Search

We have recently reported that the intra-tumoral injection of adrenomedullin (AM) antagonist (AMA; AM (22-52)) peptides significantly reduced the in vivo growth of a pancreatic cancer cell line in severely combined immunodeficient (SCID) mice. In the present study, we examined the effects of intra-tumoral and intra-muscular transfers of naked DNA encoding AMA on the in vivo growth of cancer cell lines. We demonstrate that these treatments induce the regression of a pancreatic cancer cell line and a breast cancer cell line inoculated in SCID mice. Furthermore, CD31-positive cells disappear completely from tumor tissues, following treatment, indicating that neo-vascularization is entirely inhibited. These results suggest that the intra-tumoral or intra-muscular transfer of naked DNA encoding AMA might be a promising alternative modality for treating human cancers.

Lentinan, an antitumor polysaccharide, has been assessed for its potential in vivo to augment erythroid progenitor cells and protect them from the cytotoxic effects of antitumor chemotherapeutics. Lentinan augmented the level of burst-forming units-erythroid (BFU-E) and accelerated the recovery of the reduced number of BFU-E in mice treated with 5-fluorouracil (5-FU); lentinan did not influence red blood cell counts or colony-forming unit-erythroid (CFU-E) numbers in the femoral marrow. A significant decrease in stem cell inhibitory factor (SCIF) activities in bone marrow and an increase in colony-forming unit-spleen (CFU-S) formation were observed in the lentinan-treated mice. The mechanism of augmented BFU-E formation may be partly due to augmented production of stem cells, giving rise to both CFU-GM and BFU-E. Furthermore, when lentinan administration was followed by administration of erythropoietin (Epo) in 5-FU-treated mice, increases in femoral marrow and splenic CFU-E formation and augmentation of reticulocyte counts were observed beyond the level observed in mice treated with Epo alone. These results suggest that lentinan may augment the effects of Epo on erythropoiesis in the course of anemia and the decreased erythropoiesis in cancer patients receiving chemotherapy.

To determine whether resistance to chemoimmunotherapy is acquired during therapy, we investigated the effects of chemotherapeutic agents and anti-tumour polysaccharide, lentinan, on the progression of Rous sarcoma virus-induced S908.D2 fibrosarcomas. The chemoimmunotherapy was effective against the parental S908.D2-bearing mice. Nearly all the mice that were treated with cyclophosphamide (CY) and lentinan achieved complete tumour regression. Only a few of the mice that achieved complete regression of the primary tumours showed a recurrence of the tumour in regional lymph nodes. S908.D2-vp.1 was established from metastatic tumours that developed in the regional lymph nodes of parental S908.D2-bearing mice during therapy. S908.D2-vp.2-or vp.3 cells were sequentially derived in a similar way from S908.D2-vp.1-or-vp.2-bearing mice respectively, in which complete tumour regression at each primary site was achieved during therapy. These lines acquired resistance to CY and lentinan and also to 5-fluorouracil (5-FU)/5'-deoxy-5-fluorouracil and lentinan. No significant difference in either the sensitivity to 5-FU or 4-deoxycyclophosphamide in vitro or in the susceptibility to immune effector cells was observed between the parental and progressed lines (S908.D2-vp1 -vp3). There was an increase in the level of prostaglandin E2 (PGE2) in the progressed lines during repeated therapy (parental, 1171 pg ml(-1); vp.1, 2199 pg ml(-1); vp.2, 5500pg ml(-1); vp3, 16187 pg ml(-1)). There was no significant increase in the production of transforming growth factor beta (TGF-beta). The amount of interleukin-2 (IL-2) produced by spleen cells isolated from the S908.D2-vp.2-bearing mice was decreased compared with the amount produced by the parental S908.D2- bearing mice. Furthermore, combination therapy with lentinan and IL-2 achieved complete tumour regression in all the mice transplanted with S908.D2 progressed tumour lines, although IL-2 alone did not show any anti-tumour effects in either the S908.D2 parental or progressed lines. The findings suggest that the reduced production of IL-2 induced an increase in the production of the PGE2 by progressed tumour lines is involved in the acquisition of resistance.

