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The role of IL-4 has often been studied, especially in the Leishmania major infection model, but not in Trypanosoma cruzi infection. In the present study, the role of IL-4 in host defense against infection with the Tulahuén strain of T. cruzi was examined by depleting IL-4 with an anti-IL-4 monoclonal antibody in vivo. In both IL-4 depleted and control C57BL/6 mice, the parasitemia showed peaks on the 21st day of infection. Both parasitemia and mortality were decreased in IL-4 depleted mice compared with control mice when IFN-γ and nitric oxide productions were increased in IL-4 depleted mice compared with control mice. The mice treated with N-nitro- L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase, showed increased susceptibility to T. cruzi infection to the same level in both IL-4 depleted and control mice. Thus, it is suggested that endogenous IL-4 induces susceptibility to T. cruzi mainly by suppressing the production of IFN-γ and nitric oxide, which has trypanocidal activity. [ABSTRACT FROM AUTHOR]

The effect of interleukin-4 (IL-4) on the induction of intestinal mast cells and cytokine profiles during Strongyloides ratti infection was studied using IL-4 knockout (IL-4 KO) mice. The antigen-specific proliferative response of mesenteric lymph node cells was not impaired in IL-4 KO mice. The number of intestinal mast cells induced in IL-4 KO mice during S. ratti infection was 2- to 3-fold lower than that observed in WT mice. Intestinal mastocytosis had disappeared in IL-4 KO mice by day 21 postinfection, when significant mastocytosis continued to be observed in WT mice. In mesenteric lymphnode of IL-4 KO, IL-3 production decreased and mice IFN-γ production significantly increased as compared with those of WT mice. The numbers of eggs excreted per gram of feces (EPG) by IL-4 KO mice were greater than those excreted by WT mice on day 6 postinfection, but no difference was observed in the subsequent period. In conclusion, intestinal mast cells are induced during S. ratti infection in the absence of IL-4, and IL-4 is not essential for protection against intestinal adult worms of S. ratti. [ABSTRACT FROM AUTHOR]

The results of previous studies indicate that only CD4+ T cells generated via the indirect pathway play an essential role in causing discordant skin xenograft rejection. The present study was conducted in an attempt to clarify further the roles of effector T cells generated via direct pathways on discordant xenograft rejection using CD45 exon-6 knockout (CD45−/−; C57BL/6 (B6): H-2b) mice. It has been strongly suggested that CD45 exon-6 knockout mice have profound impairment in T-cell functions via an indirect pathway. When human skin was grafted onto untreated normal C57BL/6 (B6; H-2b) mice, rejection occurred within 12 days; however, in the CD45 exon-6 knockout mice, the grafts lasted for slightly longer as in fully allogeneic C3H (H-2k) skin rejection, with a mean survival time ± SD of 19.4 ± 1.5 days and median survival times of 19 days. The difference in survival periods between the human and C3H skin grafts in the CD45 knockout mice was not statistically significant. Both CD4+ and CD8+ T cells seemed activated in the spleens of these CD45 exon-6 knockout mice 10 days after the human skin grafting. These results suggest that effector T cells generated via a direct pathway can cause discordant skin xenograft rejection, and that CD45 exon-6 knockout mice can generate effector T cells via a direct pathway to reject discordant skin xenografts, similarly to fully allogeneic skin allografts. [ABSTRACT FROM AUTHOR]

The effect of local injections with streptococcal preparation OK432 on the antitumor effect induced by adoptive immunotherapy (AIT) was investigated. Draining lymph node cells on day 14 after B7-P815 inoculation were used for AIT after in vitro stimulation. AIT on days 7 and 10 showed no effect on the growth of s.c. established P815 mastocytoma, but local injections with OK432 into the tumor sites on days 3, 6 and 9 resulted in a moderate antitumor effect. On the other hand, the combination therapy significantly suppressed tumor growth, and the tumor-bearing mice survived longer than those receiving only one of the treatment modalities. The significant infiltration of CD4+ or CD8+ T cells and multiple necrosis in the tumor sites were observed only when the tumor-bearing mice were treated with the combination therapy. In addition, a transfer experiment using labeled effector cells revealed these infiltrated CD8+ T cells and CD4+ T cells to be derived from the donor and the host respectively. More importantly, the combination therapy clearly led to higher expression of the mRNA for Th1-type cytokines and CXC3 chemokines in the tumor sites than resulted from each of the treatment modalities alone. Collectively, these results indicate that local injections with OK432 can help the infiltration of adoptively transferred CD8+ T cells into the tumor sites and synergistically induce the local production of the Th1-type cytokines and CXC3 chemokines. [ABSTRACT FROM AUTHOR]

