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Mannan-binding lectin (MBL), a circulating C-type lectin, is an important member of the defense collagen family. It exhibits a high potential for recognizing broad categories of pathogen-associated molecular patterns and initiating complement cascade responses. DCs are well-known specialist antigen-presenting cells that significantly trigger specific T cell-mediated immune responses. In our previous study, it was observed that high concentrations of MBL significantly attenuate LPS-induced maturation of monocyte-derived DCs (MoDCs). In the current study, it was postulated that MBL at similar supraphysiological concentrations would affect early differentiation of MoDCs in some way. CD14+ monocytes from human peripheral blood mononuclear cells were cultured with granulocytemacrophage colony-stimulating factor and IL-4 in the presence or absence of physiological (1μg/mL) and supraphysiological concentrations (20μg/mL) of MBL protein, respectively. Phenotypic analysis indicated that the differentiated DCs incubated with high concentrations of MBL expressed MHC class II and costimulatory molecules (e.g., CD80 and CD40) more weakly than did control groups. The secretion of IL-10 and IL-6 increased markedly, whereas their mixed lymphocyte reaction-stimulating capacity decreased. Members of the signal transducer and activator of transcription family were also found to be differentially regulated. Thus, beyond the role of MBL as an opsonin, our data reveal a possible inhibitory effect of MBL at high concentrations in monocyte-DC transition, which probably provides one way of regulating adaptive immune responses by strict regulation of DCs, making MBL a better prospect for controlling relevant pathological events such as autoimmune diseases. [ABSTRACT FROM AUTHOR]

Objective: To detect the plasma levels of mannan?binding lectin (MBL) and MBL?associated serine protease?2 (MASP-2) in patients with hepatocellular carcinoma (HCC) and explore their role in the tumorigenesis and progression of HCC., Methods: The plasma levels of MBL and MASP?2 were detected by enzyme?linked immunosorbent assay in 64 HCC patients and 30 healthy control subjects. The correlation of MBL and MASP?2 with the clinical parameters of HCC patients were analyzed., Results: The plasma levels of MBL (P=0.014) and MASP?2 (P=0.002) were significantly higher in HCC patients than in the healthy controls, but the MBL?to?MASP?2 ratio did not differ significantly between the two groups. In HCC patients, plasma MBL level was positively correlated with vascular invasion (r=0.253, P=0.047) and total bilirubin level (r=0.283, P=0.024). The plasma level of MASP?2 was positively correlated with TNM stage (r=0.276, P=0.027) and negatively correlated with plasma albumin level (r=0.?0.317, P=0.015). ROC curve analysis revealed an area under curve of 0.665 for MBL (P=0.010) and 0.694 for MASP?2 (P=0.003). The sensitivities of MBL and MASP?2 were 50% and 89.1% in the diagnosis of HCC, respectively., Conclusion: MBL and MASP?2 are associated with the inflammatory state and disease progression in patients with HCC.

Objective: To study the effect and mechanism of soluble dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (sDC-SIGN) on the phagocytosis of Staphylococcus aureus (S. aureus) by immature dendritic cells (imDCs)., Methods: Flow cytometry was employed to examine the effect of sDC-SIGN on the phagocytosis of S. aureus by imDCs. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the binging of sDC-SIGN to S. aureus, lipoteichoic acid (LTA) and lipopolysaccharides (LPS) and investigate the effect of the ligands mannan and LTA and anti-DC-SIGN antibodies 1C6 and 4H3 on the binging of sDC-SIGN to S. aureus., Results: sDC-SIGN inhibited the phagocytosis of S. aureus by imDCs. sDC-SIGN bound to S. aureus in a Ca(2+)-dependent manner. sDC-SIGN concentration-dependently bound to LTA, but not to LTA, and the binging of sDC-SIGN to S. aureus was blocked by mannan, LTA, 1C6 and 4H3., Conclusion: sDC-SIGN preferentially binds to the carbohydrate constituents on S. aureus to affect the binding between membrane-bound DC-SIGN and S. aureus, thus suppressing the phagocytosis of S. aureus by imDCs.

