Repolarization of glioblastoma macrophage cells using non-agonistic Dectin-1 ligand encapsulating TLR-9 agonist: plausible role in regenerative medicine against brain tumor.

AIM OF THE STUDY: Glioblastoma multiforme (GBM) is the most severe forms of brain cancer, eventually becoming the leading cause of brain cancer-related death worldwide. Owing to the bleak surgical interventions and resistance to the different treatment regime, GBM is a parlous disease demanding newer therapeutical perspective for its treatment. Toll-like receptors (TLRs) are well-known members of pathogen recognition receptors (PRRs) and have been extensively explored for their therapeutic and prophylactic potential in an array of disease including cancer. Recent trends in drug delivery research has shown shift towards delivering short DNA sequences (CpG DNA) to endosomal TLR9 within immune cells (macrophages, dendritic cells, etc.) for the activation of desired inflammatory response using non-agonistic ß-glucan particles; a well-known ligand for Dectin-1 receptors. Our study is therefore focused to explore the role of nano-encapsulated CpG ODN as critical players in polarizing M2 scavenging to much desired pro-inflammatory type. MATERIALS AND METHODS: The nanoparticles entrapping CpG ODN 1826 were prepared by using a fungal polymer Schizophyllan (SPG). The constructed nanoparticles were characterized and assessed for their efficacy on rat glioblastoma cells (C6). RESULTS: The constructed Schizophyllan (SPG) nanoparticles entrapping CpG ODN 1826 (95.3%) were of 25.49 nm in diameter and thus capable of crossing blood-brain barrier. The rat glioblastoma (C6) cells evaluated for intracellular oxidative burst and cytokine levels pre- and post-incubation with nanoparticles exhibited marked elevation in the expression of intracellular ROS and IFN-γ as well as IL-1ß post treatment. CONCLUSION: The findings indicate towards potentiality of repolarizing the M2 macrophages to much desired M1 phase by inducing higgh levels of oxidative burst and inflammatory cytokines. Consequently, the apoptosis was induced in glioblastoma cells establishing the suitablity of CpG ODN carrying nanoformulations as emerging therapeutic intervention for GBM.

Leishmanial CpG DNA nanovesicles: A propitious prophylactic approach against visceral leishmaniasis.

Unmethylated CpG motifs with phosphothioate backbone trigger TLR9 to elicit innate immune response characterized by the production of Th1 cytokines. The use of CpG DNA as an adjuvant has established its role in potentiating the humoral and cell mediated vaccine specific immune response. However, none of the synthetic oligodeoxynucleotides (ODNs) know and used till date are associated with the parasite itself. Our group identified a novel CG rich sequence of 14 base pairs from Leishmania donovani genome (Ld CpG ODN) and established it as a TLR9 agonist. The present study was designed to ascertain the adjuvanticity of Ld CpG ODN with soluble leishmanial antigen in experimental model of L. donovani. During the study Schizophyllan (SPG), a fungal polymer was used for encapsulating Ld CpG ODN for efficient endosomal delivery. The synthesized nanovehicles were of nearly 100 nm and localized within endosomes as confirmed by confocal microscopy. Immunization studies displayed the superior ability of synthesized nanovehicles co-administered with parasite antigen in augmenting innate immune response in comparison to ODN, nanoparticles or soluble antigen alone. The response included generation of ROS, NO and iNOS expression followed by proinflammatory cytokine milieu with reduced parasitic load within liver, spleen and bone marrow. These immune-tailored particles in combination with parasitic antigens elicited significant generation of cell mediated response owing to the presence of high levels of CD8+ T-cells and lymphocyte proliferation. Moreover, vaccination regime with synthesized adjuvant also activated humoral immunity by escalating the levels of IgG2 followed by reduced levels of anti-leishmanial IgG and IgG1 antibodies. The findings support the efficacy of Ld CpG ODN as a potential adjuvant against visceral leishmaniasis.

Dietary schizophyllan reduces mitochondrial damage by activating SIRT3 in mice.

