Immunotoxicity of N,N-diethylaniline in mice: effect on natural killer activity, cytotoxic T lymphocyte activity, lymphocyte proliferation response and cellular components of the spleen

We previously found that N,N-diethylaniline increased the frequency of sister chromatid exchange (SCE) of human lymphocytes to about five times that of the control value, and was as toxic as cyclophosphamide used as a positive control for SCE. To explore whether N,N-diethylaniline affects the function of lymphocytes, we evaluated its immunotoxicity using CBA/N mice. The mice were divided into four groups and received 0, 100, 200, or 400 mg/kg body weight of N,N-diethylaniline by subcutaneous injection. The following items were investigated on days 3 and 7 after injection: body weight, weight of spleen, number of splenocytes, natural killer (NK) and cytotoxic T lymphocyte (CTL) activities, and concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated lymphocyte proliferation using splenocytes. The following splenocyte phenotypes were also quantified by flow cytometry: (1) B cells; (2) total T cells; (3) CD4⁺ and CD8⁺ T cells; (4) NK; (5) macrophages and (6) nucleated erythrocytes. The splenic NK and CTL activities in exposed groups significantly decreased compared to the control in a dose-dependent manner and lymphocytes from the 200 and 400 mg/kg groups showed significantly higher spontaneous proliferation. The weight of the spleen and number of splenocytes were significantly higher in exposed groups than in the control. N,N-Diethylaniline also increased the percentages of macrophages, nucleated erythrocytes and B cells in the spleen. On the other hand, N,N-diethylaniline did not affect LPS-stimulated B cell and Con A-stimulated T cell proliferation, or the percentages of NK, total T, and CD4⁺ and CD8⁺ T cells in the spleen or the body weight of mice. The above findings indicated that N,N-diethylaniline selectively inhibited splenic NK and CTL activity and this inhibition was due to decreased NK and CTL functions, but not due to changes in the numbers of splenic NK and T cells.     

3-Methylindole-induced splenotoxicity: biochemical mechanisms of cytotoxicity.

3-Methylindole (3-MI) is a pneumotoxic metabolite of L-tryptophan that can form in the digestive tracts of humans and ruminants as a result of microbial protein metabolism. Alternatively, human lungs can be directly exposed to 3-MI formed during protein pyrolysis and inhalation of tobacco smoke. 3-MI has been shown to cause acute lung injury in both ruminants and rodents. The present studies demonstrate that the spleen is also a target for 3-MI-induced toxicity. A dose-dependent decrease in splenic weight (24-75%) and nucleated splenic cell number (22-68%) was observed 24 hr after intraperitoneal injection of 3-MI (50-300 mg/kg) to intact and adrenalectomized rats. These findings were associated with significant alterations in splenic histopathology. Mice appeared less affected by 3-MI than rats as no splenotoxicity was observed at doses less than 200 mg/kg. Other mono- and dimethyl-substituted indoles did not decrease mouse spleen cell numbers when administered in vivo. Phenobarbital pretreatment in vivo protected against 3-MI-induced splenotoxicity, suggesting a role for cytochrome P450-mediated metabolism of 3-MI in the splenotoxicity of this compound. Exposure of rat or mouse splenic cells to 3-MI (1 mM) in vitro resulted in toxic changes over 24 hr. However, equimolar concentrations of the structurally related mono- and dimethylindoles were also toxic in vitro, and preincubation with a variety of inhibitors of cytochrome P450 or prostaglandin synthase in vitro failed to protect against 3-MI-mediated toxicity to splenic cells in culture. These results suggest mechanisms of 3-MI splenotoxicity also exist that do not require bioactivation, and indicate a possible role for alkylindoles in suppression of immune function.     

Acute immunotoxicity of p-chloronitrobenzene in mice: II. Effect of p-chloronitrobenzene on the immunophenotype of murine splenocytes determined by flow cytometry.

