Cardiac-specific renalase overexpression alleviates CKD-induced pathological cardiac remodeling in mice.

Introduction: CKD-induced pathological cardiac remodeling is characterized by myocardial hypertrophy and cardiac fibrosis. The available therapeutic options are limited, it is thus urgently needed to identify novel therapeutic targets. Renalase (RNLS) is a newly discovered protein secreted by the kidney and was found beneficial in many renal diseases. But whether it exerts protective effects on cardiac remodeling in CKD remains unclear. Methods: RNLS knockout (KO) and wild-type (WT) mice were both used to build CKD models and the adeno-associated virus (AAV9) system was used to overexpress RNLS cardiac specifically. Echocardiography was performed to detect cardiac structural changes every 6 weeks until 18 weeks post-surgery. High throughput sequencing was performed to understand the underlying mechanisms and the effects of RNLS on cardiac fibroblasts were validated in vitro. Results: Knockout of RNLS aggravated cardiac remodeling in CKD, while RNLS cardiac-specific overexpression significantly reduced left ventricular hypertrophy and cardiac fibrosis induced by CKD. The following RNA-sequencing analysis revealed that RNLS significantly downregulated the extracellular matrix (ECM) receptor interaction pathway, ECM organization, and several ECM-related proteins. GSEA results showed RNLS significantly downregulated several profibrotic biological processes of cardiac fibroblasts which were upregulated by CKD, including fibroblast proliferation, leukocyte migration, antigen presentation, cytokine production, and epithelial-mesenchymal transition (EMT). In vitro, we validated that RNLS reduced the primary cardiac fibroblast proliferation and α-SMA expression stimulated by TGF-ß. Conclusion: In this study, we examined the cardioprotective role of RNLS in CKD-induced cardiac remodeling. RNLS may be a potential therapeutic factor that exerts an anti-fibrotic effect in pathological cardiac remodeling.

Nicotinamide retains Klotho expression and ameliorates rhabdomyolysis-induced acute kidney injury.

OBJECTIVES: Acute kidney injury (AKI) is a severe complication of rhabdomyolysis that significantly increases mortality. Unfortunately, the therapeutic approach is limited. Inflammation plays a critical role in the pathogenesis of rhabdomyolysis-induced AKI, which is a potential therapeutic target. Nicotinamide, a form of vitamin B3 and a precursor of nicotinamide adenine dinucleotide, has been shown to have potent antiinflammation effects. Klotho is a tubular highly expressed renoprotective protein. Therefore, we explored the effect of nicotinamide on rhabdomyolysis-induced AKI and the underlying mechanisms. METHODS: We intramuscularly injected glycerol to induce rhabdomyolysis, and intraperitoneally administrated nicotinamide to observe the effect on kidney injury. Interleukin-1 beta, tumor necrosis factor alpha, nuclear factor kappa B (NF-κB), and Klotho were determined by Western blot. Chromatin immunoprecipitation was used to assess the interaction of NF-κB, nuclear receptor corepressor, and histone deacetylase 1 with Klotho promoters. Small interfering RNA was used to evaluate the role of Klotho in nicotinamide-related renoprotection. RESULTS: The results showed that nicotinamide attenuated renal pathologic morphology, kidney functional abnormalities, and kidney inflammatory response in rhabdomyolysis. Moreover, nicotinamide effectively blocked the recruitment of NF-κB, nuclear receptor corepressor, and histone deacetylase 1 to the promoter of Klotho, and preserved Klotho expression. More importantly, the renoprotection effect of nicotinamide was abrogated when Klotho was knocked down by small interfering RNA in rhabdomyolysis mice. CONCLUSIONS: Our study demonstrated that Klotho preservation is essential for the renoprotection effect of nicotinamide, and provides a new preventive strategy for rhabdomyolysis-induced AKI.

Dl-3-n-butylphthalide pretreatment attenuates renal ischemia/reperfusion injury.

