Basic studies on a labeled anti-mucin antibody detectable by infrared-fluorescence endoscopy

Background. We developed a fluorescent dye, indocyanine green (ICG)-sulfo-OSu, which was excited by infrared rays and conjugated to various antibodies. We attempted to clarify the staining patterns of anti-sulfomucin and anti-MUC1 antibodies in gastrointestinal cancer. We then evaluated the potential of the dye as a fluorescent label for antibodies specific to cancer, to be used as a diagnostic method for microcancer, with infrared fluorescence endoscopy. Methods. Paraffin sections of samples collected from 10 patients with esophageal cancer, 30 patients with gastric cancer, and 20 patients with colorectal cancer were immunohistologically stained using an anti-sulfomucin antibody and an anti-MUC1 antibody, and the staining patterns were examined. If a section had a high staining intensity, it was reacted with the ICG-suflo-OSu-labeled antibody and evaluated with infrared fluorescence imaging. Results. The staining patterns with the antibodies varied depending on the organs and the histological types and depth of the cancers, but the staining was generally good and the staining on the mucosal surface of cancer tissues was retained. Good images of cancer cells could be obtained by infrared fluorescence observation using the ICG-sulfo-OSu-labeled anti-MUC1 antibody. Conclusions. The anti-MUC1 antibody stained gastrointestinal cancer cells well, and nearly specific infrared fluorescence in cancer tissues was observed using the labeled anti-MUC1 antibody. The ICG-sulfo-OSu-labeled anti-MUC1 antibody has possible usefulness for the screening of cancer via infrared fluorescence endoscopy. Received: December 8, 2000 / Accepted: September 28, 2001     

Mapping the HLA-A24-restricted T-cell epitope peptide from a tumour-associated antigen HER2 / neu: possible immunotherapy for colorectal carcinomas

HER2 / neu is a potential antigen candidate for immunotherapy because of its correlation to a poor prognosis and high expressions in many kinds of epithelial tumours. Especially in the colorectal carcinomas, the higher expression of HER2 / neu is recognized in metastatic regions as well as in primary sites. Several CTL epitopes restricted by HLA-A2.1 and -A3 were identified so far, however epitopes restricted by HLA-A24, that is one of the most common allele in Japanese and Caucasians, have not been identified. In this paper, we showed identification of a CTL epitope peptide of HER2 / neu restricted by HLA-A24. HLA-A24 binding peptides selected by an analysis based on HLA-A24 binding motifs were determined for their binding affinities to HLA-A24 molecules. The peptide with a sequence of RWGLLLALL (position 8-16) named HE1 showed the highest affinity. We induced CTLs from CD8(+)cells of HLA-A24 healthy donors by stimulation with HE1-pulsed autologous dendritic cells. The CTLs showed cytotoxic activity against not only the peptide-pulsed target cells but also HLA-A24 colorectal tumour cell lines that endogenously overexpressed HER2 / neu. The antigen-specificity was confirmed by cold target inhibition assay using HE1-pulsed target cells. In summary, HER2 / neu peptide, RWGLLLALL, may contribute to the induction of antitumour immunity with the peptide-based immunotherapy for the colorectal carcinomas.     

OF4949, new inhibitors of aminopeptidase B. II. Elucidation of structure

The structures of OF4949-I, II, III and IV were identified by analysis of the products of their chemical degradation and by 1H NMR, 13C NMR, and mass spectrometry. These compounds were new cyclic peptides containing diphenyl ether as a chromophore. OF4949-I had two amino acids, beta-hydroxy-L-asparagine and 4-methylisodityrosine. The structural differences between I and II and between III and IV lay solely in the diphenyl ether moiety; the phenolic hydroxyl group in II and IV was methylated in I and III. OF4949-III and IV contained L-asparagine instead of the beta-hydroxy-L-asparagine moiety of I and II.     

Demalonyl derivatives of guanidylfungin A and copiamycin: their synthesis and antifungal activity

Guanidylfungin A was chemically modified by alkylation, reduction and/or demalonylation. Demalonylmethylguanidylfungin A became soluble in water and showed approximately eight-fold higher activity against fungi and Gram-positive bacteria than guanidylfungin A along with strongly fungicidal effect. Similarly, copiamycin was converted to demalonylmethylcopiamycin, which also showed higher antifungal activity than copiamycin itself.     

