The presence of high level soluble herpes virus entry mediator in sera of gastric cancer patients

The development of gastric cancer (GC) is closely related to chronic inflammation caused by Helicobacter pylori infection, and herpes virus entry mediator (HVEM) is a receptor expressed on the surface of leukocytes that mediates potent inflammatory responses in animal models. However, the role of HVEM in human GC has not been studied. Previously, we showed that the interaction of HVEM on human leukocytes with its ligand LIGHT induces intracellular calcium mobilization, which results in inflammatory responses including induction of proinflammatory cytokine production and anti-bacterial activities. In this study, we report that leukocytes from GC patients express lower levels of membrane HVEM (mHVEM) and have lower LIGHT-induced bactericidal activities than those from healthy controls (HC). In contrast, levels of soluble HVEM (sHVEM) in the sera of GC patients were significantly higher than in those of HC. We found that monocyte membrane-bound HVEM is released into the medium when cells are activated by proinflammatory cytokines such as TNF-alpha and IL-8, which are elevated in the sera of GC patients. mHVEM level dropped in parallel with the release of sHVEM, and release was completely blocked by the metalloprotease inhibitor, GM6001. We also found that the low level of mHVEM on GC patient leukocytes was correlated with low LIGHT-induced bactericidal activities against H. pylori and S. aureus and production of reactive oxygen species. Our results indicate that mHVEM on leukocytes and sHVEM in sera may contribute to the development and/or progression of GC.

Expression of 4-1BB and 4-1BBL in thymocytes during thymus regeneration

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide-induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4+CD8+ double-positive (DP) thymocytes. Furthermore, the CD4loCD8lo, CD4+CD8lo and CD4loCD8+ thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4+CD8+ DP thymocytes present during thymus regeneration were TCR(hi) and CD69+ unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCRhi/int and CD69+/int phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4+CD8+ DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.

Chemokine Lkn-1/CCL15 enhances matrix metalloproteinase-9 release from human macrophages and macrophage-derived foam cells

Atherosclerosis is characterized by a chronic inflammatory disease, and chemokines play an important role in both initiation and progression of atherosclerosis development. Leukotactin-1 (Lkn-1/CCL15), a new member of the human CC chemokine family, is a potent chemoattractant for leukocytes. Our previous study has demonstrated that Lkn-1/CCL15 plays a role in the initiation of atherosclerosis, however, little is currently known whether Lkn-1/CCL15 is associated with the progression of atherosclerosis. Matrix metalloproteinases (MMPs) in human coronary atherosclerotic lesions play a crucial role in the progression of atherosclerosis by altering the vulnerability of plaque rupture. In the present study, we examined whether Lkn-1/CCL15 modulates MMP-9 release, which is a prevalent form expressed by activated macrophages and foam cells. Human THP-1 monocytic cells and/or human peripheral blood monocytes (PBMC) were treated with phorbol myristate acetate to induce their differentiation into macrophages. Foam cells were prepared by the treatment of THP-1 macrophages with human oxidized LDL. The macrophages and foam cells were treated with Lkn-1/CCL15, and the levels of MMP-9 release were measured by Gelatin Zymography. Lkn-1/CCL15 significantly enhanced the levels of MMP-9 protein secretion from THP-1 monocytic cells-derived macrophages, human PBMC-derived macrophages, as well as macrophage-derived foam cell in a dose dependent manner. Our data suggest that the action of Lkn-1/CCL15 on macrophages and foam cells to release MMP-9 may contribute to plaque destabilization in the progression of atherosclerosis.

Stimulation of Bactericidal and Fungicidal Activity of Neonatal Monocytes by Immunoactivating Peptide

PURPOSE: To evaluate the function of Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) in human neonatal monocytes. METHODS: The peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), was synthesized, purified, and prepared in the Peptide Library Support Facility at Pohang University of Science and Technology. Female Sprague-Dawley rats (200+/-10 g) were preinfected with S. aureus and treated with WKYMVm through femoral vein. At various time points, blood samples were obtained by puncture of femoral artery and the serum was plated on the nutrient agar plate. The number of viable bacteria was determined by counting the number of bacterial colonies. In addition, using S. aureus and C. albicans, we evaluated the bactericidal and fungicidal activities of neonatal monocytes, which were separated from umbilical cord blood by Ficoll gradient. RESULTS: The numbers of bacteria in the blood of WKYMVm-treated rats were rapidly decreased with time, as compared with those of the untreated rats. The peptide treatment enhanced the bactericidal activity in vivo within 10 minutes. In neonatal monocytes, WKYMVm stimulated the intracellular killing of S. aureus in a dose dependent manner, showing the maximum effect at 100 nM. WKYMVm stimulated the phagocytic and fungicidal activities against C. albicans in a dose dependent manner, with the maximum effect at the 100 nM. CONCLUSION: These results suggest that WKYMVm may be an effective agent against the neonatal infections.

