Footpad Reaction Research Articles

Humoral and cell-mediated immunity in mice after immunization with live oral vaccines of Salmonella typhimurium: auxotrophic mutants with two attenuating markers

Persistence and clearance of the vaccinal strains, humoral and cell-mediated immune response and protective immunity were assessed in ICR mice, immunized intraperitoneally or intragastrically with double-marker auxotrophic mutants of Salmonella typhimurium strains 1771 and 3334 (his − pur −). Strain 3334 possesses in addition the antiepidemic Hst (high sensitivity to tensides) marker which confers decreased survival in the gut and environment. The results showed that the strains are not overattenuated. They revealed preserved multiplication capacity, established carrier state for 28–30 days and induced humoral and cell-mediated immunity as measured by passive haemolysis and micro-agglutination or footpad swelling tests, respectively. The immune response was determined by biological features of the strains. Strain 1771 induced synthesis of O- and H-antibodies 7–11 days earlier than strain 3334. Footpad reactions appeared 3–4 days after immunization and a week before the serum antibodies. Cell-mediated immunity showed clear correlation with the protection against challenge with a virulent strain. The present results suggest that the double-marker auxotrophic mutants of S. typhimurium might prove very useful in the development of a live vaccine in further studies on livestock.

Effect of suplatast tosilate (IPD-1151T) on types I-IV allergic reactions

We examined the effect of suplatast tosilate (IPD-1151T), which exhibits a class-specific suppression of IgE antibody production, on types I-IV allergic reactions. 1) Type I reaction: IPD-1151T dose-dependently inhibited 48-hr homologous passive cutaneous anaphylaxis (PCA) when the agent was given p.o. 30 min prior to the antigen challenge. To observe the time course of the inhibitory activity, IPD-1151T in a dose of 50 mg/kg was given orally at various times prior to the antigen challenge. IPD-1151T showed inhibitory activity when it was given at 0.5 to 2 hr prior to the antigen challenge, and the maximum inhibition was found when the IPD-1151T pretreatment was 2-hr before the challenge. IPD-1151T also suppressed the antigen-induced degranulation of mesenteric mast cells and histamine release from peritoneal exudate cells of rats. 2) Type II reaction: Only a high dose of IPD-1151T given orally inhibited reversed cutaneous anaphylaxis in rats, whereas the agent did not affect Forssman shock in guinea pigs. 3) Types III and IV reactions: IPD-1151T neither affected the Arthus reaction in rabbits nor the picryl chloride-induced contact dermatitis and sheep erythrocytes-induced footpad reaction in mice. The results obtained here indicate that IPD-1151T shows a relatively specific suppression of the type I allergic reaction with the inhibition of degranulation and histamine release from mast cells.

Effects of suplatast tosilate (IPD-1151T) on antibody formations in mice

We examined the effects of suplatast tosilate (IPD-1151T) on antibody formation in mice, and the following results were obtained: 1) IPD-1151T clearly increased the productions of IgM and IgG-hemolytic plaque forming cells (PFC) in mice immunized with sheep red blood cells (SRBC) when the agent was given p.o. for 5 days from the day of immunization. 2) IgM and IgG-PFC productions in old mice, 60 weeks of age, were clearly lower than those in adult mice, 10 weeks of age; and an oral administration of IPD-1151T significantly recovered the decayed PFC-production in the old mice. 3) IPD-1151T clearly suppressed the production of anti-dinitrophenyl (DNP)-IgE antibody in mice when the agent was given p.o. for 5 days from the day of immunization, whereas the agent did not affect anti-DNP-IgM and IgG antibody productions. 4) IPD-1151T did not affect the induction phase of the cellular immune reaction of picryl chloride-induced contact dermatitis and the SRBC-induced footpad reaction. Our present results suggest that IPD-1151T has a class-specific suppressive effect on the production of IgE antibody, but does not suppress the other immune responses.

Recombinant human granulocyte colony-stimulating factor improves the compromised state of recipient mice without affecting the induction of specific tolerance in the cyclophosphamide-induced tolerance system.

