Suppression of Helicobacter pylori-induced interleukin-8 production in vitro and within the gastric mucosa by a live Lactobacillus strain.

BACKGROUND: Accumulating evidence indicates that interleukin-8 (IL-8) plays a major role in the mucosal inflammation caused by Helicobacter pylori infection. The purpose of the present study was to examine whether Lactobacillus gasseri OLL2716 (LG21) can inhibit the H. pylori-induced production of IL-8. METHODS: A coculture system including MKN45 cells, H. pylori, and LG21 was established for an in vitro analysis. Biopsy specimens were obtained from H. pylori-infected human subjects consisting of 19 men and six women. RESULTS: When LG21 was 1/100 less than H. pylori in a coculture system, LG21 significantly suppressed both the IL-8 mRNA and protein generation in the coculture. Live, but not heat- or UV-treated LG21, could exert the suppressive effect. However, this amount of LG21 could not suppress either the adhesion of H. pylori to the cell surface or the IL-8 production by tumor necrosis factor-alpha, which induces IL-8 generation through the activation of the transcription. These results thus suggest that LG21 suppresses an event leading to IL-8 production, which is specific for H. pylori-induced IL-8 generation, and this event is located upstream from the IL-8 transcription but downstream from the adhesion. The measurement of the IL-8 level using gastric biopsy specimens from H. pylori-infected subjects demonstrated that LG21 also suppresses the production of IL-8 in the gastric mucosa. CONCLUSIONS: Live LG21 were found to suppress H. pylori-induced IL-8 production in both a gastric cell line and within gastric mucosa.

Doxorubicin-conjugated anti-midkine monoclonal antibody as a potential anti-tumor drug.

BACKGROUND: Midkine is a heparin-binding growth factor preferentially expressed in tumor cells. The present study was performed to utilize anti-midkine antibody for tumor therapy. METHODS: A monoclonal antibody to midkine was raised by immunizing mice deficient in the midkine gene. The binding site of the antibody was studied by using N-terminal half and C-terminal half of midkine, both of which were chemically synthesized. Doxorubicin (DOX)-conjugate of the antibody was produced by chemical conjugation. The effects of the antibody and the conjugate on cell growth were examined using a midkine-secreting tumor cell, i.e. human hepatocellular carcinoma cell (HepG2). RESULTS: The monoclonal antibody bound to the N-terminal half of midkine. The antibody did not inhibit the growth of HepG2 cells probably because the active domain of midkine is in the C-terminal half. We produced the antibody conjugated with DOX with the hope that the conjugate would be internalized accompanied with midkine. Indeed, the antibody-DOX conjugate significantly inhibited the growth of HepG2 cells compared with DOX-conjugated control IgG. CONCLUSION: The result raises the possibility of using anti-midkine antibody conjugated with DOX for cancer therapy.

Serum midkine concentrations and gastric cancer.

Midkine (MK) is one of a family of heparin-binding growth factors, and increased MK expression is reported in various types of human carcinomas. To clarify the association between serum MK (S-MK) concentrations and gastric cancer, we examined S-MK concentrations of gastric cancer patients (n=275) and healthy controls (n=275). S-MK concentrations of all subjects were measured by enzyme-linked immunosorbent assay (elisa). The medians (25th and 75th percentiles) of S-MK were 192 (123 and 314) pg/mL in the cases and 170 (81 and 273) pg/mL in the controls (P <0.01). We also compared S-MK concentrations in each group divided by the progression stage or histological type of cancer. A difference was observed in the median S-MK concentrations between early and advanced cancers [182 (105 and 301) pg/mL vs 203 (139 and 331) pg/mL, P=0.07], but not between intestinal and diffuse type cancers [185 (121 and 306) pg/mL vs 198 (127 and 323) pg/mL, P=0.51]. We found that those progression stages affect S-MK concentration more strongly than the histological types in gastric cancer patients. Because S-MK seems to reflect the progression stage of gastric cancer, it may serve as a useful marker in the clinical follow-up of gastric cancer patients.

Efficient production of recombinant human pleiotrophin in yeast, Pichia pastoris.

Approximately 260 mg/l of authentic recombinant human pleiotrophin (rhPTN) was expressed into the medium of high-cell density fermentation using a Pichia pastoris protein expression system. The prepro-sequence of yeast alpha-mating factor was used successfully. The recombinant hPTN was efficiently recovered from the medium by expanded bed adsorption, and purified using successive column chromatography steps. In the purified rhPTN preparation, modified rhPTN were scarcely detected. Circular dichroism measurement of the purified PTN showed the presence of the characteristic beta-structures in the protein.

High levels of urinary midkine in various cancer patients.

Midkine (MK) is a heparin-binding growth factor, which promotes growth, migration, and survival of various cells, and MK expression is increased in many human carcinomas. We determined the urinary MK level by enzyme-linked immunoassay. Taking 311pg/mg creatinine as a cut-off level, 70% of patients with various carcinomas (n=142) gave positive values, while only 5.5% of healthy volunteers (n=330) did. In case of gastric carcinoma, 17 out of 21 patients with stage 1 tumor were positive. Urinary MK levels are expected to become a convenient marker as an aid in detection of tumors.