Does chemotherapy augment anti-tumor immunotherapy by preferential impairment of regulatory T cells?

Chemotherapy, the treatment modality of choice for advanced cancers, is considered immunosuppressive due to its depletion of immune cells. Hence, chemotherapy is traditionally thought to adversely affect anti-tumor immune responses and antagonistic to tumor immunotherapy. Contrary to conventional belief, recent studies have shown that combining chemotherapy with immunotherapy resulted in enhanced anti-tumor immunity and improved therapeutic outcome. The mechanisms by which the use of chemotherapy paradoxically benefits immunotherapy await elucidation. CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are a lymphocyte subset which plays a crucial role in inhibiting tumor-reactive effector cell functions and suppressing anti-tumor immunity. We hypothesize that chemotherapy benefits immunotherapy by preferentially impairing Treg, in effect eliminating immunosuppressive elements and augmenting the immune function of anti-tumor effector cells. Clarification of how chemotherapy exerts its immunomodulatory effects will aid in the development of better combination strategies of chemoimmunotherapy in the treatment of cancer.

[Complete sequence determination and phylogenetic analysis of FKN among seven higher primates including homonids and Old World Monkeys].

To obtain full-length FKN nucleotide sequences of homonids including human, chimpanzee, gorilla, orangutan and gibbon, and Old World Monkeys including macaque and leaf monkey and make phylogenetic analysis, three exons of FKN were amplified by degenerated PCR using obtained peripheral blood cells DNA as template which was extracted from homonids and Old World Monkeys. After extracting and purifying from agarose gels, PCR products were sequenced and then spliced by using BioEdit. All the FKN sequences were aligned and compared the percent identity by using DNAStar. The phylogenetic tree was constructed using maximum evolution approach in Mega. The negative selection sites were analyzed by using Datamonkey. There is an apparent 30 bp nucleotides deletion mutation in homonids FKN comparing to that of Old World Monkeys besides other point mutations. Homology of nucleotide sequence between human and chimpanzee, gorilla, orangutan, gibbon, macaque and leaf monkey is 99.2%, 98.4%, 98.1%, 96.5%, 95.9% and 93.8%, respectively. Homology of amino acid sequence of them is 98.5%, 98.0%, 97.7%, 94.7%, 93.7% and 90.5%, respectively. In the same time, the genealogical relationship of human is a lot closer to chimpanzee than it is to gorilla and other apes. It is generally agreed that the evolution rule of FKN gene is in accord with that of the higher primates. In addition, Datamonkey shows that there are 3 negative selection sites 53Q, 84D and 239N in FKN. The full-length FKN gene of human, chimpanzee, gorilla, orangutan, gibbon, macaque and leaf monkey were sequenced successfully, and the FKN sequences analysis lays the foundation for further studying its evolution in immunological function in higher primates and the relation between its structure and function.

[Inoculation of the cells transfected with IP10 inhibited experimental tumor metastases in vivo].