The involvement of granulocytes in the host early defense against the nematode, Strongyloides ratti, was studied. It was confirmed that granulocytes were effectively depleted for 4 days by anti-granulocyte monoclonal antibody (anti-Gr-1). To examine the involvement of granulocytes in the host defense against migrating larvae, 2000 S. ratti infective larvae (L3) were inoculated subcutaneously 1 day after antibody treatment. The number of S. ratti eggs secreted in feces (EPG) was higher in the granulocyte-depleted group than in the control group. The number of migrating larvae also increased in the granulocyte-depleted group in accordance with the increase in EPG. Therefore granulocytes are crucial for the host early defense against migrating larvae of S. ratti. Next, the involvement of granulocytes in the intestinal early defense was examined. Mice were treated with the antibody on day 3 post-infection. On that day, almost all inoculated larvae reached the intestine and molted to become adults. EPG on day 5 post-infection was increased by the antibody treatment, but no effect was observed on intestinal worm numbers. The fecundity (EPG/worm number) of S. ratti adult worms in the granulocyte-depleted group was higher than that in the control group. Thus granulocytes are also involved in the intestinal early defense through suppressing fecundity of the adult worms. On the other hand, the depletion of granulocytes had no effect on the late adaptive response against S. ratti adult worms (e.g. number of intestinal mucosal mast cells, time of worm expulsion). These results suggest that granulocytes are mainly involved in the host early defense against parasites. [ABSTRACT FROM AUTHOR]

In 3031 inbred mice, 3093 allogeneic free skin grafting procedures, including 62 double grafts, were performed using trunk skin as the graft tissue. We developed a new method of graft bed preparation, using corneal scissors for the incision of the square frame of the graft bed and gauze sponges held in a pair of mosquito forceps for the blunt dissection of recipient skin. This method effectively preserved the panniculus carnosus of the graft beds. Grafts from the donor trunk skin were also prepared by a new method in which the skin was spread on aluminium foil, cleaned of superfluous tissues using large gauze sponges held in forceps, cut into appropriately sized grafts with a scalpel, and marked by scoring in order to designate the original direction of the grafts. The graft was approximated to the graft bed by 8 interrupted sutures of 5-0 silk, then covered with a bandage and protective tape for 7 days. The overall operative and postoperative mortality, in most cases attributable to overdoses of anesthetic drugs, was 3.8 per cent (114/3031) and the overall graft failure rate, in most instances due to incorrect covering of the grafts, infection or inside-outgrafting, was 1.2 per cent (36/2979). [ABSTRACT FROM AUTHOR]

The effects of splenectomy on tumor growth following inoculation with a relatively large number of cells (1×10) and a smaller number of cells (5×10) of Meth I tumor were studied. When 1×10 tumor cells were inoculated, tumor growth in splenectomized mice was depressed, while tumor in sham-operated mice grew progressively. On the contrary, when 5×10 tumor cells were inoculated, the tumor take was lower in sham-operated than in splenectomized mice. The spleen cells from mice inoculated with either a large or small number of tumor cells, showed an equally potent cytotoxic activity, but no detectable suppressor cell activity. On the other hand, the activity of immunosuppressive factor was detected in sera from mice inoculated with 1×10 tumor cells, but not in those given 5×10 cells. The effect of splenectomy on tumor growth is, thus, bidirectional, depending on the dose of tumor cells inoculated. [ABSTRACT FROM AUTHOR]

A skim-milk fraction and a whey-protein concentrate (WPC) fraction were prepared from the cows that had been immunized with E. coli isolated from the mouse intestine. The antibacterial effect of these fractions against E. coli was examined. They contained antibody with a high affinity for E. coli strain 48, a representative strain in the mouse intestine, which is composed of a large amount of IgG and smaller amouns of IgA and IgM. Although these fractions showed no bactericidal or bacteriostatic activity against E. coli 48 directly in vitro, they exhibited strong agglutination and opsonization activities against the bacteria in vitro. The bacteria opsonized with the WPC fraction were taken up more effectively by liver macrophages in vivo, compared with unopsonized E. coli, after an intravenous injection into mice. Oral administration of the skim-milk fraction to mice significantly reduced the susceptibility to the lethal toxicity of 5-fluorouracil (5 FU). The increase in the population levels of E. coli in the intestinal tract after administration of 5 FU was inhibited by oral administration of the skimmilk fraction. These results strongly suggest that specific antibody may be effective in the prophylaxis against the indigenous infection with gram-negative bacteria such as E. coli after a period of chemotherapy in cancer patients. [ABSTRACT FROM AUTHOR]

