Prevention of inflammation-mediated acquisition of metastatic properties of benign mouse fibrosarcoma cells by administration of an orally available superoxide dismutase.

Weakly tumorigenic and nonmetastatic QR-32 cells derived from a fibrosarcoma in C57BL6 mouse are converted to malignant cells once they have grown after being coimplanted with a gelatine sponge which induces inflammation. We administered a newly developed peroral superoxide dismutase (SOD), oxykine, and as control vehicle, gliadin and saline, starting 2 days before the coimplantation and continued daily throughout the experiment. In the oxykine group, tumour incidence was lower (41%) than in the gliadin or saline group (83 and 79%, respectively). The inhibitory effect of oxykine was lost when an individual component of oxykine was administered, that is, SOD alone and gliadin alone. The effect was also abolished when administered by intraperitoneal route. When perfused in situ with nitroblue tetrazolium, an indicator of superoxide formation, the tumour masses from gliadin and saline groups displayed intense formazan deposition, whereas, those from oxykine group had less deposition. Enzymatic activity of SOD was also increased in oxykine group. Arising tumour cells in gliadin and saline groups acquired metastatic phenotype, but those in oxykine group showed reduced metastatic ability. These results suggested that the orally active SOD derivative prevented tumour progression promoted by inflammation, which is thought to be through scavenging inflammatory cell-derived superoxide anion.

Immunogenicity of various beta-lactam antibiotic-protein conjugates and cross-reactivity of the antibodies produced in guinea pig.

Immunogenic properties of bovine serum albumin (BSA) conjugates of various beta-lactam antibiotics (ABs) and cross-reactivity of the produced antibodies were investigated in guinea pigs. Each AB-BSA conjugate was immunized with complete Freund's adjuvant. Following the second injection, AB-specific antibodies were detected with high titers. Produced antibodies showed concentration-dependent inhibition by the corresponding AB. Cross-reactivity between the produced antibodies and ABs was evaluated with two methods, i.e. by testing for reactivity with the various AB-ovalbumin conjugates, and by assaying for inhibition of enzyme-linked immunosorbent assay (ELISA) by the various ABs. The latter method could detect minor cross-reactions more sensitively than the former one. The results suggested that similarity of the acyl side chain of a cephalosporin nucleus or a penicillin nucleus contributes to immunological cross-reactivity.

Drug-specific immune responses induced by immunization with drugs in guinea pigs and mice.

In order to develop a system for evaluating the allergenicity of drugs in clinical use, we tested drugs for the ability to induce drug-specific immune responses in guinea pigs and mice. Test drugs were benzylpenicillin, procainamide, hydralazine, isoniazid, alpha-methyldopa, D-penicillamine, captopril, sulfamethoxazole and 2,4-dinitrochlorobenzene (DNCB), which are known to induce allergic responses in man including hypersensitivity reactions and drug-induced auto-immune responses. Guinea pigs were immunized with an emulsion of complete Freund's adjuvant (CFA) and 25 mg of each drug. Mice were immunized with an emulsion of CFA and 2 mg of each drug or a mixture of aluminum hydroxide gel and 2 mg of each drug. In order to examine drug-specific immune responses, we employed detection of antibodies by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis tests, active systemic anaphylaxis (ASA) tests and delayed type hypersensitivity (DTH) tests. In guinea pigs, drug-specific antibodies were detected following immunization with benzylpenicillin, procainamide, hydralazine, isoniazid, captopril, sulfamethoxazole or DNCB. Some of these drugs were also positive in DTH tests and/or ASA tests. In mice, however, only DNCB gave positive results. Therefore, our system involving immunization of guinea pigs with CFA emulsion of a drug and detection of drug-specific immune responses is considered to be an effective test method for evaluating drug allergenicity.

Immunogenic properties of structurally modified human tissue plasminogen activators in chimpanzees and mice.