BACKGROUND: Renal ischemia reperfusion injury (IRI) has become a growing concern in clinical practice with high morbidity and mortality rates. There is currently no effective prophylactic regimen available to prevent its occurrence and to improve its clinical prognosis. Dl-3-n-butylphthalide (NBP) has been used for stroke treatment in China for years. Little is known about its role in preventing kidney injury. METHODS: The kidneys of male C57BL/6J mice were subjected to 33 min of ischemia followed by 24 h of reperfusion. NBP was administered by gavage prior to surgery. The reno-protective effect of NBP was evaluated by serum creatinine, kidney injury markers and renal pathological changes. Furthermore, the inflammation, oxidative stress, and apoptosis markers in kidney tissue were examined. In vitro, HK2 cells were treated prophylactically with NBP and then exposed to hypoxia/reoxygenation (H/R). Cell viability and apoptosis related protein were quantified to verify the protective effect of NBP. Pro-inflammation genes expression as well as ROS generation were further investigated also. RESULTS: NBP pretreatment significantly improved renal dysfunction and alleviated pathological injury, renal inflammation response, oxidative stress and cell apoptosis. Consistently, NBP attenuated H/R induced increases in ROS, pro-inflammatory genes expression, apoptosis and cleaved caspase-3 levels in HK2 cells. CONCLUSION: Our promising results validated for the first time that NBP could ameliorate renal IRI via attenuating inflammation, oxidative stress, and apoptosis, which indicated that NBP might be a good candidate against AKI.

Wnt5a promotes renal tubular inflammation in diabetic nephropathy by binding to CD146 through noncanonical Wnt signaling.

Immune and inflammatory factors have emerged as key pathophysiological mechanisms in the progression of diabetic renal injury. Noncanonical Wnt5a signaling plays an essential role in obesity- or diabetes-induced metabolic dysfunction and inflammation, but its explicit molecular mechanisms and biological function in diabetic nephropathy (DN) remain unknown. In this study, we found that the expression of Wnt5a and CD146 in the kidney and the level of soluble form of CD146 (sCD146) in serum and urine samples were upregulated in DN patients compared to controls, and this alteration was correlated with the inflammatory process and progression of renal impairment. Blocking the activation of Wnt5a signaling with the Wnt5a antagonist Box5 prevented JNK phosphorylation and high glucose-induced inflammatory responses in db/db mice and high glucose-treated HK-2 cells. Similar effects were observed by silencing Wnt5a with small-interfering RNA (siRNA) in cultured HK-2 cells. Knockdown of CD146 blocked Wnt5a-induced expression of proinflammatory cytokines and activation of JNK, which suggests that CD146 is essential for the activation of the Wnt5a pathway. Finally, we confirmed that Wnt5a directly interacted with CD146 to activate noncanonical Wnt signaling in HK-2 cells. Taken together, our findings suggest that by directly binding to CD146, Wnt5a-induced noncanonical signaling is a contributing mechanism for renal tubular inflammation in diabetic nephropathy. The concentration of sCD146 in serum and urine could be a potential biomarker to predict renal outcomes in DN patients.

Sitagliptin ameliorates renal tubular injury in diabetic kidney disease via STAT3-dependent mitochondrial homeostasis through SDF-1α/CXCR4 pathway.

Mitochondrial abnormalities play critical roles in diabetic tubular injury progression. Dipeptidyl peptidase-4 (DPP4) inhibitors are widely used antihyperglycemic agents that exert renal protective and positive effects against mitochondrial dysfunction in diabetic kidney disease (DKD). However, their underlying mechanism remains unclear. In this study, DPP4 upregulation, mitochondrial fragmentation, and altered mitochondrial dynamics-associated protein expression were observed in the tubules of DBA2/J (D2) diabetic mice with unilateral nephrectomy and in albumin-stimulated tubular cells. The inhibition of DPP4 by sitagliptin (Sita) ameliorated these mitochondrial perturbations both in vivo and in vitro, whereas DPP4 overexpression aggravated mitochondrial fusion-fission disorder and tubular cell injury in albumin-treated HK-2 cells. Downstream of DPP4, the SDF-1α/CXCR4 pathway was significantly suppressed in diabetic tubules. After Sita treatment, this signaling pathway was restored, and the mitochondrial dynamics was improved. Furthermore, a direct interaction between STAT3 and OPA1 was found in the mitochondria of tubular cells, and this effect was weakened by overloading albumin and by CXCR4 siRNA treatment, suggesting a possible link between DPP4-mediated SDF-1α/CXCR4/STAT3 signaling and mitochondrial dysfunction in diabetic tubular cells. The results suggest that a novel mechanism links the DPP4 enzyme to impaired mitochondrial dynamics homeostasis during tubular injury in DKD and highlight that the SDF-1α/CXCR4/STAT3 signaling pathway could become a potential target for managing DKD.

