T cell regulation of the thymus-independent antibody response to trinitrophenylated-Brucella abortus (TNP-BA)

We have previously observed a reduction of the T cell-dependent primary antibody response to dinitrophenylated keyhole limpet hemocyanin, and an enhancement of the T cell-independent response to trinitrophenylated Brucella abortus (TNP-BA) in BALB/c mice after treatment with total lymphoid irradiation (TLI). To elucidate the relative contribution of T and B cells to the enhanced T cell-independent antibody responses after TLI, a syngeneic primary adoptive transfer system was utilized whereby irradiated hosts were reconstituted with unfractionated spleen cells or a combination of purified T and B cells from TLI-treated and untreated control mice. Antibody responses of purified splenic B cells from TLI-treated BALB/c mice (TLI/B) to TNP-BA were enhanced 10-fold as compared with those of unfractionated (UF) spleen cells or B cells from normal (NL) BALB/c mice (NL/UF and NL/B, respectively). Splenic T cells from normal animals (NL/T) suppressed the anti-TNP-BA response of TLI/B by more than 100-fold. NL/T neither suppressed nor enhanced the response of NL/B. On the other hand, T cells from TLI-treated mice (TLI/T) enhanced by 100-fold the anti-TNP-BA response of NL/B, but neither suppressed nor enhanced the response of TLI/B. Thus, T cells can regulate the "T cell-independent" antibody response to TNP-BA. However, experimental manipulation of the T and B cell populations is needed to demonstrate the regulatory functions.     

Immunosuppressive activity of human seminal plasma. I. Inhibition of in vitro lymphocyte activation.

A high molecular weight fraction prepared from human seminal plasma by gel filtration chromatography suppresses human lymphocyte transformation and DNA synthesis induced by mitogens (PHA, Con A, PWM), antigens (Candida albicans, tetanus toxoid), and allogenic cells. This same fraction also suppresses the stimulated response of mouse lymphocytes to allogenic cells and to various mitogens, including T cell-dependent and T cell-independent mitogens. The induction, but not the expression, of cell-mediated cytotoxicity is also suppressed. Similar high molecular weight fractions suppress the in vitro humoral response of mouse spleen cells to both a T cell-dependent (SRBC) and a T cell-independent (DNP-F) antigen. The high m.w. fraction exhibited in vitro suppressive activity at concentrations of 0.1 to 1.0 mg/ml which corresponds to a 1/50 or greater dilution of human seminal plasma. These observations support the concept that a local immune response against sperm in the female reproductive tract is actively suppressed by a component in seminal plasma.     

Hierarchy of T cell dependency in antibody response among different antigens.

T cell dependency of antibody response to polyvinylpyrrolidone (PVP), sheep red blood cells (SRBC), bovine gamma globulin (BGG), and bovine serum albumin (BSA) was examined. PVP and the other three are known as a T cell-independent antigen and T cell-dependent antigens respectively. Adult mice were thymectomized, X-irradiated, reconstituted with syngeneic bone marrow cells (TxXB mice), with bone marrow cells plus thymus cells (TxXBT mice), or with bone marrow cells treated with anti-Thy-1.2 serum and complement (TxXB-theta mice) and used as experimental animals. The anti-PVP response of TxXBT mice was significantly lower than that of TxXB mice, suggesting that T cells exerted a suppressive effect on the response to PVP. Both IgM and IgG responses to SRBC and BGG occurred even in TxXB-theta mice with the aid of bacterial lipopolysaccharide (LPS). However, a significant response to BSA was not observed in TxXB mice even in the presence of LPS or several other adjuvants. These results indicate that the T cell dependency of antigens is different among so called thymus-dependent antigens, that antibody response less dependent on the helper action of T cells can be supported by LPS in the absence of T cells, and that anti-BSA response seems to be extremely T cell dependent.     

