YAP-galectin-3 signaling mediates endothelial dysfunction in angiotensin II-induced hypertension in mice.

BACKGROUND: Vascular endothelial dysfunction is regarded as an early event of hypertension. Galectin-3 (Gal-3) is known to participate in various pathological processes. Whilst previous studies showed that inhibition of Gal-3 effectively ameliorates angiotensin II (Ang II)-induced atherosclerosis or hypertension, it remains unclear whether Ang II regulates Gal-3 expression and actions in vascular endothelium. METHODS: Using techniques of molecular biology and myograph, we investigated Ang II-mediated changes in Gal-3 expression and activity in thoracic aortas and mesenteric arteries from wild-type and Gal-3 gene deleted (Gal-3-/-) mice and cultured endothelial cells. RESULTS: The serum level of Gal-3 was significantly higher in hypertensive patients or in mice with chronic Ang II-infusion. Ang II infusion to wild-type mice enhanced Gal-3 expression in the aortic and mesenteric arteries, elevated systolic blood pressure and impaired endothelium-dependent relaxation of the thoracic aortas and mesenteric arteries, changes that were abolished in Gal-3-/- mice. In human umbilical vein endothelial cells, Ang II significantly upregulated Gal-3 expression by promoting nuclear localization of Yes-associated protein (YAP) and its interaction with transcription factor Tead1 with enhanced YAP/Tead1 binding to Gal-3 gene promoter region. Furthermore, Gal-3 deletion augmented the bioavailability of nitric oxide, suppressed oxidative stress, and alleviated inflammation in the thoracic aorta of Ang II-infused mice or endothelial cells exposed to Ang II. CONCLUSIONS: Our results demonstrate for the first time that Ang II upregulates Gal-3 expression via increment in YAP nuclear localization in vascular endothelium, and that Gal-3 mediates endothelial dysfunction contributing to the development of hypertension.

AMPK upregulates KCa2.3 channels and ameliorates endothelial dysfunction in diet-induced obese mice.

The opening of endothelial small-conductance calcium-activated potassium channels (KCa2.3) is essential for endothelium-dependent hyperpolarization (EDH), which predominantly occurs in small resistance arteries. Adenosine monophosphate-activated protein kinase (AMPK), an important metabolic regulator, has been implicated in regulating endothelial nitric oxide synthase activity. However, it was unclear whether AMPK regulated endothelial KCa2.3-mediated EDH-type vasodilation. Using bioinformatics analysis and myograph system, we investigated the regulation by AMPK of KCa2.3 in human umbilical vein endothelial cells (HUVECs) or mouse second-order mesenteric resistance arteries. In HUVECs, AMPK activation either by activators (AICAR, A769662 and MK-8722) or expression of the constitutively active form of AMPK significantly upregulated KCa2.3 expression. Such effects were abolished by AMPK inhibitor (compound C) or AMPK α1-/α2-siRNA, extracellular-signal-regulated-kinase 5 (ERK5) inhibitor (ERK5-IN-1), and specific siRNA to myocyte-enhancer factor 2 (MEF2) or krüppel-like factor 2/4 (KLF2/4). KCa2.3 expression was significantly reduced in mesenteric resistance arteries in AMPKα2 knockout mice when compared with littermate control mice. Furthermore, in high-fat diet fed mice, 2-week treatment with AICAR restored endothelial KCa2.3 expression in mesenteric resistance arteries with improved endothelial dysfunction. Our results demonstrate that activation of AMPK upregulates KCa2.3 channel expression through the ERK5-MEF2-KLF2/4 signaling pathway in vascular endothelium, which contributes to benefits through KCa2.3-mediated EDH-type vasodilation in mesenteric resistance arteries.

Gal-3 (Galectin-3) and KCa3.1 Mediate Heterogeneous Cell Coupling and Myocardial Fibrogenesis Driven by ßAR (ß-Adrenoceptor) Activation.