Comparison of Kidney Transcriptomic Profiles of Early and Advanced Diabetic Nephropathy Reveals Potential New Mechanisms for Disease Progression.

To identify the factors mediating the progression of diabetic nephropathy (DN), we performed RNA sequencing of kidney biopsy samples from patients with early DN, advanced DN, and normal kidney tissue from nephrectomy samples. A set of genes that were upregulated at early but downregulated in late DN were shown to be largely renoprotective, which included genes in the retinoic acid pathway and glucagon-like peptide 1 receptor. Another group of genes that were downregulated at early but highly upregulated in advanced DN consisted mostly of genes associated with kidney disease pathogenesis, such as those related to immune response and fibrosis. Correlation with estimated glomerular filtration rate (eGFR) identified genes in the pathways of iron transport and cell differentiation to be positively associated with eGFR, while those in the immune response and fibrosis pathways were negatively associated. Correlation with various histopathological features also identified the association with the distinct gene ontological pathways. Deconvolution analysis of the RNA sequencing data set indicated a significant increase in monocytes, fibroblasts, and myofibroblasts in advanced DN kidneys. Our study thus provides potential molecular mechanisms for DN progression and association of differential gene expression with the functional and structural changes observed in patients with early and advanced DN.

Ischemic Duration and Frequency Determines AKI-to-CKD Progression Monitored by Dynamic Changes of Tubular Biomarkers in IRI Mice.

Ischemia reperfusion injury (IRI) is one of the most common causes of acute kidney injury (AKI). However, the pathogenesis and biomarkers predicting the progression of IRI-induced AKI to chronic kidney disease (CKD) remain unclear. A side-by-side comparison between different IRI animal models with variable ischemic duration and episodes was performed. The dynamic changes of KIM-1 and NGAL continuously from AKI to CKD phases were studied as well. Short-term duration of ischemia induced mild renal tubule-interstitial injury which was completely reversed at acute phase of kidney injury, while long-term duration of ischemia caused severe tubular damage, cell apoptosis and inflammatory infiltration at early disease stage, leading to permanent chronic kidney fibrosis at the late stage. Repeated attacks of moderate IRI accelerated the progression of AKI to CKD. Different from serum and urine levels of KIM-1 that increased at acute phase of IRI then declined gradually in chronic phase, NGAL increased continuously during AKI-to-CKD transition. Severity and frequency of ischemia injury determines the progression and outcome of ischemia-induced AKI. Inflammation, apoptosis and fibrogenesis likely participate in the progression of AKI to CKD. Both KIM-1 and NGAL enable noninvasive and early detection of AKI, but NGAL is associated better with the process of AKI-to-CKD progression.

Expression of Endothelial Cell Injury Marker Cd146 Correlates with Disease Severity and Predicts the Renal Outcomes in Patients with Diabetic Nephropathy.