Objective: To explore the clinical value of determination of ATP levels in CD4(+) cells of patients with cytomegaloviral pneumonia after kidney transplantation., Methods: Twenty-eight patients with cytomegaloviral pneumonia following kidney transplantation and 30 healthy volunteers were enrolled in this study. ATP-bioluminescence assay (ATP-CVA) was used to assess the immune response of CD4(+) cells to phytohemagglutinin (PHA) stimulation in the normal volunteers and the recipients (before and at 1, 2, and 4 weeks after renal transplantation, before and at 2 and 4 week after the treatment)., Results: ATP concentration in CD4(+) cells of the recipients was 402-/+58 ng/ml before the operation, significantly lower than that in normal volunteers (458-/+196 ng/ml, P<0.05), and reached the lowest level in the first week after operation especially in the recipients with antibody-inducing therapy; ATP level increased slowly since week 2 post-operation, but still remained significantly lower than the preoperative by the fourth week (266-/+87 ng/ml, P<0.05), especially in the recipients receiving antibody-inducing therapy. In the event of cytomegaloviral pneumonia, ATP level underwent a mild reduction to 152-/+78 ng/ml in comparison with the postoperative level at the first week (P>0.05), and was significantly lower than preoperative level (P<0.01); the decrease was especially obvious during the exacerbation of the condition. ATP level then increased slowly after effective treatment, but was still lower than the preoperative level at 4 weeks after the operation (336-/+92 ng/ml, P<0.05)., Conclusion: The determination of ATP level in CD4(+) cells allows more accurate assessment of the cellular immunity in the renal transplant recipients with cytomegaloviral pneumonia to help in the clinical treatment of the patients.

Aim: To obtain the intact encoding gene of human DC-SIGN and express its extracellular region in E.coli., Methods: The intact cDNA encoding human DC-SIGN was amplified from total RNA of placenta of healthy parturient by RT-PCR, and its extracellular region was inserted into prokaryotic expression vector pET-41a. The recombinant plasmid pET-41a-sDC-SIGN was transformed into E.coli BL21(DE3). The expressed product was purified by GST affinity chromatography and identified by SDS-PAGE and Western blot., Results: The DNA fragment of about 1 300 bp was amplified by RT-PCR, and cloned into pGM-T vector to obtain the recombinant plasmid pGM-DC-SIGN. The DNA fragment encoding the extracellular region of human DC-SIGN was amplified from pGM-DC-SIGN plasmid and the recombinant expression vector pET-41a-sDC-SIGN was constructed. The component of M(r); 66 000 in the purified recombinant product was found to be recognized by anti-DC-SIGN antibody., Conclusion: The cDNA of human DC-SIGN is cloned and the protein of its extracellular region is obtained successfully, which lay the foundation for further research on functions of DC-SIGN.

Objective: To express the carbohydrate recognition domain (CRD) of Balb/C mouse mannan binding lectin A (MBL-A) in E.coli., Methods: The target gene fragment was obtained by PCR from the plasmid pmMBL-A harboring mouse MBL-A gene. The PCR product was recombined with the prokaryotic expression vector pET-41a(+) and the resulting recombinant plasmid was identified by PCR, restriction analysis and sequencing before transformation into E.coli BL21(DE3) cell for expression of the target protein. After washing and renaturation, the protein was purified on GST-Tag purification resins and analyzed by SDS-PAGE, Western blotting and enzyme-linked immunosorbent assay (ELISA)., Results: A DNA fragment of about 450 bp was amplified by PCR and the recombinant plasmid pET41a-mMBL-A-CRD was constructed by linking the fragment with pET41a(+) vector. The result of restriction enzyme analysis and sequencing of the selected clones were consistent with those by computer analysis. The recombinant vector was expressed in E.coli BL21(DE3), and the expressed protein existed mainly as inclusion bodies, whose relative molecular mass was about 47,000 by SDS-PAGE analysis. After washing, renaturation and purification, the purity of recombinant protein was about 90%. Western blotting suggested immunoreactivity of the purified protein with anti-GST antibody, and its sugar binding activity was verified by ELISA., Conclusion: We have successfully obtained mouse MBL-A CRD protein, which provides the base for further functional study of the MBL-A molecule.