Immunogenic properties of second generation human tissue plasminogen activator (tPA) derivatives were examined in chimpanzee and mouse systems. Five species of modified tPAs (mtPAs) (designated 2660, 2663, 2810, 8000, and 9200), recombinant native tPA or bovine serum albumin (BSA) as a positive control were subcutaneously injected nine times at suitable intervals into chimpanzees, genetically the closest species to man. These animals were tested for antigen(Ag)-specific antibodies to the corresponding proteins by means of enzyme-linked immunosorbent assay and Western blot analysis. Neither 9200, one of the five mtPAs tested, nor tPA was immunogenic, although BSA and the other four mtPAs were immunogenic under these conditions. Thus, an antigenic determinant was not exposed by the modification on 9200 and this modified tPA is expected not to be immunogenic in humans. In the mouse studies, mice were immunized with mtPAs. Serum samples from these animals were tested for antibodies to the mtPAs which did not concomitantly recognize native tPA by immune adsorption of the antibodies to tPA. The amount of such antibodies after the elimination of native tPA-reactive antibodies was little or none when the serum samples from 9200 and from the other mtPAs, except 8000, were tested. Taking into consideration the results of the chimpanzee studies, it can be concluded that Ag-specific antibodies are dominantly produced to unchanged epitopes present in modified proteins in the mouse system, in which the native protein is immunogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-specific immune responses induced by procainamide, hydralazine and isoniazid in guinea-pigs.

The drug-induced graft vs host reaction (GVHR) hypothesis requires, as its first step, specific T-cell immune responses to the drug-modified self. Procainamide, isoniazid and hydralazine are known to provoke various allergic reactions including GVHR-like adverse effects in man. We now report that drug-specific immune responses can easily be induced by these drugs in guinea-pigs. Twenty-five milligrams of each of these drugs and penicillin G, which is known to make covalent bonds with proteins and to also induce drug-specific immune responses, were mixed with complete Freund's adjuvant (CFA) and subcutaneously (s.c.) injected twice at an interval of 2 weeks into female Hartley guinea-pigs. The antibodies to these drugs were assessed by means of an enzyme-linked immunosorbent assay (ELISA). Two weeks after the last injection, all animals treated with isoniazid, hydralazine and penicillin G produced high titers of antibodies to these drugs. Antibodies to procainamide were also detected, although their antibody titers were low. The specificity of the antibodies produced were tested by the inhibition of ELISA and concentration-dependent inhibition was observed. Delayed type hypersensitivity (DTH) reactions were also observed in the animals treated with procainamide, isoniazid and hydralazine 2 weeks after the last injection. These results suggest that the allergic reactions observed in clinical use are related to the inducing potential of drug-specific immune responses in an animal system. Therefore, immunization of guinea-pigs with test drugs and CFA may give useful information for predicting the occurrence of allergic reactions in man.

Popliteal lymph node enlargement induced by procainamide.

The ability of procainamide (PA) to induce primary local popliteal lymph node (PLN) reactions has been investigated. We employed an in vitro drug-metabolizing system, based on the hypothesis that the negative result for PA in a PLN assay (PLNA) was due to insufficient metabolizing activity at the reaction site. PA was incubated previously in vitro with the S-9 mixture derived from rat liver. The reactants were ultrafiltered in order to eliminate high molecular-weight molecules, and then the low molecular-weight fraction was subcutaneously (s.c.) injected into the hind footpad of mice. PLN reactions were assessed by weighing the popliteal lymph node of the injected side. The reactants of more than 5 mg of PA and S-9 mixture induced PLN enlargement in C3H/He mice 8 days after injection. BALB/c and C57BL/6 mice were also susceptible to PLN reaction in response to the reactants of PA (10 mg) and S-9 mixture. PLN reactions to PA were induced 2 days after the injection and sustained until 18 days. Contact of PA with the S-9 mixture for 30 min at 37 degrees C was sufficient to induce PLN enlargement. However, contact for 24 h reduced the peak reaction. On the other hand, PA which had not been incubated with the S-9 mixture and acetylprocainamide (acetylPA) gave little or no reaction. Local PNA has not been considered to be suitable for the detection of drugs with the potential to induce immune disorders in cases where a metabolite contributes to the adverse reaction. However, the employment of an in vitro drug-metabolizing system may overcome that defect.

Ganglioside-induced inhibition of in vivo immune response in mice.