Studies on the immune response to fixed antigens. III. Induction of helper function for antibody-dependent cellular cytotoxicity responses

Heavy trinitrophenylated sheep red cells (TNP128SRC) and glutaraldehyde-treated SRC (G-SRC) could not induce cellular cytotoxicity against 51Cr-SRC. In contrast, the native antigen SRC could stimulate a cytolytic response against the radiolabeled homologous target cell. However, fixed SRC could stimulate a priming function that accelerated and augmented the secondary cytotoxic response to SRC. Such fixed antigens could stimulate a delayed-type hypersensitivity (DTHS) response also. Thus, the immunologic memory to the chemically modified antigen, as well as the DTHS response, are completely dissociated from the primary cytotoxic responses. The primary and the secondary cytotoxic responses that were developed in the spleens of the injected mice were mediated by antibody-dependent cellular cytotoxicity (ADCC), since the active supernatant that was released from the spleen cells could lyse the target cells in the presence of normal splenocytes. The active supernatant was identified as antibody. We suggest that B effector cells cytolyzed the antibody-coated target cells. Normal cells from nude mice could mediate the cytolytic process as efficiently as spleen cells from other strains of mouse. The results are discussed in terms of selective stimulation of T cell subpopulations.     

Cellular basis for a hapten-specific state of tolerance induced in vitro.

A hapten-specific unresponsive state was induced in vitro by the incubation of normal murine spleen cells with highly conjugated dinitrophenylated bovine gamma-globulin (DNP-BGG) or a dinitrophenylated copolymer of D-glutamic acid and D-lysine (DNP-D-GL) for 24 hr. After this incubation period spleen cells were washed and cultured for 4 days with the thymic-independent antigen dinitrophenylated polyacrylamide beads (DNP-PAA) or the thymic-dependent antigen trinitrophenylated burro the erythrocytes (TNP-BRBC). Preincubation with either DNP-BGG or DNP-D-GL led to a specific depression of the in vitro anti-hapten plaque-forming cell response. The degree of depression was dependent upon the concentration of the tolerogen and the duration of preincubation. The response to DNP-PAA or TNP-PAA beads was depressed to a greater degree than was the response to TNP-BRBC. The cellular basis of the immunologic unresponsiveness induced by DNP-BGG was attributable to an inhibition of B cell function whereas the unresponsive state induced with DNP-D-GL was due to both a specific inhibition of B cell function and the activation of antigen-specific suppressor T cells.     

Opposite effects of total lymphoid irradiation on T cell-dependent and T cell-independent antibody responses

The effect of total lymphoid irradiation (TLI) on the primary antibody response to the dinitrophenylated heterologous protein, keyhole limpet hemocyanin (DNP-KLH), in complete Freund's adjuvant (CFA), and to the trinitrophenylated polysaccharide antigen, Brucella abortus (TNP-BA), was studied in BALB/c mice. The antibody response to both antigens was diminished in comparison with nonirradiated mice when antigens were injected within 3 days after TLI. When the mice were immunized 30 days after completion of TLI the antibody response to DNP-KLH in CFA was still diminished, but the antibody response to TNP-BA was enhanced 5- to 10-fold as compared with that of control animals. The opposite effect of TLI on the two antibody responses was also observed in a syngeneic primary adoptive transfer system.     

Effect of Haemophilus influenzae polysaccharide outer membrane protein complex conjugate vaccine on macrophages.

