Neurotrophic and Neuritogenic Effects of Water Extracts of Rhizoma of Coptis chinensis Franch in PC12 Cells

Coptis chinensis (CC) is one of the traditional herbs used in Oriental medicine for the treatment of gastrointestinal disorders, anxiety, and insomnia. In this study, neurotrophic and neuritogenic effects of CC on rat pheochromocytoma (PC12) cells were evaluated. Pretreatment of PC12 cells with water extracts of CC (120microgram/ml) produced considerable outgrowth of neurites that is comparable to the effect of nerve growth factor (NGF). Therefore, neurite outgrowth was quantified and expression of NGF mRNA was examined. Furthermore, characteristics of neurites were immunocytochemically confirmed using axon and dendrite-specific antibodies. These results suggest that water extracts of CC contain components that have neurotrophic and neuritogenic properties.

FKBP-12 Exhibits an Inhibitory Activity on Calcium Oxalate Crystal Growth in Vitro

Urolithiasis and calcium oxalate crystal deposition diseases are still significant medical problems. In the course of nephrocalcin cDNA cloning, we have identified FKBP-12 as an inhibitory molecule of calcium oxalate crystal growth. lambdagt 11 cDNA libraries were constructed from renal carcinoma tissues and screened for nephrocalcin cDNA clones using anti-nephrocalcin antibody as a probe. Clones expressing recombinant proteins, which appeared to be antigenically cross-reactive to nephrocalcin, were isolated and their DNA sequences and inhibitory activities on the calcium oxalate crystal growth were determined. One of the clone lambdagt 11 #31-1 had a partial fragment (80 bp) of FKBP-12 cDNA as an insert. Therefore, a full-length FKBP-12 cDNA was PCR-cloned from the lambdagt 11 renal carcinoma cDNA library and was subcloned into an expression vector. The resultant recombinant FKBP-12 exhibited an inhibitory activity on the calcium oxalate crystal growth (Kd=10(-7) M). Physiological effect of the extracellular FKBP-12 was investigated in terms of macrophage activation and proinflammatory cytokine gene induction. Extracellular FKBP-12 failed to activate macrophages even at high concentrations. FKBP-12 seems an anti-stone molecule for the oxalate crystal deposition disease and recurrent stone diseases.

DNA-mediated immunization of mice with plasmid encoding HBs antigen

In order to develop an experimental DNA vaccine for the prevention and treatment of hepatitis B virus infection, hepatitis B virus surface antigen (HBsAg) DNA was subcloned into an E. coli-eukaryotic cell shuttle vector and was expressed in the Baculovirus expression system. Intramuscular, intradermal, and intraperitoneal injections of 30 microg of the plasmid DNA expressing HBsAg induced humoral and cellular immune responses in ICR mice. The first IgG antibodies were detected after ten days and specific IgG antibody titers peaked after two months of a single intramuscular DNA injection. Anti-HBs antibody titers gradually increased and peaked at four months following intradermal DNA injection, and in case of intraperitoneal injection they peaked at seven months. Generation of HBs-specific helper T lymphocytes was also investigated through the production of interleukin-2 by T helper cells. Boosting effects of HBs DNA were investigated without much results. In general, DNA-mediated HBs immunization induced humoral and cellular immune responses in mice that appears to simulate immune responses in human during the course of HBV vaccination.

Induction of Dendritic Cell and Cytokine Gene Expression by In situ Delivery of Flt3 Ligand Plasmid / 대한면역학회지

Dendritic cell (DC)s are protessional antigen presenting cells and they have been used for antitumor immunotherapy or cell vaccines. However therapy using DC is restricted because the number of DC available from tissue is very low. Flt3 ligand (FL) has been known as a hematopoietic growth factor that increases proliferation of hematopoietic stem cells and progenitor cells, and recently it showed inducibility of dendritic cell (DC)s and signiticant antitumor effects in vivo. Thus FL will be frequently used for DC induction and antitumor immunotherapy in future. Here we constructed FL plasmid and studied its in vivo effect. FL plasmids were made by cloning of partial FL cDNA into pcDNA3 plasmid, and gene expression and protein producibility of FL plasmid were confirmed in Renca cells transfected with FL plasmid. Mice were injected with FL plasmid (100ug/mouse) three times and 20 days later mouse spleens were harvested for staining and RT-PCR. There were lots of blastogenic cells in the spleen of mice treated with FL plasmid. FL plasmid also induced DEC205, IL-12 and GM-CSF gene expression in mouse splenocyte. All these data suggest FL plasmid may be used for induction of DC and antitumor therapy as DNA adjuvant.

Study on the Production and Function of Macrophage Migration Inhibitory Factor ( MIF ) : Effect of Recombinant MIF and MIF cDNA on the Induction of Cytokine mRNA / 대한면역학회지

In order to study the functions of migration inhibitory factor (MIF) as macrophage activating cytokine and to investigate the possibility of MIF cDNA as gene therapeutic agent or adjuvant, we produced recombinant MIF (rMIF), anti-MIF antibody and pcDNA I plasmid containing mMIF cDNA (mMIF plasmid). We have investigated the effects of recombinant mMIF or mMIF plasmid on the expression of immune response-related gene in the mouse peritoneal macrophage or splenocyte. Recombinant mMIF produced by Baculovirus expression system was biologically active; it increased mRNA expression of tumor necrosis factor (TNF)-a, Interleukin (IL)-1, IL-6, granulocyte monocyte-colony stimulating factor (GM-CSF), nitric oxide synthase (NOS), Fas and Bcl-x when applied to the cultures of mouse peritoneal macrophage. Anti-mMIF antibody blocked these effects of mMIF on macrophage. Plasmid DNA carrying MIF cDNA inoculated into mouse peritoneal cavity also increased mRNA transcriptions from TNF, IL-1, IL-6, IL-12, GM-CSF, NOS genes of peritoneal macrophage. It enhanced proliferation of splenocyte stimulated with phorbol myristate acetate and IL-2 mRNA expression of splenocytes. Frorn these results, we conclude that rMIF is a strong macrophage activating factor and especially MIF plasmid can be used as an immune potentiating DNA drug in gene therapy for cancer or DNA adjuvant in vaccination in future.

Identification of CD44 splice variant in Korean colorectal cancers and cell lines

CD44 is a glycoprotein expressed in a wide variety of cell types. Recently expression of some alternatively-spliced variants of CD44 transcripts (CD44v) has been suggested to play a potential role in tumor metastasis and the detection of CD44v containing exon 6 to 11 may be helpful for the diagnosis of cancers. Expressions of CD44v containing exon 6 to 11 were investigated in 20 human colorectal cancer samples, peripheral blood leukocytes isolated from colorectal cancer patients, and 4 colorectal cancer cell lines using reverse transcription-polymerase chain reaction and Southern blot analysis. The standard form of CD44 transcripts was expressed in all samples tested. CD44v containing exon 6 to 11 was expressed in 18 cases of colorectal cancers (sensitivity = 90%), 3 out of 4 cell lines, and one normal tissue (specificity = 95%). These results suggest that the expression of CD44v containing exon 6 to 11 can be regarded as tumor specific and that this marker may be helpful for the early diagnosis of colon cancers, if specimens from the early stage are available.