BACKGROUND/AIMS: Glomerular endothelial cell injury plays a crucial role in the development of diabetic nephropathy (DN). CD146, an endothelial marker, was shown to increase in chronic kidney disease (CKD), but its role in DN remains unknown. We aim to assess whether CD146 could be used to evaluate disease severity and predict renal outcomes in DN at early stages. METHODS: 159 non-dialysis type 2-DN patients from 2008 to 2015 were enrolled to measure the plasma concentration of soluble CD146 (sCD146). 94 type 2 diabetes mellitus patients without DN and 100 healthy subjects were used as controls. The patients with CKD stage 1-3 were referred as early stage patients. Another independent cohort of 48 patients with biopsy-proved DN was used for the immunohistochemistry study of CD146. Renal outcomes were defined as doubling of serum creatinine, initiation of renal replacement therapy or death. RESULTS: We found that plasma level of sCD146 was upregulated and associated with renal function in DN patients. sCD146 was proved to be a more optimal marker than urine albumin creatinine ratio to evaluate disease severity in these DN patients. The kidney expression of CD146 was co-localized with endothelial marker CD31 and increased in DN. CD146 staining in kidney was correlated with the severity of pathological changes in DN patients. Survival analysis suggested that both plasma and biopsy expression of CD146 were correlated with renal outcomes. CONCLUSIONS: CD146 is associated with kidney injury and could be a good marker to predict renal outcomes in patients with early stages of DN.

Rtn1a-Mediated Endoplasmic Reticulum Stress in Podocyte Injury and Diabetic Nephropathy.

We previously reported a critical role of reticulon (RTN) 1A in mediating endoplasmic reticulum (ER) stress in kidney tubular cells and the expression of RTN1A correlates with the renal function and the severity of kidney injury in patients with diabetic nephropathy (DN). Here, we determined the roles of RTN1A and ER stress in podocyte injury and DN. We used db/db mice with early unilateral nephrectomy (Unx) as a murine model of progressive DN and treated mice with tauroursodeoxycholic acid (TUDCA), a specific inhibitor of ER stress. We found increased expression of RTN1A and ER stress markers in the kidney of db/db-Unx mice. Treatment of TUDCA not only attenuated proteinuria and kidney histological changes, but also ameliorated podocyte and glomeruli injury in diabetic mice, which were associated with reduction of RTN1A and ER stress marker expression in the podocytes of TUDCA-treated mice. In vitro, we showed RTN1A mediates albumin-induced ER stress and apoptosis in human podocytes. A positive feedback loop between RTN1A and CHOP was found leading to an enhanced ER stress in podocytes. Our data suggest that ER stress plays a major role in podocyte injury in DN and RTN1A might be a key regulator of ER stress in podocytes.

Inhibition of Reticulon-1A-Mediated Endoplasmic Reticulum Stress in Early AKI Attenuates Renal Fibrosis Development.

Several animal studies have shown an important role for endoplasmic reticulum (ER) stress in AKI, whereas human studies are lacking. We recently reported that Reticulon-1A (RTN1A) is a key mediator of ER stress and kidney cell injury. Here, we investigated whether modulation of RTN1A expression during AKI contributes to the progression to CKD. In a retrospective study of 51 patients with AKI, increased expression of RTN1A and other ER stress markers were associated with the severity of kidney injury and with progression to CKD. In an inducible tubular cell-specific RTN1A-knockdown mouse model subjected to folic acid nephropathy (FAN) or aristolochic acid nephropathy, reduction of RTN1A expression during the initial stage of AKI attenuated ER stress and kidney cell injury in early stages and renal fibrosis development in later stages. Treatment of wild-type mice with tauroursodeoxycholic acid, an inhibitor of ER stress, after the induction of kidney injury with FA facilitated renoprotection similar to that observed in RTN1A-knockdown mice. Conversely, in transgenic mice with inducible tubular cell-specific overexpression of RTN1A subjected to FAN, induction of RTN1A overexpression aggravated ER stress and renal injury at the early stage and renal fibrosis at the late stage of FAN. Together, our human and mouse data suggest that the RTN1A-mediated ER stress response may be an important determinant in the severity of AKI and maladaptive repair that may promote progression to CKD.

Febuxostat attenuates ER stress mediated kidney injury in a rat model of hyperuricemic nephropathy.