Lentinan was administered for gastric cancer in order to determine what type of changes would occur as a consequence. In an experimental study, the intraperitoneal administration of lentinan on cancerous tissues caused a marked development of reticular fibers, along with an enhanced interstitial response. Findings which support the relationship between a marked development of reticular fibers and an anti-tumor effect, were also obtained. Furthermore, lentinan was administered for human gastric cancer to evaluate whether or not an increase in interstitial response would be observed. As a result, reticular fibers developed in tumor sites with a resultant fragmentation of cancer cell nests. Many T lymphocytes infiltrated cancer sites where the lentinan was administered. These findings suggest that the intratumor administration of lentinan enhanced an interstitial response and also activated an anti-tumor immune response in the human gastric cancer tissues.

Five anti-Id mAb (Ab2) were prepared from a BALB/c mouse immunized with anti-carcinoembryonic Ag (CEA) mAb MA208 (Ab1) in a syngeneic system. These anti-Id mAb appear to recognize unique idiotopes at the combining site of mAb MA208, because they were specifically reactive with mAb MA208 and showed the inhibitory activity against the binding of mAb MA208 to CEA. These anti-Id mAb were divided into three groups: group 1 (M7-625), group 2 (M7-413, M7-914), and group 3 (M7-049, M7-418), according to the analysis of anti-anti-Id antibodies (Ab3) induced with each anti-Id mAb (Ab2). Anti-anti-Id mAb M7-625 antisera (Ab3) reacted with purified CEA in binding assay and in Western blot analysis, and competed with Ab1 binding to CEA. Furthermore, the binding of anti-Id mAb M7-625 (Ab2) to mAb MA208 (Ab1) was inhibited with CEA, indicating that Ab2 mimicks the structure of the epitope in CEA which was recognized with Ab1. These serologic findings suggest that anti-Id mAb M7-625 carries the internal image of the Ag. According to the amino acid sequences of CDR 1, 2, and 3 of the mAb M7-625 variable region, there exists a homology of amino acid sequences between CDR2 in the H chain (5 amino acids of 10) and CDR3 in the L chain (3 amino acids of 9) of mAb M7-625 and domain III of CEA (545-554).

The neu oncogene product, p185neu, is a tyrosine kinase receptor with structural similarity to the epidermal growth factor (EGF) receptor. We have recently described that coexpression of EGF receptors and high levels of normal p185c-neu lead to transformation of rodent fibroblasts. Anti-EGF receptor and anti-p185neu monoclonal antibodies inhibited tumorigenic growth of these transformants implanted into nude mice. These monoclonal antibodies also suppressed focus formation of the cells transformed by the synergistic action of these receptor proteins in vitro. However, EGF enhanced focus formation and stimulated cell growth when added to cells transfected just with the EGF receptor encoding cDNA. These data suggest that receptor specific effectors may have potentially useful applications in cancer therapy for neoplasms which demonstrate increased receptor densities. In addition the data suggest novel differences in the actions of tyrosine kinases when acting alone or in concert with other receptors.

HTLV-I transformed T cells not only express a large number of interleukin-2 receptors [IL-2R/p55(Tac)], but also produce a factor named ATL-derived factor (ADF) that augments the expression of IL-2R/p55(Tac). Based on a partial N-terminal amino acid sequence, complementary DNA (cDNA) clones for human and mouse ADF were isolated and sequenced. Recombinant ADF produced by COS-7 monkey kidney cells showed IL-2R/Tac inducing activity on YT cells, which are sensitive for ADF. ADF mRNA was strongly expressed in HTLV-I(+) T cells lines, but not in inactivated cells (THP-1, unstimulated PBMC). Furthermore, in normal human peripheral blood mononuclear cells, the expression of ADF mRNA was enhanced by mitogens or phorbol myristate acetate, suggesting a possible involvement of ADF in the lymphocyte activation. Homology analysis revealed an unexpected relationship between ADF and dithiol-reducing enzyme, thioredoxin, involved in many important biological reactions such as the conversion of ribonucleotides into deoxyribonucleotides, or the stabilization of glucocorticoid receptors. The biological significance of the generation of a redox potential in lymphocyte activation, and the possible involvement of dithiol reduction in the induction of IL-2R/Tac are discussed.

We describe monoclonal antibodies (mAbs: anti-MaG-1, TGI-1, TGI-5, and TGI-6) that block the proliferation of AML-193 cells in response to GM-CSF or IL-3 and do not affect the proliferation of AML-193 cells in response to G-CSF and IL-2-driven proliferation of Kit 225 cells. However, none of the mAbs tested had any stimulative effect on the proliferation of AML-193 cells. The mAbs (anti-MaG-1, TGI-1, -5, and -6) could inhibit the binding of [125I)GM-CSF to AML-193 cells. We were able to purify MaG-1 Ag by anti-MaG-1 affinity chromatography. Thus, the MaG-1 Ag and the Ags recognised by mAbs (TGI-1, -5, and -6) may be associated with the receptor for GM-CSF or IL-3 or a structure close to the receptor for GM-CSF or IL-3.