The effects of recombinant human granulocyte CSF (rhG-CSF) on cyclophosphamide (CP)-induced tolerance was studied. In the recipient C57BL/10 Sn Slc (B10) mice given 1 x 10(8) B10.BR Sg Sn Slc (B10.BR) spleen cells (SC) on day -2 followed by 200 mg/kg CP on day 0, the number of leukocytes and neutrophils in the periphery declined to their minimum levels on day 4. When rhG-CSF in a dose of 200 micrograms/kg was given daily for 5 days to the B10 mice, which had been treated with B10.BR SC and CP, starting one day after the administration of CP, the leukocyte and neutrophil counts declined to the same levels as those in the B10 mice treated with B10.BR SC and CP alone on day 2. On day 4, however, the counts recovered to their normal levels. The nucleated cell count of the spleen in the B10 mice given B10.BR SC and CP followed by rhG-CSF decreased less and recovered faster than that in the B10 mice given B10.BR SC and CP. The case was found to be the same in bone marrow, and the difference did not reach statistical significance. When the recipient mice were inoculated i.p. with 4 x 10(4) Pseudomonas aeruginosa (GNB-139) on day 4, the survival of the B10 mice treated with B10.BR SC and CP was markedly improved by rhG-CSF administration. The administration of rhG-CSF did not affect either the prolongation or the specificity of skin allograft survival, as shown in an H-2 mis-matched combination of B10.BR----B10 and in an H-2 identical combination of AKR/J Sea(AKR)----C3H/HeN Crj (C3H). The tolerant state, which was demonstrated by various immune responses, such as CTL, delayed footpad reaction, and antibody, was also not affected by rhG-CSF. Furthermore, the basic mechanisms for inducing a long-lasting skin allograft tolerance in this system--namely, the specific destruction of Ag-stimulated and then proliferating mature T cells in the periphery, the establishment of mixed chimerism, and the intrathymic clonal deletion of immature T cells--were preserved even when rhG-CSF was given to C3H mice previously made tolerant of AKR.

Immunological Changes in Babesia rodhaini Infected BALB/c Mice after Treated with Anti-Babesial Drug; Diminazene Diaceturate.

A model system capable of investigating immunological changes was first established in Babesia rodhaini infected mice with an aid of a drug, diminazene diaceturate (DD). Intraperitoneal (ip) inoculation with B. rodhaini resulted in acute death in euthymic (nu/+) and athymic (nu/nu) BALB/c mice. Treatment with DD at an early stage of infection saved both mice from acute death. Parasitemia recurred in some of them but resulted in death only in nu/nu mice. A re-challenge with 10(5) parasitized erythrocytes (PE) on the surviving mice on day 28 post infection revealed resistance in nu/+ but not in nu/nu mice. The results suggested a participation of the thymus in the protective mechanisms. Immunological changes were then observed on nu/+ and nu/nu mice which were inoculated ip with 10(4)PE and treated with the drug, and then challenged with 10(5)PE ip on day 28. An antibody response was measured with immediate reaction by footpad injection of a soluble antigen of B. rodhaini and by ELISA of serum antibody using the antigen and protein A, on day 10 and later, and further a pronounced response was detected after re-challenge in nu/+ mice. No response was detected by ELISA in nu/nu mice. Delayed footpad reaction was seen in nu/+ mice by day 14 and later but it was suppressed after the re-challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Difference in the induction of macrophage interleukin-1 production between viable and killed cells of Listeria monocytogenes

T-cell-mediated immunity to Listeria monocytogenes in mice, as determined by delayed-type hypersensitivity and acquired resistance, was induced by immunization with viable bacteria but not with killed bacteria, even when killed cells were injected in a high dose or repeatedly. T cells obtained from mice immunized with viable L. monocytogenes were readily stimulated with killed-bacterial antigens, resulting in T-cell proliferation in vitro and expression of a delayed footpad reaction in vivo. After immunization with killed-bacterial vaccine, T-cell responsiveness to interleukin 2 (IL-2) never developed but a lower level of responsiveness to IL-1 appeared later than with T cells from mice immunized with viable bacteria. When IL-1 production by macrophages was examined in vitro, viable L. monocytogenes stimulated a high level of IL-1 release while killed bacteria did not. Avirulent strains which were ineffective in the induction of T-cell mediated immunity were incapable of inducing IL-1 production as well. The impaired ability of killed bacteria to stimulate IL-1 production was confirmed by the level of IL-1 mRNA expression. These results suggested that the ineffectiveness of killed L. monocytogenes vaccine is not due to loss or lack of antigenic epitopes but may be ascribed to insufficient induction of IL-1 production in the initial stage of the immune response in vivo.

