Okayama University Medical School

We investigated the antitumor effect of purified natural human tumor necrosis factor-beta (nHuTNF-beta) produced by human acute lymphoblastic leukemia BALL-1 cells stimulated with HVJ on pulmonary metastatic tumors of Lewis lung carcinoma (3LL) transplanted into BDF1 mice. nHuTNF-beta showed antiproliferative effects on metastatic tumors in a dose-dependent manner when administered daily for 10 days by the intravenous route. Histological examination of the tumors treated with nHuTNF-beta revealed that the tumor size and number of metastases were much reduced. Lytic cellular changes, including cytoplasmic vacuolation, loosening of the intercellular junction and both cytoplasmic and nuclear swelling, were found, but tumor necrosis was not. These findings indicate a therapeutic effect of Grade IIa according to the histological criteria of Shimosato and Ohboshi. In addition, synergistic augmentation of the antiproliferative effects of nHuTNF-beta by natural murine interferon-alpha/beta (nMu-IFN-alpha/beta) or recombinant murine interferon-gamma (rMuIFN-gamma) was recognized by median effect plot analysis. The results suggested that nHuTNF-beta may well deserve clinical trial as a new immunotherapeutical agent for human cancer.

The development of useful therapy for intraabdominal carcinomatosis originating from gastrointestinal cancer is an important theme in cancer therapy. We developed recently an experimental model of intraabdominal carcinomatosis in nude mice by intraperitoneal transplantation of human colon cancer cells (RPMI 4788). Using this model, we investigated the antitumor effects of recombinant human interferon (rIFN)-beta and rIFN-gamma administered singly or in combination. Treatment was initiated 2 days after CD-1 nude mice were inoculated intraperitoneally with 5 X 10(6) RPMI 4788 cells. Intraperitoneal administration for 10 consecutive days of either rIFN-beta (2.5 X 10(5) IU/mouse/day) or rIFN-gamma (2.5 X 10(5) JRU/mouse/day) resulted in a significant prolongation of survival compared with the saline control group [survival in the control: 41.8 +/- 5.6 days (mean +/- SD)]. Combined administration of rIFN-beta and rIFN-gamma for 10 days yielded a marked synergistic effect on the prolongation of survival (114.0 +/- 8.2 days). However, combined administration of rIFN-beta and rIFN-gamma in a single dose equal to the total dose given fractionally over 10 days did not yield a synergistic effect. These results suggest that daily administration of rIFN-beta and rIFN-gamma combined may provide a highly potent antitumor effect against human peritoneal carcinomatosis.

Using C3H/He mice, the antitumor effect of lipopolysaccharide (LPS) alone and in combination with Lentinan extracted from Lentinus edodes was studied. The influence of LPS on cellular immunity and the antitumor effect of the tumor necrosis factor (TNF) were also examined. LPS, which was administered into mice with tumor, induced hemorrhagic necrosis of the tumor within 48 h, demonstrating a high antitumor effect. When LPS was used in combination with Lentinan, the tumor growth was significantly inhibited as compared to that in the control mice. The combination of LPS and Lentinan prevented a decrease in the delayed type hypersensitivity in tumor bearing mice. Application of rabbit serum containing TNF resulted in hemorrhagic necrosis of the tumor within 48 h, as with LPS.

Antitumor activities of five platinum analogs, including cisplatin, carboplatin, 254-S, DWA2114R, and NK121, were compared using five human lung cancer cell lines and 19 tumor specimens obtained from lung cancer patients. The antitumor activity was evaluated by determining the ratio of the maximum tolerated dose of each drug to the 70% tumor growth inhibitory concentration in a colony assay. Cisplatin was the most potent agent, followed by 254-S and carboplatin. DWA2114R and NK121 were less potent than cisplatin and 254-S. Cross-resistance to adriamycin was also investigated using an adriamycin-resistant small cell lung cancer subline, SBC -3/ADM30. SBC-3/ADM30 was 1.7- to 4.0-fold more resistant to cisplatin, carboplatin, NK121, and DWA2114R, than was the parent line, SBC-3, and the subline was 2.0-fold more sensitive to 254-S. Using SBC-3, in vitro combination effects of etoposide and cisplatin, carboplatin, or 254-S were evaluated by the median-effect principle. Synergism was noted when cisplatin and etoposide were combined at a fixed molar ratio of 1:1. Combination of carboplatin and etoposide showed an additive effect. The combination of 254-S and etoposide was antagonistic at low concentrations, but was markedly synergistic at higher concentrations. These data suggested the efficacy of 254-S in the treatment of lung cancer.