Heart failure is associated with sympatho-ßAR (ß-adrenoceptor) activation and cardiac fibrosis. Gal-3 (galectin-3) and KCa3.1 channels that are upregulated in diverse cells of diseased heart are implicated in mediating myocardial inflammation and fibrosis. It remains unclear whether Gal-3 interacts with KCa3.1 leading to cardiac fibrosis in the setting of ßAR activation. We tested the effect of KCa3.1 blocker TRAM-34 on cardiac fibrosis and inflammation in cardiac-restricted ß2-TG (ß2AR overexpressed transgenic) mice and determined KCa3.1 expression in ß2-TG×Gal-3-/- mouse hearts. Mechanisms of KCa3.1 in mediating Gal-3 induced fibroblast activation were studied ex vivo. Expression of Gal-3 and KCa3.1 was elevated in ß2-TG hearts. Gal-3 gene deletion in ß2-TG mice decreased KCa3.1 expression in inflammatory cells but not in fibroblasts. Treatment of ß2-TG mice with TRAM-34 for 1 or 2 months significantly ameliorated cardiac inflammation and fibrosis and reduced Gal-3 level. In cultured fibroblasts, Gal-3 upregulated KCa3.1 expression and channel currents with enhanced membrane potential and Ca2+ entry through TRPV4 (transient receptor potential V4) and TRPC6 (transient receptor potential C6) channels leading to fibroblast activation. In conclusion, ßAR stimulation promotes Gal-3 production that upregulates KCa3.1 channels in noncardiomyocyte cells and activates KCa3.1 channels in fibroblasts leading to hyperpolarization of membrane potential and Ca2+ entry via TRP channels. Gal-3-KCa3.1 signaling mobilizes diverse cells facilitating regional inflammation and fibroblast activation and hence myocardial fibrosis.

KCa3.1 channel mediates inflammatory signaling of pancreatic ß cells and progression of type 2 diabetes mellitus.

Chronic islet inflammation is associated with development of type 2 diabetes mellitus (T2DM). Intermediate-conductance calcium-activated K+ (KCa3.1) channel plays an important role in inflammatory diseases. However, the role and regulation of KCa3.1 in pancreatic ß cells in progression of T2DM remain unclarified. In the present study, we evaluated the effect of the specific KCa3.1 channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) on diabetic phenotype in the db/db model. In diabetic mice, blockade of KCa3.1 significantly improved glucose tolerance, enhanced secretion of postprandial insulin level, and reduced loss of ß-cell mass through attenuating the expression and secretion of inflammatory mediators. Furthermore, in cultured pancreatic ß cells, exposure to high levels of glucose or palmitic acid significantly increased expression and current density of the KCa3.1 channel as well as secretion of proinflammatory chemokines, and the effects were similarly reversed by preincubation with TRAM-34 or a NF-κB inhibitor pyrrolidinedithiocarbamate. Additionally, expression of KCa3.1 in pancreas islet cells was up-regulated by activation of NF-κB with IL-1ß stimulation. In summary, up-regulated KCa3.1 due to activation of NF-κB pathway leads to pancreatic inflammation via expression and secretion of chemokines and cytokines by pancreatic ß cells, thereby facilitating progression of T2DM.-Pang, Z.-D., Wang, Y., Wang, X.-J., She, G., Ma, X.-Z., Song, Z., Zhao, L.-M., Wang, H.-F., Lai, B.-C., Gou, W., Du, X.-J., Deng, X.-L. KCa3.1 channel mediates inflammatory signaling of pancreatic ß cells and progression of type 2 diabetes mellitus.

KCa3.1 Channels Promote Cardiac Fibrosis Through Mediating Inflammation and Differentiation of Monocytes Into Myofibroblasts in Angiotensin II -Treated Rats.