Haemophilus influenzae type b polysaccharide-conjugate vaccines elicit protective antibody responses in young infants. One of these conjugates, polysaccharide linked to outer membrane protein complex (PRP-OMPC), is produced by linking the capsular polysaccharide to an outer membrane protein complex derived from group B Neisseria meningitidis. The outer membrane protein complex contains T cell carrier epitopes that elicit T cell-dependent antibody responses. OMPC also has been shown to increase the antibody response to other proteins administered concurrently that are not covalently linked (i.e., acts as an adjuvant). In this study PRP-OMPC immunized mice demonstrated significant increases in spleen size as well as in splenocyte number as compared to saline controls (p < 0.01, p < 0.001, respectively). No such increase was noted after immunization with another H. influenzae type b-conjugate vaccine, oligosaccharide linked to a variant of diphtheria toxin. By analytic flow cytometry, the mice immunized with PRP-OMPC demonstrated an increase in large splenocytes expressing the Ag Mac-1 (CD11b, CR3). Furthermore, the spleens on histologic examination were characterized by an increase in the red pulp area consisting predominantly of cells of macrophage morphology. By immunohistochemical staining, the cells were identified as macrophages due to expression of Mac-1 and p150,95 (CD11C) Ag. After PRP-OMPC immunization, severe combined immunodeficient mice also demonstrated significant splenomegaly with an increase in macrophages identified by expression of Mac-1 and MHC class II Ag. Thus PRP-OMPC vaccine resulted in T cell-independent splenomegaly with an increase number of macrophages. We propose that this unique property may confer increased immunogenicity to PRP-OMPC through macrophage activation and cytokine release. Furthermore, the effect on macrophages may explain the "adjuvant" capacity of OMPC.     

Macrophages as effector cells of protective immunity in murine schistosomiasis. VI. T cell-dependent, lymphokine-mediated, activation of macrophages in response to Schistosoma mansoni antigens

Macrophages from Schistosoma mansoni-infected mice kill significant numbers of skin stage schistosomula and murine fibrosarcoma cells in vitro. In order to determine whether the macrophage tumoricidal and larvicidal activation observed in mice as a result of S. mansoni infection are mediated through T cell-dependent (lymphokine) or B cell-dependent (antibody or immune complex) mechanisms, the development of macrophage populations with cytotoxic activity against schistosome larvae or tumor cells was monitored in S. mansoni-infected nude or mu-suppressed mice. Whereas peritoneal cells from S. mansoni-infected congenitally athymic mice had no activity in either assay, cells from mu-suppressed S. mansoni-infected mice showed cytotoxic activity equivalent to that of cells from untreated S. mansoni-infected counterparts. Cells from mu-suppressed uninfected mice were not activated. The mu-suppressed animals had no detectable nonspecific IgM or specific antischistosome IgM, IgG, or IgE antibodies and showed a 90% reduction in numbers of splenic IgM+ cells upon fluorescence activated cell sorter analysis. These results indicate that antibody is not required for in vivo activation of macrophages during S. mansoni infection. Further experiments showed that lymphoid cells from S. mansoni infected mice respond in culture with various specific antigens (such as living or dead whole schistosomula or soluble adult worm antigens) by production of factors capable of activating macrophages from uninfected control mice to kill schistosomula or tumor cells in vitro. Macrophage-activating factors were produced by T cell-enriched, but not T cell-depleted or B cell-enriched, populations from spleens of schistosome-infected mice in response to schistosome antigen. Similar lymphokines may be responsible for the macrophage activation observed during chronic murine schistosomiasis. These observations emphasize the potential contribution of T cell-mediated immune mechanisms in resistance to S. mansoni infection.     

Studies on the cellular site of action of macrophage RNA-antigen complexes.

Nonadherent spleen cell populations exposed in vitro to ribonucleic acid-rich preparations from mouse macrophages that had been incubated with human γ-globulin (RNA : HGG) were able to produce specific antibody, as measured by rosette-forming cells, in lethally irradiated (800 R), reconstituted, syngeneic mice. Exposure of the RNA to anti-HGG serum abrogated its ability to initiate antibody synthesis, as did monospecific anti-human γ chain and anti-human κ chain serum. Normal rabbit serum, anti-bovine albumin serum, anti-human μ chain or anti-human λ chain serum, when substituted for anti-HGG serum had no effect. Thus, the presence of both γ heavy chains and κ light chains of the antigen in the RNA moiety was indicated. Although both an adherent and a nonadherent cell were required by HGG to stimulate rosetteforming cells in irradiated mice, the need for the adherent cell was eliminated when RNA : HGG was substituted for HGG. In addition, anti-θ-treated bone marrow cells exposed to the RNA : HGG were capable of rosette-cell formation, suggesting that RNA attachment converted a T cell-dependent antigen to a T cell-independent antigen.     