Schizophyllan (SPG), produced by Schizophyllum commune, is an exopolysaccharide with multiple academic and commercial uses, including in the food industry and for various medical functions. We previously demonstrated that SPG conjugated with c-Src peptide exerted a significant therapeutic effect on mouse models of the acute inflammatory diseases polymicrobial sepsis and ulcerative colitis. Here we extended these results by investigating whether SPG exerted a protective effect against mitochondrial damage in the liver via sirtuin 3 (SIRT3) induction, focusing on the deacetylation of succinate dehydrogenase A (SDHA) and superoxide dismutase 2 (SOD2). Liver damage models induced by alcohol or conjugated linoleic acid (CLA, which simulates lipodystrophy) in SIRT3-/-, SOD2-/-, and SDHA-/- mice were used. Results showed that dietary supplementation with SPG induced SIRT3 activation; this was involved in mitochondrial metabolic resuscitation that countered the adverse effects of alcoholic liver disease and CLA-induced damage. The mitochondrial SIRT3 mediated the deacetylation and activation of SOD2 in the liver and SDHA in adipose tissues, suggesting that SPG supplementation reduced ethanol-induced liver damage and CLA-induced adverse dietary effects via SIRT3-SOD2 and SIRT3-SDHA signaling, respectively. Together, these results suggest that dietary SPG has a previously unrecognized role in SIRT3-mediated mitochondrial metabolic resuscitation during mitochondria-related diseases.

Designing an immunocyte-targeting delivery system by use of beta-glucan.

A ß-1,3-d-glucan called Schizophyllan (SPG) can form a novel complex with homo oligodeoxynucleotides (ODNs) via the combination of hydrogen bonding and hydrophobic interactions. Dectin-1 is a major receptor involved in the recognition of ß-1,3-d-glucans and expressed on antigen presenting cells (APCs) including macrophages, dendritic cells, monocytes, neutrophils, and a subset of T cells. Therefore, the SPG/ODN complex can be used as APCs cell-specific delivery of functional ODNs including unmethylated CpG sequences (CpG-ODNs). In fact, CpG-ODN/SPG complex induced high antibody titers when it was administered to cynomolgus monkeys as adjutant of influenza vaccine. These results indicate that SPG can be an excellent immunocyte-targeting drug delivery system.

Induction of humoral and cellular immune response to hepatitis B virus (HBV) vaccine can be upregulated by CpG oligonucleotides complexed with Dectin-1 ligand.

A persistent hepatitis B virus (HBV) infection is characterized by a lack of or a weak immune response to HBV, which may be reflective of tolerance to HBV. Efficient induction of HBV-specific immune response leads to the clearance of HBV in patients with a chronic HBV infection. CpG oligodeoxynucleotides (ODN) has a powerful adjuvant effect in HBV vaccination. A recent report demonstrated that the immunization by B/K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand, schizophyllan (SPG), namely K3-SPG, was more effective in the induction of antigen-specific immune response than that by K3. In this study, we examined the efficacy of K3-SPG as a HBV vaccine adjuvant. Wild-type (WT) mice and HBV transgenic (HBV-Tg) mice were subcutaneously immunized with hepatitis B surface antigen (HBsAg) alone, HBsAg and K3, or HBsAg and K3-SPG. The vaccination with HBsAg and K3-SPG significantly enhanced humoral and cellular immune response to HBV antigen compared to the other vaccinations in WT and HBV-Tg mice. K3-SPG induced the accumulation of dendritic cells (DCs) into draining lymph node and the activation of DCs. The expression of cytokines and chemokines related to Th1 and Th2 responses was upregulated after the vaccination including with K3-SPG. In conclusion, these results indicated that the vaccination using K3-SPG may overcome tolerance even in patients with chronic HBV infection.

The targeted delivery of the c-Src peptide complexed with schizophyllan to macrophages inhibits polymicrobial sepsis and ulcerative colitis in mice.

