Expression of cytokines in mouse hepatitis B virus X gene-transfected model.

The expression profile in the mouse hepatitis B virus X (HBx)-transfected model was investigated in order to lay a foundation for further study on the implication of cytokines expression in hepatitis B virus (HBV) infection. Hydrodynamic injection method via the tail vein was used to establish the animal HBx-transfected model. By using microassay, the differential expression of gene in each group was analyzed, which was further confirmed by using real-time PCR and semi-quantitative PCR. Most of chemokine genes such as Ccl2, Ccl5, Ccl9, MIG and IP-10 were up-regulated in the HBx-transfected mouse model versus the control mice, which was coincided with the microarray results. Western blotting and immunohistochemistry were applied to detect the expression of MIG and IP-10 in the liver tissues. Simultaneously, ELISA was adopted to measure the content of IFN-γ in the liver tissues. DNA microassay revealed that the expression of 611 genes changed in HBx-transfected mice as compared with that in pCMV-tag2B-transfected mice, and most of the screened chemokines were up-regulated (including MIG and IP-10). Additionally, IFN-γ protein levels were increased by 20.7% (P<0.05) in pCMV-tag2B-HBx-transfected mice as compared with the untreated mice. IFN-γ protein levels were reduced by 53.9% (P<0.05) in pCMV-tag2B-transfected mice as compared with the untreated mice, which was consistent with the up-regulation of MIG and IP-10. It was suggested HBx transfection could induce the expression of MIG and IP-10 in the liver tissues, which might play the roles in HBV-related liver immunity and cytokines-mediated antiviral effect.

Tumor cell-released TLR4 ligands stimulate Gr-1+CD11b+F4/80+ cells to induce apoptosis of activated T cells.

Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

Hepatitis B virus protein X-induced expression of the CXC chemokine IP-10 is mediated through activation of NF-kappaB and increases migration of leukocytes.

Interferon-gamma inducible protein 10 (IP-10) involves inflammatory cell recruitment and cellular immune damage during virus infection. Although an increase of the peripheral IP-10 level is known in HBV-infected patients, the molecular basis of HBV infection inducing IP-10 expression has remained elusive. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IP-10 expression in a dose-dependent manner. Transfection of the HBx-expressing vector into HepG2 cells results in nuclear translocation of NF-kappaB, which directly binds the promoter of IP-10 at positions from -122 to -113, thus facilitating transcription. The addition of the NF-kappaB inhibitor blocks the effect of HBx on IP-10 induction. In parallel, increase of NF-kappaB subunits p65 and p50 in HepG2 cells also augments IP-10 expression. Furthermore, we show that HBx induces activation of NF-kappaB through the TRAF2/TAK1 signaling pathway, leading to up-regulation of IP-10 expression. As a consequence, up-regulation of IP-10 may mediate the migration of peripheral blood leukocytes in a NF-kappaB-dependent manner. In conclusion, we report a novel molecular mechanism of HBV infection inducing IP-10 expression, which involves viral protein HBx affecting NF-kappaB pathway, leading to transactivation of the IP-10 promoter. Our study provides insight into the migration of leukocytes in response to HBV infection, thus causing immune pathological injury of liver.

Transforming growth factor-beta1 upregulates the expression of CXC chemokine receptor 4 (CXCR4) in human breast cancer MCF-7 cells.

AIM: To investigate whether rhTGF-beta1 or a recombinant vector encoding a fusion protein comprising an extracellular domain of TGF-beta receptor II and an IgG Fc fragment) affects the regulation of CXC chemokine receptor 4 (CXCR4) expression in MCF-7 human breast cancer cells. METHODS: MCF-7 breast cancer cells were treated with rhTGF-beta1 or transfected with a recombinant vector, pIRES2-EGFP-TbetaRII-Fc. Expression of CXCR4 in these cells was then analyzed at the mRNA and protein levels by quantitative RT-PCR and flow cytometry assay, respectively. A transwell assay was used to measure the chemotactic response of these cells to SDF-1alpha. RESULTS: CXCR4 mRNA and protein expression were upregulated in TGF-beta1-treated MCF-7 cells. These cells also demonstrated an enhanced chemotactic response to SDF-1alpha. In MCF-7 cells transiently transfected with pIRES2-EGFP-TbetaRII-Fc, a fusion protein named TbetaRII-Fc (approximately 41 kDa) was produced and secreted. In these transfected cells, there was a reduction in CXCR4 expression and in the SDF-1alpha-mediated chemotactic response. CONCLUSION: TGF-beta1 upregulated CXCR4 expression in MCF-7 cells, which subsequently enhanced the SDF-1alpha-induced chemotactic response. The results suggest a link between TGF-beta1 and CXCR4 expression in MCF-7 human breast cancer cells, which may be one of the mechanisms of TGF-beta1-mediated enhancement of metastatic potential in breast cancer cells.