Aim: To express the N-terminal fragment of human mannan-binding lectin (MBL) associated serine proteases-1 (MASP1-N) in E.coli., Methods: The target sequence in pGEM-MASP1 plasmid that contains human MBL-MASP1 cDNA was amplified by PCR, inserted into prokaryotic expression vector pGEX4T-1 and identified by restriction mapping and sequencing. The recombinant expression vector was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by GSTrap Immobilized Metal Affinity Chromatography(IMAC) and identified by SDS-PAGE and Western blot assay, its binding-activity with the collagen-like region of human MBL(MBL-CLR) and with recombinant human MBL was analyzed by an indirect enzyme-linked immunosorbent assayèELISAé., Results: The DNA fragment of 860 bp, which encode the N-terminal region of human MASP1, was amplified from pGEM-MASP1 plasmid and the recombinant expression vector, pGEX4T-MASP1-N, was constructed, whose restriction maps and sequence were consistent with those expected. The component of M(r) 60 000 in the purified recombinant product was found by SDS-PAGE and could be recognized by anti-GST antibody in Western blot assay. The purified recombinant product could react with human MBL-CLR and human MBL in the indirect ELISA., Conclusion: The prokaryotic cell strain that expresses efficiently recombinant human MASP1-N(rhMASP1-N) protein and the purified rhMASP1-N protein were successfully obtained, which provides the basis for further research of MASP1 molecule.

Objective: To obtain highly purified tetanus toxin fragment C (TTC) with good immunogenicity., Methods: The gene fragment encoding TTC was amplified from Clostridium tetani plasmid DNA by PCR, inserted into the vector pET43.1a (+) and expressed in E. coli BL21(DE3)plysS. After purification using Ni2+-chelate affinity chromatography, the expressed fusion protein was digested by thrombin and the resultant TTC protein was purified with Ni2+-chelate affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purifed TTC protein was then used to immunize mice to test its immunogenecity., Results: The 1373-bp gene fragment encoding TTC was obtained, and the constructed recombinant expression vector pET43.1a (+)-TTC was successfully expressed in E. coli BL21(DE3)plysS. SDS-PAGE identified a recombinant fusion protein with relative molecular mass (Mr) of 117 000, which accounted for 22% of the total bacterial protein. The TTC protein with Mr of 50 000 was obtained after purification of the thrombin digestion products of the fusion protein, with a purity reaching 95.5%. Both the fusion protein and TTC protein could be recognized by anti-tetanus toxin antibody as shown by Western blotting. The titer of the anti-serum from mice immunized with the TTC protein was 1:25 600, and the anti-serum could specifically bind to tetanus toxin., Conclusion: Highly purified and immunogenetic TTC protein has been successfully obtained, which provides a good model antigen for studying antigen presentation and immune responses in vivo.

Objective: To prepare a single-epitope polyclonal antibody against mouse long peptidoglycan recognition protein (mPGRP-L)., Methods: B cell dominant epitopes of mPGRP-L predicted by bioinformatics were synthesized, and the immunogen was prepared by conjugation of the synthetic peptide and the carrier protein key-hole limpet hemocyanin (KLH) by MBS method. The single-epitope polyclonal antibody was obtained by immunizing rabbits with the KLH-peptide conjugate, purified by SPG affinity columns or antigenic peptide affinity columns, and identified by ELISA and Western blotting., Results and Conclusion: A dominant epitope in N-terminal region of mPGRP-L, with amino acid sequence of NH(2)-(C)DPHSLSPELQALISEVAQHD-COOH, was chosen and synthesized. The titer of anti-serum of the rabbits immunized with the KLH-peptide conjugate was 1:256,000. The polyclonal antibody purified with SPG affinity columns and antigenic peptide affinity columns were named as mPGRP-Ln1 and mPGRP-Ln2, respectively. Western blotting demonstrated that the antibody mPGRP-Ln1 could recognize the recombined N-terminal fragment of mPGRP-Ln with a clear band at relative molecular mass of about 29,000, suggesting the successful preparation of the single-epitope polyclonal antibody against the N-terminal region of mPGRP-L.

