Biomedical subjects

Yuzhang Wu

Publications and source records attributed to Yuzhang Wu.

At least 19 recordsLinked to original sources

PubMed2007

Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization.

Sublytic C5b-9 has been described as a pro-inflammatory mediator that triggers cell activation rather than inducing cell death. Dendritic cells (DC) play a critical role in controlling antigen-specific immune responses. Although DC maturation induced by various stimuli has been well characterized, the role of C5b-9 in DC function has not been described. In this report, we use in vitro assembled functional C5b-9 based on purified distal complement protein to show that DC maturation is promoted by sublytic C5b-9. This was demonstrated by up-regulation of CD83, HLA-antigens and costimulatory molecules, including CD80, D86, B7-H1, B7-H3, B7-H4 and BTLA. In addition, secretion of cytokines such as interleukin (IL)-12 and tumor necrosis factor-alpha was increased while the capacity for antigen uptake (FITC-Dextran and Lucifer Yellow) was reduced in C5b-9-treated DC. Mixed lymphocyte reactions indicated that C5b-9-activated DC acted as stimulators that significantly promoted CD4+ T cell activation and elicited production of cytokines, including interferon-gamma and IL-2. Interestingly, C5b-9-treated DC also orient CD4+ CD45RA+ naïve T cells toward Th1 polarization. Our results are the first to report that DC are potential immunoregulatory targets of C5b-9, suggesting that C5b-9 bridges innate and acquired immunity by inducing DC maturation.

Cell Differentiation↗

PubMed2006

An altered peptide ligand for naïve cytotoxic T lymphocyte epitope of TRP-2(180-188) enhanced immunogenicity.

Tyrosinase-related protein-2 (TRP-2) is a non-mutated melanocyte differentiation antigen. The TRP-2-recognizing CD8(+) T cells can evoke immune responses to melanoma in both humans and mice. Developing epitopes with amino acid replacements in their sequences might improve the low immunogenicity against this 'self' tumor antigen. We designed altered peptide ligands (APLs) of TRP-2((180-188)) (SVYDFFVWL) with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. These APLs were screened for MHC-affinity by affinity prediction plots and molecular dynamics simulation, and analyzed in vitro for stability and binding-affinity to molecular HLA-A*0201. We also investigated the CTLs activities induced by TRP-2 wild-type epitope and the APLs both in vitro in human PBMCs and HLA-A2.1/K(b) transgenic mice. The results indicate that TRP-2 2M analog simultaneously had stronger binding-affinity and a lower dissociation rate to HLA-A*0201, than wild-type peptide. In addition, the analog 2M was superior to other APLs and wild-type epitope in terms of immunological efficacy ex vivo as measured by the ELISPOT assays of IFN-gamma and granzyme B. These results demonstrate that TRP-2 2M is an agonist epitope that can induce anti-tumor immunity superior to its wild-type epitope, and has potential application in peptide-mediated immunotherapy.

Amino Acid Substitution↗

PubMed2006

Expression of small hairpin RNAs for S100A9 used in the protein function research.

To study the function of S100A9 protein in HL-60 cells treated with all-trans retinoic acid (ATRA), we had designed and constructed retroviral vectors for expression of small hairpin RNAs (shRNAs), and silenced S100A9 expression in HL-60 cells treated with ATRA. The silence efficiency of siRNA was detected with RT-PCR and Western blotting. The differentiation of HL-60 was monitored by nitro blue tetrazolium (NBT) reduction experiment. Western blot showed that shRNAs remarkably reduce of S100A9 expression in HL-60 cells when they were induced to differentiation by ATRA. But NBT positive percentage of differentiated HL-60 cells was no remarked change with S100A9 expression. The data indicated S100A9 could be no important action during differentiation of HL-60 treated with ATRA.

Calgranulin B↗

PubMed2006

Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice.

This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can.

Animals↗

PubMed2005

Expression of B7-H1 in inflammatory renal tubular epithelial cells.