OBJECTIVE: To explore the inhibitory effects of IP10 on the experimental tumor metastases of a mammary carcinoma cell line 4T1 in vivo. METHODS: 4T1 cells were transfected with pcDNA3-IP10 plasmid and the positive clones (IP10-4T1) were screened in the presence of G418. The parental 4T1 cells and 4T1 cells transfected with pcDNA3 (pcDNA3-4T1) were used as controls. The expression of IP10 mRNA was examined with RT-PCR. The chemotactic activity of the cell-cultured supernatant for the activated lymphocytes was assessed with chemotaxis assay. All BALB/c mice were divided into 3 equal groups: IP10-4T1, parental 4T1 and pcDNA3-4T1 (6 mice in each group). Seven days after mice were inoculated with 2 x 10(6) 4T1, pcDNA3-4T1 or IP10-4T1 cells, 1 x 10(5) 4T1 cells were injected into mice in tail vein. On day 14 tumors were sectioned. On day 28 mice were sacrificed and the lung weight, metastatic forci on the lung surface, disseminated metastases in the lungs and the number of clonogenic metastases of 4T1 cells were observed. The splenocytes were isolated from tumor-bearing mice, and the cytotoxicity of the splenocytes was evaluated by CFSE/7-AAD method. RESULTS: The transcription of IP10 mRNA increased in IP10-4T1 cells compared to parental 4T1 and pcDNA3-4T1 cells (P = 0.002). Moreover, accumulated lymphocytes migrated to the supernatants of IP10-4T1 cells, which can be abrogated by anti-CXCR3 (P < 0.05). Compared to controls, inoculation of IP10-4T1 cells resulted in the decreased disseminated metastases as indicated by dramatically reduced lung metastatic forci on the surface of the lungs; the lung weight was lighter (IP10-4T1, 0.27 +/- 0.02 g v.s. 4T1, 0.48 +/- 0.08 g and pcDNA3-4T1, 0.43 +/- 0.16 g, P = 0.021); the number of clonogenic metastatic 4T1 cells enumerated in ten fields decreased greatly (IP10-4T1, 2.6 +/- 1.7 v.s. 4T1, 34 +/- 6.3 and pcDNA3-4T1, 33 +/- 2.3, P < 0.05); histological assay showed that metastasis was not found in the lungs of the 4/6 of mice in IP10-4T1 group, and was seen in all the mice of parental 4T1 and pcDNA3-4T1 groups. The splenocytes from the mice inoculated with IP10-4T1 cells exhibited stronger cytotoxic activity against 4T1 target cells at different ratios of effector versus target cells (3:1, 9:1, 27:1) than controls (P < 0.05). CONCLUSION: IP10 expressed locally could inhibit disseminated metastasis of circulating 4T1 tumor cells by enhancing anti-tumor cellular immune responses.

Heat Shock Protein 47 Promotes Glioma Angiogenesis.

Heat shock protein 47 (HSP47) is a collagen-binding protein, which has been recently found to express in glioma vessels. However, the expression profile of HSP47 in glioma patients and the underlying mechanisms of HSP47 on glioma angiogenesis are not fully explored. In the current study, we found that expression of HSP47 in glioma vessels was correlated with the grades of gliomas. HSP47 knockdown by siRNAs significantly decreased cell viability in vitro and tumor volume in vivo; moreover, it reduced the microvessel density (MVD) by CD31 immunohistochemistry in vivo. HSP47 knockdown significantly inhibited tube formation, invasion and proliferation of human umbilical vein endothelial cells (HUVECs). Furthermore, conditional medium derived from HSP47 knockdown cells significantly inhibited HUVECs tube formation and migration, while it increased chemosensitivity of HUVECs cells to Avastin. Silencing of HSP47 decreased VEGF expression in glioma cells consistently, and reduced glioma vasculature. Furthermore, HSP47 promoted glioma angiogenesis through HIF1α-VEGFR2 signaling. The present study demonstrates that HSP47 promotes glioma angiogenesis and highlights the importance of HSP47 as an attractive therapeutic target of GBM.

Involvement of MicroRNA-210 Demethylation in Steroid-associated Osteonecrosis of the Femoral Head.

Angiogenesis is an important event in steroid-associated osteonecrosis of the femoral head (SONFH). Here we performed miRNA microarray with SONFH tissues (ONs) and the adjacent normal tissues (NLs) to select the angiogenic miRNA. The results showed that miR-210 was differentially expressed in SONFH versus normal tissues. Unexpectedly, its specific transcription factor, hypoxia-inducible factor-1α, was shown of no significant changes in ONs compared with NLs. Further Bisulfite sequencing revealed that miR-210 is embedded in a CpG island and miR-210 gene has 2 CpG sites with lower methylation percentage in ONs compared with NLs. Additionally, ONs with lower miR-210 gene methylation exhibited higher miR-210 expression. Next, we found that the endothelial cells treated with demethylating agents could significantly increase the expression of miR-210, along with promoted cell viability and differentiation. Some angiogenic genes (VEGF, bFGF, TNF-α and PCNA) were up-regulated as well. In addition, the supernatant of the cells after demethylation treatment displayed an enhanced ability of recruiting new microvessels in vivo. Taken together, our study not only provides novel insights into the regulation of angiogenesis in this disease, but also reveals a therapeutic opportunity for treatment of SONFH patients with demethylating agents.