Aim: To construct pET32a/His MBL-CLR recombinant prokaryotic expression plasmid and to express mannan-binding lectin-CLR (MBL-CLR) protein in E.coli, Methods: The human MBL-CLR gene was amplified by PCR from pGEM-MBL plasmid, and was inserted into prokaryotic expression vector pET32a. After identified by restriction mapping and sequencing, the recombinant plasmid pET32a/His MBL-CLR was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by Immobilized Metal Affinity Chromatography (IMAC) and identified by SDS-PAGE, Western blot and indirect enzyme-linked immunosorbent assay (ELISA) using the antibody from BALB/c mice immunized with the recombinant human MBL protein., Results: The cDNA fragment of 180 bp was amplified from pGEM-MBL plasmid and the recombinant expression vector pET32/His MBL-CLR was constructed. The recombinant plasmid was consistent with those expected by restriction maps and sequence. Three components of relative molecular mass 30,000, 60,000 and 120,000 in the purified recombinant product were detected by SDS-PAGE and all the components could be recognized by anti-6His antibody in Western blot assay. The three components were correspondingly with the band of the monomer and oligomer of the fusion protein. The purified recombinant product could react with the antibody against the recombinant human MBL protein in the indirect ELISA., Conclusion: The prokaryotic expression strains that efficiently express recombinant human MBL-CLR and the recombinant human MBL-CLR-Trx fusion protein were obtained successfully, which will help the further structure-function research of MBL molecule.

Objective: To investigate the frequencies of three point mutations, CGT52TGT, GGC54GAC and GGA57GAA, in exon 1 of mannan-binding lectin (MBL) structural gene in Chinese Uyghur population., Methods: Blood samples were collected from a Uyghur population in Xinjiang Uyghur Autonomous Region, and the genomic DNA was extracted from the leucocytes and the target gene fragment amplified by PCR. The three point mutations in exon 1 of MBL gene were detected by fluorogenic probe hybridization technique with visual monitoring., Results: In 95 Uyghur individuals, 2 were identified as homozygous for codon 54 mutations, 28 were heterozygous for codon 54 mutation, and no CGT52TGT and GGA57GAA point mutations were found., Conclusion: The frequencies of CGT52TGT, GGC54GAC and GGA57GAA mutant alleles in exon 1 of MBL structural gene are 0, 0.168 and 0 respectively in the Chinese Uyghur population.

Objective: To clone the gene coding for the peptidoglycan recognition protein (PGRP) domain (PGRPd) of mouse long PGRP (mPGRP-L) and express the protein in E. coli., Methods: The cDNA fragment encoding PGRPd of mPGRP-L was obtained by RT-PCR from the total RNA of Balb/C mouse liver cells and cloned into pUCm-T vector. The recombinant plasmid were identified by PCR, restriction endonucleases and sequence analysis. The PGRPd gene fragment was amplified by PCR from the recombinant plasmid, inserted into pQE-30 vector and transformed into E. coli strain M15, and the expressed PGRPd protein was purified., Results: A cDNA fragment of about 500 bp was amplified by RT-PCR and the recombinant plasmid, pmPGRPd, was constructed by linking the fragment to pUCm-T vector. The results of restriction mapping of the recombinant vector were consistent with those of computer analyses. Sequence analysis showed that the cloned gene fragment (518 bp) had identical sequence with the gene encoding PGRPd of mPGRP-L gene in GenBank. The recombinant expression vector pQE-PGRPd was constructed and expressed in E. coli M15. SDS-PAGE showed that the expressed product existed mainly in the lysate supernatant as a soluble protein with relative molecular mass of 29 kD., Conclusion: The PGRPd cDNA of mPGRP-L has been successfully cloned and expressed in E. coli, which provides the basis for further study of PGRP molecule.