Neonatally thymectomized (NTx) mice, sham-operated control mice and congenitally athymic nude mice were immunized with viable Listeria monocytogenes and their spleen cells examined for the capacity to transfer both delayed footbad reaction and protection against challenge at the site of local transfer. Cells from immune NTx mice conferred significant degrees of delayed footpad reaction and protection comparable to sham mice, while cells from immune nude (nu/nu) mice did not. This abilty was completely eliminated by the treatment of cells with anti-Thy1, anti-Lytl or anti-L3T4 antibody plus complement but not with anti-Lyt2 antibody plus complement. These results indicated that NTx mice can normally mount the immunity to L. monocytogenes by generating Lyt12, L3T4 T cells. Immune competence of NTx mice and thymus dependency of various immune responses are discussed. [ABSTRACT FROM AUTHOR]

We recently reported that regional lymph node lymphocytes (RLNL) from patients with primary lung cancer were in a more highly activated state than peripheral blood lymphocytes (PBL) when the activation-related molecules were studied by FACS analysis. To identify whether or not RLNL had the ability to respond to autologous tumor cells (AT), in the present study a mixed lymphocyte tumor cell reaction (MLTR) either with or without recombinant interleukin 2 (rIL-2) was performed in 41 cases with primary lung cancer. Significant proliferative responses to AT were found in RLNL from 20 of 41 cases (48.8%) without IL-2, and in 23 of 41 cases (56.1%) with IL-2. On the other hand, such responses were observed in the PBL from 8 of 30 cases (26.7%) without IL-2, and from 11 of 30 cases (36.7%) with IL-2. No significant correlation between MLTR and such clinical factors as tumor size, metastasis to lymph node, histology and stage of the disease was found. To further analyze the anti-AT response of RLNL, the cytokine production of RLNL was investigated after stimulation by AT. An increase in interferon-γ (IFN-γ) production was observed in RLNL with a positive reaction of MLTR, while no such increase was found in PBL. Finally, to elucidate whether the expression of MHC class II molecules was a key point in MLTR, tumor cells in primary lesions were examined for the expression of MHC class II by immunohistochemical staining, and the blocking assay of MLTR was performed with anti-MHC class II monoclonal antibody. Data suggested that there was a positive correlation between MHC class II expression of the tumor and MLTR and that MLTR were partially blocked by anti-MHC class II monoclonal antibody. These results demonstrated that RLNL were in a more highly activated state against AT than were PBL, and this finding is considered to be helpful in enhancing our understanding of the role of RLNL in lung cancer patients. [ABSTRACT FROM AUTHOR]

A B16 melanoma-specific CD8+ T cell line (AB1) was established from the spleen cells of C57BL/6 mice cured of B16 melanoma with interleukin (IL)-12 treatment. The AB1 line exclusively used T cell receptor Vβ11. The AB1 cells exhibited a cytolytic activity against both syngeneic B16 melanoma and allogeneic P815 mastocytoma, whereas a cold inhibition assay revealed specificity of the AB1 cells against B16 melanoma. Their lostability to kill a class I loss variant of B16 melanoma was restored by the transfection of H-2Kb gene. In addition, their interferon (IFN)-γ production was significantly suppressed by the addition of anti-H-2Kb monoclonal antibody, and RT-PCR analysis showed that the AB1 line expressed the mRNA encoding IFN-γ, but not IL-4 or IL-10. The experiment using synthetic peptides of tyrosinase-related protein-2 (TRP-2) revealed that the AB1 cells could recognize TRP-2181–188 peptide. Moreover, the AB1 cells showed an in vivo antitumor effect against established pulmonary metastases of B16 melanoma. Overall, these results indicate that the Tc1-type Vβ11 + AB1 cells exert an antitumor activity against syngeneic B16 melanoma through recognition of TRP-2181–188 peptide in an H-2Kb-restricted manner. [ABSTRACT FROM AUTHOR]

Both natural killer (NK) cells and macrophages are thought to be the main effectors responsible for early antitumor defense. In this study, we investigated the role of tumor-infiltrating NK cells in initiating nitric oxide (NO) production by tumor-associated macrophages (TAM). The in vivo depletion of NK cells prior to the i.p. inoculation of melanoma cells resulted in a significant decrease in the NO production of the TAM prepared from the peritoneal exudate cells (PEC). Such prior NK cell depletion also decreased the ability of TAM to show any antitumor activity in vitro. The addition of NG-monomethyl- L-arginine (Me- L-Arg) to the culture partially inhibited the ability of TAM to suppress the proliferation of melanoma cells and also decreased their cytolytic activity against melanoma cells. These results suggest that the TAM exhibited both cytostatic and cytolytic activities through their NO production. In an in vivo assay, the administration of Me- L-Arg permitted the more rapid growth of i.p. inoculated melanoma cells compared with the control. On the other hand, the decreased NO production of TAM, resulting from the prior NK cell depletion, was restored by the i.p. administration of interferon γ (IFNγ). In addition, the in vivo administration of anti-IFNγ mAb into mice inoculated i.p. with melanoma cells also significantly decreased the NO production of TAM in peritoneal exudate cells. Furthermore, the tumor-infiltrating NK cells produced a considerable level of IFNγ. Overall, these results indicate that early-appearing tumor-infiltrating NK cells play an important role in the NO production of TAM through their IFNγ production. [ABSTRACT FROM AUTHOR]