Single B or T-cell epitope-based DNA vaccine using modified vector induces specific immune response against hepadnavirus.

Epitope-based DNA vaccine is an effective and powerful approach against a variety of pathogens or tumors. In present study, we reconstructed a vector that could effectively express short B and T-cell epitope of duck/hepatitis B virus, and investigated the role of the epitope-based DNA vaccination. The pUC19 was modified by inserting the compact transient framework (CTF), including HCMV IE1 promoter, enhancer, Kozak sequence, dual stop codon and 3' terminal bovine growth hormone terminal signal and so on. This modified vector was designated pEC(K) and supposed to effectively express short peptide. A well-defined single B-cell and T-cell epitope encoding gene of duck/hepatitis B virus has been synthesized as candidate epitope and cloned into pEC(K) plasmid, respectively. Transfection of the recombinant DNA into C(2)C(12) cell showed that modified plasmid could effectively express both the single B-cell and T-cell short epitope in the culture supernatant as confirmed by dot immunoblot assay (DIA). The recombinant single B and T-cell epitope-based DNA vaccine was administrated to C57BL/6 mice and could greatly induce specific humoral and CTL response. In addition, the specific antibody against B epitope could specifically bind to the DHBV particles. This report demonstrated that single epitope-based DNA vaccine using modified plasmid vector pEC(K) could induce effective specific immune responses and could be of great use for DNA vaccines.

CXCL16 participates in pathogenesis of immunological liver injury by regulating T lymphocyte infiltration in liver tissue.

AIM: To investigate the role of CXCL16 in the pathogenesis of immunological liver injury and to explore the possible mechanism of T lymphocyte infiltration regulated by CXCL16. METHODS: Immunological liver injury in murine model was induced by Bacille Calmette-Guerin and lipopolysaccharide. Expression pattern and distribution of CXCL16 were examined by real-time quantitative RT-PCR and immunohistochemical analysis. Anti-CXCL16 antibody was administrated in vivo to investigate its effect on T-cell recruitment and acute hepatic necrosis. The survival of murine model was also evaluated. RESULTS: The murine immunological liver injury model was successfully established. CXCL16 expression increased and predominantly distributed in periportal areas and vascular endothelia in injured liver tissues. Administration of anti-CXCL16 Ab protected the mice from death and acute liver damage. Approximately 70% of the mice survived for 72 h in the anti-CXCL16 Ab treatment group, whereas 80% died within 72 h in control Ab group. The number of liver-infiltrating T lymphocytes was significantly reduced from 1.01 x 10(7) to 3.52 x 10(6)/liver, compared with control Ab treatment. CONCLUSION: CXCL16 is involved in immunological liver injury by regulating T lymphocyte infiltration in liver tissue.

[Anti-tumor effect of anti-dsDNA autoantibodies].

OBJECTIVE: To investigate effects of anti-dsDNA autoantibodies on growth of tumor in vitro and in vivo. METHODS: BALB/c mice were inoculated with inactivated tumor cells and challenged s.c. with SP 2/0 and Wehi 164 tumor cells four weeks after the last inoculation. The naïve mice were inoculated with SP 2/0 tumor cells immediately after incubating with sera derived from the immunized mice at week 6. Then the tumor size was examined. In vitro, the cytotoxicity of anti-dsDNA autoantibodies to tumor cells was analysed. Furthermore, apoptosis of SP 2/0 and Wehi 164 tumor cells induced by anti-dsDNA autoantibodies was examined by FACS. RESULTS: In vivo study showed that the growth of SP 2/0 and Wehi 164 tumors were inhibited in mice with anti-dsDNA autoantibodies, but not in mice lack of anti-dsDNA autoantibodies. In vitro, apoptosis of SP 2/0 and Wehi 164 tumor cells was induced when the tumor cells were incubated with the sera containing anti-dsDNA autoantibodies. Statistical analysis showed that the ability of anti-dsDNA autoantibodies to induce apoptosis of SP 2/0 and Wehi 164 tumor cells was significantly correlated with affinity (r = 0.990, P < 0.01 and r = 0.901, P < 0.05). CONCLUSION: Anti-dsDNA autoantibodies have inhibitory effect on tumor cells via inducing apoptosis.