Objective: To construct the standard recombinant plasmids for 7 common haplotypes of mannan-binding lectin (MBL) gene., Methods: The DNA samples with known haplotypes and genotypes of MBL gene were used as the templates for amplifying the fragments of MBL gene haplotypes including the promoter region and exon 1 with sequence-specific primer-polymerase chain reaction (SSP-PCR) method. The amplified fragments were cloned into T vector and the bases located at codon 52 and codon 57 of exon 1 in MBL gene were mutated respectively by site-directed mutagenesis. All the 7 recombinant plasmids were identified by PCR and direct sequence analysis., Results: From the DNA samples with known haplotypes and genotypes of MBL gene, the standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA and LYPB of MBL gene were constructed by SSP-PCR and molecular cloning technique. From the recombinant plasmids of HYPA and LYQA, the standard plasmids of haplotypes HYPD and LYQC of MBL gene were constructed by site-directed mutagenesis, respectively., Conclusion: The constructed standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA, LYPB, HYPD and LYQC of MBL gene provide standard controls for detecting the SNPs, haplotypes and genotypes of MBL gene with such genotyping methods us SSP-PCR and real-time PCR.

Objective: To explore the role of complement C3 in delayed-type hypersensitivity (DTH)., Methods: After inducing DTH reaction in the footpads of C3 knockout C3(-/-) and wild-type (C3(+/+) ) mice with ovalbumin (OVA), the thickness of the footpad was measured and HE and immunohistochemical staining preformed to identify the number and types of the infiltrating mononuclear cells in the footpad tissues. T lymphocytes were separated from the spleens of the mice and incubated in 96-well plates with serial dilutions of OVA or mitogens in the presence of mitomycin C-treated macrophages, and the proliferation of the T cells was assessed by (3)H-TdR incorporation assay., Results: The footpad thickness of DTH-C3(-/-) mice was significantly smaller than that of DTH-C3(+/+) mice. The number of the infiltrating mononuclear cells in the footpad tissue of C3(-/-) mice was obviously decreased in comparison with that of C3(+/+) mice, and the cells were characterized mainly as CD4(+) T lymphocytes. No significant difference in the proliferation of mitogen-stimulated splenic T cells was noted between C3(-/-) and C3(+/+) mice, but after stimulation with the specific antigen OVA, significant reduction in the proliferation of splenic T cells from C3(-/-) mice was observed as compared with the T cell proliferation in C3(+/+) mice., Conclusion: C3 defect results in impaired DTH responses in mice, which indicates the important role of C3 in DTH reaction.

Objective: To investigate the expression of endogenetic matrix metalloproteinases-9 (MMP-9) and transforming growth factor-beta(TGF-beta) and their role in the wound healing of blast injury., Methods: Rat models of blast injury under a humid and hot environment were established and the effusion from the wound surface was collected at 4, 24, 48 h and 5, 7, 14, 21 and 28 days after injury, respectively. The contents of MMP-9 and TGF-beta in the effusion of the wound were measured by zymography and enzyme-linked immunosorbent assay (ELISA), respectively., Results: During the wound healing of blast injury, MMP-9 and TGF-beta exhibited changes that followed a regular pattern, both reaching the peak value at 48 h after the injury. TGF-beta content reached the another peak on day 7. TGF-beta value and MMP-9 contents decreased in the second week after injury and their reduction was no longer parallel. Administration of tissue inhibitor of metalloproteinase (TIMP) in the early phase of injury showed no obvious effect, but during the 2 weeks after the injury, its administration caused decrease in MMP-9 content and increase in TGF-beta content in the effusion., Conclusions: In the early phase of wound healing, the elevation of MMP-9 and TGF-beta accelerated cell migration to promote the clearance of the inflammatory necrosis tissues, which might be one of the wound healing mechanisms. But overexpression of MMP-9 in the wound may hinder wound healing, and appropriate use of TIMP can accelerate the delayed wound healing.