In order to enhance the antitumor vaccination effect of dendritic cells (DC) pulsed with class I tumor peptide, we tried to utilize the local cytokine help of CD4+ T cells reactive to a streptococcal preparation OK432. DC were prepared from murine bone marrow cells by culture with both granulocyte/macrophage-colony-stimulating factor and interleukin(IL)-4. The peritumoral injections of OK432 induced OK432-reactive CD4+ T cells in the draining lymph nodes, and their in vitro production of interferon γ was thus significantly enhanced by restimulation with OK432-pulsed DC. In addition, anti-P815 mastocytoma cytotoxic T lymphocytes were generated from the in vivo OK432-treated P815-draining lymph node cells only when the lymph node cells were restimulated in vitro with the DC pulsed with both P1A peptide and OK432. Moreover, the peritumoral injections of OK432 and the subsequent vaccination of the DC, pulsed with both OK432 and P1A peptide, significantly suppressed the growth of s.c. inoculated P815. Interestingly, a significant level of IL-12 was detected in the coculture supernatant of both OK432-pulsed DC and OK432-reactive CD4+ T cells. Collectively, our results suggest that the antitumor vaccination effect of DC pulsed with class I tumor peptide could thus be effectively augmented by locally utilizing the Th1-type cytokines from OK432-reactive CD4+ T cells. [ABSTRACT FROM AUTHOR]

A glycoprotein extract (CVS), derived from the unicellular green alga Chlorella vulgaris, strain CK22, exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis in mice. Inhibition of tumor metastasis was enhanced when intratumor administration of CVS was followed by s.c. injection of CVS. Anti-metastatic immunopotentiation was observed in euthymic mice, but not in athymic nude mice. The antitumor activity of CVS was reflected in antigen-specific, T-cell-mediated immunity. Both CD4 and CD8 T cells contributed to the antimetastatic effects, as shown by in vivo depletion experiments with anti-T-cell subset antibodies. Furthermore, CVS caused the recruitment of T cells to the regional lymph nodes and their proliferation in these organs. The CD4-positive population, following CVS injection at the time of tumor rechallenge, displayed a pronounced increase in the proportion of T cells that were CD18 bright, CD44 bright, CD25+, CD54+, CD69+ or CD71+ in the lymph nodes. Thus, CVS induces T cell activation in peripheral lymph nodes in tumor-bearing mice. We conclude that CVS augments antimetastatic immunity through T cell activation in lymphoid organs and enhances recruitment of these cells to the tumor sites. Presurgical treatment with CVS might prevent metastasis or tumor progression. [ABSTRACT FROM AUTHOR]

In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 - 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8 cells and CD56 cells and a lower proportion of CD4 cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinomas (SCC) are often infiltrated by a large number of T lymphocytes. To clarify the nature of the tumour-infiltrating lymphocytes (TIL), we examined T cell receptor (TCR) Vα and Vβ gene usage by TIL and peripheral blood mononuclear cells (PBMC) obtained from 10 patients with oral SCC. We obtained RNA from TIL and PBMC, synthesized complementary DNA, and used the polymerase chain reaction (PCR) method with a panel of primers specific for the V gene segment subfamily (Vα1 - 18/Vβ1 - 20). We thus found that TIL showed more restricted usage of Vβ gene families in contrast to PBMC of the same patients while two unique Vβ gene (Vβ6 and Vβ5.2) segment transcripts were overexpressed in the TIL of more than half of the patients. On the other hand, no major difference was observed in the Vα gene usage between the TIL and PBMC of most patients. To characterize these T cell subpopulations with unique Vβ gene segment transcripts further, we sequenced the complementarity-determining region 3 in Vβ6-Cβ and Vβ5.2-Cβ PCR products derived from TIL and PBMC of two selected patients in each case. Although no usage of the conserved amino acid sequence by TIL was detected, the frequent use of Vβ6/Jβ1.1 in one patient and the Vβ6/Jβ2.7 gene segments in another patient was observed. Regarding the Vβ5.2 transcripts, obtained from the other two patients, no preferential usage of specific Jβ gene segments by TIL was observed. These results suggest that the unique T cell populations are amplified in patients with oral SCC, possibly as a consequence of an in situ immune reaction. [ABSTRACT FROM AUTHOR]