Selection for Anti-transferrin Receptor Bispecific T-cell Engager in Different Molecular Formats.

Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody (BsAb). To choose an ideal format of anti-CD3 × anti-transferrin receptor (TfR) bispecific antibodies for clinical application, we constructed TfR bispecific T-cell engager (BiTE) in two extensively applied formats, including single-chain tandem single-chain variable fragments (scFvs) and double-chain diabodies, and evaluated their functional characterizations in vitro. Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+ HepG2 cells. However, compared to two-chain diabodies, scFvs were more efficient in antigen binding and TfR+ target killing. Furthermore, different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies. Thus, the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.

Transcriptional upregulation of HSP70-2 by HIF-1 in cancer cells in response to hypoxia.

Heat shock protein 70-2 (HSP70-2) can be expressed by cancer cells and act as an important regulator of cancer cell growth and survival. Here, we show the molecular mechanisms by which hypoxia regulate HSP70-2 expression in cancer cells. When cells were subjected to hypoxia (1% O2), the expression of HSP70-2 had a significant increase in cancer cells. Such increase was due to the direct binding of hypoxia-inducible factor to hypoxia-responsive elements (HREs) in the HSP70-2 promoter. By luciferase assays, we demonstrated that the HRE1 at position -446 was essential for transcriptional activation of HSP70-2 promoter under hypoxic conditions. We also demonstrated that HIF-1alpha binds to the HSP70-2 promoter and the binding is specific, as revealed by HIF binding/competition and chromatin immunoprecipitation assays. Consequently, the upregulation of HSP70-2 enhanced the resistance of tumor cells to hypoxia-induced apoptosis. These findings provide a new insight into how tumor cells overcome hypoxic stress and survive, and also disclose a new regulatory mechanism of HSP70-2 expression in tumor cells.

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells.

AIM: Somatostatin receptor subtype 2 (SSTR2) is the principal mediator of somatostatin's (SST) antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved. METHODS: SSTR2 expression levels were determined by qRT-PCR in several tumor cell lines. Then, a plasmid pIRES2-EGFP-SSTR2 (pSIG) was constructed and stably transfected into MCF-7 cells (MCF-7/pSIG). After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor (EGFR) expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT. RESULTS: The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G(1)/S cell cycle arrest. Furthermore, the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSIG cells. CONCLUSION: Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G(1)/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.Acta Pharmacologica Sinica (2009) 30: 1053-1059; doi: 10.1038/aps.2009.59.

Rapid and sensitive detection of the vanA resistance gene from clinical Enterococcus faecium and Enterococcus faecalis isolates by loop-mediated isothermal amplification.

OBJECTIVES: Vancomycin resistance in Enterococcus spp., mediated mainly by the vanA resistance gene, has become a major health concern as it has spread worldwide. Therefore, a rapid method is urgently required to detect the vanA gene for timely and appropriate antimicrobial control of resistant Enterococcus infections. METHODS: The loop-mediated isothermal amplification (LAMP) assay was optimised for vanA detection in Enterococcus spp. isolates. RESULTS: The LAMP primer set designed in this study could reliably recognise seven distinct regions of the vanA gene and amplify the gene within 25min at an isothermal temperature of 65°C with high specificity. The sensitivity of the optimised assay was high, with a detection limit for vanA as low as 100pg/µL, which is 100-fold more sensitive than the PCR assay. A special advantage of this optimised LAMP method is that the vanA gene could be detected directly from clinical specimens. CONCLUSION: This optimised LAMP assay has great application potential for efficient detection of vanA in clinical diagnosis and epidemiological studies.

CTLA4-Ig-modified dendritic cells inhibit lymphocyte-mediated alloimmune responses and prolong the islet graft survival in mice.