Role of T cell subsets in the maternal-to-neonatal transmission of immunity against Trichinella spiralis during lactation in rats

We have previously demonstrated the maternal-to-neonatal transfer of immunity to T. spiralis during lactation and have shown that antigen-specific T lymphocytes, when injected into the mother or orally fed to neonates, can mediate this transfer. To further analyze the T cell subsets involved in conferring this protection, T lymphocytes were isolated from the mesenteric lymph nodes of syngeneic donor rats infected 4–6 days earlier with T. spiralis. The T cells were incubated in vitro with either mouse-anti-rat OX8 or W3/25 monoclonal antibody, “panned” on plates coated with goat-anti-mouse Ig, and the non-adherent T helper or T cytotoxic/suppressor cells harvested. 100 × 10 6 T helper cells were injected i.v. into mothers once in early lactation and again two days prior to challenging their pups (200 T. spiralis larvae) at 2 weeks of age. This resulted in significant passage of immunity from the mothers to their suckling neonates, worm counts being 59% and 73% of control values 3 and 8 days post-challenge ( P < 0.01). Injection of T-cytotoxic/suppressor cells using the same regimen resulted in significant suppression of immunity in challenged pups, who retained worm counts that were 105% and 145% of control values at 3 and 8 days post-challenge. Synergy between recombined panned T-helper and T cytotoxic/suppressor cells without Ly1 +2 +3 + amplifier cells was tested by recombining non-adherent panned 0X8 and W3/25 cells. This resulted in no significant expressions of immunity in the pups when compared to controls. The presence of transferred maternal T cells within the neonate was evidenced by the fact that neonates (nursing on immune mothers) had significant ( P < 0.01) delayed footpad reactions to a crude T. spiralis antigen preparation, as compared with neonates nursing on non-immune controls.

Effects of Stimulated or Immunologically Activated Macrophages on the Induction of Immune Responses to Listeria monocytogenes

The influences of peritoneal macrophages induced by proteose peptone, Corynebacterium parvum (C. parvum) or Bacillus Calmette Guérin (BCG) on the initiation and development of immune responses and protection against Listeria monocytogenes infection were studied in mice. Mice treated intraperitoneally (i.p.) with proteose peptone 4 days previously showed much the same level of protection against an intraperitoneal infection with Listeria as untreated mice. Mice treated i.p. with C. parvum 4 days previously, of which peritoneal macrophages had increased abilities for intracellular killing of Listeria and 0 - 2 generation as compared with peptone-elicited macrophages, exhibited an enhanced resistance against the listerial infection. The degree of immune responses, as assessed by delayed footpad reaction (DFR), was rather depressed in these mice because C. parvum-activated macrophages acting as scavenger cells reduced the amount of effective antigenic stimulation. BCG-activated peritoneal macrophages from mice treated i.p. with BCG 14 days previously showed a strong ability for antigen presentation in correlation with increases in the number of la-bearing macrophages and in the level of interleukin 1 (IL 1) production. These mice showed an early appearance of DFR response and a markedly enhanced resistance against the listerial infection. These results suggested that the differences in macrophage activities as scavenger cells, cytokine-secreting cells and antigen presenting cells may account for the differences in the responsiveness against listerial infection in peptone-, C. parvum- and BCG-treated mice.

BBN誘発マウス膀胱癌に関する研究

To evaluate the genetic difference in induction of bladder cancer by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), BBN was given to two different strains of mice, i.e. A/J (BCG resistant) strains and C57BL/6 (BCG susceptible) strains. The influence of Freund complete adjuvant (FCA), which includes BCG, was similarly examined along with associated cellular immunity as evaluated by footpad reaction (FPR). The results were as follows: 1. The incidence of bladder cancer was significantly higher in C57BL/6 strains (90.0%) than A/J strains (54.5%). 2. FCA reduced the incidence of bladder cancer significantly in C57BL/6 strains. 3. FPR, which reflects the delayed type hypersensitivity, was activated significantly in FCA treated C57BL/6 strains. These data suggest that the strain differences in mice probably explains the inhibition of carcinogenesis by FCA and cellular immunity.