Characterization of a Vibrio parahaemolyticus Phage Isolated from Marine

A novel bacteriophage, designated as VPP97, that infects the strains of Vibiro parahaemolyticus (hallophilic, Gram-negative bacterium) isolated most commonly from marine environments, has been discovered, and several of its properties have been determined. The plaques were clear and sized 0.6-1.0 mm in diameter. The virion forms a single band on 70% sucrose gradient and p1.50 CsC1 gradient by sucrose gradient centrifugation and CsCI gradient centrifugation respectively. It has a hexagonal head and a relatively long tail, as shown by electron microscopy. Vibrio alginolyticus, Vibrio fluvialis and Vibrio furnissii were also sensitive to this phage It was almost totally inactivated at 70 degree C and at pH below 5 or over 10. The nucleic acid of VPP97 is composed of DNA. The VPP97 had 9 specific structural proteins sized between 21.5 kDa and 97.4 kDa on SDS-PAGE. When V. parahaemolyticus cultures were treated with either phage VPP97 or one of the several antibiotics for 2 hours, the viable number of V. parahaemolyticus treated with the phage VPP97 is lower than that treated with chloramphenicol, erythromycin or penicillin, but not lower than that treated with tetracycline. Mice that have responded to the phage treatment revealed the lower numbers of V. parahaemolyticus in small intestine and less damage on small intestine compared to the untreated mice. Therefore, we suggest that the phage treatment appears effective to the infection by V. parahaemolyticus.

Purification Siderophore from Vibrio mimicus ATCC 33653 and its Effect to Bacterial Pathogenecity

Growth under conditions of iron-restriction and the production of siderophore was examined in Vibrio mimicus ATCC 33653. This strain grew and multiplied in the presence of the high-affinity iron chelators ethylenediamine-di (o-hydroxyphenylacetic acid). Chrorne azurol S (CAS) agar and solution were used to detect the production of siderophore under these condition. Siderophore could be detected in the iron-rcstricted culture supernatants. The siderophore was extracted from iron-restricted culture supernatants by phenol-chloroform-ether method and purified by Dowex ion-exchange and Sephadex G-25 gel filtracton chromatography. The purified siderophore was confirmed by paper chromatography and HPLC. The Purified siderophore enhanced the growth of V. mimicus when the bacterium was grown in iron limited medium. Injection of both the siderohore and the bacteria to mice resulted in more rapid death than that of the only bacteria. However, the siderophore did not show lethality to mice and any toxicity to cell line like HeLa and U937.

The Effect of Iron Limmted Condition on Outer Membrane of Vibrio mimicus

Vibrio mimicus, marine bacteria pathogenic for fish, can causes acute gastroenteritis in human. Iron limmited condition like in human body, may change the surface structure of V. mimicus. In this study we obse'rved the effect of iron limmited condition on outer membrane protein of V. mimicus. Ethylenediamine-di (O-hydroxy-phenylacetic) acid (EDDA), an iron chelator, delayed the time to reach expotential growth of V. mimicus in brain heart infusion medium from 3 hours to 20 hours. Outer membrane protein of V. mimicus-CON (cultured in BHI) and V. mimicus-EDDA (cultured in BHI contain EDDA) were seperated by 1% sarcosine from total cell envelop. SDS-PAGE of V. mimicus-EDDA and V. mimicus-CON showed similar protein profiles contain 37 kDa major protein but 86 and 90 kDa protein were induced differently. Immunological properties of above protein were determined by ELISA and western blotting. 86 kDa EDDA- specific OMP was induced in V. mimicus (isolate 96-1), V. parahaemolyticus (serotype 09), V. alginolyticus (isolate 95-1), E. coli (human isolate) and V. vulnificus ATCC 27562 in iron limmited condition.

Electromicroscopic Characterization about Vibrio alginolyticus Phage Isolated from Marine Products

The study of bacteriophage began by F.W. Twort in 1915 and the lytic cycle recognized by d'Herellel in 1917. It repeated about the marine bacteriophage containing Vibrio phage by Smith, Spencer and Ju. Authors isolated 2 virulent phages for the pathogenic V. alginolyticus from marine products. These 2 phages were examined their ultrastructure & host-infection by elecron microscopy and in vivo test using skin of rats. V. alginolyticus phages(VAPs) fomed plaques about 0.5 - 0.9mm in diameter and bands 50 - 60% in sucrose density gradient. VAP had 50 - 120nm tail and 40 - 90nm head in diameter. In vivo test, using rat skin, as well as in vitro test VAP had the activity to V. alginolyticus isolated.

Pathogenicitic Characterization of Purified beta-Hemolysin - Produced by Vibrio mimicus

In order to investigate the main factor of pathogenicity in V. mimicus, we have studied the toxic effects of j3-hemolysin produced by V. mimicus. The purified hemolysin of V. mimicus was active erythrocytes from three animal species including mouse, rabbit and rat, but the hemolysin was most active against rabbit erythrocyte. The hemolysin lysed cultured cell and killed mouse. Rapid death of mouse was observed with rather small doses of the toxin. Intravenous injection of 20 mg of the purified toxin killed mice within 25 sec. The hemolysin also had a lethal effect on intraperitoneal injection into mice although less than on intravenous injection. Purified hemolysin injected rabbits had large morphological change in jejunum. In electron micrograph of thin sections of the human erythrocytes, cells were threated with the hemolysin at 37 C for 5 min., significant changes were not observed. But after 10 min., hemolysis was observed and after 60 min., complete degradation of human erythrocyte was observed.