Hideyuki Nagasawa

Contribution of extrathymic gamma delta T cells to the expression of heat-shock protein and to protective immunity in mice infected with Toxoplasma gondii.

We demonstrated that γδ T cells contribute to protective immunity against Toxoplasma gondii by inducing the expression of a 65u2003000u2003MW heat‐shock protein (hsp 65) in host macrophages. Here we examined the role of extrathymic and intrathymic γδ T cells in protective immunity and hsp 65 expression in mice infected with T. gondii. Intrathymic γδ T cells were obtained from severe combined immunodeficiency (SCID) mice grafted with syngeneic fetal thymus (TG‐SCID), in which only T cells derived from the donor thymus developed, whereas extrathymic γδ T cells were obtained from nude mice that lack thymus. Extrathymic γδ T cells from T. gondii‐infected nude mice differed from intrathymic γδ T cells of infected TG‐SCID mice, in terms of Thy1.2 expression and V‐region gene usage of T‐cell receptor (TCR) γδ. Extrathymic γδ T cells expressed extremely high levels of Thy1.2, and had Vγ7 repertoire but lacked Vγ5,6 and Vδ1,5. On the other hand, intrathymic γδ T cells express intermediate and low levels of Thy1.2. These cells possessed Vγ5,6 and Vδ1,5 but failed to rearrange the Vγ7 gene. Peritoneal macrophages from infected nude mice contained hsp 65, whereas this protein was scarcely expressed in those of infected TG‐SCID mice. Transfer of extrathymic, but not of intrathymic γδ T cells to SCID mice enabled their macrophages to express hsp 65. Athymic nude mice were significantly resistant to the infection compared with SCID mice which lack γδ T as well as αβ T cells. The resistance was dependent upon extrathymic γδ T cells, since nude mice depleted of γδ T cells using a corresponding monoclonal antibody became extremely susceptible. These results indicated that extrathymic rather than intrathymic γδ T cells play some crucial roles in protection against T. gondii and in hsp 65 expression.

Role of L3T4+ and Lyt-2+ T Cell Subsets in Protective Immune Responses of Mice against Infection with a Low or High Virulent Strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt‐2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt‐2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt‐2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite‐immune mice with anti‐Lyt‐2 antibody, but not anti‐L3T4 antibody, before challenge significantly increased mortality.

Combined treatment of autoimmune MRL/MP-lpr/lpr mice with a herbal medicine, Ren-shen-yang-rong-tang (Japanese name: Ninjin-Youei-To) plus suboptimal dosage of prednisolone

Therapeutic effects of combined treatment with a Chinese medicine prescription, Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NYT) and suboptimal doses of prednisolone (PSL) on pathological findings of autoimmune-prone MRL/lpr mice were examined. Six-week-old MRL/lpr mice were treated orally with 1000 mg/kg of NYT, 0.5 or 2 mg/kg of PSL, 1000 mg/kg of NYT plus 0.5 or 2 mg/kg of PSL (combined treatment) or solvent only (control) six times per week. The rates of signs and symptoms of autoimmune disease (lymphadenopathy, proteinuria, dermatitis, loss of hair) were suppressed significantly in groups given PSL (2 mg/kg) alone, NYT alone and combined treatment with PSL (2 mg/kg) plus NYT (1000 mg/kg) compared with control, respectively, whereas treatment with PSL (0.5 mg/kg) alone did not inhibit their occurrence. ConA response and IL-2 production were also improved significantly in lymphocytes of mice given the combined treatment. Interestingly, treatment with NYT alone enhanced further the augmented IFN-gamma production in MRL/lpr mice but the combined treatment suppressed such an augmented production. The combined treatment dramatically reduced the level of anti-DNA antibodies in serum of MRL/lpr mice. By contrast, NYT alone treatment had no effect on autoantibodies production. These results suggest that combined treatment with NYT plus a suboptimal dose of PSL could be effective for systemic lupus erythematosus without severe side-effects.

Treatment effect of a traditional Chinese medicine, Ren-shen-yang-rong-tang (Japanese name: Ninjin-Youeito), on autoimmune MRL/MP-lpr/lpr mice

Abstract Autoimmune MRL /lpr mice were i.p. treated with 200 mg/kg Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NYT), a traditional Chinese herbal medicine (Japanese name: Kampo), from 8 weeks of age every 3 days before the onset of autoimmune disease. Compared to age-matched control MRL /lpr mice, the serum IL-6 concentration in NYT-treated mice was decreased, their serum IFN-γ concentration was increased, and the proliferative responses of whole and enriched CD4 + cells in their spleen and lymph nodes stimulated with ConA in vitro were restored. FACS analysis revealed that the rate of decreased CD4 + CD8 + T-cell population in the thymus was decreased in MRL /lpr mice but recovered by NYT treatment. Further, adult thymectomized (ATX) MRL /lpr mice were treated with 200 mg/kg NYT similarly. NYT treatment prolonged the survival of sham-operated MRL /lpr mice and ameliorated their proteinuria but did not improve such autoimmune manifestations in ATX-MRL /Ipr mice. These findings suggest that NYT plays an important role in the abrogation of autoimmune-prone T cell differentiation and that the therapeutic effect of NYT is dependent on the thymus in MRL /Ipr mice.