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice. I. Inhibition of proliferation of lymphocytes in primary antibody responses

The suppressor cells induced by Toxoplasma infection were shown to be macrophages, since they adhered to plastic, and their suppressive activity in anti-sheep erythrocytes (SRBC) antibody responses was abrogated by treatment with silica or carrageenan, which are selectively cytotoxic for macrophages. The suppressor macrophages strongly inhibited the uptake of tritiated thymidine ( [3H]TdR) by normal mouse spleen cells in the responses to SRBC and Toxoplasma antigens. Supernatant fluids from the suppressor macrophages could not passively transfer the suppressive effect on anti-SRBC antibody responses. Furthermore, when the suppressor macrophages were isolated by a cell-impermeable membrane from normal mouse spleen cells, the antibody responses of normal spleen cells were not suppressed. These results indicate that suppression of antibody responses in Toxoplasma-infected mice is caused by an inhibitory effect of the suppressor macrophages upon proliferation of lymphocytes via direct contact with responder target cells. The suppressive effect of the macrophages was not counteracted by indomethacin, a potent inhibitor of prostaglandin synthesis, or catalase, a catabolic enzyme for hydrogen peroxide (H2O2).     

Lectin-induced regulation of human thymus-independent anti-TNP responses in vitro

Recently we showed that human hapten-specific T cell-independent (TI) antibody responses could be elicited in vitro with the antigen trinitrophenylated Brucella abortus (TNP-Ba). Although by definition T cells are not required for TI responses, they have also shown capable of regulating such responses in the mouse. In this study we examine the ability of the T cell lectins Con A and PHA to modulate TI responses of human tonsil cells. Addition of the lectins to cultures on day 0 or day 3 resulted in inhibition (greater than 90%) or enhancement (greater than 150%) of the anti-TNP PFC response, respectively. The inhibition was only apparent if the lectins were added at the onset of culture but not 24 hr later, and was T cell-dependent, because T cell-depleted cultures (less than 0.5% E-rosetting cells) could not be inhibited. The enhancement observed was even greater in T cell-depleted cultures and was antigen-specific because TNP-SRBC but not PC-SRBC targets were lysed and the PFC were inhibited by soluble TNP (greater than 70%). This finding suggested that anti-TNP PFC precursors were proliferating more vigorously or that additional, previously antigen-insensitive cells were being recruited in the presence of lectin.     

A novel role for macrophage: antigen discrimination of distinct carbohydrate bonds.

Velocity sedimentation-derived subpopulations of peritoneal exudate macrophages have been pulsed with a variety of TNP-coupled carbohydrate/protein antigens and investigated for their ability to present those antigens in immunogenic form to whole spleen cell populations. The data presented indicate that while no difference is seen in the ability of the various subpopulations examined to present protein antigens for an antibody response, different cells are certainly involved in the antigen handling of carbohydrate antigens for an antibody response. Moreover, it seems that individual macrophage subpopulations can only interact with certain subpopulations of B lymphocytes to induce antibody responses to the same immunogenic determinants.     

Streptococcal serotype carbohydrate represents a novel class of type 2 antigen which is T-independent

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyclonal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb-3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.     

Nature of T lymphocyte recognition of macrophage-associated antigens. IV. Inhibition of peptide antigen presentation by brief treatment of macrophages with anti-Ia serum

To examine the role of macrophage la antigens in T-lymphocyte stimulation, guinea pig macrophages were briefly treated with anti-Ia serum before or after antigen pulsing with the peptide antigen human fibrinopeptide B (hFPB). To assess their antigen-specific stimulatory capacity, the variously treated macrophages were added to culture with hFPB-immune guinea pig T cells and stimulation was determined by the incorporation of [³H]thymidine. Macrophages treated with anti-Ia serum before antigen pulsing stimulated T-cell responses equivalent to those observed with antigen-pulsed macrophages treated with normal serum. By contrast, brief anti-Ia treatment of macrophages immediately following a 2-hr antigen pulse reduced subsequent T-cell responses by 45 to 70%. Similar treatment of macrophages pulsed with antigen for only 1 hr produced only modest inhibition of T-cell responses. However, if macrophages pulsed for 1 hr with antigen were incubated several hours before brief anti-Ia serum treatment, the subsequent T-cell responses were reduced by 40 to 60%. This inhibition was specific for antiserum directed against Ia antigens of the guinea pig MHC, since brief macrophage treatment with antiserum directed against B.1 antigens, the guinea pig equivalent of murine H-2K and H-2D antigens, produced no inhibition of their T-cell stimulatory capacity. These results are discussed with respect to the formation of the immunogen presented by macrophages for T-cell recognition.     