Hyper-inflammatory responses triggered by intracellular reactive oxygen species (ROS) can lead to a variety of diseases, including sepsis and colitis. However, the regulators of this process remain poorly defined. In this study, we demonstrate that c-Src is a negative regulator of cellular ROS generation through its binding to p47phox. This molecule also competitively inhibits the NADPH oxidase complex (NOX) assembly. Furthermore, we developed the schizophyllan (SPG)-c-Src SH3 peptide, which is a ß-1,3-glucan conjugated c-Src SH3-derived peptide composed of amino acids 91-108 and 121-140 of c-Src. The SPG-SH3 peptide has a significant therapeutic effect on mouse ROS-mediated inflammatory disease models, cecal-ligation-puncture-induced sepsis, and dextran sodium sulfate-induced colitis. It does so by inhibiting the NOX subunit assembly and proinflammatory mediator production. Therefore, the SPG-SH3 peptide is a potential therapeutic agent for ROS-associated lethal inflammatory diseases. Our findings provide clues for the development of new peptide-base drugs that will target p47phox.

Immunization with antigenic peptides complexed with ß-glucan induces potent cytotoxic T-lymphocyte activity in combination with CpG-ODNs.

The induction of antigen-specific immune responses requires immunization with not only antigens, but also adjuvants. CpG oligonucleotides (CpG-ODNs) are well-known ligands for Toll-like receptor 9 and a potent adjuvant that induces both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte responses. We previously demonstrated that ß-glucan schizophyllan (SPG) can form complexes with CpG-ODNs with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses by co-administration of antigenic proteins, namely ovalbumin (OVA). Immunization with antigenic peptides, OVA257-264, did not induce these antigen-specific immune responses even in combination with CpG-dA/SPG, indicating that peptides require a carrier to antigen presenting cells. In this study, we prepared conjugates comprising OVA257-264 and dA40, and made complexes with SPG. Immunization with OVA257-264-dA/SPG induced peptide-specific immune responses in combination with CpG-dA regardless of complexation with SPG both in vitro and in vivo. When splenocytes from immunized mice were incubated with E.G7-OVA tumor model cells presenting OVA peptides, the number of cells drastically decreased after 24h. Furthermore, mice pre-immunized with OVA257-264-dA/SPG and CpG-ODNs exhibited a long delay in tumor growth after tumor inoculation. Therefore, these peptide-dA/SPG and CpG-dA/SPG complexes could be used as a potent vaccine for the treatment of cancers and infectious diseases.

Molecular function of macrophage migration inhibitory factor and a novel therapy for inflammatory bowel disease.

Macrophage migration inhibitory factor (MIF) is a unique protein that participates in inflammation, immune responses, and cell growth. An array of in vitro and in vivo experiments has demonstrated that MIF is profoundly involved in the pathogenesis of acute and chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Blockade of MIF bioactivities by either neutralizing anti-MIF antibodies or antagonists prevents inflammatory cytokine cascade, which strongly suggests that an anti-MIF therapeutic strategy is feasible for treatment of IBD. Recently, we developed a new therapeutic approach for IBD by administration of antisense MIF oligonucleotides in conjugation with schizophyllan (SPG), a member of the glucan family. SPG specifically binds Dectin-1 expressed in antigen-presenting cells (APCs), and the antisense MIF/SPG complex is incorporated into the cells. In in vivo experiments of colitis models in mice, we found that intraperitoneal administration of the complex ameliorated the clinical signs of colitis and improved the histological scores. This novel therapy designed to knock down the MIF production in APCs is expected to be clinically applicable for the treatment of IBD.

Schizophyllan inhibits the development of mammary and hepatic carcinomas induced by 7,12 dimethylbenz(α)anthracene and decreases cell proliferation: comparison with tamoxifen.