To evaluate the immunotoxicity of p-chloronitrobenzene (p-CNB), we investigated its effect on the immunophenotype of murine splenocytes. BDF1 male mice were randomly divided into exposed and control groups: the exposed group received p-CNB at 300 mg/kg dissolved in olive oil, while the control group received only olive oil, by a single intraperitoneal (i.p.) or subcutaneous (s.c.) injection. On days 3, 5, 7, and 10 after the injection, splenocytes were harvested from both groups, and the following cell phenotypes were quantified by flow cytometry: (1) B cells (CD45R/B220); (2) T cells (CD3e); (3) T-cell subsets (CD4 and CD8a); (4) natural killer (NK) cells (NK-1.1); (5) macrophages (CD11b; Mac-1); (6) nucleated erythrocytes (Ter-119); and (7) dead cells with propidium iodide (PI). The percentages and numbers of B, T, subsets of T (CD4 and CD8), and NK cells in the exposed mice significantly decreased as compared with the respective control. On the other hand, macrophages (Mac-1+ cells), nucleated erythrocytes (Ter-119+ cells), and dead cells in the exposed mice markedly increased as compared with the respective control after i.p. injection of p-CNB. The above findings indicate that p-CNB has an immunotoxic effect on mice.     

Splenic cell targets in gallium arsenide-induced suppression of the primary antibody response

In vivo exposure of female B6C3F1 mice to gallium arsenide (GaAs) was evaluated for its effect on the in vitro IgM antibody-forming cell (AFC) response. In vivo exposure to a single intratracheal dose of GaAs (2.5-200 mg/kg) resulted in a dose-dependent decrease in the in vitro IgM AFC response to the T-dependent antigen sheep red blood cells (SRBC) with a 97% decrease at 200 mg/kg when compared to vehicle controls. The response to the T-independent antigen DNP-Ficoll was significantly reduced at 100 and 200 mg/kg. Spleen cellularity decreased in a dose-related manner with a 54% decrease at 200 mg/kg. Enumeration of splenic subpopulations following GaAs (200 mg/kg) indicated a 58, 61, and 30% decrease in the total number of Thy 1.2 (T cells), Ig (B cells), and F4/80 (macrophages) positive cells, respectively, with no alterations in the percentages of these cells. Mitogenic responsiveness of splenocytes from GaAs-exposed mice was unaltered. To identify the splenic cell populations targeted by GaAs, the AFC response to SRBC was evaluated following cell separation/reconstitution of splenocytes from GaAs- (200 mg/kg, 24-hr exposure) and vehicle-exposed mice. Results demonstrated AFC suppression was due to functional alterations in both adherent (AD; macrophages) and nonadherent, (both T and B lymphocytes) cell populations. Further investigation focused on alterations in the AD population. Separation/reconstitution experiments demonstrated AFC suppression to SRBC was dependent on the concentration of macrophages from GaAs-exposed mice. This macrophage-mediated suppression of the in vitro AFC response could not be attributed to the presence of suppressor macrophages or release of prostaglandins.     

Tumor site-dependent differential modulation of systemic immunity in RENCA bearing mice

Renal cell carcinoma (RCC) is a highly metastatic cancer which is known to be immunogenic and responsive to immunotherapies using cytokines. The effect of tumor site (organ microenvironment) on the biologic behavior of murine RCC cell line (RENCA) was examined by observing systemic immunomodulations. Reduction of the spleen size observed in tumor bearing mice correlated with a significant decrease in splenic nucleated cell numbers. In the mice bearing RENCA cell tumors in the renal subcapsule, a significant increase of splenic T cell blastogenesis against Con-A (234% of naive control) was observed with reduction of NK activity (45% of naive control). The changes in T cell blastogenesis and NK activity observed in subcutis (s.c.) inoculated RENCA tumor bearers were less significant. However, macrophage functioning and proportions in the spleen were significantly increased only in s.c. tumor bearers. Data indicated tumor site-dependent differential modulation of systemic immunity in RENCA bearing mice which suggests that immunotherapy for RCC should consider not only the tumor but also the tumor's microenvironment.     

Modulation of immune response following dietary genistein exposure in F0 and F1 generations of C57BL/6 mice: evidence of thymic regulation.