犬の住血原虫Ｂａｂｅｓｉａ ｇｉｂｓｏｎｉの免疫に関する研究 Ｂｏｒｄｅｔｅｌｌａ ｂｒｏｎｃｈｉｓｅｐｔｉｃａの免疫増強効果

Four species of bacteria, Corynebacterium anaerobium 578, Actinobacillus pleuropneumoniae G-4, Mycobacterium bovis BCG, and Bordetella bronchiseptica A-2, were injected intravenously into mice (5 weeks old, ICR-SPF). The clearance of carbon from the blood stream and the weights of the spleen and liver were determined as indicators of RES stimulation. Mouse footpad reaction was assessed as an indicator of delayed-type hypersensitivity to each species of bacteria. The immuno-stimulative activity of each species of bacteria against bovine serum albumin was monitored by passive hemagglutination assay and the macrophage migration-inhibition test in guinea pigs. Based on the results of the experiments described above, B. bronchiseptica was selected as an immunostimulator (Ims) for immunization trials of the hemo-protozoan parasite, Babesia gibsoni, with inactivated merozoites of B. gibsoni (BgK). Twelve dogs, pointers about 6 months old, were divided into four groups of three dogs each. Group 1 dogs were initially injected with Ims, and later injected with BgK and Ims (BgK+Ims) after a 3-week interval. Group 2 and Group 3 dogs were injected twice, at a 3-week interval, with BgK+Ims and BgK, respectively, and Group 4 served as a control. As the results, the serum antibody titres of Group 1 and 2 were several times higher than that of Group 3, and the cell-mediated immunity to parasites was noticeably stimulated by immunization with BgK+Ims. The peak level of parasitemia following the challenge were over 10% for Group 4 and 4.5% for Group 3, while levels for Group 1 and 2 were 2.5% and less than 1%, respectively. No such major clinical signs of babesiosis as jaundice and anemia were observed in Group 1 or 2.

&lt;I&gt;Salmonella enteritidis&lt;/I&gt; 2547株SPAの免疫学的研究

Studies were carried out to analyze the antigenicity of Soluble Protective Antigen (SPA) separated from culture supernatant fluids of Salmonella enteritidis strain 2547. Mice injected with anti-SPA mouse serum were capable of tolerating a challenge dose of 100 LD50 S. enteritidis. After absorption of the anti-SPA mouse serum with lipopolysaccharide (LPS) prepared from strain 2547, no protective effect was observed. Ouchterlony immunodiffusion analysis showed that the P1 fraction obtained from Sephadex G-50 gel filtration of strain 2547 or 2822 LPS reacted with antiserum to SPA, but no reaction was observed with the P2 or P3 fraction. The LPS from strain 2547 gave 80% mouse protection against challenge with 100 LD50 of the homologous bacteria, while the P1 from strain 2547 LPS afforded 40% protective immunity. When P1, LPS and SPA were transferred into the footpad of SPA-immunized mice, a positive delayed footpad reaction was elicited. Similarly, macrophage migration inhibitory activity was observed when SPA-induced peritoneal exudate cells were cultured in medium containing P1, LPS and SPA. These results suggest that the antigenic determinant of SPA exists in the O-antigenic components of LPS.

The immunotoxicity of triphenyltin chloride in mice.

The immunotoxicity of triphenyltin chloride (TPTC) in mice was investigated. 1) When TPTC was injected intraperitoneally (i.p.) into mice at doses between 1 and 10 mg/kg for 14 d, a reduction in the weights of thymus and spleen was noticed, however, the body weight was not changed significantly. 2) The production of hemolytic plaque forming cells in the spleen of mice immunized with sheep red blood cells (SRBC) was inhibited by the administration of 10 mg/kg of TPTC. 3) The i.p. injection of TPTC at a dose of 10 mg/kg for 5 d after the primary immunization of mice with dinitrophenylated ascaris antigen resulted in suppression of immunoglobulin E antibody formation in primary immune response, but did not affect the secondary immune response. 4) The production of antibody against polyvinylpyrrolidone (T cell independent antigen) was not affected by the administration of TPTC. 5) The production of effector T cells which are able to cause SRBC- or tuberculin-induced footpad reaction in mice and the induction of cytotoxic T cell in local graft versus host reaction in mice were also inhibited by TPTC. These results indicate that TPTC inhibits both the T cell dependent humoral and cellular immune responses in mice.

