Cancer cell-derived IL-1alpha induces IL-8 release in endothelial cells.

PURPOSE: Cancer cells release a multitude of cytokines and growth factors that influence neighboring cells and help establish a favorable environment for tumor development. As part of our studies designed to elucidate the complex cellular interactions within the tumor microenvironment that facilitate tumor development, we investigated cancer cell-induced changes in gene expression in endothelial cells. METHODS: After treatment of human umbilical vein endothelial cells (HUVEC) with conditioned medium (CM) of SNUC5 colon cancer cells, gene expression profile in HUVEC was analyzed using cDNA microarray. Neutralizing antibodies against pro-inflammatory cytokines were used to identify the major effecter in SNUC5 CM. RESULTS: IL-8 was one of the four genes up-regulated over fourfold, and IL-1alpha in SNUC5 CM was revealed as a major effecter of IL-8 over-expression and release, which was nearly completely neutralized by anti-IL-1alpha antibody. Constitutive secretion of IL-1alpha was confirmed in many other human cancer cells. CONCLUSIONS: IL-1alpha is constitutively expressed in many human cancer cells and directly induces IL-8 secretion in neighboring endothelial cells.

A neutralizable epitope is induced on HGF upon its interaction with its receptor cMet.

A new conformational neutralizable epitope is created on heptocyte growth factor (HGF), when it interacts with its receptor, cMet. By immunizing rabbits with HGF-cMet complex, we successfully generated a monoclonal antibody (SFN68) that inhibits HGF-cMet interaction, and blocks the biological function mediated by HGF. To define the epitope, we screened out an epitope-mimicking peptide, KSLSRHDHIHHH, from a phage display of combinatorial peptide library. In molecular mimicry this peptide bound to cMet and inhibited HGF-cMet interaction. No humoral response was induced to this epitope-mimicking peptide when immunization was done with HGF alone.

Metabolic loading of guanosine induces chondrocyte apoptosis via the Fas pathway.

Although the apoptosis of chondrocytes plays an important role in endochondral ossification, its mechanism has not been elucidated. In this study, we show that guanosine induces chondrocyte apoptosis based on the results of acridine orange/ethidium bromide staining, caspase-3 activation, and sub-G1 fraction analysis. The potent inhibitory effect of dipyridamole, a nucleoside transporter blocker, indicates that extracellular guanosine must enter the chondrocytes to induce apoptosis. We found that guanosine promotes Fas-Fas ligand interaction which, in turn, leads to chondrocyte apoptosis. These findings indicate a novel mechanism for endochondral ossification via metabolic regulation.

Detection of abnormally high amygdalin content in food by an enzyme immunoassay.

Amygdalin is a cyanogenic glycoside compound which is commonly found in the pits of many fruits and raw nuts. Although amygdalin itself is not toxic, it can release cyanide (CN) after hydrolysis when the pits and nuts are crushed, moistened and incubated, possibly within the gastrointestinal tract. CN reversibly inhibits cellular oxidizing enzymes and cyanide poisoning generates a range of clinical symptoms. As some pits and nuts may contain unusually high levels of amygdalin such that there is a sufficient amount to induce critical CN poisoning in humans, the detection of abnormal content of amygdalin in those pits and nuts can be a life-saving measure. Although there are various methods to detect amygdalin in food extracts, an enzyme immunoassay has not been developed for this purpose. In this study we immunized New Zealand White rabbits with an amygdalin-KLH (keyhole limpet hemocyanin) conjugate and succeeded in raising anti-sera reactive to amygdalin, proving that amygdalin can behave as a hapten in rabbits. Using this polyclonal antibody, we developed a competition enzyme immunoassay for determination of amygdalin concentration in aqueous solutions. This technique was able to effectively detect abnormally high amygdalin content in various seeds and nuts. In conclusion, we proved that enzyme immunoassay can be used to determine the amount of amygdalin in food extracts, which will allow automated analysis with high throughput.

Screening of LPS-specific peptides from a phage display library using epoxy beads.

The selection of identical or highly homologous peptides from phage display combinatorial peptide libraries has been unsuccessful in biopanning experiments using microtiter plates. In the present study, by biopanning on LPS-conjugated epoxy beads, we repeatedly enriched clones encoding AWLPWAK and NLQEFLF. These peptides were found to interact with the polysaccharide moiety of LPS, which is highly variable among gram negative bacterial species. In addition, phages encoding these peptides preferentially bound to the LPS of Salmonella family. AWLPWAK-conjugated beads absorbed Salmonella enteritidis from solution and showed a preference for S. enteritidis over Escherichia coli. In summary, this study shows for the first time that a peptide screened from phage displays of combinatorial peptide libraries can be synthesized on beads and be used practically to concentrate bacterial cells from solution.

Generation and characterization of IgG monoclonal antibodies specific for malondialdehyde.

Numerous studies have indicated that the oxidative modification of low-density lipoprotein (LDL) plays a critical role in the pathogenesis of atherosclerosis. Malondialdehyde-modified LDL (MDA-LDL) is one of the candidate oxidative products. Therefore, to allow the assessment of oxidized LDL in human serum, we developed monoclonal antibodies for MDA-LDL. Two of these-MDA1 and MDA2-bound to oxidized LDL but not to native LDL by Western blot analysis. The murine monoclonal antibodies to oxidized LDL have potential clinical implications, as imaging agents, for defining the compositions of atherosclerotic vessels in vivo.

Identification of antigenic peptide recognized by the anti-JL1 leukemia-specific monoclonal antibody from combinatorial peptide phage display libraries.

PURPOSE: In the present study an antigen-mimetic peptide of the anti-JL1 leukemia-specific monoclonal antibody (mAb) was identified and characterized. METHODS: From combinatorial peptide phage display libraries displaying the random linear heptapeptides and dodecapeptides, we selected clones with affinity to anti-JL1 mAb through repeated rounds of panning on a mAb-coated ELISA plate. The antigenicity and immunogenicity of the peptide epitopes were then studied using chemically synthesized peptides. RESULTS: The selected clones had the LXPSIP consensus sequence. Two synthetic peptides LPPSIPFGLTVGGGGS and LLPSIPNQAYLGGGGS specifically reacted with anti-JL1 mAb in ELISA. These two peptides were found to inhibit the interaction between anti-JL1 mAb and JL1 antigen-positive Molt-4 cells. Although the immune sera raised against the keyhole limpet hemocyanin-conjugated peptides failed to react with Molt-4 cells, it showed strong reactivity to the peptide epitope. However, one mAb raised by peptide immunization successfully bound to Molt-4 cells. CONCLUSION: An epitope-mimetic peptide of anti-JL1 mAb was found using combinatorial peptide phage display libraries. It induced strong humoral response against itself, but only a limited fraction of this humoral response was cross-reactive with the original JL1 antigen.