The effect of a local injection with a streptococcal preparation OK432 on the antitumor vaccination with tumor cells was investigated. Natural killer (NK) cells, which were detected by anti-NK1.1 monoclonal antibody (mAb), increased in the peritoneal exudate cells after an intraperitoneal (i.p.) injection with syngeneic B16 melanoma cells. Furthermore, a concurrent i.p. injection with OK432 efficiently sustained the locally infiltrating NK cells. The OK432 treatment also sustained the augmented NK and lymphokine-activated killer activities in the peritoneal exudate cells. This treatment also increased the ability of the locally infiltrating NK cells to produce interferon γ in response to the tumor cells. In addition, the concurrent i.p. injection with OK432 in combination with the tumor cells enhanced the capacity of the spleen cells to turn into anti-(B16 melanoma) cytotoxic T lymphocytes after in vitro restimulation. This augmenting effect of OK432 was dependent on NK cells. Moreover, the concurrent injection with OK432 at the time of antitumor vaccination significantly enhanced the protective immunity against B16 melanoma at the rechallenge. Taken together, these findings indicate that a concurrent local injection with OK432 in combination with tumor cells efficiently augments the antitumor vaccination effect, in part, by sustaining the locally infiltrating activated NK cells. [ABSTRACT FROM AUTHOR]

An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4 - 9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent. [ABSTRACT FROM AUTHOR]

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor γII)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon γ, but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL. [ABSTRACT FROM AUTHOR]

To establish an efficient cell-culture system for adoptive immunotherapy, we attempted to use lipopolysacharide (LPS)-activated B cells (LPS blasts) as costimulatory-signal-providing cells in the in vitro induction of antitumor effector cells. Both normal and tumor-draining lymph node cells were efficiently activated by both anti-CD3 monoclonal antibody (mAb) and LPS blasts, and subsequently expanded by a low dose of interleukin-2 (IL-2; anti-CD3 mAb and LPS blasts/IL-2). The expanded cells were predominantly CD8 T cells and showed a low level of tumor-specific cytotoxic T lymphocyte (CTL) activity. The adoptive transfer of B16-melanoma-draining lymph node cells expanded by anti-CD3 mAb and LPS blasts/IL-2 showed significant antitumor effect against the established metastases of B16 in combination with intraperitoneal injections of IL-2. This treatment cured all B16-bearing mice. In addition, these mice also showed tumorspecific protective immunity against B16 at the rechallenge. Considering that activated B cells express several kinds of costimulatory molecules, these findings thus indicate an efficacy of costimulation that is derived from activated B cells for the in vitro induction of tumor-specific CTL, in co-operation with anti-CD3 mAb. The culture system presented here may thus be therapeutically useful, providing potent effectors for adoptive immunotherapy against various types of cancer. [ABSTRACT FROM AUTHOR]

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8 T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8 CTL in vivo. [ABSTRACT FROM AUTHOR]

The growth of MethA tumor was significantly inhibited by oral administration of the α-glucan SPR-901 in BALB/c (+/+) mice but not in nude mice. Mice treated orally with SPR-901 exhibited an augmentation of antigen-specific resistance against rechallenge with the tumor cells. The tumor-neutralizing activity of regional lymph node cells from MethA-bearing mice against the tumor was augmented by oral administration of SPR-901. The tumor-neutralizing activity of lymph node cells from SPR-901-treated mice mainly appeared in Lyt2+cells. Furthermore, lymphokine-activated killer activity of these cells was enhanced by administration of SPR-901. The antitumor effect of SPR-901 was abrogated in mice depleted of either L3T4+ or Lyt2+ cells, and in cyclosporin-A-treated mice. These results suggest that Lyt2+ cells are important effector cells in MethA-bearing mice orally adminstered SPR-901 and that functional exertion of both Lyt2+ and L3T4+T cells is necessary for the antitumor effect of orally administered SPR-901 in vivo. [ABSTRACT FROM AUTHOR]

RS-0481, (4 R)-3-benzoyl- N-[(1 R)-phenylethyl]-4-thiazolidinecarboxamide, is a compound that can re-establish the function of certain lymphoid cell populations impaired by the presence of a growing tumor in an animal. The compound markedly augmented the tumorspecific cytotoxic T lymphocytes, Tdth (delayed-type hypersensitivity T cells), and the nonspecific lymphokine-activated-killer-cell-like cell responses. It also enhanced the tumor-inhibitory effect of macrophages in tumor-bearing mice, but not in normal mice, indicating that it enhances the antitumor immune responses. Lymphocytes from RS-0481-treated tumor-bearing mice released significantly higher amounts of macrophage-activating factor(s) (MAF) and interleukin-2(IL-2)-like factors in culture compared with lymphocytes from untreated animals. Also, sera from treated tumor bearers showed elevated colony-stimulating factor (CSF) activity. Although the compound did not influence the factor-producing activity in mice without tumor, it enhanced the responsiveness of their bone marrow cells, T cells, and macrophages to CSF, IL-2, and MAF. It seems therefore possible that the compound enhances the responsiveness of immunocompetent cells to cytokines, resulting in a marked augmentation of antitumor T cell responses in tumor-bearing mice. Consistently it inhibited the development of lymph node metastasis of transplanted X5563 plasmacytoma, and we showed that T cells play a decisive role in this inhibition. The compound also counteracted the development of suppressor T cell activity in the spleen of tumor-bearing mice. [ABSTRACT FROM AUTHOR]