Background Cardiac fibrosis is a core pathological process associated with heart failure. The recruitment and differentiation of primitive fibroblast precursor cells of bone marrow origin play a critical role in pathological interstitial cardiac fibrosis. The KCa3.1 channels are expressed in both ventricular fibroblasts and circulating mononuclear cells in rats and are upregulated by angiotensin II . We hypothesized that KCa3.1 channels mediate the inflammatory microenvironment in the heart, promoting the infiltrated bone marrow-derived circulating mononuclear cells to differentiate into myofibroblasts, leading to myocardial fibrosis. Methods and Results We established a cardiac fibrosis model in rats by infusing angiotensin II to evaluate the impact of the specific KCa3.1 channel blocker TRAM -34 on cardiac fibrosis. At the same time, mouse CD 4+ T cells and rat circulating mononuclear cells were separated to investigate the underlying mechanism of the TRAM -34 anti-cardiac fibrosis effect. TRAM -34 significantly attenuated cardiac fibrosis and the inflammatory reaction and reduced the number of fibroblast precursor cells and myofibroblasts. Inhibition of KCa3.1 channels suppressed angiotensin II -stimulated expression and secretion of interleukin-4 and interleukin-13 in CD 4+ T cells and interleukin-4- or interleukin-13-induced differentiation of monocytes into fibrocytes. Conclusions KCa3.1 channels facilitate myocardial inflammation and the differentiation of bone marrow-derived monocytes into myofibroblasts in cardiac fibrosis caused by angiotensin II infusion.

[Preparation and identification of type specific and conformation dependent HPV16 L1 protein monoclonal antibody.].

AIM: To generate type specific and conformation dependent monoclonal antibodies against human papillomavirus 16 major capsid L1 protein (HPV16 L1). METHODS: HPV16 L1 protein was expressed by modified insect-baculovirus expression system and purified by ProBond(TM); purification system in nature conditions. BALB/c mice were immunized with the purified HPV16 L1 protein. Hybridoma technique was used to prepare the hybridoma cell lines. Positive colonies were screened by ELISA. The non-denaturing electrophoresis and Western blot methods were used for detecting the conformation dependent antibodies. Immunocytochemistry was used to confirm the type specific of the antibodies. The sub-class, isotype and titer were also identified respectively. RESULTS: One hybridoma cell line named 2E3 was obtained which can constantly secret type-specific and conformation-dependent HPV16 L1 mAb. The titers of the antibody are 1:800 in the cell culture medium and 1:12 800 in ascetic fluid. The sub-class and isotype were IgG1kappa. The antibody could selectively recognize non-denaturing HPV16 L1 protein and there was no cross-reaction with HPV18, HPV58 and HPV11 L1 protein. CONCLUSION: One cell line which secretes type specific and conformation dependent HPV16 L1 monoclonal antibody is successfully generated. This monoclonal antibody can be used in study HPV16 L1 protein epitope.

[Clinicopathological significance of cytotoxic lymphocytes in breast cancer and draining lymph nodes].

OBJECTIVE: To analyze retrospectively the quantity and activation status of the tumor infiltrating cytotoxic lymphocytes in breast cancer and the draining lymph nodes, and its relation to the clinical pathological significance. METHODS: Seventy-four breast cancer samples with their corresponding axillary lymph nodes were histologically typed and staged. Cytotxic lymphocytes were analyzed by immunohistochemistry with the monoclonal antibodies against CD8, CD56, granzyme B and perforin. RESULTS: The number of infiltrating CD8(+) T cells in the cancerous interstitial tissue were much higher than that in the tumor parenchyma. Compared with the metastatic tumor samples, the CD8(+) T cells were more intensive in the primary tumors (35.7 +/- 16.0 vs. 23.7 +/- 9.6). The tumor infiltrating CD8(+) T cells of patients with 5 years survivals were more than that of the dead cases in this follow-up series death (32.9 +/- 14.1 vs. 20.1 +/- 9.9). There was no significant difference of activated tumor infiltrating cytotoxic T cell analyzed by using the activation marker granzyme B(+) and there was also no significant correlation between the intensity of CD8(+), CD56(+) cells and the clinicopathological stages. However, percentages of the activated cytotoxic lymphocytes in Stage I groups were significantly higher than those in stage III and IV. Moreover, the number of perforin(+) cells was significantly less than that of granzyme B(+) cells, particularly in the cancerous tissue, indicating a dysfunctional status of tumor infiltrating cytotoxic lymphocytes. CONCLUSIONS: Activated cytotoxic lymphocytes may play a significant role against the tumor progression and is associated with a favorable prognosis to some extent. However, a putative dysfunctional status of cytotoxic lymphocytes at tumor site may compromise the host immunity against cancer.