In vitro immunomodulatory properties of gangliosides have been well characterized such as the ganglioside-induced modulation of CD4 on T lymphocytes and inhibition of lectin-induced proliferative response of lymphocytes. These findings have led to an interesting suggestion that gangliosides play a role as in vivo immune modulators, although this possibility is not clearly defined yet. We then first confirmed in vitro effects of gangliosides on murine immunocytes and examined in vivo effects of gangliosides on immune response in mice. Murine spleen cells that were treated with a ganglioside mixture (GS) purified from bovine brain exhibited a marked decrease in CD4 expression, while CD8 expression was slightly suppressed. Transplantation of GS-untreated control immunocytes that were isolated from syngeneic mice into the immune suppressed mice by X-ray irradiation restored in vivo immune responses, while GS-treated cells could not. Immune response was assayed by the evaluation of footpad swelling which was induced by immunization with sheep erythrocytes as antigens. Moreover, intramuscular administration of gangliosides into mice suppressed both immediate (Arthus)-type and delayed-type allergic reactions. These results suggest that gangliosides would be potential in vivo immune modulators.

Cytotoxic activity of leukotoxin, a neutrophil-derived fatty acid epoxide, on cultured human cells.

Leukotoxin (LX: 9,10-epoxy-12-octadecenoate) which was previously found to be biosynthesized by neutrophils, exhibited a dominant cytotoxic activity toward human tumor cells as well as toward normal cells. At the concentration of 50-100 micrograms LX/ml cell culture medium, LX completely blocked the growth of the cells of several transformed cell lines (HeLa, Hep-2, MG-63, Chang liver, Wish, FL-Amnion) as well as the normal (Flow 2000 and Flow 13000). From the results, a part of inhibition of tumor growth by neutrophils can be attributed to LX.

Induction of antitumor immunity by tumor cells treated with abrin.

Abrin is known as a cytotoxic lectin. Immunization with Meth-A tumor cells which were treated in vitro with abrin induced a strong antitumor immunity in syngeneic BALB/c mice. The immunizing effect was stronger than that produced by an irradiated Meth-A tumor cell vaccine. Studies on the mechanisms of the immunizing effect with the abrin-treated tumor cells demonstrated that abrin acts as an immunoadjuvant. Furthermore, the regression of a growing Meth-A tumor was observed after abrin was injected into the tumor, while the induction of a strong antitumor immunity also occurred. It appears, therefore, that the antitumor effects of abrin are attributable to two kinds of activity: cytotoxicity and adjuvant activity.

A new method for extraction of extravasated dye in the skin and the influence of fasting stress on passive cutaneous anaphylaxis in guinea pigs and rats.

A method for quantitative extraction of extravasated dye from the skin was studied in guinea pigs and rats. A simple method with a low cost and good recovery was established as follows; A piece of the skin containing extravasated dye was soaked overnight in a stoppered glass tube containing 1 ml of 1 N KOH at 37 C. Then, 9 ml of mixed solution of 0.6 N H3PO4 and acetone (5:13) was added to the tube. The tube was shaken vigorously for a few seconds and centrifuged at 3,000 rpm for 15 min. Absorbance of supernatant was measured at 620 nm. The recovery rate of the dye was about 95 per cent both in guinea pigs and rats. Using this method we observed that fasting stress significantly reduced the intensity of skin reaction induced by chemical mediators, heterologous PCA and especially homologous PCA in guinea pigs.

Common protective antigen (OEP) of Pseudomonas aeruginosa.

Prior immunization of mice with the protein moiety of the endotoxin (OEP) of Pseudomonas aeruginosa isolated by Homma and his associates [1, 2], can protect mice against infection with any of 21 standard strains belonging to Fisher's immunotypes and Homma's serotypes. It is also confirmed that there is found a significant protection in mice each injected with 0.1 ml of anti-OEP rabbit serum against challenge with each of 7 strains of Fisher's immunotypes. It has been revealed through the OEP-HA, agar gel diffusion and indirect fluorescent antibody tests that OEP is an antigen existing serologically in common in P. aeruginosa.