BACKGROUND: Renal tubular epithelial cells (TECs) function as antigen-presenting cells (APCs) as they constitutively express MHC-II molecules and have the capacity to present peptide antigen to T cells. Nevertheless, co-stimulatory signals provided by TECs for regulating T cell activation have not been fully characterized. We therefore investigated the expression of B7-H1, a member of the B7 superfamily, on TECs under normal or pathologic conditions in vivo and analyzed the regulation and functional role of it after proinflammatory factors treatment in vitro. METHODS: Immunohistological staining for B7-H1 on cryostat sections of core needle biopsies from patients with different renal diseases was examined. Furthermore, we also detected B7-H1 protein expression on cultured human TECs stimulated by various inflammatory factors and performed TEC/T-cell co-cultured experiment to determine TEC-associated B7-H1 in regulating CD4+ T cell activation as well as antigen presentation. RESULTS: Significant B7-H1 protein was detected in TECs of diseased renal samples. Although the presence of B7-H1 does not have any correlation with clinicopathological variables, marked B7-H1 expression on sections without interstitial inflammation revealed that B7-H1 has some protective function. In vitro, the expression of B7-H1 on TECs was increased after TECs were stimulated with IL-1alpha, LPS, TNF-alpha, IFN-gamma or anti-CD40. Co-cultured experiments revealed that TEC-related B7-H1 was identified as a strong inhibitor of CD4+ T-cell activation as assessed by increased cytokine production (interleukin-2 and interferon-gamma) and expression levels of the T cell activation marker (CD69) in the presence of a neutralizing antibody against B7-H1 (clone MIH1). Interestingly, IL-2 production by C10 T cells after antigen presentation by murine TECs was also enhanced when the B7-H1/PD-1 pathway was interrupted. CONCLUSION: This study clearly shows that B7-H1 is an inducible renal tubular epithelial antigen that inhibits T cell activation. It is speculated that B7-H1/PD-1 pathway might play a role in protecting tubular epithelium from immune-mediated damage and active delivery of the B7-H1 inhibitory signal represents a novel therapeutic strategy in autoimmune renal diseases.

Animals↗

PubMed2005

Administration of MIP-3alpha gene to the tumor following radiation therapy boosts anti-tumor immunity in a murine model of lung carcinoma.

Dendritic cells (DC), the most potent antigen presenting cells (APC), have been shown able to process apoptotic tumor cells and necrotic tumor cells for antigen presentation. Apoptosis and necrosis are the two common final pathways through which the tumors are killed by chemotherapy or radiation therapy. The tumor cells receiving radiation often produce the "danger signal" cytokines such as TNF-alpha and IL-1. Another cytokine MIP-3alpha that is able to attract DC to the tumor site is normally not secreted. We hypothesize that if artificial introduction of a large number of DC to the necrotic tumor site after radiation therapy by transfecting any cells at the tumor site to secrete DC-tropic MIP-3alpha, an anti-tumor immune response would be initiated. C57BL/6J mice bearing a well-known Lewis lung carcinoma are used to assess efficacy of this modality. The plasmid DNA containing pcDNA3.1/MIP-3alpha was injected into the subcutaneous tumors after radiation treatment. We demonstrate a detectable local expression of MIP-3alpha and local accumulation of DC. Tumor infiltrating lymphocytes after the treatment are predominantly CD8+ T-cells with rare CD4+ T-cells. The anti-tumor immune response was also measurable, which contributes at least in part to the finding that the treated mice have smaller tumor and prolonged survival, comparing to the control groups. This study suggests a potential new means of immune modulation and provides us a new concept of immunotherapy of cancer.

Animals↗

PubMed2005

Triptolide inhibits B7-H1 expression on proinflammatory factor activated renal tubular epithelial cells by decreasing NF-kappaB transcription.

Triptolide has been used extensively in China for the treatment of autoimmune diseases and tumor for many centuries. Nevertheless, little is known about its exact immunosuppressive and anti-inflammatory properties. Increasing recognition of the importance of renal tubular epithelial cells (TECs) in renal diseases raises the question whether triptolide can regulate TEC activity. In this study, various cultured human and murine TECs were exposed to tumor necrotic factor-alpha (TNF-alpha) and triptolide, followed to examine the expression of B7-H1 and B7-DC. Flow cytometric analysis revealed that B7-H1 but not B7-DC constitutively expresses on TECs, and the B7-H1 protein expression was profoundly up-regulated by the stimulation of TNF-alpha with a dose-dependent manner. However, triptolide under non-cytotoxic concentration could down-regulate B7-H1 expression on activated TECs at both mRNA and protein level. This effect was transcription factor NF-kappaB dependent. Interestingly, the significant damping effect of triptolide on B7-H1 signal could promote interleukin-2 production by T cell hybridoma (C10) after antigen presentation and enhance cytokine (IFN-gamma and IL-2) secretion by anti-CD3 activated T cells. Our results indicated that triptolide could regulate TEC activity via B7-H1, in addition to previously reported it directly affects the production of some inflammatory factors by T cells, tumor cells and peripheral blood mononuclear cells.

Animals↗

PubMed2005

Comprehensive antibody epitope mapping of the nucleocapsid protein of severe acute respiratory syndrome (SARS) coronavirus: insight into the humoral immunity of SARS.