Hyperuricemia contributes to kidney tubular injury and kidney fibrosis. However, the underlying mechanism remains unclear. Here we examined the role of RTN1A, a novel endoplasmic reticulum (ER)-associated protein and ER stress in hyperuricemic nephropathy. We first found the expression of RTN1A and ER stress markers was significantly increased in kidney biopsies of hyperuricemia patients with kidney injury. In a rat model of hyperuricemic nephropathy (HN) established by oral administration of a mixture of adenine and potassium oxonate, increased expression of RTN1A and ER stress was shown in tubular and interstitial compartment of rat kidneys. Treatment of Febuxostat, a new selective inhibitor of xanthine oxidase (XO), not only attenuated renal tubular injury and tubulointerstitial fibrosis, but also reduced uric acid crystals deposition in HN rat kidneys. In vitro, Febuxostat also reduced ER stress and apoptosis in uric acid treated tubular epithelial cells. Our data suggest that RTN1A and ER stress mediate tubular cell injury and kidney fibrosis in HN. Urate-lowering therapy (ULT) with Febuxostat attenuates uric-acid induced ER stress in renal tubular cells and the progression of HN.

Co-delivery of LIGHT expression plasmid enhances humoral and cellular immune responses to HIV-1 Nef in mice.

The immunogenicity and efficacy of a DNA vaccine can be greatly enhanced when a gene adjuvant is used. LIGHT, a member of TNF superfamily, can function as a costimulatory molecule for human naïve T cells to proliferate and can be a potential gene adjuvant. In the current study, the eukaryotic expression plasmid pcDNA-nef was constructed by inserting a full-length nef gene into pcDNA3.1(+), and an in vitro transfection experiment suggested that the nef gene could be expressed successfully in mammalian cells. BALB/c mice were immunized with HIV-1 nef DNA vaccine plasmids alone or in combination with LIGHT expression plasmids, and the specific humoral and cellular immune responses were measured. The data showed that HIV-1 nef DNA vaccine plasmids could induce anti-Nef antibodies, Nef-specific lymphocyte proliferation and CTL activity, whereas stronger specific immune responses were induced in mice when co-immunizing with HIV-1 nef DNA vaccine plasmids and LIGHT expression plasmids, suggesting that the eukaryotic expression vector encoding HIV-1 nef is capable of inducing specific immune responses towards HIV-1 Nef and that LIGHT could be considered as a gene adjuvant for HIV-1 DNA vaccination.

Improvement in efficacy of DNA vaccine encoding HIV-1 Vif by LIGHT gene adjuvant.

DNA vaccine can induce the prolonged immune responses against the encoded antigen with the appropriate adjuvant. To study the immunogenicity of the HIV-1 vif DNA vaccine in inducing the humoral and cellular immune responses and the immunoadjuvant effect of LIGHT, which is a member of TNF superfamily and can stimulate the proliferation of naïve T cells as a co-stimulatory molecule, DNA vaccine plasmid pcDNA-Vif was constructed by inserting HIV-1 vif gene into the downstream of CMV promoter in eukaryotic expression vector pcDNA3.1(+). In vitro expression of HIV-1 Vif in pcDNA-Vif-transfected HeLa cells was confirmed in transcriptional and protein level by RT-PCR and Western blot, respectively. After BALB/c mice were injected muscularly with DNA vaccines for three times, the specific immune responses were analyzed. The data showed that anti-Vif antibody response, Vif-specific T cell proliferation, and CTL activities were induced in the mice that were inoculated with HIV-1 vif DNA vaccine plasmid. Interestingly, stronger humoral and cellular immune responses were detected in mice that were immunized with plasmid pcDNA-Vif and pcDNA-LIGHT together compared to the single immunization with plasmid pcDNA-Vif alone. Together, the results of the study suggest that candidate HIV-1 DNA vaccine can elicit HIV-1 Vif-specific immune responses in mice and that LIGHT plays the role of immunoadjuvant in co-immunization with DNA vaccine.

