Impact of minimal tumor burden on antibody response to vaccination.

Four randomized phase III trials conducted recently in melanoma patients in the adjuvant setting have been based in part on the correlation between antibody responses in immunized patients and improved survival. Each of these randomized trials demonstrated no clinical benefit, although again there was a significant correlation between antibody response after vaccination and disease free and overall survival. To better understand this paradox, we established a surgical adjuvant model targeting GD2 ganglioside on EL4 lymphoma cells injected into the foot pad followed by amputation at variable intervals. Our findings are (1) comparable strong therapeutic benefit resulted from treatment of mice after amputation with a GD2-KLH conjugate vaccine or with anti-GD2 monoclonal antibody 3F8. (2) The strongest correlation was between antibody induction in response to vaccination and prolonged survival. (3) Antibody titers in response to vaccination in tumor challenged mice as compared to unchallenged mice were far lower despite the absence of detectable recurrences at the time. (4) The half life of administered 3F8 monoclonal antibody (but not control antibody) in challenged mice administered was significantly shorter than the half life of 3F8 antibody in unchallenged controls. The correlation between vaccine-induced antibody titers and prolonged survival may reflect, at least in part, increased tumor burden in antibody-negative mice. Absorption of vaccine-induced antibodies by increased, although not detected tumor burden may also explain the correlation between vaccine-induced antibody titers and survival in the adjuvant clinical trials described above.

Limited effect of CpG ODN in preventing type 1 diabetes in NOD mice.

Type 1 diabetes is considered as Th1 cell mediated autoimmune disease and the suppression of Th1 cells or the activation of Th2 cells has been regarded as a plausible immunologic intervention for the prevention of type 1 diabetogenesis in a rodent model. CpG ODN is an immunostimulatory sequence primarily present in bacterial DNA, viral DNA and BCG. CpG ODN is conventionally classified as a Th1 cell activator, which has been clinically applied to cancer, allergy and infectious disease. Recently, there was a promising report of that CpG ODN administration suppressed the development of type 1 diabetes in NOD mice by inducing Th2 cell mediated cytokine. However, the antidiabetogenic effect of CpG ODN on NOD mice is controversial. Thus, two studies were serially undertaken with various kinds of CpG motif to find a more optimal sequence and administration method. In the first study, CpG ODN was vaccinated four times and pancreatic inflammation and the quantity of serum insulin subsequently evaluated. In the second study, the amounts of IFN gamma and IL-4 in sera were measured as representative cytokines of Th1 and Th2 cells, respectively. As a result, vaccination or continuous injection of CpG ODN failed to show a preventive effect on type 1 diabetogenesis in NOD mice. Structural differences of CpG ODN also had no affect on the result. CpG ODN also consistently showed affect on the pancreatic pathology. The productions of IFN gamma and IL-4 were detected only in the K and D type CpG ODN administration groups. Comparison of the two cytokines leads to the conclusion that CpG ODN generated a Th1-weighted response in both study groups. It was assumed that CpG ODN failed to produce Th2-weighted cytokine milieu, which can overcome the genetically determined phenotype of NOD mice. Given these results, it was concluded that the immunotherapeutic application of CpG ODN on Type 1 diabetes had clear limitations.

In vitro antibacterial activity of echinomycin and a novel analogue, YK2000, against vancomycin-resistant enterococci.

The in vitro inhibitory and bactericidal activity of echinomycin and its the novel synthetic analogues of echinomycin,YK2000 and YK2005, were evaluated using 93 clinical isolates of vancomycin-resistant enterococci (VRE). In agar dilution tests, the MIC(90) of echinomycin and YK2000 were 0.125 and 8 mg/l, respectively, using Mueller-Hinton II agar, while that of YK2005 was 32 mg/l. Bactericidal activity of echinomycin and YK2000 were two to four times higher than the MIC in time-kill assay experiments. These results suggest that echinomycin and its analogues might be useful as anti-VRE drugs.

Susceptibility of several strains of mice to Echinostoma hortense infection.

Susceptibilities of 5 different mice strains, including C3H/HeN, BALB/c, C57BL6, FvB and ICR, to Echinostoma hortense infection, was evaluated. The worm expulsion rate, worm size and egg production were observed from 1 to 8 weeks after infection with 30 metacercariae. C3H/HeN and ICR mice showed the highest worm maturation rates. The worm recovery rate and the number of eggs per gram (EPG) of feces was also higher in C3H/HeN and ICR mice than in BALB/c, C57BL6, and FvB mice. It is suggested that E. hortense is highly infectious to ICR and C3H/HeN mice, but not to the other strains of mice. Based on the results obtained, we believe that the susceptibility of different mouse strains to E. hortense infection is dependent on the genetic and immunologic background of mice.

Echinomycin and a novel analogue induce apoptosis of HT-29 cells via the activation of MAP kinases pathway.

Echinomycin, in typical DNA minor groove binder, had comparable efficacy compared to 5-FU in the phase II trail of colon cancer treatment. To improve echinomycin's drawback (hydrophobicity, toxicity), we synthesized the YK-2000 series (echinomycin analogues). Among these, YK-2000 had the best in vitro cytotoxicity on six different human solid cancer cell lines. Echinomycin and YK-2000 were enabled to induce the apoptosis on the HT-29 colorectal cancer cell line. The hypothesis that apoptosis in the HT-29 cell was triggered by echinomycin and YK-2000 were supported through DNA laddering, poly-(ADP-ribose) polymerase (PARP) cleavage, and flow cytometric analysis. In order to explore the signaling pathway of echinomycin and YK-2000, we examined the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAP kinase. However, what the mechanism of cancer cell death would be induced by echinomycin and YK-2000 is unknown. Here, we present some evidence that one of the major apoptotic signaling pathways induced by echinomycin and YK-2000 is possibly the MAP kinases pathway in HT-29 human colon cancer cells.

Synthesis and biological activity of new quinoxaline antibiotics of echinomycin analogues.

Novel quinoxaline antibiotics having the methylenedithioether bridge as an analogue of echinomycin have been synthesized by insertion of methylene moiety between -S-S- bond. The compound 1a shows remarkable cytotoxicities against human tumor various cell lines, and is active VRE (vancomycin-resistant enterococci) within MIC range 0.5-8 microg/mL. According to the eukaryotic or prokaryotic data, 1a might be a first analogue to replace echinomycin.

Antitumor activity of novel deoxoartemisinin monomers, dimers, and trimer.

The first primary amines 9 and bromoalkyl analogues 7 of deoxoartemisinin with nonacetal functionality at C-12 are prepared as versatile intermediates for the synthesis of various derivatives. Eight C-12 nonacetal type dimers and one trimer of deoxoartemisinin were prepared using novel chemistry. Dimers, particularly 12a, 18a,b, and trimer 17, were especially potent and selective at inhibiting the growth of certain human cancer cell lines and were comparable to that of clinically used anticancer drugs. The linker with one amide- or one sulfur-centered two ethylene groups of the dimers is essential for high anticancer activity. Trimer 17 shows very potent activity against most of the human cancer cell lines tested.