BACKGROUND: Breast cancer is one of the leading causes of cancer mortality among women. Some anticancer compounds have been isolated from mushrooms. The aim of the present work was to study the anticancer effects of schizophyllan (SCH), a ß-D: -glucan extracted from the mushroom Schizophyllum commune alone or in combination with tamoxifen (TAM) on 7, 12 Dimethylbenz(α)anthracene (DMBA)-induced carcinomas in mice. METHODS: We isolated SCH from S. commune. Female mice received DMBA, SCH, DMBA+SCH, DMBA+TAM or DMBA+TAM+SCH or vehicles. We studied mice survival, tumour incidence, histopathology, oestrogen receptor (ER) expression, cell proliferation by immunohistochemical detection of proliferating cell nuclear antigen (PCNA), apoptosis by TUNEL assay, as well as caspase-3 expression. RESULTS: DMBA treatment resulted in mammary and hepatocellular carcinomas (HCC). Both SCH and TAM reduced the incidence of DMBA-induced mammary tumours by 85 and 75 %, respectively, and equally decreased the PCNA labelling index relative to DMBA. TAM treatment increased the incidence of- and PCNA index in HCCs relative to DMBA, while SCH suppressed these effects. TAM was more effective than SCH in the induction of apoptosis in both mammary and hepatic carcinomas. Caspase-3 levels correlated with the apoptotic index in most experimental groups. CONCLUSIONS: Only one dose of SCH had similar therapeutic effects against DMBA-induced mammary carcinomas as 4 weeks of TAM treatment. This coupled with the ability of SCH to suppress hepatic lesions associated with TAM treatment provides the rationale for further investigating the combined therapeutic effects of TAM+SCH in preclinical models of ER-positive breast cancer, as well as in liver cancer.

A new therapeutic approach using a schizophyllan-based drug delivery system for inflammatory bowel disease.

Antisense technologies for the targeted inhibition of gene expression could provide an effective strategy for the suppression of inflammation. However, the effective use of antisense oligonucleotides (ODN) has been limited because of several problems. Therefore, a delivery system for antisense ODNs that enhances antisense stability, while maintaining the specificity of antisense for its target RNA or DNA is needed. We have developed a delivery system for antisense ODN using schizophyllan (SPG), a polysaccharide that belongs to the ß-(1-3) glucan family. This system has several advantages enabling the effective suppression of targeted RNA or DNA: the SPG complex is stable in vivo and does not dissolve in the presence of deoxyribonuclease, and the SPG complex is effectively taken up into macrophages by phagocytosis through Dectin-1. Macrophage-migration inhibitory factor (MIF), which is mainly produced by macrophages has been shown to have a pathogenetic role in inflammatory bowel disease (IBD). We developed a technique to create an SPG complex that highly conformed to the antisense MIF. The administration of antisense MIF/SPG complex effectively suppressed MIF production and significantly ameliorated intestinal inflammation. Our result demonstrated a possible new therapeutic approach, i.e., the administration of antisense MIF/SPG complex, for the treatment of IBD.

A polysaccharide carrier to effectively deliver native phosphodiester CpG DNA to antigen-presenting cells.

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy.

A polysaccharide carrier for immunostimulatory CpG DNAs to enhance cytokine secretion.

A beta-(1 --> 3)-d-glucan schizophyllan (SPG) forms a stoichiometric complex with some polynucleotides. This communication describes our attempt to apply the SPG complex to deliver CpG DNA to endosomes to enhance cytokine secretion. To increase cellular uptake, we introduced spermine, arginine-glycine-aspartic acid tripeptide, octaarginine, or cholesterol to the SPG side chain. The chemically modified SPG showed essentially no cytotoxicity. When CpG DNA complex made therefrom was exposed to macrophages, dramatic enhancement in the cytokine secretion was observed. It increased 5-10 times from the naked dose and 100 times from the background. This performance promises that SPG can be an excellent carrier for CpG DNA.

Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency.

Schizophyllan is a natural beta-(1-->3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-phosphodiester polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. Furthermore, we have already demonstrated that one of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. This work describes a versatile and universal modification technique which enables us to introduce various functional groups only to the side chain of schizophyllan. This technique consists of periodate oxidation of the glucose side chain (it does not react with the main chain because of the absence of the 1,2-diol group in beta-(1-->3)-glucan) and subsequent introduction of the functional groups into the formyl terminate. In the present work, the introduced functional groups were spermine, octa-arginine (R8), arginine-glycine-aspartic acid tripeptide (RGD) and some amino or alpha-amino acid compounds. Using these compounds, we made the complexes and carried out an in vitro antisense assay for them, administrating a phosphorothioate AS ODN to the melanoma A375 or leukemia HL-60 cell lines to depress their c-myb mRNA. When we used the R8 or RGD modified schizophyllan as the antisense carrier, the antisense effect was most enhanced among others. Their superiority can be ascribed to enhancement of endocytosis due to these functional peptides. Furthermore, the cytotoxicity for these two modified schizophyllans was negligibly as small as the natural (unmodified) schizophyllan. One of the peculiar features of our system is that the complex (i.e., carrier+AS ODN) is charged negatively in total, which is different from the conventional systems. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers.