[Expression of VP0 protein of enterovirus 71 in Escherichia coli and generation of the corresponding polyclonal antibodies in guinea pigs].

AIM: To obtain recombinant VP0 protein of enterovirus 71, and generate the corresponding VP0-specific polyclonal antibodies, for molecular detection and characterization of EV71. METHODS: The VP0 gene was amplified by PCR and cloned into vector pET26b to make pET-VP0 for the prokaryotic expression of VP0. The recombinant VP0 protein was expressed in E.coli BL21 harboring pET-VP0, purified from inclusion bodies, renatured, and subsequently used to immunize guinea pigs. The resultant antisera were evaluated for anti-VP0 titer, binding capacity and specificity by ELISA, immunofluorescence staining and Western blot assays. RESULTS: Recombinant protein VP0 was efficiently produced in E.coli. Immunization of guinea pigs with recombinant VP0 elicited high-titer (1:10(6)) VP0-specific antibodies. Western blot analysis showed the resultant anti-VP0 sera reacted with E.coli-expressed VP0 as well as EV71 propagated in Vero cells. Moreover, the antisera positively recognized EV71 infected cells by immunofluorescence staining. CONCLUSION: The recombinant VP0 and the corresponding polyclonal antibodies can be used to identify and characterize EV71, and therefore represent useful agents and tools for the development of new diagnostic methods and vaccines for EV71.

[Cloning and expression of the extracellular region of human LIGHT gene in E.coli.].

AIM: To construct a recombinant prokaryotic expression vector containing the extracellular region of human LIGHT gene and perform the express in E.coli. METHODS: Total RNA was extracted from human immature bone marrow-derived dendritic cells. The extracellular region of human LIGHT gene was amplified by RT-PCR and cloned into pET32a(+) vector, the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing. After the recombinant plasmid was transformed into E.coli BL21 and induced with IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot. RESULTS: A 543 bp of the extracellular region of human LIGHT gene was obtained and the sequence was confirmed correct by DNA sequencing. SDS-PAGE and Western blot analysis showed that a protein with molecular weight of 41 000 was expressed in E.coli BL21. CONCLUSION: The extracellular region of LIGHT gene is cloned successfully and expressed in E.coli BL21 and the elementary expression conditions were obtained, which lays a basis on the further functional research of LIGHT.

B7-DC-silenced dendritic cells induce stronger anti-HBV immunity in transgenic mice.

Hepatitis B virus (HBV) is a noncytopathic DNA virus and is the pathogen of acute and chronic hepatitis. Interferon and nucleotide analogues such as lamivudine and adefovir are the current treatment strategies of HBV infection; however, it is still a serious disease. Therefore, the development of new therapeutic options against HBV is needed. In the present study, we have investigated whether the vectors carrying short hairpin RNA (shRNA) targeting the murine B7-DC gene could silence the expression of B7-DC and analyzed the function of gene-modified dendritic cells (DCs) by mixed lymphocyte reaction. The results demonstrated that two shRNA vectors efficiently suppressed the expression of B7-DC. The MLR assay showed that shRNA-B7-DC-transfected DCs induced markedly higher allogeneic lymphocyte proliferation than transfected DCs with the vector plasmid pAS and untreated DCs at all dilutions. The most efficient shRNA plasmid vector against B7-DC was then used to silence the expression of B7-DC on DCs, the gene-modified DCs were pulsed with HBV-specific peptides, and HBV transgenic mice were immunized. After three rounds of immunization, the splenocytes were stimulated in vitro and tested for cytotoxicitic T lymphocyte activity, while the sera were used to detect the level of HBsAg and HBV DNA. The data demonstrated that blockade of B7-DC on DCs augmented the cytolytic activity induced by immunization with peptide-pulsed DCs and significantly reduced the concentration of serum HBsAg and HBV DNA, suggesting that silencing of B7-DC is of potential value in DC-based therapy of HBV infection.

Engineering enhancement of the immune response to HBV DNA vaccine in mice by the use of LIGHT gene adjuvant.