Neocarzinostatin (NCS), an antibiotic with a high molecular weight, showed an inhibittory effect on Rauscher mouse leukemia. In normal mice, no significant changes were found in peri· pheral blood pictures except a tendency of lymphocytopenia, when O. 05mg/kg/day and O.50mg/kg/day of NCS were injected intraperi. toneally to two groups of mice for three days. On the other hand, peripheral nucleated cells of Rauscher leukemic mice decreased after intraperitoneal administration of NCS in a dose over O.25mg/kg/day for three days. The cells affected by NCS were mainly erythroblasts and smudged cells. Spleens of Rauscher leukemic mice treated with NCS have been reduced in weight, and histological examinations of livers showed a signicant decrease of infiltrating cells. In three groups treated with 0.25mg/kg/day of NCS for seven days, O.25mg/kg/day for three days and O.50mg/kg/day for three days, the.5()% survival time was longer than in the control group. Particularly, the 50% survival time in Rauscher leukemic mice treated with 0.50 mg/kg/day for three days was over twice that of the control group.

The present experiment was undertaken to study what types of human cancers are responsive to the antiproliferative effects of suramin. The human malignant cells used were as follows: cervical cancer (HeLa), mammary cancer (MCF-7), bladder cancer (EJ), hepatoma (HuH-7, PLC/PRF/5), embryonal carcinoma (PA-1), in vitro transformed fibroblasts (KMST-6, SUSM-1, VA-13), five myeloma cell lines (KMM-1, KMS-5, KMS-11, KMS-12, RPMI 8226), Burkitt's lymphoma (Raji), acute promyelocytic leukemia (HL-60), chronic myelocytic leukemia (K562), Epstein-Barr virus nuclear antigen positive lymphoblastoid cells (KMS-9). The cells were treated with 25 to 100 micrograms/ml suramin for 72h. Proliferation of HuH-7 and two human myeloma cells (KMS-11 and KMS-12) was remarkably inhibited, and that of PA-1, PLC/PRF/5, KMST-6, two other myeloma cell lines (KMM-1 and KMS-5), Raji and HL-60, was moderately inhibited. In order to confirm part of the results obtained from in vitro experiments, in vivo experiments were also undertaken. The growth of HuH-7 cells transplanted subcutaneously into nude mice was significantly suppressed by intravenous injection of suramin. We discussed the possibility that certain types of human cancers, the growth of which seemed to be more or less dependent on polypeptide growth factors, might be sensitive to the antiproliferative effects of suramin.

Chinese ant extract preparations (CAEP) are a Chinese traditional medicine which is mainly used as a health food or drink for the treatment of rheumatism, rheumatoid arthritis, chronic hepatitis, sexual hypofunction, and antiaging in China. The effects on free radicals were examined by electron spin resonance spectrometry using the spin trapping agent 5.5-dimethyl-1-pyrroline-1-oxide (DMPO). Superoxide radicals (3.35 x 10(15) spins/ml) were quenched 50% by the extract at 0.5 mg/ml. The CAEP extract at 0.7 mg/ml inhibited 50% of hydroxyl radicals (52.0 x 10(15) spins/ml) generated by the Fenton reaction. Against DPPH radical, the scavenging action of CAEP was observed at 1.8 mg/ml of the extract and 50% of the DPPH radicals (8.14 x 10(15) spins/ml) were quenched. In vitro tests showed that CAEP inhibited the production of thiobarbituric acid-reactive substances, an index of lipid peroxidation, in rat brain homogenate.

The antimycotic action of 1, 4-bis-(m, m'-amidinophenoxymethyl)-cyclohexane dilactate (MAC), a synthetic diamidine compound, on Candida albicans was studied. The minimum inhibitory concentration (MIC) ranged from 3.31 to 6.25 micrograms/ml against both standard and clinically isolated strains. MAC was fungistatic at MIC and weakly fungicidal at the concentration of 100 micrograms/ml. MAC did not affect the cell wall or cause cell lysis. Intracellular constituents, such as 260 nm and 280 nm absorbing materials, were released from the cells by treatment with MAC indicating that MAC affected membrane permeability. The release of 260 nm absorbing material was inhibited by the presence of Ca2+ and Mg2+. Acidic phospholipids such as cardiolipin and phosphatidylglycerol inhibited the anti-Candida activity of MAC, but sterols and lecithin were not inhibitory, indicating that MAC interacted with acidic phospholipids of the cell membrane. Freeze-fracture electron microscopy showed that MAC caused aggregation of membrane particles and patch formation on the P face, which may indicate that MAC is a membrane disrupting agent. It appeared that MAC affected C. albicans at the cell membrane by interacting with acidic phospholipids and caused disorganization of the membrane structure resulting in the release of intracellular constituents without lysis.