The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In this study, we transfected CTLA4-Ig gene into dendritic cells (DCs), and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on pancreatic islet allograft in mice. An IDDM C57BL/6 murine model induced by streptozotocin is as model mouse. The model mice were transplanted of the islet cells isolated from the BALB/c mice to their kidney capsules, and injected of CTLA4-Ig modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation and induce its apoptosis; whereas, unmodified DCs (umDCs) promoted the murine lymphocyte proliferation. Compared with injection of umDCs and IgG1 modified DCs, the injection of mDCs prolonged IDDM mice's allograft survival, and normalized their plasma glucose (PG) levels within 3 days and maintained over 2 weeks. The level of IFN-gamma was lower and the level of IL-4 was higher in mDCs treated recipient mice than that in control mice, it indicated that mDCs led to Th1/Th2 deviation. After 7 days of islet transplantation, HE stain of the renal specimens showed that the islets and kidneys were intact in structure, and islet cells numbers are increased in mDCs treated mice. Our studies suggest that DCs expressing CTLA4-Ig fusion protein can induce the immune tolerance to islet graft and prolong the allograft survival through the inhibition of T cell proliferation in allogeneic mice.

An anti-transferrin receptor antibody enhanced the growth inhibitory effects of chemotherapeutic drugs on human non-hematopoietic tumor cells.

Transferrin receptor (TfR) has been used as a target for antibody-based therapy of cancer. Combining anti-TfR antibodies with chemotherapeutic drugs shows potential as one of the strategies for cancer therapy. In this study, we investigated the effects of anti-TfR monoclonal antibody 7579 alone or in combination with chemotherapeutic drugs (5-fluorouracil or doxorubicin) on non-hematopoietic tumor cells (HepG2 and MCF-7) in vitro. We found that 7579 mAb alone could dramatically down-regulate surface TfR expression on tumor cells. Consequently, marked S phase arrest and apoptosis were observed in 7579 mAb-treated tumor cells. In combination with 5-fluorouracil or doxorubicin, 7579 mAb enhanced the growth inhibitory effects of chemotherapeutic drugs on tumor cells. Results of 7AAD/Annexin V staining demonstrated that 7579 mAb enhanced the cytotoxic effects of chemotherapeutic drugs on tumor cells by mainly promoting tumor cell necrosis. Using the median-effect/combination-index isobologram method, we further evaluated the nature of 7579 mAb/chemotherapeutic drug interactions. Synergistic interaction was observed for 7579 mAb combined with 5-fluorouracil whereas additive efficacy was observed for 7579 mAb plus doxorubicin. Our study provided the basis to further develop 7579 mAb-containing chemoimmunotherapy for non-hematopoietic malignancies.

Uric acid enhances T cell immune responses to hepatitis B surface antigen-pulsed-dendritic cells in mice.

AIM: To study the induction of T cellular immune responses in BALB/c mice immunized with uric acid and dendritic cells (DCs) pulsed with hepatitis B virus surface antigen (HBsAg). METHODS: DCs were generated from bone-marrow cells of BABL/c mice, and then pulsed or unpulsed with HBsAg protein (HBsAg-pulsed-DCs or unpulsed-DCs) in vitro. BABL/c mice were immunized with HBsAg-pulsed-DCs (1 x 10(6)) and uric acid, injected through the tail vein of each mouse. The mice in control groups were immunized with HBsAg-pulsed-DCs alone, unpulsed-DCs alone or 200 microg uric acid alone or PBS alone. The immunization was repeated 7 d later. Cytotoxic T lymphocytes (CTLs) in vivo were determined by the CFSE labeled spleen lysis assay. Spleen cells or spleen T cells were isolated, and re-stimulated in vitro with HBsAg for 120 h or 72 h. Production of IFN-gamma and IL-4 secreted by spleen cells were determined by ELISA method; proliferation of spleen T cells were detected by flow cytometry. RESULTS: The cytotoxicities of HBsAg-specific-CTLs, generated after immunization of HBsAg-pulsed-DCs and uric acid, were 68.63% +/- 11.32% and significantly stronger than that in the control groups (P < 0.01). Compared with control groups, in mice treated with uric acid and HBsAg-pulsed-DCs, the spleen T cell proliferation to HBsAg re-stimulation was stronger (1.34 +/- 0.093 vs 1.081 +/- 0.028, P < 0.01), the level of IFN-gamma secreted by splenocytes was higher (266.575 +/- 51.323 vs 135.223 +/- 32.563, P < 0.01) , and IL-4 level was lower (22.385 +/- 2.252 vs 40.598 +/- 4.218, P < 0.01). CONCLUSION: Uric acid can strongly enhance T cell immune responses induced by HBsAg-pulsed-DCs vaccine. Uric acid may serve as an effective adjuvant of DC vaccine against HBV infection.