Anti-CD3 scFv-B7.1 fusion protein expressed on the surface of HeLa cells provokes potent T-lymphocyte activation and cytotoxicity.

The targeting of tumor cells by cytotoxic T lymphocytes is a promising strategy for biotherapy, but T cells require 2 signals via the T-cell receptor - CD3 complex and CD28 molecules for activation. To bridge the gap between cytotoxic T lymphocytes and tumor cells, our objective in this study was to describe the construction and the cell surface-anchored expression of a fusion protein, anti-CD3 scFv-B7.1, derived from inserting a fusion gene encoding anti-CD3 scFv and the extra-cellular domain of B7.1 fused by the splicing by overlap extension method into a mammalian expression vector, pDisplay. Transfection of the recombinant vector by electroporation into HeLa cells resulted in the production of protein migrating at approximately 57 kDa under reducing conditions. The expressed fusion protein could bind to T lymphocytes and induce strong T-cell activation. Meanwhile, a potent cytotoxicity was induced in the mixed culture of T-cell-modified tumor cells in a 96 h methyl-thiazolyl-diphenyl tetrazolium bromide assay. Our results indicate that this bifunctional protein, through activating T lymphocytes to lyse homologous human carcinomas, may be of potential value for T-cell-based immunotherapeutical treatment protocols in vivo.

[In situ analysis of distribution of immunocompetent cells in tumor's local draining lymph nodes].

AIM: To observe the distribution of immunocompetent cells (ICCs) in tumor's local draining lymph nodes (LDLN) at different stages of disease including non-metastasis, micro-metastasis and late metastasis. METHODS: 71 LDLN from 22 breast cancer, 28 LDLN from 7 gastric carcinoma were analysed by using catalyzed signal amplification (CSA) immunohistochemical staining. Monoclonal antibodies (mAbs) to perforin, granzyme B, CD8, CD56, CD68, S-100, CD134 and CD25 were used to detect the amount and functional change of ICCs. RESULTS: Paracortical hyperplasia and sinus histocytosis was mainly observed in tumor's LDLN. As it was reported, the density of S100(+) dendritic cells (DCs) was decreased as the draining lymph nodes became tumor-containing from the status of tumor free (P<0.05), and the morphology of DCs turned to be inactivated. As the lymph nodes were invaded by tumors, the densities of CD134(+) lymphocytes and CD25(+) cells were significantly increased (P<0.01). Furthermore, there was a significant trend of decreasing in the number of activated cytotoxic T lymphocytes (granzyme B(+) cells) in LDLN as tumor progressed from non/micro-metastasis, early metastasis to advanced stage metastasis (P<0.01). There was no obvious difference in distribution of ICC between non/micro-metastasis sentinel lymph nodes and non-sentinel lymph nodes. CONCLUSION: The dynamic change of the ICCs in LDLN implied that immune response of tumor bearing host tends to be inhibited with the progress of tumors.

[Immunopotentiation of novel adjuvant SWZY on the poorly immunogenic murine melanoma vaccine].