Studies on new antifungal antibiotics, guanidylfungins A and B. I. Taxonomy, fermentation, isolation and characterization

Guanidylfungin A, C58H103N3O18, and guanidylfungin B, C57H101N3O18, were isolated from the mycelia of Streptomyces hygroscopicus No. 662 by means of silica gel absorption and reversed phase liquid chromatographies. The guanidylfungins were active against fungi and Gram-negative bacteria.     

Apoptosis in placentas from human T-lymphotropic virus type I-seropositive pregnant women: a possible defense mechanism against transmission from mother to fetus.

OBJECTIVE: The mechanism by which the placenta serves as the barrier against mother-to-fetus transmission of microorganisms remains to be elucidated. Programmed cell death, apoptosis, is considered a cellular defense mechanism against infection. The hypothesis of this study is that apoptosis of human T-lymphotropic virus type I (HTLV-I)-infected placental villous cells is involved in the defense mechanism against mother-to-fetus transmission of HTLV-I. METHODS: Apoptosis was compared in term placentas from eight HTLV-I-seropositive pregnant women and eight HTLV-I-seronegative pregnant women by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method. In addition, an in vitro cocultivation with an HTLV-I-infected lymphocyte cell line (MT-2 cells) was performed to examine whether placental villous cells were infected with HTLV-I and apoptosis was induced. RESULTS: The incidence of apoptosis-positive cells (nuclei) in placentas from the HTLV-I-seropositive pregnant women was higher than in the HTLV-I-seronegative pregnant women (P < .02). Cocultivation with MT-2 cells showed that trophoblast cells were able to be infected with HTLV-I and that apoptosis was induced in the placental villous cells. CONCLUSION: HTLV-I infection induces apoptosis in the placenta. We speculate that apoptosis may be involved in the defense mechanism of the placenta against mother-to-fetus transmission of HTLV-I.     

Dendritic cell-based vaccination in metastatic melanoma patients: Phase II clinical trial

Metastatic and chemoresistant melanoma can be a good target of immunotherapy because it is an intractable cancer with a very poor prognosis. Previously, we tested a dendritic cell (DC)-based phase?I vaccine, and confirmed that it was safe. In the present study, we performed a phase?II trial of a DC vaccine for metastatic melanoma patients with mainly the HLA-A24 genotype, and investigated the efficacy of the vaccine. Twenty-four patients with metastatic melanoma were enrolled into a phase?II study of DC-based immunotherapy. The group included 19 HLA-A24-positive (A*2402) patients and 3 HLA-A2-positive (A*0201) patients. The protocol for DC production was similar to that in the phase?I trial. Briefly, a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-A2, MAGE-A3 and MART-1 or MAGE?A1) restricted to HLA-A2 or A24 and KLH were used for DC pulsing. Finally, DCs were injected subcutaneously (s.c.) into the inguinal region in the dose range of 1-5x107 per shot. The DC ratio (lin-HLA-DR+) of the vaccine was 38.1±13.3% and the frequency of CD83+ DCs was 25.7±20.8%. Other parameters regarding DC processing were not different from phase?I. Immune response-related parameters including the ELISPOT assay, DTH reaction to peptide or KLH, DC injection numbers were shown to be related to a good prognosis. The ELISPOT reaction was positive in 75% of the patients vaccinated. The increase of anti-melanoma antigen antibody titer before vaccination was also shown to be a prognosis factor, but that post-vaccination was not. Based on immunohistochemical analysis, CD8 and IL-17 were not involved in the prognosis. Adverse effects of more than grade?III were not seen. Overall survival analysis revealed a significant survival prolongation effect in DC-given melanoma patients. These results suggest that peptide cocktail-treated DC vaccines may be a safe and effective therapy against metastatic melanoma in terms of prolongation of overall survival time.     

FP therapy for controlling malignant ascites in advanced pancreatic cancer patients

BACKGROUND/AIMS: Malignant ascites is one of the poor prognostic factors for pancreatic cancer, and causes serious symptoms and treatment-related toxicity. We conducted a retrospective analysis to evaluate the efficacy of 5-fluorouracil (5-FU) plus cisplatin (FP therapy) for controlling malignant ascites in patients with advanced pancreatic cancer. METHODOLOGY: This analysis was based on 28 consecutive chemotherapy-naive advanced pancreatic cancer patients with cytologically proven malignant ascites who were treated with FP therapy from November 1991 to April 2003. RESULTS: No patients achieved measurable tumor responses. The objective improvement of ascites was seen in 35.7% of the patients (N = 10/28, 95% confidence interval, 18.0 to 53.4%), but there was no patient with complete disappearance of ascites. The median time to disease progression and the median survival time were 1.7 months and 2.7 months, respectively. In all pretreatment variables, the presence of distant metastasis other than peritoneal dissemination was an unfavorable predictive factor for the objective improvement of ascites (Fisher's exact test: P = 0.002). CONCLUSIONS: FP therapy was modestly effective for controlling malignant ascites but insufficient in shrinking for measurable metastatic lesions. Systemic chemotherapy for controlling malignant ascites might be worth while for palliative management in advanced pancreatic cancer patients, especially in patients without distant metastasis.     