Five different beta(1 leads to 3) glucans were tested for immune adjuvant activity on the in vivo induction of alloreactive murine cytotoxic T-lymphocytes (CTL). The beta(1 leads to 3) glucans, lentinan, pachyman, pachymaran, and two differently substitute hydroxyethylated pachymans strongly enhanced the in vivo generation of alloreactive CTL. The augmenting effect of i.p.-administered beta(1 leads to 3) glucans exhibited a clear dose-response relationship and was strictly dependent on the injection schedule used. Injection of high doses of beta(1 leads to 3) glucans as well as the injection during the late phase of the immune response markedly suppressed the magnitude of the lytic CTL activity induced. When the optimal conditions for enhanced CTL responses were chosen, the augmented CTL activity within spleen cells and mesenteric lymph node cells persisted for more than 25 days. Since beta(1 leads to 3) glucans are chemically defined substances without obvious toxic side effects, they may be of potential use to augment in vivo antigen-specific T-cell responses.

The chemical structure and characteristics, the antitumour activity, the antibacterial, antiviral, antifungal, antiparasitic activities, immunological properties and mode of action of immunomodulators of microbial origin, especially antitumour polysaccharide lentinan, have been discussed immunopharmacologically in comparison with pachymaran, schizophyllan, DiLuzio's yeast glucan, Coriolus preparation PS-K, Streptococcal preparation OK-432, Nocardia rubra cell wall skeleton N-CWS together with BCG and Corynebacterium parvum. Lentinan exerts its inhibitory action not only on allogeneic tumours but also on syngeneic and autologous tumours, and prevents chemical and viral carcinogenesis. The phase III randomized control study of lentinan in cancer patients with gastric and colo-rectal cancer showed the clinical efficacy of lentinan in prolonging the life span and improving host immune responses. Experimental and clinical application demonstrated the advantage of N-CWS over BCG-CWS, and a recent study on OK-432 showed similar results to those obtained with C. parvum. In future work on microbial immunomodulators, it will be essential to find a parameter of the host-tumour relationship which will indicate the protocol for application of immunomodulators, and to find a new-type of selective immunostimulant. For this purpose, exhaustive studies on lymphokines, monokines and lymphocyte tropic hormones are indispensable.

Influenza virus A/Kumamoto haemagglutinin was found to induce type I, III, and IV hypersensitivity in mice. MRL/Mp-I pr/lpr (MRL/l) mice are known to be lower responders to, and poor inducers of, interleukin-2 (IL-2). Recombinant IL-2 was found to augment the type III reaction in BALB/c mice and to suppress the reaction in MRL/I mice in vivo. Cyclophosphamide 100 mg/kg had a selective suppressive effect on the suppressor T cells responsible for type IV reaction, and subsequently the delayed type hypersensitivity (DTH) was augmented. Higher doses of cyclophosphamide suppressed both suppressor and effector T cells in DTH, and subsequently the DTH was suppressed markedly. This experimental model system employing viral haemagglutinin may provide a new screening method for the development of immunomodulators.

p185neu, the protein product of the neu gene, is a tyrosine kinase receptor with structural similarity to the epidermal growth factor (EGF) receptor. The cognate ligand for the p185neu receptor remains unknown. We have defined: 1) stage and tissue-specific expression patterns of the neu gene product in developing tissues; 2) p185neu phosphorylation and the regulation of p185neu tyrosine kinase activity by EGF. 3) Synergistic interactions of cellular rat p185neu and EGF receptor leading to cell transformation; 4) structural and functional differences of normal and oncogenic p185neu. These observations explain some features of how p185neu is involved in normal development and neoplastic transformation.