An extract of seeds fromAeginetia indica L., a parasitic plant, induces potent antigen-specific antitumor immunity in Meth A-bearing BALB/c mice

SummaryThe antitumor activity of an extract of seeds fromAeginetia indica L., a parasitic plant, was investigated. BALB/c mice, inoculated i.p. 1 × 105 syngeneic Meth A tumor cells, were administered 2.5 mg/kgA. indica extract i.p. every 2 days from day 0. The untreated mice died of an ascitic form of tumor growth within 21 days, whereas all the treated mice completely recovered from tumor challenge without any side-effects. The extract did not exert direct cytotoxic activity against Meth A in vitro. Mice that survived after the first challenge as a result ofA. indica treatment overcame the rechallenge with homologous Meth A without additional administration of the extract. On the other hand, those mice could not survive after rechallenge with Meth 1 tumor cells, which were also established in BALB/c mice but were different in antigenicity from Meth A, suggesting the development of antigen-specific concomitant immunity in theA. indica-cured mice. In the induction phase of antitumor resistance in this system, CD4+ T cells appeared to be the main contributors, since in vivo administration of anti-CD4 mAb completely abolished such resistance. In contrast, anti-CD8 mAb administration did not influence the effect ofA. indica. The importance of CD4+ T cells in antitumor immunity was again clarified by Winn assay; that is, spleen and lymph node cells depleted of CD4+ T cells in vitro prior to assay abolished antitumor activity on co-grafted Meth A tumor cells in vivo.

Seed extract of Aeginetia indica L induces cytokine production and lymphocyte proliferation in vitro

We previously reported that the extract of seeds from Aeginetia Indica L (AIL), a parasitic plant, induces potent antitumor immunity in tumor-bearing mice and that CD4+ T cells appear to be the main contributors in the induction of antitumor resistance. The present study was set up to investigate the in vitro effects of AIL on various lymphoid cells. Spleen cells from mice pretreated with AIL every 2 days for 1 week produced interleukin 2 (IL-2), interferon gamma (IFN gamma), tumor necrosis factor (TNF) and interleukin 6 (IL-6) when these cells were stimulated in vitro by AIL. Further, we found that CD4+ T cells were main producers of IL-2 and TNF upon the stimulation with ALL in vitro, while both CD4+ and CD8+ T cells secreted IFN. On the other hand, ALL was mitogenic in vitro to T enriched splenic lymphocytes as well as B enriched splenic lymphocytes. Moreover, AIL also proliferated thymocytes and this activity was potently synergistic with a suboptimal dose of concanavalin A (Con A). Lipopolysaccharide (LPS) contamination in AIL preparation was negligible since proliferative activity of AIL to B enriched splenic lymphocytes was not influenced in the presence of an endotoxin antagonist, polymyxin B sulfate (PMB). Further, B cell mitogenic activity of AIL seems to be mediated by different mechanism(s) from that of LPS since ALL could proliferate B enriched lymphocytes of C3H/HeJ mice which do not respond to the stimulation with LPS. A well known biological response modifier (BRM), Krestin (PSK), had no ability in inducing either T or B lymphocyte activation in vitro as shown by AIL.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation between the mitogenic effect and antitumor activity of seed extract from Aeginetia indica L.