Immunotoxicity of soluble beta-glucans induced by indomethacin treatment.

(1 --> 3)-Beta-D-Glucan (beta-glucan) is a biological response modifier that regulates host immune response. However, the side effects of this drug have not been extensively examined. In this study, we found that the combination of a beta-glucan and a nonsteroidal anti-inflammatory drug, indomethacin, induced lethal toxicity in mice. Lethal toxicity of orally administered indomethacin (multiple administration to ICR mice; once a day for 2 weeks) was 0/8 (2.5 mg kg(-1)) and 5/8 (5 mg kg(-1)) (death/total) over 2 weeks. The toxicity was enhanced to 3/8 and 8/8 in mice treated with a clinical beta-glucan preparation, sonifilan (250 microg/mouse, single i.p. administration on day 0). A similar effect was observed for other beta-glucans, including SSG, grifolan, zymosan A and zymocel. Enhanced lethal toxicity resulted from a single p.o. administration of indomethacin on day 5 to day 9 after multiple beta-glucans administration. Interferon-gamma, interleukin-6 and colony stimulating factor concentrations in sera of indomethacin/beta-glucan-treated mice were significantly elevated. These results strongly suggest that indomethacin/beta-glucan treatment induces lethality in mice by maladjusting the cytokine network.

[Effect of 5'-DFUR used concurrently in radiotherapy and immunotherapy uterine cervical cancer--pilot study. Study of 5'-DFUR for Uterine Cervical Cancer].

We conducted a preliminary controlled study in order to evaluate 5'-DFUR dose dependency in efficacy and safety in combination therapy of radiotherapy, 5'-DFUR and SPG for patients with uterine cervical cancer, which was regarded as suitable for cases of radiotherapy. The patients were randomly allocated into group A (5'-DFUR 600 mg/body/day) and group B (5'-DFUR 800 mg/body/day), who underwent radiotherapy with simultaneous administration of 5'-DFUR and SPG (20 mg twice/week or 40 mg/ week). Those enrolled were 33 patients in stage II, III or IV a with histologically diagnosed primary squamous cell carcinoma of uterine cervix. CR was shown in 19, PR in 7, NC in 1, and PD in 2 out of 29 efficacy-evaluable cases, so the overall response rate was 89.7% (26/29, 95% CI 72.7%-97.8%). Regarding safety, some side effects were observed in 26 out of 33 safety-evaluable cases (81.3%, 95%, CI 63.6%-92.8%), but no serious cases. No significant difference in efficacy and safety was observed between the two treatment groups. These results suggested that the combination therapy of radiotherapy, 5'-DFUR and SPG might be one of the therapies whose effectiveness must be confirmed for advanced squamous cell carcinoma of uterine cervix. To confirm dose dependency of 5'-DFUR, it seems further consideration with more patients is needed.

Intratumoral administration of sizofiran activates Langerhans cell and T-cell infiltration in cervical cancer.

In order to ascertain a correlation between infiltration of Langerhans cells (LCs) or T-cells in tumor tissues and the intratumoral administration of a biological response modifier, Sizofiran (SPG) was analyzed in cancer of the uterine cervix. Cancer specimens of 45 patients with stage II-III invasive cervical cancers were analyzed. SPG was administered to the cervical tumor at high and low concentrations (Strong SPG and weak SPG) as well as by intramuscular injection twice a week. LC and T-cell infiltrations to tumor tissues of the uterine cervix were studied immunohistochemically and electron microscopically. Of 10 patients with systemic but no local immunization, 1 (10.0%) showed an increase in LC infiltration and 2 (20.0%) showed a decrease. Of 15 patients with strong SPG immunization, 2 (13%) showed an increase and 5 (33%) showed a decrease. In contrast, of 20 patients with weak SPG immunization, the incidence of increase in LC infiltration was 55% (11 patients), significantly greater than the above-mentioned groups and none showed a decrease. Of the 20 patients with weak SPG administration, 3 (15%) showed T-cell infiltration before SPG administration, and 12 (60%) showed an increase in T-cell infiltration after SPG was given. Up on electron microscopy, Birbeck granules in the cytoplasm of LC significantly increased after SPG immunization, indicating activation of LC. In conclusion, the present study suggested that the LC and T-cell infiltrations in cancer tissues were augmented by intratumoral SPG administration at a certain concentration. Intratumoral administration of SPG may be applied to improve the prognosis after multidisciplinary treatment of advanced cervical cancer.