Analysis of the antigen- and mitogen-induced differentiation of B lymphocytes from asymptomatic human immunodeficiency virus-seropositive male homosexuals. Discrepancy between T cell-dependent and T cell-independent activation

Five asymptomatic human immunodeficiency virus (HIV)-seropositive male homosexuals were immunized with the recall antigens tetanus toxoid (TT) and the three types of poliovirus present in diphtheria, tetanus, and polio vaccine. Four weeks after immunization, the in vivo response to booster immunization, the in vitro pokeweed mitogen (PWM)-induced IgG secretion, and the in vitro T cell-dependent and T cell-independent antigen-induced antibody response were assayed. Increase in serum antibody titer to TT and poliovirus was low and normal, respectively. In all five subjects studied, a high rate of spontaneous IgG production, including antibodies directed toward HIV was observed. Addition of PWM to the cultures induced suppression of the spontaneous IgG secretion. Only one donor showed a slightly increased IgG production after stimulation with PWM. Peripheral blood mononuclear cells of four of the five HIV-seropositive individuals did not produce TT, or poliovirus-specific antibodies when stimulated with the respective T cell-dependent antigens. However, stimulation of these peripheral blood mononuclear cells with TT coupled to agarose beads, which was shown to be T cell-independent, resulted in the generation of IgG anti-TT antibody-forming cells.     

Enhancement of in vivo immune response by tumor necrosis factor

Interleukin 1 (IL-1) has been shown to regulate several immunologic functions. Since tumor necrosis factor (TNF) shares many biologic properties with IL-1, we have investigated here the role of TNF in the modulation of the immune response. We have thus tested low doses of human recombinant TNF-alpha (hu rTNF-alpha) for its capacity to enhance the in vivo antibody responses evaluated at the cellular level in the hemolytic plaque assay. It was found that hu rTNF-alpha, like human IL-1 beta, is able to enhance the immune response to a T cell-dependent antigen (sheep red blood cells). Interestingly, at variance with human recombinant IL-1 beta, hu rTNF-alpha was not able to enhance the in vivo antibody response to a T cell-independent antigen (type III pneumococcal polysaccharide). These results suggest that low levels of TNF may have a role in the modulation of the immune response in vivo and shed new light on the biologic significance of this mediator.     

Mouse splenic macrophage cell lines with different antigen-presenting activities for CD4+ helper T cell subsets and allogeneic CD8+ T cells.

A panel of seven mouse splenic macrophage cell lines, derived from cloned progenitors, was compared for their ability to present antigen to Th1 or Th2 helper T cell lines and hybridomas, as well as to naive T cells, and to provide accessory cell function for the synthesis of antibody from primed B cells. One of the cell lines expressed MHC class II molecules and was the only line with constitutive antigen-presenting activity for Th1 cells. It may represent a subset of splenic macrophages responsible for the activation of naive Th1 helper cells in situ. The remaining six cell lines responded to INF-gamma by up-regulating their class II expression and acquiring Th1 antigen-presenting activity. They may represent cells which, in situ, lack constitutive antigen-presenting activity but are promoted to presenting status by Th1-derived INF-gamma. Five of the cell lines provided accessory cell function to Th2 cells, as indicated by antibody synthesis in suspensions of spleen cells from primed mice depleted of their antigen-presenting cells. One of the cell lines lacking accessory cell activity had constitutive antigen-presenting activity for Th1 cells. This reciprocal expression of antigen-presenting activity supports the idea that Th1 and Th2 helper cells are activated by different antigen-presenting cells. Finally, the cell lines differed in their ability to constitutively induce an allogeneic response; a response that was limited to CD8+ T cells occurred in a CD4+ helper cell-independent manner and was unaffected by the addition of INF-gamma. The alloantigen-presenting macrophage cell lines also possessed the most efficient accessory cell activity for antibody synthesis. These cell lines, which represent a spectrum of antigen-presenting activities in the spleen afford models for defining the roles of macrophages in the induction of immune responses and for resolving issues concerning their development.     

