Oncofetal antigen-I (OFA-I) relative antigenicity on melanoma cells

Summary The oncofetal antigen-I (OFA-I) has been defined as an immunogenic antigen that is expressed on human cancer cells and is cross reactive with fetal brain tissue. Quantitative variations in the expression of OFA-I among different cultured melanoma cell lines were determined by absorption techniques based on functionally monospecific anti-OFA-I serum. Allo-antibodies were removed by absorption with lymphoblasts autologous to an OFA-I-positive target cell. Functional monospecificity toward OFA-I was confirmed by complete absorption with a specimen of fetal brain but not by liver from the same fetus.Of 14 melanoma cell lines tested, two did not express OFA-I, whereas 12 expressed the antigen to varying degrees. Five of the cell lines were highly antigenic, and serum absorbed with 5×10⁵ of any of these cell lines could reduce the anti-OFA-I titer (1 : 96) at least four-fold. OFA-I was detected on biopsied melanomas autologous to the antigenic cultured cells. The ability to select highly antigenic cell lines could be useful in further attempts to characterize OFA-I and to monitor tumor immunity in vitro. Antigenic cell lines may improve the response of patients treated in trials of immunotherapy.     

Reaction of S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine with sulfite: Synthesis of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid and application to the determination of sulfite

A procedure for the simultaneous preparation of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid is described. The method is based on the quantitative reaction between sulfite and S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. The yield was 95% for S-sulfo-l-cysteine and 91% for l-alanine 3-sulfinic acid. The reaction was also applied to the quantitative determination of sulfite in biological materials. In this procedure, sulfite reacts with S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. Separation of the reaction product, S-sulfo-l-cysteine, is done by ion-exchange fractionation, and it is determined with acid ninhydrin reagent 2 (M. K. Gaitonde, 1967, Biochem. J.104, 627–663). The recovery was 96.8 ± 0.3%.     

Preparation of new ω-aldehydroalkyl 1-thio-d-glycopyranosides,and their coupling to bovine serum albumin by reductive alkylation

Synthesis of two ω-aldehydoalkyl 1-thioglycosides of d-glucopyranose and of d-galactopyranose is described. 3-Oxopropyl and 2-oxoethyl 1-thioglycosides were prepared by treating a tetra-O-acetyl-1-thioaldose with either acrolein or 2-bromoacetaldehyde, followed by O-deacetylation under mild conditions. These ω-aldehydoalkyl 1-thioglycosides were successfully attached to bovine serum albumin (BSA) by reductive alkylation as described previously. With the 3-oxopropyl 1-thioglycosides, much higher levels of sugar attachment (e.g., ～80 mol of sugar per mol of BSA) were attained than hitherto possible with any sugar derivative tested.     

Interrelation of Two Forms of Ferredoxin-NADP+ Reductase with Different Molecular Weights

The heavier form of ferredoxin-NADP⁺ reductase was extractedfrom spinach leaves or chloroplasts and isolated as the majorfraction at high ionic strengths with ammonium sulfate or sodiumchloride. At low ionic strengths, the form with the higher molecularweight was relatively unstable and was converted gradually intothe lower form. We concluded that the enzyme exists in vivoas the form with the higher molecular weight. ¹Present address: Market Development Department, Shionogi &Co. LTD., 5-Sagisu, Fukushima-ku, Osaka 553, Japan. (Received December 16, 1980; Accepted January 19, 1981)     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen-I (OFA-I)

Summary IgG anti-OFA-I found in melanoma patients was tested for its ability to lyse human tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC). Sera from 89 stage II melanoma patients which contained non-HLA-related IgG antibody to an OFA-I-positive melanoma cell line (M14) as tested by indirect membrane immunofluorescence (IMI) were originally chosen as possible sources of IgG anti-OFA-I. Of those tested for specific IgG activity to OFA-I by IMI, anti-OFA-I was found only in those patients immunized with OFA-I-positive tumor cells. When the same sera were tested in ADCC, no non-HLA-related activity could be demonstrated. This result was confirmed with purified IgG fractions that could, nevertheless, show anti-OFA-I reactivity in a complement-dependent cytotoxicity assay. The fact that naturally occurring IgG anti-OFA-I antibody was not readily detectable in patients' sera and that induced IgG anti-OFA-I did not participate in ADCC indicates that OFA-I-related tumor cell lysis via ADCC is an unlikely phenomenon in cancer patients.     

Macrophage activation for tumor cytotoxicity: Control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines

Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.     

Distribution of cis-Vaccenic Acid in Chinese Hamster V79-R Cells

V79-R Cells grown in lipid-free medium contained octadecenoic acids as the major fatty acids esterified to lipids. Octadecenoic acids were composed of two positional isomers, oleic and cis-vaccenic acids. The distribution of oleic and cis-vaccenic acids was altered by the addition of various fatty acids to the medium. There was no difference in the distribution of oleic and cis-vaccenic acids in phospholipids between mitochondria and microsomes. Cardiolipin contained higher amounts of palmitoleic and cis-vaccenic acids than did other lipids.     

Axial chirality in biaryl N,N-dialkylaminopyridine derivatives bearing an internal carboxy group

Axial chirality in N,N-dimethylaminopyridines as well as N,N-dipropylaminopyridines bearing an internal carboxy group were evaluated based on their racemization barriers and circular dichroism spectra. The half-life of racemization of N,N-dipropylaminopyridine derivative 2 was estimated to be 19.7 days at 20°C. Its enantiomers isolated as optically active forms showed positive-negative and negative-positive Cotton effects for (+)- 2 and (−)- 2 , respectively, from 310 to 210 nm. Furthermore, (−)- 2 was applied as a chiral nucleophilic catalyst and exhibited asymmetric induction in acylative kinetic resolution of 1-(1-naphthyl)ethane-1-ol.     

Decreased Expression of Matrix Metalloproteinase-9 and Increased Expression of Tissue Inhibitors of Matrix Metalloproteinase-1 in Paratuberculosis-Infected Cattle in the ELISA-Negative Subclinical Stage

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24 hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.     

Norovirus Binding to Intestinal Epithelial Cells Is Independent of Histo-Blood Group Antigens

Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le^b-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.