Role of L3T4+ lymphocytes in protective immunity to systemic Candida albicans infection in mice

Protective immunity to lethal Candida albicans challenge in vivo and activation of splenic macrophages with highly candidacidal activity in vitro were detected in mice infected with low-virulence agerminative yeast cells of the variant strain PCA-2, at a time when a strong delayed-type hypersensitivity (DTH) reaction to C. albicans occurred in the footpads of PCA-2-treated mice. The DTH reaction was transferable with spleen cell populations from these animals, and enrichment of splenic lymphocytes in L3T4+ cells significantly increased the footpad swelling. The reactivity transferred by L3T4+ cells was a radiosensitive (2,500 rads in vitro) phenomenon that required collaboration with radioresistant, silica-sensitive syngeneic cells in the host and was inhibited by treatment of recipient mice with antibodies to the L3T4 antigen or murine gamma interferon. In vitro, the PCA-2-immune L3T4+ cells produced various lymphokine activities upon incubation with C. albicans, including gamma interferon and granulocyte-macrophage colony-stimulating factor. Anti-L3T4 monoclonal antibody treatment of PCA-2-infected mice significantly impaired their footpad reaction and resistance to C. albicans, as shown by increased recovery of yeast cells from the kidneys of anti-L3T4-treated mice. These results suggested that the mechanisms of anti-Candida resistance induced by PCA-2 may involve specific induction of a DTH response mediated by inflammatory L3T4+ T cells and lymphokine-activated phagocytic effectors. However, the survival rate of the PCA-2-immune mice challenged with C. albicans was not significantly modified by administration of the anti-L3T4 antibody, thus allowing for the conclusion that compensatory mechanisms lead to considerable anti-Candida resistance when the activity of L3T4+ cells is deficient.

The Effect of T Cell Depletion from Spleen Cell Allografts on Graft-versus-Host Disease and Long-Term Immune Reconstitution in H-2 Haplotype-Identical Murine Combinations

Graft-versus-host disease (GVHD) and states of immune reconstitution in allogeneic chimera mice across minor histocompatibility antigens were analyzed in excess of 9 months after injecting AKR/JSea (AKR) spleen cells into irradiated C3H/HeSlc (OH) mice.When T cell-depleted AKR spleen cells were used as inoculum cells, neither graft failure nor GVHD was seen for 9 months postgrafting in the OH mice irradiated with 660 rad or more.In an AKR - C3H (850 rad) model, Thy1.1 + or L3T4 + T cell depletion from donor AKR spleen cells abolished both acute and chronic GVHD in lethally irradiated C3H mice.Lyt2 + T cell depletion, however, resulted in acute and chronic GVHD in more than half of the recipient OH mice.Moreover, actual existence of donor(AKR)-type T cells with L3T4 phenotype, but not Lyt2 phenotype, was always observed in the spleen of the OH mice suffering from acute GVHD. In addition, the C3H mice that were irradiated with 850 rad, grafted with AKR spleen cells depleted of Lyt2.1 + T cells, escaped from acute GVHD and survived for more than 10 mo postgrafting, showed impaired activities of immune responses such as delayed footpad reaction to sheep red blood cells, antibody production tested by IgM plaque forming cells and reactivity to an intracellular bacterium Listeria monocytogenes as compared with the C3H mice reconstituted with syngeneic C3H spleen cells or Thy1.1 + or L3T4 + T cell-depleted AKR spleen cells. These results suggest that L3T4 + T cells, rather than Lyt2 + T cells, contained in the grafted cells not only cause acute GVHD but also a long-term immunodeficient state (chronic GVHD) in recipient mice in the H-2-identical murine combinations examined here.