The effects of Chlorella vulgaris extract (CVE-A) on the recovery of leukocyte number and the augmentation of resistance to bacterial infection were examined in CDF1 mice made neutropenic by cyclophosphamide (CY). They were treated intraperitoneally with CY (150 mg/kg) on day 0, and were given CVE-A (50 mg/kg) subcutaneously (s. c.) every other day from day 1 to day 13 after CY treatment. CVE-A accelerated the recovery of polymorphonuclear leukocytes (PMN) in the peripheral blood in CY-treated mice. The number of granulocyte/monocyte-progenitor cells (CFU-GM) in the spleen increased rapidly and highly after the administration of CVE-A in CY-treated mice, in contrast to the absence of change due to CVE-A in the number of bone marrow cells in CY-treated mice. Administration of CVE-A in CY-treated mice enhanced the accumulation of PMN in the inflammatory site and the activity of the accumulated leukocyte cells in luminol-dependent chemiluminescence. The mice became highly susceptible to an intraperitoneal infection with E.coli on day 4 after CY treatment, whereas the mice given CVE-A showed an enhanced resistance against E.coli infection, irrespective of the timing of challenge. The bacterial number in CY-treated mice increased explosively after inoculation, resulting in death within 24 h. A progressive elimination of bacteria was observed from 6 h in the peritoneal cavity, spleen and liver of CY-treated mice given CVE-A s.c. These results indicate that CVE-A can be used as a potent stimulant of nonspecific resistance to infection in neutropenic mice. [ABSTRACT FROM AUTHOR]

To examine the clinical efficacy and the mechanism of action of polysaccharide K (PSK), a proteinbound polysaccharide extracted from a Basidiomycetes fungus, a randomized double-blind trial was performed by administering PSK to 56 patients and a placebo to another group of 55 patients after surgical operations on their colorectal cancers. The rate of patients in remission (or disease-free) was significantly higher in the PSK group than in the placebo group; the difference between both groups was statistically significant at P <0.05 by the logrank test. The survival rate of patients was also significantly ( P <0.05) higher in the PSK group than in the control group. The most significant laboratory finding was that polymorphonuclear leukocytes from PSK-treated patients showed remarkable enhancement in their activities, such as random and/or chemotactic locomotion, and phagocytic activity, when compared with those in the control group. In conclusion, PSK was useful as a maintenance therapy for patients after their curative surgical operations for colorectal cancer. The beneficial effects were probably due to the activation of leukocyte functions as one of the many biological-response-modifying (activities induced by PSK). [ABSTRACT FROM AUTHOR]

Effector mechanisms responsible for resistance against ectromelia virus including antiviral activity of non-immune macrophages, antiviral antibody, delayed footpad reaction to viral antigen, and interferon induction after viral infection were depressed in BALB/c mice bearing syngeneic Meth A tumor. The degree of viral growth correlated well with the depression of delayed footpad reaction, antibody production, and interferon induction. Therefore, modification of macrophage functions by a tumor-bearing state and treatment with PSK may contribute to this modification of antiviral resistance, at an early phase of infection. Cytotoxic activity may not be the principal effector, since the cytotoxicity was induced in normal and tumor-bearing mice to almost the same extent yet an extensive viral growth occurred only in the latter. [ABSTRACT FROM AUTHOR]

After inoculation of tumor cells (methylcholanthrene-induced sarcoma), the number of Thy 1 cells and PNA (peanut agglutinin) binding cells, which were shown to be different subpopulations were increased in the spleen of thymus-intact mice, in contrast this increase was not observed in adult thymectomized mice. In experiments performed concurrently with splenic cell analysis, we found that the plasma PGE levels declined in parallel with the tumor growth. Prevention of such a decline of plasma PGE level by replenishment with exogenous PGE inhibited the splenic cell increase in tumor bearers. In the tumorbearing mice, cell traffic systems from the thymus to the periphery was ascertained by injecting fluorescein diacetate (FDA) into the thymus and observing fluorescein positive cells in the periphery. We suggest that increased recruitment of thymic cells to the periphery may be mediated by PGE in the presence of a tumor. [ABSTRACT FROM AUTHOR]