Both the cornea and muscle cornins have no effect at all on oxygen uptake of tissue, and likewise they catalase affect Qctivity not in any way. The corneacornin has an effect to reduce P/O ratio to about one half, but the muscle cornin does not show such an effect. Both comins decrease thc incorporation of P³² into nucleic acid fraction and DNA synthesis. In the ultracentrifugal analysis of nucleic acids during development of sea urchin eggs, cornins inhibit the polymerization of nucleic acids. In addition, both of these comins depress the incorporation of P³² into DNA and ribosome RNA of regenerating rat liver. Both comins inhibit the increase of -SH quantities before the cleavage of sea urchin eggs.

We succeeded in the extraction of a substance from beef cornea and rabbit muscle, that markedly inhibits mitosis of sea urchin eggs. The substance extracted from beef cornea is non-dialysable and it can be separated into three fractions by DEAE-cellulose column. Although everyone of these fractions has an antimitotic action, that of fractions II and III is especially marked. These fractions are one of nucleoproteins that have adenine as base. The substance extracted from rabbit muscle is dialysable, and when it is fractionated through DEAE-cellulose column into three fractions, fraction I has no antimitotic effect but fractions II and III have it. Fraction II is one of nucleoproteins that have hypoxanthine as base. Carnin obtained from beef cornea or from rabbit muscle shows a typical protein wave, but after being treated with gas by passing oxygen through cornin solution the wave height is lowered. Carnin, however, is a very stable substance when kept dry in a desiccator.

Angiotensin I-converting enzyme (ACE) inhibitory and hypotensive effects of 7 peptide fractions (Frs) of royal jelly protein hydrolysate (RJPH) were studied in comparison with those of RJPH alone. Fr 4 and Fr 5 were the highest in ACE inhibitory activity and yield, respectively. Molecular weights (MWs) of RJPH and Fr 1-Fr 7 were distributed from 100 to 5,000 and those of Fr 1-Fr 7 increased in order from Fr 1 to Fr 7. RJPH, Fr 3 and Fr 4 at doses of 10, 30 and 100mg/kg i.v. and Fr 5 and Fr 6 at doses of 30 and 100mg/kg i.v. caused transiently significant hypotensive effects in spontaneously hypertensive rats (SHR). Fr 3, Fr 4, Fr 5 and Fr 6 at a dose of 1,000mg/kg also caused significant hypotensive effects 3h, 4-5h, 7-8h and 8h after oral administration in SHR, respectively. RJPH caused a long-lasting hypotensive effect in proportion to the magnitude of the MWs of RJPH fractions. The hypotensive pattern of RJPH was similar to the combined pattern of Fr 3-Fr 6. From these results, it can be concluded that the long-lasting hypotensive effect of oral administration of RJPH is dependent on the MWs of its ACE inhibitory peptides and the time required to digest them.

Antigenicity of clozapine was studied in rabbits, comparing with that of chlorpromazine as control. The results indicate that chlorpromazine produces antibody in rabbit as revealed by passive hemagglutination test, giving the titer of 1 : 2, 000 or higher in all the five cases observed, though specific precipitin lines has not been obtained and PCA test proves to be negative. Clozapine failed to produce anti.clozapine antibody giving negative passive hemagglutination test, passive cutaneous anaphylaxis and precipitin reaction, in all forms tested. Some remarks were made on the possible close relation between the antigenicity of the drug and its affinity to protein.

Marked IgE-mediated histamine release from rat mast cells sensitized in vitro with mouse antiserum occurs in the presence of added Ca++ and phosphatidylserine (PS), although a considerable degree of antigen-induced histamine release which may utilize intracellular or cell-bound calcium is also observed. The decay in the responsiveness to Ca++ of the sensitized cells stimulated by antigen in Ca++-free medium in the presence of PS is relatively slow, and maximum release is produced by Ca++ added 1 min after antigen. Histamine release also occurs when Ca++ is added after PS in the absence of antigen to the sensitized cells suspended in Ca++-free medium. Unlike the antigen-induced release, the intensity of this non-antigen-induced release varies depending on both mast-cell and antiserum pools. A heat-labile factor(s), which is different from antigen-specific IgE antibody and is also contained in normal mouse serum, is involved in this reaction. In the antigen-nondependent (PS + Ca++)-induced release, no decay in the responsiveness to Ca++ is observed after PS addition. Both the antigen-induced and non-antigen-induced release are completed fairly rapidly and are dependent of temperature, pH and energy.

A significant amount of anticoagulant substance was released along with histamine, when human lung mast cells were stimulated with anti-IgE and Ca-ionophore A23187. Its activity was lost by heparinase, not by chondroitin-ABC lyase or chondroitin-AC lyase, and also inhibited by Polybrene, suggesting it would be heparin.