A Low Degree of Requirement for Ia-Positive Macrophages and IL 2 in the Induction Phase of Listeria monocytogenes-Specific T Cells in vitro

We have analyzed the priming process of Listeria-specific T cells using an in vitro primary culture system. Listeria-specific T cells mediating delayed footpad reaction (DFR) and acquired cellular resistance (ACR) upon passive local transfer into naive recipients were generated from non-immune mouse spleen cells when stimulated with viable Listeria monocytogenes primarily in vitro. The effectors were detected on the third day of culture, and culturing for 5 days was sufficient for the generation of effectors mediating the peak level of DFR and ACR. The requirement of T cell subsets, la antigen and interleukin 2 (IL 2) for inducing effectors was studied. Presence of macrophages (M) and their contact to T cells were required for priming of Listeria-specific T cells in vitro. The presence of Ia antigens on macrophages was absolutely required for priming, but this requirement was lower than that in secondary immune response of Listeria-specific T cells. Effectors could not be generated when L3T4 + cells were depleted, but effectors capable of conferring a full level of DFR and ACR were induced even after the depletion of Lyt2 + cells. Contribution of IL 2 to the generation of effectors during early phase of priming was not observed. IL 2 was not produced in the supernatant of the in vitro primary culture. Precursor cells of the effectors did not respond to exogenously added recombinant IL 2 (rIL 2). Some mechanisms operating in the induction phase of Listeria-specific T cells were clarified in this study.

The nature of tolerance in adult recipient mice made tolerant of alloantigens with supralethal irradiation followed by syngeneic bone marrow cell transplantation plus injection of F1 spleen cells.

The length of time after syngeneic bone marrow reconstitution when tolerance to alloantigens can be induced in adult mice during T cell differentiation from bone marrow cells was studied by exposing those T cells to (recipient x donor)F1 spleen cells. Supralethally irradiated C3H/He Slc(C3H; H-2k) mice were reconstituted with 1 x 10(7) syngeneic T cell-depleted bone marrow cells and then injected intravenously with 5 x 10(7) (C3H x C57BL/6[B6])F1 (B6C3F1; H-2bxk) or (C3H x AKR/J[AKR])F1 (AKC3F1; H-2kxk) spleen cells at various intervals. In the fully allogeneic combination of B6C3F1----C3H, EL-4 tumor originating from B6 was accepted, and survival of grafted B6 skin was significantly prolonged in the tolerant C3H mice treated with irradiation on day -1 followed by injection of syngeneic bone marrow cells on day 0 plus B6C3F1 spleen cells on days 0, 5, or 10, in a tolerogen-specific manner. In the multiminor histocompatibility antigen-disparate combination of AKC3F1----C3H, AKR skin grafts were permanently accepted in the tolerant C3H mice treated with AKC3F1 spleen cells on days 0, 5, 10, or 15. Immunological parameters, including cytotoxic T lymphocyte activity and delayed foot-pad reaction (DFR), were almost completely suppressed in C3H mice made tolerant of B6 or AKR antigens. A chimeric assay using a direct immunofluorescence method revealed that the tolerant C3H mice given B6C3F1 spleen cells on day 0 were mixed-chimeric for at least 8 weeks after syngeneic bone marrow reconstitution, but not definitely chimeric thereafter. The C3H mice given AKC3F1 spleen cells on day 0 were chimeric even 43 weeks after syngeneic bone marrow reconstitution, but the C3H mice given AKC3F1 spleen cells on day 15 showed temporal chimerism that disappeared within 43 weeks. The untolerant mice were never detectably chimeric. These data suggest that the earlier the timing of the injection of F1 spleen cells after syngeneic bone marrow reconstitution was, the more profound tolerance was induced. Moreover, the stronger the antigenic disparity between donor and recipient, the earlier the injection of F1 spleen cells was required to induce tolerance.

Anti-tumor cytostatic mechanism and delayed-type hypersensitivity against a syngeneic murine tumor. Comparison between neonatally thymectomized mice and congenitally athymic nude mice.

We studied the anti-tumor mechanism against a syngeneic tumor using a BALB/c-MA tumor system by cytolysis and cytostasis assays in vitro comparing mice neonatally thymectomized at 1 day or 7 days after birth (NTx-1, NTx-7), sham-operated (sham) mice, and congenitally athymic nude BALB/c mice. NTx-1 mice showed more rapid tumor growth and a slightly lower degree of strong cytostatic activity in peritoneal exudate cells (PEC) than NTx-7 or sham mice. Nude mice showed more rapid MA growth than NTx-1 mice and no cytostatic activity in PEC. After immunization with mitomycin C-treated MA (MMC-MA), NTx-1 mice acquired an immunoprophylactic capacity against MA and showed cytostatic activity and delayed footpad reaction (DFR) to MA, however, nude mice showed no acquisition of such an immunity, or cytostatic activity, or DFR to MA. These differences between NTx-1 and nude mice could be well-explained by less capacity of nude mice to produce a macrophage-activating factor, which activates macrophages to exert cytostasis and DFR. However, NTx-1 mice could not reject MA by immunization with MMC-MA in CFA (MMC-MA/CFA), although such immunized sham mice could eliminate MA completely. Both PEC and spleen cells from Sham mice immunized with MMC-MA/CFA showed cytostatic activity, whereas NTx-1 mice showed cytostatic activity of the same level in PEC and less in spleen cells compared to Sham mice. Cytolytic activity was never detected throughout this study in a BALB/c-MA system. These data suggest that cytostasis plays an important role in antitumor immunity against a syngeneic MA tumor and that two types of cytostasis is included from the standpoint of thymus-dependency of ontogenic development, relatively low and high.