Ability of chicken B cells from different compartments to respond to TNP-Ficoll

The responsiveness of chicken B cells from various compartments to T-independent antigens was studied by immune transfers of spleen and bursa cells into immunosuppressed recipients. Bursa cells from 8- to 10-wk-old donors failed to respond to trinitrophenylated Ficoll (TNP-F) even when thymus cells or splenic T cells were added. Spleen cells from the same donors transferred responses, as judged both by anti-TNP plaque-forming cells (PFC) per spleen and serum anti-TNP titers. In contrast, responses to TNP-Brucella abortus (TNP-BA) were transferred at least as well as by bursa as by spleen cells. Rabbit anti-chicken T cell serum plus complement treatment of the spleen cells reduced their ability to transfer responses to sheep erythrocytes, but either did not affect or enhanced serum antibody responses to TNP-BA and TNP-F. In intact animals, responsiveness to i.v. injected TNP-F was found to develop slowly after hatching in the chicken. At the age of 2 and 3 mo, PFC/spleen on day 4 after TNP-F injection were only 20% and 40%, respectively, of the adult response. Thymectomy at hatching further delayed this development, resulting in 12% and 45% of the adult control response at ages of 3 and 4 mo. It is concluded that responsiveness to the TI-2 antigen, TNP-F, develops slower than that to the TI-1 antigen, TNP-BA, and is restricted to the splenic B cell compartment. In addition, this development appears to be faster in the presence rather than in the absence of the thymus. In view of the previously shown effect of thymus on bursa development, these data suggest that the maturation of TI-1 antigen (TNP-F)-respondent chicken B cells requires residence in both the bursa and spleen before the development of responsiveness to such antigens.     

The suppressive effect of immunization on the proliferative responses of rat T cells in vitro. II. Abrogation of antigen-induced suppression by selective cytotoxic agents.

Rats given large i.v. doses of ovalbumin or sheep erythrocytes manifest suppressed spleen cell responses (3H-thymidine incorporation) to PHA within hours. Removal of glass wool-adherent cells totally restores responsiveness to that of normal nonadherent spleen cell cultures. Carrageenan, selectively toxic for macrophages, partially restores responses of antigen-suppressed spleen cells in culture, suggesting a supportive role for macrophages in the suppression phenomenon. Treatment of donors with low doses of cyclophosphamide (20 to 50 mg/kg) at the time of antigen injection abrogates the ability of their spleen cells to suppress the responses of normal cells to PHA. The low dose of cyclophosphamide required indicates a target other than the B cell or macrophage and suggests the possibility that cyclophosphamide eliminates the suppressor T cell component of the macrophage-T cell complex.     

Immunotoxicity of phosphamidon following subchronic exposure in albino rats

Effect of subchronic doses of phosphamidon exposure on humoral and cell mediated immune (CMI) responses were studied in male albino rats using SRBC, ovalbumin and KLH as antigens. Humoral immune responses were assessed by estimating antibody titre against antigen and splenic plaque forming cells (PFC) assay. CMI responses were studied by using leucocyte migration inhibition (LMI), macrophage migration inhibition (MMI) and delayed type hypersensitivity (DTH) response. Results obtained in the present study revealed marked suppression of humoral and CMI responses in a dose dependent pattern. Hence, suppression of immune responses by phosphamidon even at subchronic doses is clearly an important aspect for its safety evaluation.