The separation and properties of a new immunopotentiator, Benincasa cerifera mitogen (BCM) fraction, were investigated. BCM fraction was separated from hot water extract of the seed of Benincasa cerifera Savi (Tohgashi) by gel filtration using Sepharose 4B. BCM fraction is a heteropolymer consisting of uronic acid, neutral sugars, protein, and phosphorus. The proliferation and differentiation of murine B cells were markedly stimulated by BCM fraction. The in vitro development of peritoneal macrophages into antitumor macrophages was also activated by the addition of BCM fraction to cultures. BCM fraction augmented the IgM and IgG antibody responses against sheep erythrocytes (SRBC) and the induction of delayed-type footpad reaction against SRBC. The antitumor activity of BCM fraction was observed in terms of prolongation of the survival period of mice bearing Meth A fibrosarcoma. After hydrolysis with 1% acetic acid at 100° C for 4 h, marked mitogenic activity was found in a precipitate composed of 29% neutral sugars, 50% uronic acid, 1% protein, and 0.1% phosphorus. The precipitate did not contain detectable amino sugar. The possibility that the biological activities of BCM fraction may be due to contamination by bacterial lipopoly-saccharide was ruled out on the basis of the results of chemical analysis and of marked mitogenicity noted in C3H/HeJ spleen cell cultures. [ABSTRACT FROM AUTHOR]

When a hot water extract of Chlorella vulgaris (CE) was injected into the peritoneal cavity of BALB/c mice inoculated with syngeneic Meth-A tumor cells, the survival times were strikingly prolonged. Furthermore, peritoneal exudate cells (PEC) rich in polymorphonuclear cells (PMN) obtained from normal mice 24 h after CE injection exhibited an antitumor effect in a Winn-type assay using normal recipients. Such an activity of PEC remained almost intact after T cell or macrophage depletion. However, such PEC did not express an antitumor effect in a Winn-type assay using irradiated recipients. It was suggested that CE-induced PEC, presumably PMN, expressed an antitumor effect in cooperation with a host- or recipient-derived element(s) sensitive to irradiation. The anti-tumor mechanism of CE may be different from that of OK-432, one of the biological response modifiers. [ABSTRACT FROM AUTHOR]

The effect of PSK on the depressed bactericidal activity of macrophages and delayed-type hypersensitivity (DTH) to Listeria monocytogenes in BALB/c mice bearing transplantable Meth A fibrosarcoma was studied. In tumor-bearing mice pretreated with PSK, L. monocytogenes was cleared rapidly from the circulating blood and bacterial growth in the liver was inhibited effectively in the early phase of infection. This resistance to the infection could be transferred with adherent peritoneal exudate cells (PEC) but not with nonadherent or adherent spleen cells of PSK-treated mice. In the early phase of infection, tumor-bearing mice developed a lower level of DTH to L. monocytogenes than nongrafted control mice. However, the control levels of DTH could be obtained by pretreatment with PSK in tumor-bearing mice. These results suggest that the restoration of DTH to L. monocytogenes by pretreatment with PSK may be attributable to the restoration of the depressed immunological responsiveness to the normal levels in tumor-bearing mice. [ABSTRACT FROM AUTHOR]

Growth of Meth-A tumor in CDF1 mice was inhibited significantly by injection of a hot water extract of a strain of Chlorella vulgaris (CE) into the tumor or into the subcutaneous tissue near the tumor. The augmentation of resistance by CE may require the participation of T cells and macrophages, since it was abolished or reduced in athymic nude mice or mice treated with carrageenan, a macrophage blocker. Mice treated with CE exhibited antigen-specific augmented resistance against rechallenge with tumor. Moreover, the antitumor effect of CE was comparable with that of Corynebacterium parvum, but its mechanism of effect might be different. [ABSTRACT FROM AUTHOR]

Restorative effects of levamisole on suppressed immune responses were observed in BALB/c mice inoculated with a syngeneic methylcholanthrene-induced fibrosarcoma and immunized with nucleated chicken erythrocytes. In tumor-bearing mice, cell-mediated cytotoxicity, delayed footpad reaction, and antibodies were suppressed, and only the delayed footpad reaction was restored by levamisole. Suppressive effects in tumor-bearing mice can be transferred with sera or spleen cells, and the expression of suppressive effects cannot be abolished by treatment of the recipients with levamisole. However, the generation of suppressor mechanisms was affected by treatment of tumor-bearing donors with levamisole. [ABSTRACT FROM AUTHOR]