AIM: To evaluate the immunopotentiation of novel adjuvant SWZY on the cancer vaccine of poorly immunogenic melanoma. METHODS: C57BL/6 mice immunized with inactivated D5 melanoma cells were divided into 6 groups: the control without adjuvants, with FCA, FCA+IL-2+GM-CSF, FIA+IL-2+GM-CSF, FIA+SWZY, and FIA+SWZY+IL-2+GM-CSF. Three days after completion of immunization, DTH response, specific killing activity of splenocytes and the level of IFN-gamma and IL-10 in serum and splenocytes' culture supernatant were assayed in half of the mice in each group. The rest were subject to live D5 tumor cell challenge. Three weeks after tumor inoculation, the same immunological parameters were measured. RESULTS: DTH response, splenocytes' killing activity of all the experimental groups were markedly higher than those in the control group (P<0.05), but decreased as the tumor grew larger. The level of IFN-gamma in serum or splenocyte culture supernatant of each experimental group was significantly higher than that of the control (P<0.05) before tumor formation, whereas IL-10 level was lower than that of the control (P<0.05). After tumor formation, the level of IFN-gamma in all groups decreased while that of IL-10 increased in general, but the levels of IFN-gamma and IL-10 in group FCA and FIA+SWZY didn't change very much. CONCLUSION: All kinds of adjuvants can enhance the cell-mediated immune response against poorly immunogenic tumor to some extent. The novel adjuvant SWZY can strengthen immunoresponses similar to FCA. With few harmful side-effect, SWZY might be a promising adjuvant for cancer vaccine.

[The in situ analysis of cytokine mRNA expression in immunological microenvironment about non-small cell lung cancer].

AIM: Using Non-small cell lung cancer (NSCLC) as subject, to explore the characteristics of immune response in immunological microenvironment at the tumor site and its effect on anti-tumor immunity. METHODS: Using in situ hybridization (ISH), the expressions of IL-2, INF-gamma, IL-12 (p40), IL-18, IL-4, IL-10, TGF-beta1, IL-1, IL-3, IL-8, GM-CSF, TNF-alpha and TGF-alpha mRNAs in lymphocytes and tumor cells from five fresh pleural effusion samples and 18 tumor tissue samples of NSCLC patients were detected by in situ hybridization with digoxin end-labeled oligonucleotide probe, using 5 tuberculous pleurisy patients as control. RESULTS: In pleural effusion and tumor tissue of NSCLC patients, the expression levels of IL-4, IL-10, TGF-alpha, and TGF-beta1 mRNAs were significantly higher than those of IL-2, IL-12, IL-18 and INF-gamma mRNAs. In contrast, the analysis of tuberculous pleural effusion samples revealed lower levels of above-mentioned cytokine mRNA. There was no significant difference among expression levels of these cytokine mRNAs. CONCLUSION: The expressions of type II cytokine mRNAs and immunosuppressive cytokine mRNAs in pleural effusion and tumor tissue of NSCLC patients occupied a dominant position, suggesting that the immunological microenvironment about tumor be in an immunosuppressive state. The present study has contributed to a better understanding the mechanisms of tumor escape and provides important experimental basis for working out the scheme for an effective immunomodulatory treatment of NSCLC patients.

B7 molecule mRNA expression in colorectal carcinoma.

AIM: To observe the status of tumor-associated B(7) molecule mRNA expression in human colorectal cancer tissue by in situ hybridization. METHODS: The mRNA expression patterns of cancer-associated B(7-1),B(7)H(1),B(7)H(2),ICOS in 22 specimens of human colorectal cancer tissue were monitored by in situ hybridization (ISH) with digoxin-labeled oligonucleotide probes. RESULTS: B(7-1),B(7)H(1),B(7)H(2),ICOS mRNA were detected in both cancer cells and tumor infiltrating lymphocytes (TIL). The mRNA expression level of these molecules in tumor cells was higher than that in TIL (0.76+/-0.54 - 1.62+/-0.82 vs 0.38+/-0.19 - 0.65+/-0.33, P<0.001). There was no relationship between expression level of tested B(7) family molecules and patients' sex, age, differentiation status of cancer and regional lymph node metastasis. CONCLUSION: Th2 cytokine predominant in tumor microenvironment might be related to the expression of B(7)H(1),B(7)H(2) co-signal molecules in tumor cells and TIL.