Contribution of C3d-P28 repeats to enhancement of immune responses against HBV-preS2/S induced by gene immunization.

AIM: To investigate whether P28 derived from C3d can enhance the immune response to HBV-preS2/S induced by directly injection of naked plasmids containing variable repeats of P28 and HBV-preS2/S in fusion form. METHODS: One to four copies of C3d-P28 coding gene, amplified by PCR and modified by restriction endonucleases digestion, were subcloned into a eukaryotic expression vector pVAON33 to construct pVAON33-P28, pVAON33-P28.2, pVAON33-P28.3 and pVAON33-P28.4 (pVAON33-P28.[1-4]). HBV-preS2/S coding sequence was then introduced into the pVAON33-P28.[1-4] and identified by both PCR and DNA sequencing. BALB/c mice were primed by intramuscular gene immunization with 100 microg different recombinant plasmids on day 0 and were boosted by subcutaneous inoculation with HBsAg protein (1 microg) 12 wk post-priming. The levels and avidity of specific IgG in sera collected at the indicated times from each group were determined by ELISA and NaSCN-displacement ELISA, respectively. RESULTS: HBsAg specific antibody response was elicited in groups primed with plasmids pVAON33-S2/S-P28.[1-4] and pVAON33-S2/S. However, the response against HBsAg in the groups primed with pVAON33-S2/S-P28.[1-4] was significantly higher than that in pVAON33-S2/S group, the highest level of the specific antibody response was observed in the groups primed with pVAON33-S2/S-P28.4 (P<0.01). After secondary immunization with specific antigen, the acceleration of antibody levels was significantly higher and faster in the mice primed with DNA expressing preS2/S-P28 fusions than that with DNA expressing preS2/S only (P<0.05). Interestingly, mice primed with DNA expressing preS2/S-P28.4 fusions maintained the highest levels of anti-HBs antibodies in all animals. The avidity assay showed that the avidity index (AI) collected at 18 wk from mice primed with pVAON33-S2/S-P28.3 and pVAON33-S2/S-P28.4 were significantly higher than that from preS2/S-DNA vaccinated mice (P<0.01). CONCLUSION: Different repeats of C3d-P28 can enhance both humoral immune response and avidity maturation of specific antibodies induced by gene immunization, in which four copies of C3d-P28 may be necessary to achieve the most modest antibody response.

Differential susceptibility of naïve versus cloned CD4+ T cells to antigen-specific and MHC-restricted anergy induction.

T cell anergy has been successfully induced under different conditions in cloned CD4(+) T cells, but induction of T cell anergy in vivo has been difficult and controversial. Due to the low frequency of naturally occurring T cell population with specificity to a defined antigen, it is very difficult to study anergy of naïve T cells without prior in vivo priming which complicates the interpretation of experimental data. To solve this problem, we adopted the HNT-TCR transgenic mice which have homogeneous antigen specific CD4(+) T cell population. In this study, we generated an influenza virus hemagglutinin (HA) peptide-specific CD4(+) T cell clone from the HNT-TCR transgenic mice and induced anergy using APCs which were treated with the crosslinker, ECDI (1-ethyl-3-3(3-dimethylaminopropyl) carbodiimide). The proliferative response of the cloned or freshly purified naïve CD4(+) transgenic T cells after treatment with ECDI-treated APCs and the HA peptide antigen was monitored as the index of anergy induction. The results showed that anergy was successfully induced in the cloned HNT-TCR transgenic CD4(+) T cells. It was determined that the induced anergy was antigen- and MHC-specific. By contrast, anergy was not observed in freshly purified naïve CD4(+) transgenic T cells under the same conditions. The results suggest that naïve CD4(+) T cells may have different anergy inducing requirements, or that cloned CD4(+) T cells may have certain priming or in vitro cloning artifact which makes them more susceptible to anergy induction. We propose that induction of T cell anergy may depend on the T cell growth, activation and differentiation state or cloning conditions. The results from the present study may have important implications for the study of the mechanism(s) underlying T cell anergy induction in vivo and for applications of immune tolerance based therapy.