Roles of TGF-ß signaling pathway in endoplasmic reticulum stress in endothelial cells stimulated with cigarette smoke extract.

To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress (ER stress) in endothelial cells stimulated with cigarette smoke extract (CSE). Human umbilical vein endothelial cells (HUVECs) were cultured and divided into 3 groups: CSE-stimulated group, CSE-stimulated with 4-PBA group, and negative control group. HUVECs were cultured and stimulated with CSE at concentrations of 5%, 10% and 20%, respectively, mRNA of CXCL-8 and GRP78 was detected by real-time PCR. ELISA was performed to test the expression of CXCL-8 protein, and neutrophils migration was detected by Transwell board test. The NF-κB, ERK, p38MAPK and transforming growth factor beta (TGF-ß) were detected by flow cytometry. The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs (P<0.05). Furthermore, it was concentration-dependent. 4-PBA significantly reduced the expression of CXCL-8 protein (P<0.05) and neutrophil migration (P<0.05). The TGF-ß, rather than the NF-κB, ERK and P38MAPK pathway might be involved in ER stress stimulated by CSE. CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells. ER stress might play a role in the effect of neutrophils migration stimulated with CSE, and TGF-ß pathway may contribute to the ER stress in HUVECs.

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase.

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase (AP). METHODS: The VH-linker-VL, namely scFv gene, was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by Sfi I and Not I, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and Not I restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

[The construction of transferrin receptor- mediated HSV-TK gene transfer system and its effect on human hepatocellular carcinoma cells in vitro].

OBJECTIVE: To construct the localization system involving anti-TfR monoclonal antibody (McAb) and AFP promoters and assess its effect on human hepatoma cell lines. METHODS: The conjugate of anti-TfR McAb and polylysine (PLL) was made by SPDP and purified by molecular screen chromatography. DNA blocking test determined that the ratio of one pEBAF/tk to six Ab-PLL was the most suitable to couple them. The pEBAF/tk recombinant plasmid bearing HSV-TK gene was coupled to Ab-PLL by noncovalent bond. The pEBAF/tk was transferred into human hepatoma cell line HepG2, SMMC7721 and pulmonary cancer cell line A549 by receptor-mediated gene delivery (Ab-PLL-DNA) and liposome procedure. The growth inhibitory rates of HepG2, SMMC7721 and A549 cells were measured by MTT assay. RESULTS: The inhibitory rates of HepG2/tk in 100 mg/L and 1 mg/L of GCV were 60.5% and 24.3%, respectively. The inhibitory rate of GCV to SMMC7721 was 23.2% in 3 days. The pulmonary cancer cell A549, A549/tk (Ab) and A549 /tk (lipo) could not be inhibited by the addition of GCV. CONCLUSION: The localization system employed in this paper has high specificity, effectiveness and safety for gene therapy. It would be a promising strategy for gene therapy.

Interleukin(IL)-4 promotion of CXCL-8 gene transcription is mediated by ERK1/2 pathway in human pulmonary artery endothelial cells.