Aim: To explore preliminarily the mechanisms of immunodeficiency resulted from the CGT52TGT point mutation of mannan-binding lectin(MBL) gene., Methods: The MBL gene containing CGT52TGT point mutation was amplified from the plasmid pMBLm52 by PCR, and then inserted into the eukaryotic expression vector pcDNA4/HisMax C. After confirmed by DNA sequencing, the recombinant expression vector was transfected into Chinese-hamster ovary(CHO) cells by electroporation. Zeocin of 800 mg/L had been used for 30 days to select electroporated CHO cells, and then 200 mg/L for another 30 days to obtain stable transfectant. The expression of mRNA was analyzed by RT-PCR, the recombinant protein was purified from the culture supernatant by Ni-NTA agarose chromatography and analyzed by SDS-PAGE under nonreducing condition and Western blot., Results: The cDNA fragment amplified from pMBLm52 plasmid by PCR was about 750 bp and the recombinant plasmid pcDNA4/HisMax C-MBLm52 was constructed and transfected into CHO cells. The expression product purified from the culture supernatant appeared mainly at the site of M(r) 60,000, indicating a much lower oligomerization level than that of the recombinant human wild MBL and human plasma-derived MBL., Conclusion: The CGT52TGT point mutation of MBL gene does not affect the secretion of its product, but a Cys introduced by the mutation could form another disulfide bond which may disrupt the structure of MBL molecule as well as its function.

Aim: To explore the effect of mannan-binding lectin (MBL) on the differentiation and maturation of human peripheral blood monocyte-derived dendritic cells (MoDCs)., Methods: After MoDCs was stimulated with human natural MBL, MoDC's morphology was observed under inverted microscope and the expressions of CD1a, CD83, CD40, CD80, CD86 and HLA-DR on MoDCs were analyzed by FACS. The ability of MoDCs to stimulate the proliferation of allogenic T cells was detected by (3)H-TdR incorporation. The ability of MoDCs to up-take antigens was evaluated by zymosan granule phagocytosis test. The levels of IL-12 and TNF-alpha in the culture supernatant of MoDCs were determined by ELISA., Results: The expressions of CD1a, CD83, CD40, CD80, CD86 and HLA-DR on the MoDCs were up-regulated by MBL. The ability of MoDCs to up-take zymosan granules decreased and the proliferation of native T cells induced by MoDCs was enhanced. MBL stimulated the production of IL-12 by MoDCs, but had no such effect on TNF-alpha secretion., Conclusion: MBL can induce differentiation and maturation of DCs in vitro, suggesting that MBL possibly participate in the adaptive immune response through modulation of functions of DCs.

Aim: To investigate the single nucleotide polymorphisms(SNP) in the promoter region of mannan-binding lectin (MBL) gene in Bais, Uars and Lahus ethnics., Methods: 71, 73 and 94 blood samples were obtained from Bais, Uars and Lahus subjects, respectively from Yunnan province. Genomic DNA was isolated from leucocytes. Polymorphism alleles -550(G/C, named H/L alleles), -220(G/C, X/Y alleles) and +4(C/T, P/Q alleles) in the promoter region of MBL gene were detected by sequence-specific primer-PCR and molecular beacon real-time PCR, and the frequencies of haplotypes and genotypes were analyzed., Results: The frequencies of genotypes in Bais, Uars and Lahus ethnics were as follows: LYP/LYP 4(5.6%), 4(5.5%)and 2(2.1%); HYP/LYQ 6(8.5%), 11(15.1%) and 12(12.8%); LYP/LYQ 21(29.6%), 14(19.2%) and 51(54.3%); LXP/LXP 1(1.4%), 2(2.7%)and 0,LYQ/LYQ 10(14.1%); 14(19.2%), 7(7.4%), LXP/LYQ 10(14.1%), 13(17.8%) and 15(16.0%); HYP/LYP 4(5.6%), 3(4.1%) and 4(4.3%); HYP/LXP 3(4.2%), 1(1.4%) and 0; HYP/HYP 12(16.9%), 11(15.1%) and 3(3.2%)., Conclusion: The distribution of alleles L/H is statistically different among Uars, Bais and Larhus(P<0.01), while X/Y and P/Q are not statistically significant(P>0.05). All studied haplotypes and genotypes are statistically different among the 3 ethnics(P<0.01). Genotypes LYP/LYQ and LXP/LYQ are common in all the 3 ethnics.