Identification of proteins of human colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry.

AIM: To conduct the proteomic analysis of human colorectal carcinoma cell line, SW480 by using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDI-TOFMS). METHODS: The total proteins of human colorectal carcinoma cell line, SW480 were separated with 2-DE by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysis software, Image Master 2D Elite. Nineteen distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis of the tryptic digest peptides mixture was performed by using MALDI-TOF MS. Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF analysis were used to search NCBI, SWISS-PROT and MSDB databases by using Mascot software. RESULTS: PMF maps of all spots were obtained by MALDI-TOF MS and thirteen proteins were preliminarily identified. CONCLUSION: The methods of analysis and identification of protein spots of tumor cells in 2-DE gel with silver staining by MALDI-TOF MS derived PMF have been established. Protein expression profile of SW480 has been obtained. It is demonstrated that a combination of proteomics and cell culture is a useful approach to comprehend the process of colon carcinogenesis.

[Expression of IgVH and B7-1 in proteome of the human colorectal carcinoma cell lines].

OBJECTIVE: To conduct a proteomic analysis of human colorectal carcinoma cell lines LS174T and SW480 by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). METHODS: The total proteins of human colorectal carcinoma cell lines LS174T and SW480 were separated with 2-DE using immobilized pH gradient strips and analyzed by MALDI-TOF-MS to obtain peptide mass fingerprints (PMFs). Proteins were identified by using Mascot software to search protein databases. RESULTS: Good resolution 2-DE maps were obtained. Some proteins including immunoglobulin heavy chain variable region (IgVH) and co-stimulatory molecule B7-1 were identified. IgVH and B7-1 were confirmed by electrospray ionization tandem spectrometry (ESI-MS/MS) and immunocytochemistry. CONCLUSION: There are IgVH and B7-1 expressions in human colorectal carcinoma cell lines LS174T and SW480. Results obtained will help to elucidate the mechanisms of tumor immune escape.

[Study of immune responses induced by human papillomavirus type 18 L1-E6 and L1-E7 chimeric gene DNA vaccines in mice].

AIM: To examine the humoral and cellular immunoresponses induced by HPV18 L1-E6 and L1-E7 chimeric gene DNA vaccines in mice. METHODS: 54 BALB/c mice were divided into 9 groups randomly, and then vaccinated with various recombinant plasmids(pVAX1-L1-E6M3 or pVAX1-L1-E7M3) and immune adjuvants (pLXHDmB7-2 or LTB) through different administration routes (intramuscular or intranasal). After the third inoculation, blood samples were taken to measure specific antibody, and footpad swelling test was used to detect delayed-type hypersensitivity(DTH). Mice were killed and spleens were taken to prepare single spleen cell suspension for lymphocyte proliferation assay and CD4(+)/CD8(+), IFN-gamma(+) or IL-4(+) T cells double staining FACS assay. And then the pathological changes of the swollen footpad were observed. RESULTS: Compared with control, significant immunoresponses were observed in different experimental groups. The level of specific serum IgG against HPV in experiment groups was much higher than that of control group, and intramuscular immunization group had the highest antibody level. The footpad from immunized mice injected with virus-like particles was swollen and harden, and a large amounts of mononuclear cells can be seen in the footpad tissues. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8(+) IFN-gamma(+) cell number, while CD4(+) IL-4(+) cell number was higher in intranasal immunization groups. The immunization groups using pLXHDmB7-2 as adjuvant were superior to other groups in immune responses. CONCLUSION: The recombinant plasmids could induce strong humoral and cellular immunoresponses in mice. Costimulatory molecule B7-2 could enhance the immune responses against HPV18 induced by the L1-E6 and L1-E7 DNA vaccines when used as an adjuvant.

[Eukaryotic expression of anti-CD3 single chain Fv antibody gene and the characterization of its bioactivities].