[Allogeneic chimerism induced by B7 antisense peptide pre-treated splenocytes prolongs the survival of allograft in mice].

OBJECTIVE: To investigate the roles of B7 antisense peptide (B7AP) in blocking the CD28-B7 pathway and inducing the allogeneic chimerism. METHODS: B7 antisense peptide was synthesized by solid phase synthetic methods and purified with HPLC. The C57BL/6 splenocytes of mice were pre-treated by B7AP, and subsequently injected in travenously to BALB/c mice. Three days later the mice were injected with fresh-made bone marrow cells derived from C57BL/6 mice. The B7 expression and allogeneic chimerism were analyzed with FACS. The lymphocyte proliferation reaction and the mice pinna cardiac transplantation model were exerted to study the relation between chimerism and prolongation of allograft in vitro and in vivo. RESULTS: Lymphoproliferation of the splenocytes derived from BALB/c mice immunized with the B7AP pretreated C57BL/6 splenocytes versus splenocytes from C57BL/6 mice was inhibited dramatically with a inhibition rates up to 43%. Under this condition, the allogeneic chimerism was successfully induced after BMT. Both the chimerism and the survival of allogeneic cardiac grafts were prolonged over 100 days (n = 6). CONCLUSION: Synthetic B7 antisense peptide can induce allogeneic chimerism in mice and consequently prolong the survival of allogeneic cardiac grafts.

Glioma-associated antigen HEATR1 induces functional cytotoxic T lymphocytes in patients with glioma.

A2B5+ glioblastoma (GBM) cells have glioma stem-like cell (GSC) properties that are crucial to chemotherapy resistance and GBM relapse. T-cell-based antigens derived from A2B5+ GBM cells provide important information for immunotherapy. Here, we show that HEAT repeat containing 1 (HEATR1) expression in GBM tissues was significantly higher than that in control brain tissues. Furthermore, HEATR1 expression in A2B5+ U87 cells was higher than that in A2B5-U87 cells (P = 0.016). Six peptides of HEATR1 presented by HLA-A∗02 were selected for testing of their ability to induce T-cell responses in patients with GBM. When peripheral blood mononuclear cells from healthy donors (n = 6) and patients with glioma (n = 33) were stimulated with the peptide mixture, eight patients with malignant gliomas had positive reactivity with a significantly increased number of responding T-cells. The peptides HEATR(1682-690), HEATR(11126-1134), and HEATR(1757-765) had high affinity for binding to HLA-A∗02:01 and a strong capacity to induce CTL response. CTLs against HEATR1 peptides were capable of recognizing and lysing GBM cells and GSCs. These data are the first to demonstrate that HEATR1 could induce specific CTL responses targeting both GBM cells and GSCs, implicating that HEATR1 peptide-based immunotherapy could be a novel promising strategy for treating patients with GBM.

Overexpression of CD88 predicts poor prognosis in non-small-cell lung cancer.