Objective: To separate mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) from human plasma., Methods: A two-step affinity chromatography on underivatized sepharose 4B was employed for purification of MBL-MASP complex, followed by gel filtration on a Sephacryl S-300 column for separation of MBL and MASPs from the complex. The purification procedures were performed at 4 degrees Celsius with the addition of two proteolytic inhibitors, phenyl methylsulfonyl fluoride and 1,10-phenanthroline during affinity chromatography but not in the gel filtration buffer., Results: Preparations of highly purified MBL and proenzyme MASPs were obtained. The purified MBL was shown by SDS-PAGE and Western blotting to be a functional multimer composed of 28,000 and 32,000 peptide chains, with high bioactivity as demonstrated by ligand-binding assay and yeast agglutination experiment., Conclusion: A simple and convenient procedure is established successfully for the purification of MBL and the proenzyme MASPs.

Objective: To explore the methods for rapid in vitro culture of the dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) under serum-free conditions and ascertain whether intracellular signal transduction pathway differs between calcium ionophore (CI) and tumor necrosis factor (TNF)- alpha during their induction of dendritic cell differentiation., Methods: PBMCs isolated from healthy donors were plated in serum-free medium supplemented with 50 ng/ml rhGM-CSF. Cells cultured overnight were induced to differentiate with 100 ng/ml A23187 or 50 ng/ml TNF-alpha, given before or 30 min after pre-treatment with 0.5 mug/ml cyclosporine A (CsA). After culture for 40 h, the cell morphology was observed under phase-contrast microscope, and the surface markers on treated PBMCs were analyzed by flow cytometry. MTT colorimetry was employed to assess the proliferation of the allogeneic T cells., Results: PBMCs of healthy donors treated with 50 ng/ml rhGM-CSF in combination with 100 ng/ml CI or 50 ng/ml TNF-alpha for 40 h exhibited typical morphology of DCs with rapidly decreased CD14 expression and increased expressions of CD83 and co-stimulatory molecules (CD80 and CD86), showing also enhanced ability of stimulating allogeneic T cell proliferation. Calcineurin antagonist CsA inhibited the differentiation induced by CI, but not that induced by TNF-alpha., Conclusions: Under serum-free conditions, both CI and TNF-alpha are capable of inducing rapid DC differentiation from human PBMCs, but the intracellular signal transduction of CI-induced differentiation is different from that induced by TNF-alpha.

Aim: To explore the intracellular signal transduction pathway in the differentiation of human peripheral blood mononucleocytes (PBMCs) towards dendritic cells (DCs) induced by calcium ionophore (CI)., Methods: PBMCs isolated from a healthy donor were cultured with A23187 plus rhGM-CSF, 100 microg/L each. In some experiments, PBMCs were cultured for 30 minutes with W-7 (10 micromol/L), CsA(0.5 mg/L) or KT5926(1 micromol/L) before addition of rhGM-CSF and A23187. After culture for 40 hours, morphological change of the cells were observed under phase contrast microscope; surface markers on treated PBMCs were analyzed by flow cytometry; the proliferation of allogeneic human T cells stimulated by the treated PBMCs was detected by MTT colorimetry., Results: PBMCs of the healthy donor cocultured with rhGM-CSF plus CI for 40 hours had the typical morphology of DCs, with decreased CD14 expression, and increased CD83, CD80 and CD86 expressions. The proliferation of allogeneic T cells stimulated by PBMCs treated with A23187 plus rhGM-CSF was strengthened. But the morphological changes, surface marker expressions and the ability to enhancing proliferation of allogeneic T cells were inhibited to different degrees by W-7, CsA or KT5926., Conclusion: The differentiation of PBMCs towards DCs by CI may be modulated by Ca (2+)/calmodulin and multiple signal transduction pathways downstream.