Effects of CKS-17, a synthetic retroviral envelope peptide, on cell-mediated immunity in vivo: immunosuppression, immunogenicity, and relation to immunosuppressive tumor products.

CKS-17 is a heptadecapeptide corresponding to a region highly conserved in retroviral transmembrane proteins such as p15E. Because a relationship had previously been determined between p15E and immunosuppressive tumor cell products, we examined the effect of CKS-17, control peptides and conjugates thereof on the expression of cell-mediated immunity (delayed-type hypersensitivity, DTH) in mice. Conjugates of CKS-17 inhibited DTH reactions to sheep erythrocytes in the feet of mice. The degree of inhibition was dose-dependent. Unconjugated CKS-17 had almost no effect, and control peptide conjugates had no inhibitory effect. Immunization of mice with CKS-17 conjugates, but not with control conjugates, rendered them resistant to the depression of DTH reactions, not only by CKS-17 conjugates, but also by products of cultured tumor cells. CKS-17 conjugates, but not control conjugates, also depressed the cellular inflammatory reactions induced in mouse footpads by concanavalin A (ConA) and immunized mice against the depression of ConA reactions by products of cultured tumor cells. Injections of globulin from sera of mice immunized with CKS-17 conjugates conferred upon normal recipients resistance to the depression of footpad reactions to ConA by products of cultured tumor cells. Globulin from sera of normal mice or control immunized mice did not confer such resistance. Thus conjugates of a synthetic peptide not only mimic the immunosuppressive effects of tumor products in vivo, but can also immunize mice against those effects.

Suppression of Interleukin 2 Production by Sera Obtained from Hypersensitivity Granuloma-Bearing Mice with Defective T Cell-Mediated Immune Responses

Hypersensitivity pulmonary granulomas were able to induce by the intratracheal injection of antigen-conjugated Sepharose 4B beads into immunized mice. The mice with hypersensitivity granulomas showed a lack of T cell-mediated immune responses to antigens both in vivo (delayed type footpad reaction) and in vitro (lymph node cell proliferation) in association with impaired interleukin 2 (IL2) production by suppressor cells in lymph nodes. However, no immunosuppressive factors were detected in the granuloma lesions. In the present study, we describe that the sera obtained from these mice contain suppressive factor(s) capable of inhibiting IL 2 production by immune lymph node cells. The appearance of serum suppressor factor(s) is correlated well with inhibition of delayed hypersensitivity in such mice. Partial characterization of suppressor factor(s) showed it is absorbed by T cells and may be proteins. The approximate molecular weight of the factor ranged from 12 to 67 kd. These results suggest that the suppressive mechanisms such as the suppressor cells and serum inhibitory factor(s) may be responsible for the impaired IL 2 production and, therefore, for the lack of both in vivo and in vitro expressions of T cell-mediated immune responses to antigens in mice with hypersensitivity granulomas.

Treatment with Krestin® Combined with Mitomycin C, and Effect on Immune Response

The combination effects of Krestin (PSK) and mitomycin C (MMC) were examined in experimental tumor models. PSK was administered either orally or intraperitoneally. Delayed-type footpad reaction and antibody formation against sheep erythrocytes were measured in hosts of which immune functions were depressed by tumor burden. Results of the experiment indicated that the simultaneous administration of PSK and MMC significantly increased the survival rate of tumor-bearing mice and restored more effectively their immune functions compared to those of nontreated tumor-bearing controls or tumor-bearing hosts treated with a single agent.