AIM: To express anti-CD3 scFv in Hela cells and investigate its biological activity. METHODS: DNA fragment encoding anti-CD3 scFv was inserted into eukaryotic expression vector pDisplay. The recombinant expression vector was sequenced and then transfected into Hela cells by electroporation method. The expression of anti-CD3 scFv was identified by in situ hybridization. In-vitro T lymphocyte activation was then detected by (3)H-TdR incoporation method. Anti-CD3 scFv gene-transfected Hela cells were co-cultured with T cells and cytotoxicity was measured by MTT colorimetry. RESULTS: Anti-CD3 scFv gene was correctly inserted into pDisplay and expressed in Hela cells. The secreted anti-CD3 scFv was able to activate T lymphocytes in the presence of anti-CD28 mAb. Cytotoxicity could be observed when anti-CD3 scFv gene-transfected Hela cells were mixed and co-cultured with T lymphocytes. CONCLUSION: Anti-CD3 scFv expressed by Hela cells can activate T lymphocytes.

[Study on immunoregulatory functions of E.coli heat-labil enterotoxin B subunit].

AIM: To explore the immunoregulatory functions of Escherichia coli heat-labil enterotoxin B subunit(LTB) and its possible mechanism. METHODS: The rLTB in engineering bacteria VSP60 was purified by Sephacryl S-100 gel filtration chromatography. BALB/c mice were immunized nasally with hen egg lysozyme(HEL) alone or in combination with various doses of LTB. After three times of immunization, specific anti-HEL IgG and IgA levels in serum and small intestinal secretory fluid were determined by ELISA. The effects of LTB on alloantigen- and ConA- mediated lymphocyte proliferation reactions were measured by (3)H-TdR incorporation assay. RESULTS: Very weak serum anti-HEL IgG response and no IgA response were detected in mice immunized with HEL alone. However, levels of both serum anti-HEL IgG, IgA and intestinal secretory fluid IgA in mice immunized with various doses of HEL+LTB were higher than those in mice immunized with HEL alone(P<0.001). In-vitro, LTB could obviously inhibit the lymphocyte proliferation induced by mitogen and alloantigen. CONCLUSIONS: The effects of LTB protein on immnune system may be bi-directional. LTB is not only a powerful mucocal immunoadjuvant in-vivo, but also a potent immunosuppressive agent In-vitro.

[Construction and expression of eukaryotic expression plasmid pcDNA3.1/hIL-18].

AIM: To construct an eukaryotic expression plasmid pcDNA3.1/hIL-18 and express it in mammalian cells. METHODS: cDNA encoding mature hIL-18 was cleavaged by enzyme digestion from mesomeric clone vector pGEM-T/hIL-18 and inserted into an eukaryotic expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pcDNA3.1/hIL-18. Then the constructed plasmid was transfected into COS-7 and Rlc310 cells by liposome-mediated gene transfer method. hIL-18 expressed in transfected COS-7 and Rlc310 cells was detected by immunohistochemical staining and level of hIL-18 mRNA in transfected Rlc310 cells was assayed by RT-PCR. RESULTS: A recombinant eukaryotic expression plasmid pcDNA3.1/hIL-18 was successfully constructed and expressed transiently in COS-7 cells and stably in Rlc310 cells. CONCLUSION: The construction and expression of pcDNA3.1/hIL-18 have been achieved successfully, which lays a foundation for further research on anti-tumor effect of IL-18.

Expression of Human Single Chain Interleukin 12 Gene in Drosophila Cells.

The hscIL-12 gene(no signal sequence), amplified by PCR, was subcloned into an expressing vector pMT/Bip/V5. The recombinant plasmid was transferedinto Drosophila cell line S2. The secreted hscIL-12 fusion protein was identified by Western blot, and the bioactivity of hscIL-12 was investigated by lymphocyte proliferation assay. The results showed hscIL-12 expressed in S2 cells could stimulate lymphocyte proliferation in dose-dependent manner. hscIL-12 has bioactivity identical to natural interleukin 12. These findings suggest that hscIL-12 is promising in theoretical reseach and in clinical use.