BACKGROUND: The epidemic outbreak of severe acute respiratory syndrome (SARS) posed a worldwide threat to public health and economic stability. Although the pandemic has been contained, concerns over its recurrence remain. It is essential to identify specific diagnostic agents and antiviral vaccine candidates to fight this highly contagious disease. METHODS: We generated 14 monoclonal antibodies (mAbs) specific to the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein and used these to thoroughly map the N protein antigenic determinants. We identified the immunodominant antigenic sites responsible for the antibodies in sera from SARS patients and antisera from small animals and differentiated the linear from the conformational antibody-combining sites comprising the natural epitopes by use of yeast surface display. RESULTS: We identified 5 conformational and 3 linear epitopes within the entire N protein; 3 conformational and 3 linear epitopes were immunodominant. The antibody responses to the N protein fragments in mammalian sera revealed that 3 regions of the N protein are strong antigenic domains. We expanded the specificity of the N protein epitope and identified 4 novel conformational epitopes (amino acids 1-69, 68-213, 212-341, and 337-422). CONCLUSION: The antigenic structures identified for the SARS-CoV N protein, the epitope-specific mAbs, and the serum antibody profile in SARS patients have potential use in the clinical diagnosis and understanding of the protective immunity to SARS-CoV.

Adult↗

PubMed2005

Identification of leukemia-associated antigens in chronic myeloid leukemia by proteomic analysis.

The immune system plays an important role in the treatment of chronic myeloid leukemia (CML). Identification of leukemia-associated antigens (LAAs) eliciting an immune response in patients is a prerequisite for specific immunotherapy of CML. To identify new LAAs in CML, We utilized a novel approach based serology and proteomics technologies. LAAs were identified by comparing the reactivity of proteins resolved by 2-DE with sera from CML patients and healthy donors. Several new LAAs were identified including alpha enolase, aldolase A, HSP70 protein8, beta-tubulin and tropomyosin isoforms. Although, the functions of these identified proteins in CML need further investigation, the detection of autoantibodies in CML may have value on CML screening, diagnosis, or follow-up. Additionally, identification of LAAs in CML may also be of vital importance in antigen-based immunotherapy.

Adolescent↗

PubMed2005

Induction of specific human primary immune responses to a Semliki Forest virus-based tumor vaccine in a Trimera mouse model.

Recombinant Semliki Forest virus (rSFV) enables high-level, transient expression of heterologous proteins in vivo, and is believed to be a superior vector for genetic vaccination, compared with the conventional DNA plasmid. Nonetheless, the efficacy of rSFV-based vaccine in eliciting human immune responses has not been tested. We used a Trimera mouse model, consisting of lethally irradiated BALB/c host reconstituted with nonobese diabetes/severe combined immunodeficiency (NOD/SCID) bone marrow plus human peripheral blood mononuclear cells (PBMCs), to characterize the in vivo immune responses against rSFV-encoded human melanoma antigen MAGE-3. MAGE-3-specific antibody and cytotoxic T lymphocyte (CTL) activity were detected by ELISA and 51Cr-release assay, respectively, and the responses were compared with those induced by a plasmid DNA vaccine encoding the same antigen. The results showed that rSFV vaccine could elicit human MAGE-3-specific antibody and CTL response in the Trimera mice, and the antitumor responses were more potent than those by plasmid DNA vaccination. This is the first report to evaluate human immune responses to an rSFV-based tumor vaccine in the Trimera mouse model. Our data suggest that rSFV vector is better than DNA plasmid in inducing protective immunity, and the Trimera model may serve as a general tool to evaluate the efficacy of tumor vaccines in eliciting human primary immune response in vivo.

Animals↗

PubMed2005

Cross-presentation of phage particle antigen in MHC class II and endoplasmic reticulum marker-positive compartments.

It has been shown that exogenous antigens can access the MHC class I pathway of professional antigen-processing cells. However, details as to how the MHC class I-peptide complex forms in the presentation pathway are still poorly understood. Here we used MHC class I-peptide-specific antibodies to investigate the formation and intracellular location of class I-peptide complexes in macrophages. We observed that the formation of class I-peptide complexes occurs within a few hours and lasts for another few hours on the cell surface of macrophages following loading with filamentous phage particles. The class I-peptide complexes in the process were co-localized with MHC class II molecules and endocytic system markers. Moreover, endosomal compartments containing class I-peptide complexes were found within intracellular organelles stained by DiOC6 and calnexin. In addition, the cross-presentation of phage particles was transporter associated with antigen processing (TAP)-dependent and sensitive to proteasome inhibitors and NH(4)Cl. These data suggest that endocytosed phage particles may be processed and cross-presented in organelles positive for phagosome and endoplasmic reticulum (ER) markers via a classical ER MHC class I loading mechanism.