Objective: To construct pcDNA4/His C-MBL recombinant eukaryotic expression plasmid and examine its expression of mannan-binding lectin (MBL) in mammary cells., Methods: The target sequence was amplified by PCR from pGEM-MBL plasmid that contains wild-type human MBL cDNA, and inserted into eukaryotic expression vector PcDNA4/His C followed by restriction mapping and sequencing. The recombinant plasmid PcDNA4/His C-MBL was transformed into Chinese-hamster ovary (CHO) cells by electroporation, and the Zeocin-resistant clones were selected for analysis of mRNA expression by reverse transcription (RT)-PCR. The expressed product was purified by immobilized metal affinity chromatography (IMAC) and identified by SDS-PAGE and Western-blot analysis, and its immunoreactivity was detected by an indirect enzyme-linked immunosorbent assay ELISA using the anti-serum from Balb/C mice immunized with the recombinant protein., Results: The cDNA fragment of 750 bp was amplified from pGEM-MBL plasmid, which was shown by restriction enzyme digestion and DNA sequencing. The mRNA expression of Zeocin-resistant CHO cell clones was detected by RT-PCR. Three components of 29, 58 and 87 kD in the purified recombinant product were found by SDS-PAGE and the 29 kD component could be recognized by anti-6His antibody in Western blot analysis. The titers of the anti-serum from immunized mice were 1: 819 200 against the recombinant protein and 1:25 600 against both the natural human MBL and the recombinant trimeric carbohydrate-recognition domain (CRD) of human MBL, as determined by the indirect ELISA., Conclusion: The cell strains that express recombinant human MBL (rhMBL) and rhMBL protein have been obtained successfully, which provides the basis for further research of MBL molecule.

Aim: To explore whether the promyelocytic leukemia cell line HL-60 may differentiate into activated dendritic cells (DCs) by A23187, a calcium ionophore., Methods: The HL-60 cells were cultured in common medium alone or with various concentrations of A23187 (25-1 600 microg/L) and rhGM-CSF (100 microg/L). After culture for 24-96 hours, the cellular morphological change was observed under light microscope and electron microscope. Surface makers on treated HL-60 cells were analyzed by flow cytometry. The proliferation of allogeneic human T cells was detected by MTT colorimetry., Results: Under the condition of a suitable dose (200 microg/L) of A23187 treatment of HL-60 cells for 24 hours, the expression of CD83 molecule, a characteristic marker on DCs, was highest. The typical dendritic outgrowth appeared at a time when HL-60 cells were treated with A23187 for 72 hours. However, when HL-60 cells were treated with A23187 for 96 hours, the expressions of CD80 (B7.1), CD86 (B7.2), MHC-class II molecule and CD54 on HL-60 cells reached peak, and marked activation of allogeneic T cells occurred., Conclusion: Calcium ionophore A23187 can induce the HL-60 cells to differentiate into activated DCs-like cells.

Objective: To investigate the single nucleotide polymorphisms (SNP) in the promoter region of mannan-binding lectin (MBL) gene in a Han population in Guangdong Province., Methods: A total of 167 blood samples were obtained from this Han population to isolate the genomic DNA from the leucocytes. The polymorphism alleles -550(G/C, named H/L alleles), -220(G/C, X/Y alleles) and +4(C/Tr, P/Q alleles) in the promoter region of MBL gene were detected by PCR with sequence-specific primers and molecular beacon real-time fluorescent PCR, and the frequencies of haplotypes and genotypes were analyzed., Results: The frequencies of several genotypes in the 167 samples were: LYP/LYP, 10(5.9%); HYP/LYQ, 7(4.2%); LYP/LYQ, 94(56.3%); LXP/LXP, 6(3.6%); LYQ/LYQ, 4(2.4%); LXP/LYQ, 29(17.4%); HYP/LYP, 3(1.8%); HYP/LXP, 2(1.2%); HYP/HYP, 12(7.2%)., Conclusion: The polymorphism genotypes in the promoter region of MBL gene in this chosen population are mostly LYP/LYQ and LXP/LYQ.

Objective: To obtain full-length cDNA encoding mouse mannan-binding lectin C(MBL-C) polypeptide., Methods: The cDNA encoding mouse MBL-C was isolated from Balb/c mouse liver cells by reverse transcriptase (RT)-PCR and inserted into pUC-T vector, and the encoding region structure of the DNA was analyzed to understand its evolutionary relationship with single human MBL homologues., Results: The amplified mouse MBL-C cDNA, which was 735 bp in length, encoded 245 amino acid residues, and its structural analysis showed 100% homology with published mouse MBL-C gene sequence and 71.4% homology with MBL gene of Chinese human., Conclusion: The gene encoding Balb/c mouse MBL-C has been successfully isolated, which may facilitate further study of the innate immune functions of MBL molecule in vivo.