CD88 (C5aR), a G-protein-coupled receptor, is well known as it functions in various inflammatory diseases, however, its role in tumorigenesis remains unclear. In this study we investigated the prognostic value of CD88 in patients with non-small-cell lung cancer (NSCLC) after surgical resection. Five NSCLC cell lines and one normal bronchial epithelial cell line were used to analyze the CD88 expression at the mRNA level. Then, the expression of CD88 and E-cadherin were further examined by immunohistochemistry (IHC) in tissue microarray (TMA) consisting of 208 cases of NSCLSs. Data revealed that CD88 expression was significantly higher in NSCLC cells than that in normal bronchial epithelial cells, and compared with the adjacent non-tumorous lung tissues, the CD88 protein overexpressed in NSCLC tissues. Furthermore, high levels of CD88 were found to be correlated with lymph node metastasis in NSCLC patients (p = 0.012). The 5-year overall survival of patients with CD88(high) was significantly lower than those in the CD88(low) group (p = 0.001), and multivariate analysis revealed that CD88 expression was an independent prognostic factor in patients' overall survival (HR = 1.614, 95% CI 1.082-2.407, p = 0.019). Finally, we confirmed the CD88 expression negatively correlated with E-cadherin expression (p < 0.001). Interference of CD88 expression impaired the migration of lung cancer cells and up-regulated the E-cadherin protein expression. Thus, our results indicate that CD88 is overexpressed in NSCLC. High levels of CD88 are associated with poor prognosis of NSCLC after resection and promote tumor metastasis via down-regulation of E-cadherin. CD88 can be a potential prognostic marker to screen patients for unfavorable prognosis.

Coxsackievirus B3 infection induced viral myocarditis by regulating the expression pattern of chemokines in cardiac myocytes.

Viral myocarditis is a common cardiovascular disease, which has greatly threatened human health. However, up to now, the pathogenesis of viral myocarditis has been unclear, which leads to the lack of its effective treatments. To investigate the role of chemokines in pathogenesis of viral myocarditis, mRNA expression for a panel of 19 chemokines detected by RT-PCR in myocardial tissue of BALB/c mice that were inoculated intraperitoneally with coxsackievirus B3. Moreover primary cultured cardiac myocytes were infected with coxsackievirus B3 following extraction of RNA, from myocytes the expression of 19 chemokines was detected by by RT-PCR. Our results showed that there was much difference in the expression pattern of chemokines in myocardial tissue between infected mice with viral myocarditis and uninfected control mice. The expression of chemokines was varied significantly in clusters in myocardium post coxsackievirus B3 infection. There were also complexity and imbalance in the change of the expression of chemokines. In the meantime, Coxsackievirus B3 infection also influenced the expression pattern of chemokines in cardiac myocytes in vitro. However the expression of monocyte chemoattractant protein-1 alone was upregulated in cardiac myocytes post coxsackievirus B3 infection in the 19 detected chemokines. The chemokine expression pattern changed in complexity and imbalance manner both in myocardium and in primary cultured cardiac myocytes after coxsackievirus B3 infection. Coxsackievirus B3 infection may start viral myocarditis by regulating the expression pattern of chemokines in cardiac myocytes. MCP-1 may be one of key chemokines in the initial stage of viral myocarditis.

The distinct effects of three tandem repeats of C3d in the immune responses against tumor-associated antigen hCGbeta by DNA immunization.

Several examples have shown that C3d3, when fused to a corresponding antigen, had a strong adjuvant effect on certain specific antibody production. In a previous study, we attempted to prove that this was the case of the human chorionic gonadotrophin beta chain (hCGbeta)-induced immunity following DNA vaccination. However, we found that C3d3 when fused to hCGbeta inhibited rather than enhanced the antigen-specific immune response. In the present study, using hCGbeta DNA vaccine preparations, we demonstrated that C3d3 inhibited the antigen-specific humoral antibody response and several other immune responses, such as the hCGbeta specific lymphoproliferation. Such inhibitory effects of C3d3 were not related to the expression level of the target protein, the gender of the test mice, or the vector used. Contrastingly, C3d3 fused with the envelope protein of hepatitis B virus (PreS2/S) used as a control system resulted in the enhancement of both humoral and cell-mediated antigen-specific immune responses against HBV-preS2/S, which was consistent with other groups' adjuvant-effect findings. We further showed that the mechanisms involved in the inhibitory effect of C3d3 might be possible due to impairing the function of antigen presenting B lymphocytes and reducing the expression of transcription factors (T-bet and GATA-3) and cytokine IL-4. Collectively, unlike its usual expected adjuvant function, the fusion of C3d3 with the tumor-associated antigen hCGbeta was found to inhibit both humoral and cell-mediated antigen-specific immune responses. These findings indicate that research concerning tumor immune escapes and vaccine designs require further extensive attention.