Ammonium Chloride↗

PubMed2005

Sinomenine inhibits B7-H1 and B7-DC expression on human renal tubular epithelial cells.

Previous studies have shown that sinomenine possesses potent immunoregulatory properties. However, little is known about its exact mechanisms of action. Increasing recognition of the importance of renal tubular epithelial cells (TECs) in renal diseases raises the question whether sinomenine can regulate TEC activity. In this study, cultured human TECs were exposed to proinflammatory factors interferon-gamma (IFN-gamma) and tumor necrotic factor-alpha (TNF-alpha) in presence or absence of sinomenine for 72 h, followed by analysis of surface expression of costimulatory molecules. Flow cytometric analysis revealed that various costimulatory molecules were expressed on TECs and that they were significantly up-regulated by the simulation of IFN-gamma and TNF-alpha. However, sinomenine especially down-regulated B7-H1 and B7-DC expression on TECs at both mRNA and protein levels. Moreover, the significant damping effect of sinomenine on B7-H1 and B7-DC signals could promote IL-2 and IFN-gamma production by co-cultured CD4(+) T cell. Our results indicated that sinomenine could regulate TECs activity via B7-H1 and B7-DC, in addition to previously reported its effects on some pro-inflammatory factors production by macrophages and peripheral blood mononuclear cells.

Antigens, CD↗

PubMed2005

H-2 Kd-restricted hepatitis B virus-derived epitope whose specific CD8+ T lymphocytes can produce gamma interferon without cytotoxicity.

It is necessary to evaluate the cytokine secretion status of CD8+ T lymphocytes and elucidate the factors influencing cytokine secretion, because the secretion of cytokines is also an important feature of CD8+ T lymphocytes, and the cytokines usually play critical roles in the outcome of diseases. We showed here that peptide AYRPPNAPI, derived from the core antigen of hepatitis B virus (HBV), could bind to H-2 Kd and induce primed splenocytes from HBcAg expression plasmid-immunized mice to produce gamma interferon (IFN-gamma) in H-2 Kd- and CD8-dependent manners instead of in a CD4-dependent manner. The induced cells were mainly CD3 and CD8 positive but had no cytotoxic effect on the corresponding target cells. When administered into HBV transgenic mice, these cells can decrease the serum HBV load without causing liver damage. These results suggest that this peptide is a special kind of CD8+ T-cell epitope, for which specific CD8+ T cells can produce IFN-gamma when antigenic stimulation is encountered but which have no cytotoxic effect on the corresponding target cells both in vitro and in HBV transgenic mice. This phenomenon indicates initially that the functional mechanisms of CD8+ T cells can be determined by their epitope specificity, which may be associated with the development of epitope-based immunotherapeutic approaches for infectious diseases and tumors.

Animals↗

PubMed2005

Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA.

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease caused by a novel coronavirus (SARS-CoV), which spread to over 30 countries in early 2003. Until recently, no specific vaccines and effective drugs have been available to protect patients from infection by this virus. To exploit a new strategy to fight this disease, we investigated the effect of interference RNA (RNAi) on the virus infection and replication with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), plaque-forming, Western-blot and real-time PCR assays. Results showed that the plasmid-derived siRNAs targeting the non-structural protein 1 (NSP1) sequence of the SARS-CoV genome could specifically inhibit the expression of the NSP1 sequence and effectively suppress the replication and propagation of SARS-CoV in cultured Vero E6 cell lines. The expression of the Spike and Nucleoprotein genes of SARS-CoV at mRNA and protein levels in small interfering (si)RNA-expressing cells was significantly less than that in controls when analysed with PCR and Western-blot assays, 3 days post SARS-CoV infection. Our study provides strong evidence that the NSP1 sequence in the SARS-CoV genome is a valid target for RNAi and the effect of the siRNAs probably mainly resulted from effects on global reduction of subgenome synthesis and subsequent protein expression of SARS-CoV.

Animals↗

PubMed2005

Protection of mammalian cells from severe acute respiratory syndrome coronavirus infection by equine neutralizing antibody.

