levels of TLR4 mRNA and the nuclear translocation of NF-kappaB were also suppressed by pretreatment with sST2-Fc. These results thus identified soluble ST2 as a negative regulator in hepatic I/R injury, possibly via ST2-TLR4 pathway.

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo.

AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with in vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.

Gene therapy using adenoviral vector encoding 4-1BBIg gene significantly prolonged murine cardiac allograft survival.

4-1BB, a member of the tumor necrosis factor (TNF) receptor superfamily, interacts with 4-1BBL expressed on APC and delivers a costimulatory signal for T cell activation and growth. In this study, we investigated the efficacy of an adenoviral vector encoding murine 4-1BB extracellular domain and human IgG1 Fc (Ad4-1BBIg) fusion gene on murine cardiac allograft survival. Abdomen heterotopical heart graft model was performed from Balb/c to C57BL/6 mice. The adenoviral vectors, Ad4-1BBIg or an adenoviral vector containing EGFP gene (AdEGFP), were administered intravenously to recipient animals after cardiac grafting. The cardiac allograft survival was monitored by daily palpation. The serum level of 4-1BBIg and graft histology was assessed. Cytokine profiles in the grafts were detected by RT-PCR. IFN-gamma producing cells in recipient spleen were examined by flow cytometry. 4-1BBIg gene expression was achieved highly level at 72 h after vector injection. The proportion of IFN-gamma producing cells in recipient spleen was significantly reduced after administration of Ad4-1BBIg, compared to the group given AdEGFP or to the untreated control group. Unlike in controls, cardiac allograft expression of mRNA coding for IL-2 and IFN-gamma remained low in the Ad4-1BBIg group. Ad4-1BBIg therapy markedly reduced T cell infiltration into the graft and significantly prolonged recipient survival time (13.5 days), compared to the untreated group (7.5 days) and the AdEGFP-treated group (8.0 days) (P < 0.05). These results indicate that blockade of 4-1BB/4-1BB ligand interactions by Ad4-1BBIg inhibited alloreactive T-cell activation and attenuated T-cell infiltration into the graft, resulting in significant prolongation of murine cardiac allograft survival. Therefore, Ad4-1BBIg may be useful for preventing allograft rejection.

[Clone and expression of murine BTLA extracellular domain gene and its effect on the expression of B7 on dendritic cells].

AIM: To study the regulatory effect of recombinant glutathione S-transferase (GST)-extracellular domain of murine B and T lymphocyte attenuator (mBTLAext) fusion protein GST-mBTLAext on the expression of B7 on murine dendritic cell (DC) line DC2.4. METHODS: cDNA of mBTLA was amplified from total RNA of murine splenocytes by RT-PCR. The recombinant prokaryotic expression vector pGEX-4T-2/mBTLAext was constructed by cloning mBTLAext into pGEX-4T-2 and then transformed into E. coli BL21 (DE3). The fusion protein GST-mBTLAext was expressed under the induction of 1 mmol/L IPTG, and then was extracted from inclusion body and purified through Glutathione Sepharose 4B chromatography column. The fusion protein was added into the culture supernatant of DC2.4 and its effect on the expression of B7-1 and B7-2 on DC2.4 was analyzed by flow cytometry. RESULTS: The recombinant prokaryotic expression vector pGEX-4T-2/mBTLAext was constructed and the fusion protein GST-mBTLAext was expressed successfully. The molecular weight of the fusion protein was 43.0 kDa, determined by SDS-PAGE, which was corresponding to expectation. GST-mBTLAext could up-regulate the expression of B7-1, but didn't alter the expression of B7-2, on DC2.4 in a dose dependent manner. CONCLUSION: BTLA had a regulatory effect on the expression of B7 on DC. It is significant to study the effect of BTLA on the biological behaviour of DC and its molecular mechanism.

[HLA-G1 molecule expressed by ECV304 cells inhibit cytotoxic activity of allogeneic NK cells].

AIM: To study the effect of HLA-G1 molecule expressed by an endothelial cell line (ECV304) on the cytotoxic activity of allogeneic NK cells. METHODS: ECV304 cells were transfected with recombinant plasmid pcDNA3-HLA-G1 by the liposome transfection, and the expressed HLA-G1 on the cell surface was detected by indirect immunofluorescent assay and flow cytometry. The cytotoxic activity of allogeneic NK cells against ECV304 cells was analyzed by the MTT method. RESULTS: HLA-G1 was expressed on the surface of the transfected ECV304 cells. The specific lysis of NK cells against plasmid pcDNA3 transfected ECV304 was (50.6+/-18.1)%, while the specific lysis against pcDNA3-HLA-G1 transfected ECV304 was (29.7+/-11.4)%, which was significantly lower than the former (P<0.001). CONCLUSION: HLA-G1 expressed by the ECV304 cells can inhibit cytotoxicity of allogeneic NK cells.

[Expression of human T-cell immunoglobulin mucin 3(TIM-3) on eukaryotic cells and establishment of stable transfectant cell line].

AIM: To construct eukaryotic expression vector of human T-cell immunoglobulin mucin 3(TIM-3) and transfect mammalian cells to establish stable cell line. METHODS: The whole coding region of TIM-3 was amplified by PCR and inserted into eukaryotic expression vector pIRES2EGFP. The recombinant plasmid was transfected into mammalian cells by Lipofectamine. The expression product was analyzed by flow cytometry and Western blot. The stable transfectant was screened and established by flow cytometry and selective medium. RESULTS: COS-7 and CHO cells were transfected with recombinant plasmid by Lipofectamine. The expression speciality was identified by Flow cytometry and Western blot. The stable transfectant of CHO cell line was established. CONCLUSION: The whole coding region of TIM-3 was successfully subcloned into eukaryotic expression vector and expressed on the surface of mammalian cells. The stable transfectant of CHO cell line was established.