The anticancer drug sensitivity of human cancers was tested by the human tumor clonogenic assay (HTCA). Of 152 human cancer specimens tested, 63 (41%) formed more than 30 tumor cell colonies in control plates and could be used to evaluate the drug sensitivity of tumor cells. In 42 (93%) of 45 clinical trials in 24 patients, a parallel correlation was observed between the in vitro anticancer drug sensitivity measured by the HTCA and the clinical response of tumors to anticancer drugs. These results suggest that the HTCA is a good technique for the in vitro test of the anticancer drug sensitivity of human cancers.

We investigated the antibody dependent cell-mediated cytotoxicity (ADCC) of lymphocytes and monocytes toward human O+ red cells coated with anti-D antibody using a 51Cr release assay. Lysis of sensitized red cells by lymphocytes occurred rapidly, but monocyte-mediated lysis occurred slowly. This difference might be due to postphagocytic 51Cr release by monocytes. ADCC of lymphocytes increased in proportion to the effector cell number, but large amounts of antibodies were required. In contrast, ADCC of monocytes was independent of the effector/target ratio and very small amounts of antibodies could produce red cell lysis. Large amounts of fluid phase IgG were required to inhibit the lymphocyte ADCC, whereas the monocyte ADCC was markedly inhibited by small amounts of IgG. Monocyte-mediated lysis was completely inhibited by the addition of 10% human AB serum, but lymphocyte-mediated lysis was only slightly inhibited. Purified IgG1 and IgG3 were much more inhibitory to the lysis by both effectors than IgG2 and IgG4 (IgG2 greater than IgG4). Erythrophagocytosis also was inhibited by IgG1 and IgG3. These studies demonstrate that lymphocytes as well as monocytes can cause the lysis of antibody sensitized red cells, and IgG1 and IgG3 subclasses are more important than IgG2 and IgG4 in causing lysis of anti-D coated red cells.

Antigenicity of the peptide of ribosomal digest in Ehrlich ascites tumor was studied. The peptide was purified by DEAE Sephadex A-50 column chromatography. The peptide was electrophoretically basic, single, and 1.32 S20w sedimentation coefficient with poor content of tyrosine and phenylalanine. The maximum absorbancy was at 267 mμ. Mice and rabbits were immunized with the mixture of the purified peptide with Freund's complete adjuvant. The inhibitory effect of immune γ-globulin on the tumor growth was demonstrated in vitro cultured JTC-11 cells. A single precipitin line was observed between rabbit antiserum and tumor cell extract of Ehrlich ascites cells in ouchterlony double diffusion chamber and immunoelectrophoresis. The sedimentation coefficient of the effective fraction in immune-serum was 17 S20w. The precipitin line was observed at β2-γ region in immunoelectrophoresis.

A unique low density lipoprotein was obtained from the tumor transplanted with a cultured cell line of Ehrlich ascites tumor, JTC-ll cell. The tumor low density lipoprotein electrophoretically migrated as a single band, and the mobility was different from that of other organs. The chemical composition of lipid, cholesterol and phospholipids in tumor low density lipoprotein were characteristic. The flotation rate was Sf 5.9, and thus the molecular weight was estimated to be about 130 x 104. The inhibitory effect on tumor growth of the immune serum was most elevated at 25th day after the intraperitoneal administration of tumor low density lipoprotein. The main fraction effective for inhibition of tumor growth existed in γ-globulin.

Helicobacter pylori (H. pylori) infection in the stomach is etiologically closely associated with chronic active gastritis, peptic ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. In this study, we examined the antibody responses and cytokine profiles of three strains of mice (BALB/c, C3H/He, and C57BL/6) infected with H. pylori. Following this, correlations between host-immune reactions and intensity of inflammation were analyzed. H. pylori (ATCC43504) was intragastrically administered once a week to the mice from 4 weeks of age, and they were sacrificed at the ages of 4 and 7 months. In these mice, we examined the histology of the stomach, antibody titers against H. pylori, and serum levels of cytokines (IL-4, IL-10, TNF-alpha, IL-2 and Interferon-gamma). In BALB/c mice, inflammation of the stomach was minimal. Inflammation was observed in 63.6% of C57BL/6 mice and 33.3% of C3h/He mice. In C57BL/6 and C3H/He mice, all the cytokines tended to increase. In contrast, BALB/c mice were inactive in cytokine production except for IL-2. Two C3H/He mice developed severe inflammation with lymph follicles; one showed a response largely typical of Th-1, and the other showed a response largely typical of Th-2. Although a definite correlation was not shown between Th-1/Th-2 response evaluated by cytokine production and intensity of inflammation, it appears that in H. pylori-induced inflammation both cell-mediated (Th-1) and humoral (Th-2) immunity play a role in pathogenesis.