In this study we activated breast cancer-specific cytotoxic T lymphocytes (CTL) from regional lymph node lymphocytes (RLNL) of HLA-A2-positive patients with breast cancer. Freshly isolated RLNL were stimulated with solid phase anti-CD3 monoclonal antibody followed by expansion with recombinant interleukin-2. Subsequently, the RLNL were stimulated with an irradiated HLA 0201 breast cancer cell line, MCF-7, at a responder/stimulator ratio of 10/1 once a week for 2 weeks. The cultured RLNL exhibited specific lysis against MCF-7 in all 5 HLA-A2-positive patients tested, but not in 2 HLA-A2-negative patients. Cytotoxicity against MCF-7 was substantially inhibited by addition of anti-HLA-A2 mAb. In 3 of 5 HLA-A2-positive patients, anti-MCF-7 CTL also exhibited a substantial level of reactivity against PC-9, an HLA-A0206-positive lung adenocarcinoma cell line. Conversely, anti-PC-9-specific CTL were inducible by multiple stimulations of RLNL with PC-9 cells in 2 of 3 patients. These results suggest that several common tumor antigens might exist among HLA-A2-positive breast cancers, some of which may be shared with lung adenocarcinomas. [ABSTRACT FROM AUTHOR]

C3H/He mice were inoculated with Pseudomonas aeruginosa by various routes 1 day after X5563 transplantation or 4 days after cyclophosphamide (CY) administration. Administration of PSK (Krestin) i.p. or p.o. to the tumor-bearing mice or CY-treated tumor-bearing mice resulted in an increase in survival rates. Viable P. aeruginosa were inoculated i.v. on day 0 into mice inoculated with tumor cells on day -12 and vaccinated with killed P. aeruginosa on day -10, or into mice inoculated with tumor cells on day -15, treated with CY on day -14 and vaccinated on day -10. Resistance to infection, which is enhanced by vaccination, was depressed by tumor burden or treatment with CY, but such depression was prevented by PSK administration. [ABSTRACT FROM AUTHOR]

Suppression of various types of immune reactions including antibody (Ab) production, cell-mediated cytolysis (CMC), and delayed footpad reaction (DFR) was compared in EL-4 lymphoma-bearing syngeneic mice. In the primary response, the suppression of Ab production and CMC was detected 4 days after tumor inoculation, reached a peak on day 8, and then disappeared. The suppression of DFR was detected on day 1 and persisted to day 12. The suppressive effects of tumor-bearing mice could be transferred with sera to normal recipients, and the degree of the suppressive effect in tumor-bearing hosts correlated with the suppressive effect of sera but not with tumor size. In the secondary response, the suppressive effect of the tumor-bearing state was detected only on DFR. Spleen cells of these hosts were able to transfer positive DFR adoptively to normal recipients, indicating the presence of sensitized lymphocytes in the absence of expression. DFR of normal mice in the primary response was suppressed by transfer of sera of tumor-bearing mice befor elicitation, at which time sensitized lymphocytes should already have been raised. The very high susceptibility of DFR to the suppressive effects of the tumor-bearing state may be ascribed to the distinct susceptibility of mononuclear cells, probably of the macrophage series, which are required as secondary cells for expression of DFR. Further studies with Sephadex G-200 fractionation of the sera from the tumor-bearing mice showed that the immunosuppressive activity appeared in the initial fraction containing relatively high-molecular-weight materials. [ABSTRACT FROM AUTHOR]

In CF1 mice thymectomized and inoculated with polyoma virus at birth, tumor formation was markedly accelerated; by 70 to 82 days after polyoma virus inoculation, tumors developed in 100% of neonatally thymectomized mice, while less than 20% of control mice developed tumors. Furthermore, grossly apparent polyoma tumors were produced in neonatally thymectomized CF1 mice when inoculated with polyoma virus at 21 days of age: of 18 neonatally thymectomized and polyoma virus inoculated-weanling mice, 3 were found to have grossly apparent tumors 100 to 150 days after the virus inoculation. No tumors were found in so treated sham-thymectomized mice, during the same observation period. Polyoma virus recovery from kidneys and antipolyoma virus antibody were almost equal in neonatally thymectomized mice and control mice. The data may indicate that anti-polyoma virus antibody has little influence on the production of polyoma tumors, and that neonatal thymectomy causes a higher incidence of polyoma tumors because of the reduced capacity of homograft immunity. By varying the days of operation and of virus inoculation after birth, it was found that operation two days after birth was as effective as thymectomy at birth, and inoculation of polyoma virus into neonatally thymectomized mice as late as 7 days after birth was as effective as inoculation at birth. The accelerating effect of neonatal thymectomy on polyoma tumor formation is suggestive that so operated mice may be used as hosts in search for oncogenic viruses. [ABSTRACT FROM AUTHOR]