[Combined effects of sizofiran and rG-CSF on myelosuppression in cancer chemotherapy].

A combination of the antitumor polysaccharide Sizofiran and rG-CSF was investigated for its effects on peripheral blood counts in ovarian cancer patients undergoing chemotherapy. We performed the analysis by using variations in peripheral blood counts and the duration of treatment as variables. In the analysis, effects on decreases in both neutrophils and platelets and their recoveries could be assessed. As a result, the decrease in neutrophils was inhibited and its recovery was promoted, suggesting that the combined use of the drugs may be useful for myelosuppression by chemotherapy for ovarian cancer.

Augmentation of antitumor effect by combined administration with interleukin-2 and sizofiran, a single glucan, on murine EL-4 lymphoma.

The antitumor effect of the combined administration with recombinant human interleukin-2 (rIL-2) and sizofiran (SPG), a single glucan of Shizophyllum commune Fries, was studied in vivo in C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The effect was evaluated by a) comparing the survival time of the mice, b) analysis of the intraperitoneal cell population in Giemsa-stained specimens, c) surface marker analysis of peritoneal exudative cells with flow cytometry, d) cytotoxic assay of cells against EL-4 and Yac-1 lymphoma, and e) elimination of some cell populations by monoclonal antibodies, to identify the antitumor-effector cells showing cytotoxic activity. The survival of mice given both rIL-2 and SPG was significantly longer than the control mice or those given SPG alone or rIL-2 alone. It was demonstrated that the administration of SPG and/or rIL-2 to the EL-4 lymphoma-bearing mice activated immune-response cells in the peritoneal cavity such as T lymphocytes, NK cells, or macrophages, which might be effective in reducing lymphoma cells. The combination of rIL-2 and SPG administration appears to activate the antitumor-immune response at the tumor site more effectively than when either agent was administered alone.

Inhibitory effect of metastasis by combined administration with interleukin-2 and sizofiran, a single glucan--immunohistochemical study.

Innovations in methods of combined administration with other BRM or chemotherapeutic drugs have been discussed. We have reported that combined administration with recombinant interleukin-2 (rIL-2) and sizofiran (SPG) is effective in prolonging survival time of C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The immunomechanisms of the combined administration were clarified investigating the intraperitoneal cell population in the primary tumor site, especially the tumor infiltrating lymphocyte (TIL) quantitatively. In the present study, to clarify the antitumor effects of combined administration with rIL-2 and SPG on the metastatic sites, the immunomechanisms of the suppressive effects of combined administration on the metastasis were studied in EL-4 lymphoma cells intraperitoneally transplanted to mice. Inasmuch as EL-4 lymphoma shows rapid hepatosplenic metastasis, we studied the metastatic foci in the liver and the spleen semiquantitatively in investigating the histopathological and immunohistochemical findings of the metastatic foci, especially the TIL. The metastatic foci were stained by hematoxylin-eosin (HE) and monoclonal antibodies (L3T4, Lyt2, asialo GM1, Mac-1, and Ia). The combined administration resulted in: 1) fewer infiltrating tumor cells, 2) more lymphocytic infiltration, and 3) more antitumor effector cells (cytotoxic T cells: Lyt2 and natural killer cells: asialo GM1), macrophages (Mac-1), helper T cells (L3T4), and cells with MHC-class-II antigen (Ia) than did administration of rIL-2 alone or SPG alone, or no administration of these two at all. Combined administration with rIL-2 and SPG appears to activate antitumor-immune response at the metastatic site more effectively than when either agent is administered alone.