The extract from seeds of Aeginetia indica L. (AIL), a parasitic plant, induces potent antitumor immunity against Meth A fibrosarcomas in BALB/c mice. AIL also possesses a thymocyte co-stimulatory effect in vitro with suboptimal dose of Con A, a B cell mitogenic effect, and stimulates AIL-primed CD4+ T cells to produce Th1-type cytokines. In this study, we investigated the relationship between mitogenicity and antitumor activity with AIL. When AIL was analyzed by SDS-PAGE, there was strong and diffuse staining in the region between 14 kDa and the bottom of polyacrylamide gel and it was unaffected when AIL was digested with proteinase K (PK) before SDS-PAGE. Some bands with different molecular mass were also found in silver-stained gel and they disappeared completely by incubating AIL with PK before SDS-PAGE. The in vitro thymocyte co-stimulatory and B cell mitogenic effects were not influenced by digesting AIL with PK but were completely suppressed by the oxidation of AIL with sodium periodate before culture. In contrast, the in vivo antitumor activity was completely abolished by PK, but it was not affected by periodate oxidation. We generated mAbs specific for AIL and investigated the influence on the antitumor activity of AIL in vivo. Around 60-80% of tumor-bearing mice failed to recover from a challenge tumor when they were treated with supernatants isolated from mAb-induced precipitation reactions. Immunoblotting (Western blotting) revealed that all the mAbs reacted exclusively with a 50-60 kDa protein and that this reactivity was not influenced after oxidizing the blots with sodium periodate. We demonstrated that AIL contains polysaccharides and proteins. The polysaccharides induced B cell mitogenic and thymocyte co-stimulatory effects in vitro, while the proteins, especially a 50-60 kDa protein containing non-carbohydrate epitopes recognized by the mAbs, mediated antitumor activity in vivo.

Expression of heat shock protein in host macrophages correlates with a protective potential against infection with Leishmania major in mice

Abstract We studied the relationship between the expression of 65-kDa heat shock protein (HSP65) and resistance of mice to infection with Leishmania major (L. major) , an obligate intracellular protozoan. C57BL/6 (B6) mice, a strain genetically resistant to L. major infection, expressed high level of HSP65 in their peritoneal and draining lymph node macrophages after infection, whereas susceptible BALB/c mice expressed only slightly at the early stage of infection. This protein was not expressed in the parasite itself and macrophages of non-infected mice. We examined the role of T cells in the expression of HSP65 by using SCID mice grafted with the fetal thymus from B6 or BALB/c mice (B6-TG or BALB-TG mice, respectively). Either BALB-TG or B6-TG mice were reconstituted with T cells derived from each grafted fetal thymus. B6-TG mice were markedly resistant against infection with L. major , compared with BALB-TG mice. Furthermore, the HSP65 expression in macrophages of thymus-grafted mice was similar to that of the thymus-donor type. That is, B6-TG mice expressed a high level of this protein, whereas BALB-TG mice did in lower level than B6-TG mice. These results show that T cells are necessary to express HSP65 and the expression correlates with a protective potential of T cells against infection with L. major .

Development of Functional Rat-Derived T Cells in SCID Mice Engrafted with the Fetal Thymus of LEC Rats Which Are Defective in CD4+ T Cells

We reported that LEC rats are genetically deficient in the development of thymic CD4+8− cells and that this defect is caused by bone marrow (BM)‐derived stem cells. To determine which BM‐derived cells are responsible for the arrest of T‐cell development in LEC rats, fetal thymuses of LEC rats, or LEA rats which bear the same major histocompatibility complex (MHC) as LEC rats but are immunologically normal, were engrafted under the kidney capsule of severe combined immunodeficiency (SCID) mice (LEC‐TG and LEA‐TG mice, respectively). We then examined the differentiation of T cells and their immunological functions in the SCID mice. A large number of rat‐derived CD4+ T cells appeared in the peripheral blood, lymph nodes (LN) and spleens in LEC‐TG mice. Furthermore, the peripheral LN cells in LEC‐TG mice appeared to be functional. These cells produced IL‐2 upon Con A stimulation, whereas LN cells from LEC rats produced no IL‐2 in the same conditions. Thymopoiesis was observed at 3 weeks in LEC‐TG as well as LEA‐TG mice. The distribution of thymocyte subsets with respect to CD4 and CD8 expression in LEC‐TG mice closely resembled that of LEA rat thymus and that in LEA‐TG mice, suggesting that normal T‐cell differentiation occurred in LEC‐TG mice. The results indicated that BM‐derived progenitor T cells of LEC rats could differentiate to functional CD4+ T cells.

Antimicrobial activity of Obioactin.

A newly developed enzyme-hydrolyzed toxoplasma-hyperimmune bovine serum preparation, Obioactin , was examined for its anti-microbial and related actions against heterologous protozoa, bacteria, and viruses both in-vivo and in-vitro. An appreciable efficacy of Obioactin in a combination with nifurtimox, Lampit was observed in experimental Trypanosoma cruzi infections in mice. But prophylactic applications of the agent alone or joint with Lampit were in vain in mice. In-vitro digestion of certain bacteria in mouse peritoneal macrophages was accelerated in the presence of the agent being three to ten times stronger than that in untreated macrophages. Inhibitory effect of Obioactin on in-vitro infectivity of viruses was suggested.