Plasma mannan?binding lectin and MBL?associated serine protease 2 in patients with hepatocellular carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>MBL and MASP?2 are associated with the inflammatory state and disease progression in patients with HCC.</p>

Human soluble dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin inhibits phagocytosis of Staphylococcus aureus by immature dendritic cells / 南方医科大学学报

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Clinical value of ATP determination in CD4+ cells of patients with cytomegaloviral pneumonia after kidney transplantation / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the clinical value of determination of ATP levels in CD4(+) cells of patients with cytomegaloviral pneumonia after kidney transplantation.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Prokaryotic expression of Balb/C mouse MBL-A carbohydrate recognition domain / 南方医科大学学报

<p><b>OBJECTIVE</b>To express the carbohydrate recognition domain (CRD) of Balb/C mouse mannan binding lectin A (MBL-A) in E.coli.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>We have successfully obtained mouse MBL-A CRD protein, which provides the base for further functional study of the MBL-A molecule.</p>

Gene cloning, optimized expression and immunogenicity evaluation of tetanus toxin fragment C / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain highly purified tetanus toxin fragment C (TTC) with good immunogenicity.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Preparation of anti-mouse PGRP-L single-epitope polyclonal antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare a single-epitope polyclonal antibody against mouse long peptidoglycan recognition protein (mPGRP-L).</p><p><b>METHODS</b>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.</p><p><b>RESULTS AND CONCLUSION</b>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.</p>

Frequencies of CGT52TGT, GGC54GAC and GGA57GAA point mutations in MBL gene in Chinese Uyghur population / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Cloning and prokaryotic expression of the gene encoding PGRP domain of mouse long peptidoglycan recognition protein / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Investigation of single nucleotide polymorphisms, haplotypes and genotypes of mannan-binding lectin gene in Bai(Pai) nationality in China / 中国免疫学杂志

Objective:To investigate the single nucleotide polymorphisms(SNP), haplotypes and genotypes of mannan-binding lectin(MBL) gene in the Bai(Pai) nationality from YunNan province, China.Methods:The three SNP sites CGT52TGT, GGC54GAC and GGA57GAA(named alleles D, B and C respectively, wildtype named A) in exon1 of MBL gene of 70 DNA samples of Bai nationality whose three SNP sites, -550G/C, -221C/G and +4C/T(named alleles H/L, X/Y and P/Q respectively), in promoter region of MBL gene had been clear, haplotypes and genotypes of MBL genes were detected and analyzed by sequence specific primer-polymerase chain reaction.Results:It was found that in Bais population, the frequency of alleles B was 0.100, there only were five haplotypes, HYPA, LXPA, LYQA, LYPA and LYPB, whose frequencies were 0.250, 0.107, 0.407, 0.135 and 0.100 respectively, the frequencies of several genotypes were LYPA/LYPA 0.043, LXPA/LYQA 0.143, LYPA/LYPB 0.014, HYPA/LYQA 0.086, LYPA/ LYQA 0.157, HYPA/LYPA 0.014, LYPB/LYQA 0.143, HYPA/LYPB 0.043, LXPA/LXPA 0.014, HYPA/LXPA 0.043, LYQA/LYQA 0.143 and HYPA/HYPA 0.157.Conclusion:In the MBL genes in Bais population, there is the allele B, the polymorphism haplotypes are mostly LYQA and HYPA, and the genotypes, LYPA/LYQA, HYPA/HYPA, LXPA/LYQA, LYPB/LYQA and LYQA/LYQA.