The aetiological agent for severe acute respiratory syndrome (SARS) has been determined to be a new type of coronavirus (SARS-CoV) that infects a wide range of mammalian hosts. Up to now, there have been no specific drugs to protect against SARS-CoV infection, thus developing effective strategies against this newly emerged viral infection warrants urgent efforts. Adoptive immune therapy with pathogen-specific heterologous immunoglobulin has been successfully used to control the dissemination of many viral infections. To investigate whether a neutralizing antibody against SARS-CoV raised in an artiodactylous host can have a protective role on primate cells, we prepared serum IgGs and their pepsin-digested F(ab')2 fragments from horses inoculated with purified SARS-CoV (BJ-01 strain). The protective effect of the F(ab')2 fragments against SARS-CoV infection was determined in cultured Vero E6 cells by cytopathic effect (CPE), MTT and plaque-forming assays and in a Balb/c mouse model by CPE and quantitative RT-PCR. The results showed the neutralization titres of F(ab')2 from three horses all reached at least 1:1600, and 50 microg of the F(ab')2 fragments could completely neutralize 1x10(4) TCID50- SARS-CoV in vivo. Additionally, we observed that F(ab')2, against BJ-01 strain could also protect cells from infection by the variant GZ-01 strain in vitro and in vivo. Our work has provided experimental support for testing the protective equine immunoglobulin in future large primate or human trials.

Animals↗

PubMed2004

Frequencies of epitope-specific cytotoxic T lymphocytes in active chronic viral hepatitis B infection by using MHC class I peptide tetramers.

Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are thought to play key roles in viral control and liver damage. We have used HLA-A*02 tetramer complex to human HBV core 18-27 (Tc 18-27), envelope 183-191 (Te 183-191), envelope 335-343 (Te 335-343), and polymerase 575-583 (Tp 575-583) epitopes to characterize HLA class I-restricted CD8+ T cells in active chronic HBV infection. The frequencies of specific epitopes circulating tetramer+ cells were determined in whole-blood samples by analysis of flow cytometry. The correlation of HBV epitope-specific CTL, between viral replication and liver damage, was analyzed by multiple regression. Our data shown that HBV-specific CD8+ T cells can be easily detected in peripheral blood of active chronic HBV infections. No significant correlation was found between either the frequency of HBV-specific CD8+ T cells and the viral load, or the frequency of HBV-specific CD8+ T cells and the levels of alanine transaminase. These results suggest that the existence of epitope-specific HBV CTLs are not directly correlated to hepatocyte injury, and the frequencies of HBV-specific T cells are not determinant of immune-mediated protection in chronic HBV infection.

Epitopes, T-Lymphocyte↗

PubMed2004

Induction of P815 tumor immunity by DNA-based recombinant Semliki Forest virus or replicon DNA expressing the P1A gene.

AIM: To compare the prophylactic and therapeutic effects of alphaviruses in the same tumor model, we used a DNA-based approach to generate a replicon DNA and recombinant Semliki Forest virus (rSFV) particles expressing P1A, the P815 mastocytoma tumor associated antigen, and compared the immune effect of each vaccine. METHODS: Six to eight-week-old female DBA/2 mice were inoculated with P1A plasmid or viral vaccines. Spleen cells were assayed for antigen-specific cytotoxic T cell activity. Tumor growth or survival rate was observed in preventive and therapeutic experiments, respectively. RESULTS: We found that the rSFV particles prevented tumor growth when delivered prior to innoculation of mice with P815 cells, and more importantly, improved survival when delivered after the initiation of tumor growth. Naked P1A replicon DNA also functioned as a protective and therapeutic vaccine, although with less potency than rSFV particles. Virus particles also elicited a stronger cellular immune response as measured by target cell lysis. CONCLUSION: rSFV particles have stronger specific prophylactic and therapeutic immune effects in mice than replicon DNA-based DNA vaccines, though the latter is more effective than traditional plasmid vectors (e.g. pCI-neo vector).

Animals↗

PubMed2004

Identification of a S100 calcium-binding protein expressed in HL-60 cells treated with all-trans retinoic acid by two-dimensional electrophoresis and mass spectrometry.

To characterize the alteration of protein expression during tumor cell differentiation induced by all-trans retinoic acid (ATRA) and to understand downstream signaling and molecular mechanism of ATRA action, we compared the protein expression profiles in HL-60 cells with ATRA treatment using two-dimensional electrophoresis (2-DE). Although many changes in protein expression were found in 2-DE maps, here we identified two protein spots remarkably expressed in the differentiated cells by nanoelectrospray ionization mass spectrometry and database searching. These two protein spots were found to be the same protein, namely S100 calcium-binding protein A9 (S100A9). Further study will be done to ascertain whether S100A9 plays a role in the regulation of differentiation or just a consequence of differentiation.

Calgranulin B↗