[Construction of an eukaryotic expression plasmid for short peptides used in DNA immunization].

AIM: To construct a plasmid which efficiently express short peptides in DNA immunization. METHODS: The plasmid containing peptide-expressing cassette (PEC) was constructed and its effect in DNA immunization was investigated, using a DHBV B cell epitope as the short peptide. The peptide in vitro was detected by DOT-EIA. The BALB/c mice were immunized with the empty plasmid or the recombinant plasmid, and the specific antibodies against the epitope in the sera of the mice were determined by ELISA. RESULTS: The plasmid containing peptide-expressing cassette (PEC) was successfully constructed. Recombinant epitope-based plasmid could efficiently express the short peptide in vitro and induce immune response against it in DNA immunization. CONCLUSION: The constructed vector provides highly efficient short peptide expression in DNA immunization.

[Expression of target gene in eukaryotic cells driven by prokaryotic T7 promoter and its RNA polymerase].

To enhance the efficiency of the expression of target gene in eukaryotic cells, one of the strongest prokaryotic expression systems, the T7 RNA polymerase and T7 promoter, was introduced into eukaryotic cells. A duel-plasmid gene expression system of T7 bacteriophage components was developed; one containing the T7 phage RNA polymerase gene under the control of eukaryotic promoter CMV (pCMV-T7pol) and the other (pT7IRES) containing the T7 promoter and T7 terminator as well as EMCV IRES. To test the feasibility of this plasmid system for eukaryotic expression, hepatitis B virus envelop HBV preS2/S was used to construct pT7IRES-HBs. The target genes were expressed efficiently by the eukaryonized prokaryotic expression system in a variety of the cells indicating C2C12, SP2/0, NIH3T3 and BALB/c 3T3, suggesting the potential applications of the expression system in gene therapy and gene immunization.

Coxsackievirus group B type 3 infection upregulates expression of monocyte chemoattractant protein 1 in cardiac myocytes, which leads to enhanced migration of mononuclear cells in viral myocarditis.

Coxsackievirus group B type 3 (CVB3) is an important cause of viral myocarditis. The infiltration of mononuclear cells into the myocardial tissue is one of the key events in viral myocarditis. Immediately after CVB3 infects the heart, the expression of chemokine(s) by infected myocardial cells may be the first trigger for inflammatory infiltration and immune response. However, it is unknown whether CVB3 can induce the chemokine expression in cardiac myocytes. Monocyte chemoattractant protein 1 (MCP-1) is a potent chemokine that stimulates the migration of mononuclear cells. The objective of the present study was to investigate the effect of CVB3 infection on MCP-1 expression in murine cardiac myocytes and the role of MCP-1 in migration of mononuclear cells in viral myocarditis. Our results showed that the expression of MCP-1 was significantly increased in cardiac myocytes after wild-type CVB3 infection in a time- and dose-dependent manner, which resulted in enhanced migration of mononuclear cells in mice with viral myocarditis. The migration of mononuclear cells was partially abolished by antibodies specific for MCP-1 in vivo and in vitro. Administration of anti-MCP-1 antibody prevented infiltration of mononuclear cells bearing the MCP-1 receptor CCR2 in mice with viral myocarditis. Infection by UV-irradiated CVB3 induced rapid and transient expression of MCP-1 in cardiac myocytes. In conclusion, our results indicate that CVB3 infection stimulates the expression of MCP-1 in myocardial cells, which subsequently leads to migration of mononuclear cells in viral myocarditis.