Aureobasidins: structure-activity relationships for the inhibition of the human MDR1 P-glycoprotein ABC-transporter

Cyclic depsipeptide cyclo-[D-Hmp(1)-L-MeVal(2)-L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle+ ++(6)-L-MeVal(7)-L-Leu(8)-L-betaHOMeVal(9)], the antifungal antibiotic aureobasidin A (AbA), was reported to interfere with ATP-binding cassette (ABC) transporters in yeast and mammalian cells, particularly the MDR1 P-glycoprotein (Pgp), a transmembrane phospholipid flippase or "hydrophobic vacuum cleaner" that mediates multidrug resistance (MDR) of cancer cells. In a standardized assay that measures Pgp function by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells and the cyclic undecapeptide cyclosporin A (CsA) as a reference MDR-reversing agent (IC(50) of 3.4 microM), AbA was found to be a more active Pgp inhibitor (IC(50) of 2.3 microM). Out of seven natural analogues and 18 chemical derivatives of AbA, several were shown to display even more potent Pgp-inhibitory activity. The Pgp-inhibitory activity was increased about 2-fold by some minor modifications such as those found in the naturally occurring aureobasidins AbB ([D-Hiv(1)]-AbA), AbC ([Val(6)]-AbA), and AbD [gammaHOMeVal(9)]-AbA). The replacement of the [Phe(3)-MePhe(4)-Pro(5)] tripeptide by an 8-aminocaprylic acid or the N(7)()-desmethylation of MeVal(7) led to only a 3.3-fold decreased capacity to inhibit Pgp function, suggesting that the Pgp inhibitory potential of aureobasidins, though favored by the establishment of an antiparallel beta-sheet between the [D-Hmp(1)-L-MeVal(2)-L-Phe(3)] and [L-aIle(6)-L-MeVal(7)-L-Leu(8)-] tripeptides, does not critically depend on the occurrence of the [L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle(6)] type II' beta-turn secondary structure. In contrast, the most potent Pgp inhibitors were found among AbA analogues with [betaHO-MeVal(9)] residue alterations, with some data suggesting a negative impact of the [L-Leu(8)-L-betaHOMeVal(9)-D-Hmp(1)] gamma-turn secondary structure on Pgp inhibitory potential. The [2,3-dehydro-MeVal(9)]-AbA was the most potent Pgp inhibitory aureobasidin, being 13-fold more potent than AbA and 19-fold more potent (on a molar basis) than CsA. Finally, there was no correlation between the SAR for the human MDR1 Pgp inhibition and the SAR for Saccharomyces cerevisiae antifungal activity, which is mediated by an inositol phosphoceramide synthase activity.     

Analysis of IgE reactivities of purified allergens from Candida albicans and Malassezia furfur among patients with atopic dermatitis]

We analyzed the reactivities of a series of purified allergens from Candida albicans (C. albicans) and Malassezia furfur (M. furfur) with IgE antibodies in sera from patients with atopic dermatitis. We compared the specific IgE antibody levels to manganese superoxide dismutase (Mn SOD), cyclophilin, enolase, secretory aspartic protease (SAP 2) and type A mannan from C. albicans and Mn SOD, cyclophilin and Mal f 2 from M. furfur in 21 sera from patients with atopic dermatitis and 20 sera from patients with asthma without atopic dermatitis. The prevalence of IgE antibodies and the mean IgE antibody levels to all of the allergens tested were higher among patients with atopic dermatitis than among those with asthma without atopic dermatitis. More than 50% of patients with atopic dermatitis were IgE antibody-positive to Mn SOD, cyclophilin and type A mannan from C. albicans, and Mn SOD and cyclophilin from M. furfur. The availability of these purified allergens will facilitate studies on the contribution of fungal allergens to the development of atopic dermatitis.