Interleukin-4 is central to allergic pulmonary inflammatory responses, but its contribution to airway neutrophilia remains controversial. The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. However, its response to IL-4 is reported to either increase or decrease the production of neutrophil chemotactic factors. We hypothesized that these conflicting findings may be due to the tissue and the size of the vessels from which endothelial cells have been derived. The expression of CXCL-8 by human primary culture umbilical veins endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and human pulmonary microvascular endothelial cells (HPMECs) when stimulated with recombinant human IL-4 (rhIL-4) was studied. The chemoattractant property of the cells' supernatants for neutrophils was evaluated using Boyden chambers. The role of the nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPK) in IL-4-induced HPAECs was studied using Western blotting and electrophoretic mobility shift assay (EMSA). We demonstrated that IL-4 increased the mRNA expression and the protein production of CXCL-8 in HPAECs, but not in HUVECs and HPMECs. The supernatants of HAPECs stimulated by IL-4 significantly promoted neutrophils migration in a dose-dependent manner, and was significantly attenuated by an inhibitor of CXCL-8. We also found that extracellular-regulated protein kinase1/2 (ERK1/2) is activated by IL-4 in HPAECs, but not JUN-N-terminal protein kinase (JNK) or p38 MAPK pathway. Furthermore, NF-κB-DNA binding activity, phosphorylation of IκBα and p65 levels were not affected by rhIL-4 in HAPECs. These findings indicate marked functional differences in the response of micro and macro-ECs to IL-4. ERK1/2, rather than NF-κB, JNK and p38 MAPK signaling, plays a role in IL-4 induced chemokine activation. Our results suggest that inhibition of ERK1/2 may be a possible target for airway neutrophilia in allergic lung diseases.

CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis.

Single-repeat R3 MYB transcription factors like CPC (CAPRICE) are known to play roles in developmental processes such as root hair differentiation and trichome initiation. However, none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis. We show here that CPC is a negative regulator of anthocyanin biosynthesis. In the process of using CPC to test GAL4-dependent driver lines, we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression. We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs. Rather, CPC expression level tightly controls anthocyanin accumulation. Microarray analysis on the whole genome showed that, of 37 000 features tested, 85 genes are repressed greater than three-fold by CPC overexpression. Of these 85, seven are late anthocyanin biosynthesis genes. Also, anthocyanin synthesis genes were shown to be down-regulated in 35S::CPC overexpression plants. Transient expression results suggest that CPC competes with the R2R3-MYB transcription factor PAP1/2, which is an activator of anthocyanin biosynthesis genes. This report adds anthocyanin biosynthesis to the set of programs that are under CPC control, indicating that this regulator is not only for developmental programs (e.g. root hairs, trichomes), but can influence anthocyanin pigment synthesis.

Engineering and characterization of a baculovirus-expressed mouse/human chimeric antibody against transferrin receptor.

Transferrin receptor (TfR) has been explored as a target for antibody-based therapy of cancer. In the previous study, we reported a murine anti-TfR monoclonal antibody (mAb) 7579 had good anti-tumor activities in vitro. In an attempt to reduce its immunogenicity and enhance its ability to recruit immune effector mechanism in vivo, we herein developed its chimera in the baculovirus/insect cell expression system based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The chimeric light and heavy chains, containing human IgG1 constant regions, were correctly processed and assembled in insect cells, and then secreted into the mediums as heterodimeric H(2)L(2) immunoglobulins. Furthermore, analyses of antigen-binding assay and competitive binding assay indicated that the chimeric antibody possessed specificity and affinity similar to that of its parental murine antibody. Results of the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assay verified that the chimeric antibody could efficiently mediate ADCC and CDC against TfR-overexpressing tumor cells. These results suggested that this baculovirus-expressed chimeric anti-TfR IgG1 might have the potential to be used for cancer immunotherapy. Meanwhile, the MAGIC strategy, facilitating the rapid generation of chimeric mAbs, could be one of the efficient strategies for antibody engineering.

HBx protein induces expression of MIG and increases migration of leukocytes through activation of NF-kappaB.

Elevated expression of monokine induced by the interferon-gamma (MIG) has been shown in HBV carriers, and it is involved in the infiltration of inflammatory cells and liver damage after HBV infection. However, the molecular mechanisms by which HBV-induced MIG expression have not been characterized. Our results indicated that HBx protein induced MIG expression in a dose-dependent manner. Such increase was due to the direct binding of NF-kappaB to the MIG promoter. By luciferase, chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that the NF-kappaB binding site at positions -147 was essential for transcriptional activation of MIG promoter by HBx protein. Chemotaxis assay showed that the up-regulation of MIG protein levels enhanced the migration of peripheral blood lymphocytes (PBLs), and inhibition of NF-kappaB significantly decreased the chemotaxis activity. Our findings provide a new insight into how leukocytes migrate to liver, and disclose a new regulatory mechanism of MIG expression after HBV infection.