Eight distinct polysaccharides (PS) of beta(1 leads to 3) glucan type were tested for their capacity to render murine peritoneal exudate cells (PEC) cytotoxic. After intraperitoneal injection of lentinan, pachymaran and HE-pachyman 3 and 4 highly cytotoxic PEC were induced. Pachyman and HE-pachyman 1 and 2 were of moderate effect, whereas CM-pachymaran and HE-pachyman 3 and 4, highly cytotoxic PEC were induced. Pachyman and HE-pachymacrophages. The induction of PEC-dependent cytotoxicity exhibited a strict dose relationship. Optimal administration of PS resulted in the induction of cytotoxicity, which persisted for more than 25 days. Surprisingly, none of the PS tested was capable of rendering normal or thioglycollate-induced PEC cytoxic under in vitro conditions. It is suggested that the capacity of PS to render in vivo macrophages cytotoxic is related to the potency of these PS to activate the alternative pathway of complement system (APC) in so far as C3b may be the essential component required to render macrophages cytotoxic.

The effects of human rIL-6/B cell stimulatory factor 2 (hrIL-6/BSF-2) from Escherichia coli on murine Ag, SRBC-specific antibody responses were examined in vitro and in vivo. HrBSF-2 was effective in augmenting the primary and the anamnestic plaque-forming cells response to SRBC in vitro. The augmentation of the primary response was apparent when B cell-enriched spleen cells (B cells) were cultured with BSF-2 in the presence of IL-2. On the other hand, hrBSF-2 alone strongly enhanced the anamnestic response in a dose-dependent manner when spleen cells from SRBC-immunized mice were used. These effects of BSF-2 were abolished completely by anti-BSF-2 antibody, but not by normal rabbit Ig. Cell depletion experiments indicated that L3T4 (CD4)+ T cells, but not Lyt-2(CD8)+ T cells, and adherent cells (macrophages) have an important role in this BSF-2-induced augmentation of the response. In addition, kinetic studies showed that hrBSF-2 acts on B cells in the anamnestic response even when added relatively late in the culture. Finally, it was determined whether BSF-2 also could be active in modulating antibody responses in vivo. BSF-2 was shown to enhance the primary and secondary antibody responses in mice. The most apparent effect of BSF-2 was observed in the secondary response.

Proteose-peptone-induced intraperitoneal neutrophils from rats were activated in terms of tumor cytotoxicity by pretreatment with culture supernatants from a human T cell leukemia line, Jurkat (culture sup). Culture sup-treated neutrophils showed cytotoxicity against various tumor cell lines. The cytotoxicity of culture sup-treated neutrophils was dependent on the number of neutrophils and the concentration of culture sup. Cytostasis by activated neutrophils was observed very early in the assay incubation period (within 6 hr), but cytolysis first occurred at 24 hr after the start of incubation. Factor(s) in culture sup responsible for the activation of cytotoxic neutrophils were stable to temperature and pH treatments, and their molecular weight was higher than 10,000. The responsible factor(s) for activation of cytotoxic neutrophils were different from interleukin-2, serum-derived factor, and bacterial lipopolysaccharide.

The antitumor and metastasis-inhibitory activities, mode of action, and clinical application of lentinan, a strictly purified beta-1,6:beta-1,3-glucan, are reviewed. Lentinan exerts a prominent antitumor effect and prevents chemical and viral oncogenesis. The antitumor action of lentinan is host-mediated. Compared to other well-known immunostimulants, such as bacille Calmette Guérin (BCG), Corynebacterium parvum, and lipopolysaccharide (LPS), lentinan appears to represent a unique class of immunopotentiator, a T cell-oriented adjuvant. Lentinan triggers the increased production of various kinds of bioactive serum factors associated with immunity and inflammation, such as IL-1, CSF, IL-3, vascular dilation inducer, and acute-phase protein inducer, by the direct impact of macrophages or indirectly via lentinan-stimulated T cells, which results in the induction of many immunobiological changes in the host. Augmented IL-1 production amplifies the maturation of immature effector cells to mature cells capable of responding to lymphokines such as IL-2 and T cell-replacing factors. Because of this mode of action, intact T cell compartments for antitumor activity of lentinan are required. Lentinan has little toxic side effects. Excellent results were obtained in a 4 year follow-up of the randomized control study of lentinan in phase III on patients with advanced and recurrent stomach and colorectal cancer.

PGE and PGF release from peritoneal exudate cells was studied in mice after injection with two beta (1-3) glucans, the antitumor active lentinan and the inactive pachyman, 4 days after injection of both polysaccharides, the spontaneous and phagocytosis-induced PGE and PGF release was markedly suppressed. However, only the immunopotentiator lentinan induced peritoneal exudate cells which exhibited a longer lasting diminished PG release. The data suggest that the T cell adjuvant lentinan may potentiate cellular immune responses by reducing synthesis of immune suppressive prostaglandins from peritoneal exudate cells.