DNA vaccines could induce protective immune responses in several animal models. Many strategies have been employed to improve the effect of nucleic acid vaccines. LIGHT is a member of the TNF superfamily and functions as a co-stimulatory molecule for T cell proliferation. In the study, the immunogenicity in the induction of humoral and cellular immune responses by HBV DNA vaccine and the adjuvant effect of LIGHT were studied in a murine model. The eukaryotic expression plasmid pcDNA-L was constructed by inserting mouse LIGHT gene into the vector pcDNA3.1(+). In vitro expression of LIGHT was detected by RT-PCR and indirect immunofluorescence assay in transfected HeLa cells. MLR assay showed that LIGHT-transfected DCs induced markedly higher allogeneic lymphocyte proliferation than pcDNA-transfected DCs and untreated DCs at all dilutions. After BALB/c mice were immunized by three intramuscular injections of the HBV DNA vaccine plasmids alone or in combination with LIGHT expression plasmids, the different levels of anti-HBV immune responses were measured comparable to the control groups immunized with parent plasmid pcDNA or PBS. The HBsAg-specific splenocytes proliferation and specific cytotoxic activities of splenic CTLs in the coinoculation group were both significantly higher than those in the HBV DNA single inoculation group, and an enhancement of antibody response was also observed in the coinoculation group compared with the single inoculation group. Taken together, coimmunization of HBV DNA vaccine plasmids and LIGHT expression plasmids can elicit stronger humoral and cellular immune responses in mice than HBV DNA vaccine plasmids alone, and LIGHT may be an effective immunological adjuvant in HBV DNA vaccination.

[Construction and expression of prokaryotic vector encoding extracellular region of murine B7-DC].

AIM: To construct a prokaryotic expression vector for the extracellular domain of murine B7-DC(B7-DC(ECD)) gene, and to express the gene in E.coli BL21. METHODS: The total RNA was extracted from murine immature bone marrow-derived dendritic cells and the extracellular fragment of B7-DC cDNA was amplified by RT-PCR. The recombinant plasmid pET32a(+)-B7-DC(ECD) was constructed by cloning the extracellular fragment of B7-DC cDNA into the prokaryotic expression vector pET32a(+). After the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing, pET32a(+)-B7-DC(ECD) was transformed into E.coli BL21 through IPTG induction to express the target protein, and the protein was analyzed by SDS-PAGE and Western blot. RESULTS: A 582 bp of extracellular fragment B7-DC cDNA was obtained and the sequence was confirmed right by DNA sequencing. SDS-PAGE and Western blot analysis showed that a protein with molecular weight of 41 000 was expressed in E.coli BL21. CONCLUSION: The extracellular fragment of B7-DC is successfully cloned into pET32a (+) and expressed in E.coli BL21, which lays a foundation for the further functional research of B7-DC.

Therapeutic potential of dendritic cell-based immunization against HBV in transgenic mice.

Hepatitis B virus (HBV) transgenic mice that express HBV envelope proteins represent a model of chronic HBV infection suitable for the development of therapeutic immunization strategies. To address immunologically therapeutic effects induced by peptide-pulsed DCs, HBV transgenic mice were immunized with peptide-pulsed DCs, and the mice were killed after three times of immunization and the splenocytes were stimulated in vitro and detected by IFN-gamma ELISPOT and cytotoxic T lymphocyte (CTL) activity. The data demonstrated that HBV-specific CD8+ T cell response could be induced and CD8+ T cells had specific CTL activity. Furthermore, ELISA and fluorescent quantitative PCR were performed to detect the level of serum HBsAg and HBV DNA and the results demonstrated that HBV-specific peptide-pulsed DCs could significantly reduce the concentration of serum HBsAg and HBV DNA. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured and no significant differences were observed between the different groups, which indicated that no hepatocellular injury occurred. Taken together, the data strongly demonstrated that CD8+ T cell responses and antiviral immunity were elicited in HBV transgenic mice, suggesting that peptide-pulsed DCs could elicit an effective antiviral immunity.