To further determine whether genistein (GEN) modulation of the immune responses was related to its endocrine-disrupting properties and time of exposure, pregnant C57BL/6 mice were exposed to GEN at 0-1250 ppm in feed starting on day 14 of gestation. The C57BL/6 offspring were exposed to GEN in utero and lactationally, and through feed after weaning until postnatal day 42. In dams, exposure to GEN increased the terminal body weight (250 and 1250 ppm), the number of splenic T cells and NK cells (250 ppm), and the activity of NK cells (250 ppm). In F(1) males, GEN increased the terminal body and spleen weights (25 and 250 ppm), the number of CD4(+)CD8(+) and CD4(-)CD8(+) thymocytes (25 ppm), and the number of splenic T cell subsets and NK cells (25 and 250 ppm). Moreover, splenic NK cell activity and anti-CD3-mediated splenocyte proliferation were increased in all treatment groups. In F(1) females, the percentages of CD4(-)CD8(+) and CD4(-)CD8(-) thymocytes (25 and 250 ppm), and CD4(+)CD8(-) and CD4(+)CD8(+) splenocytes (25 and 250 ppm) were increased. In contrast, the percentage and number of CD4(+)CD8(+) thymocytes were decreased (250 ppm). Exposure to GEN decreased the percentages of splenic NK cells in all treatment groups, and decreased the activity of splenic NK cells at the 25 ppm concentration. Additionally, evaluation of CD25(+) and CD44(+) expression by thymocytes indicated that the decrease in the percentage of CD44(+)CD25(+) thymocytes was at least partially responsible for the decrease in the percentage of CD4(-)CD8(-) thymocytes in F(1) male mice. Overall, the results demonstrate that GEN can modulate the immune system in both adult and developing C57BL/6 mice in a dose-specific manner. The gender-specific effects of GEN on the immune responses in F(1) mice suggest that GEN may modulate the immune system by functioning as either an estrogen agonist or antagonist. The differential effects of GEN on thymocytes in F(1) male and female mice indicate that GEN immunomodulation might be related to its effect on thymus.     

绞股蓝总皂甙对Lewis肺癌荷瘤小鼠肿瘤生长及免疫功能的影响

观察绞股蓝总皂甙对Ｌｅｗｉｓ肺癌荷瘤小鼠肿瘤生长,脾淋巴细胞数及ＮＫ活性的影响。方法整体动物的抑瘤试验,脾淋巴细胞计数及ＮＫ活性测定。结果：ＧＰｓ对荷瘤小鼠Ｌｅｗｉｓ肺癌细胞具有明显的抑制作用,在剂量１０、２０、４０ｍｇ／ｋｇ腹腔注射给药条件下,其抑瘤率分别为（３０．７±１．２）％,（５１．５±２．５）％（Ｐ〈０．０１）。同时,ＧＰｓｉｐ给药后荷瘤小鼠脾淋巴细胞总数明显增加,外周血淋巴细胞ＮＫ活性     

Immunomodulatory activity of orphan drug Elmiron® in female B6C3F1/N mice

Interstitial cystitis (IC) is a chronic disorder characterized by bladder discomfort and urinary urgency in the absence of identifiable infection. Despite the expanding use in IC treatment and other chronic conditions, the effects of Elmiron® treatment on immune system remain unknown. Therefore, female B6C3F1/N mice were orally administered Elmiron® daily for 28-days at doses of 63, 125, 250, 500 or 1000 mg/kg to evaluate its immunomodulatory effects. Mice treated with Elmiron® had a significant increase in absolute numbers of splenic macrophages (63, 500 and 1000 mg/kg) and natural killer (NK) cells (250 and 1000 mg/kg). Elmiron® treatment did not affect the humoral immune response or T cell proliferative response. However, innate immune responses such as phagocytosis by liver macrophages (1000 mg/kg) and NK cell activity were enhanced (500 and 1000 mg/kg). Further analysis using a disease resistance model showed that Elmiron®-treated mice demonstrated significantly increased anti-tumor activity against B16F10 melanoma cells at the 500 and 1000 mg/kg doses. Collectively, we conclude that Elmiron® administration stimulates the immune system, increasing numbers of specific cell populations and enhancing macrophage phagocytosis and NK cell activity in female B6C3F1/N mice. This augmentation may have largely contributed to the reduced number of B16F10 melanoma tumors.     

Immunotoxicity of nivalenol after subchronic dietary exposure to rats.

Immunobiological effects of nivalenol (NIV), a trichothecene mycotoxin produced by Fusarium nivale, were examined in male F344 rats after 90-day dietary exposure at doses of 0, 0.4, 1.5, and 6.9 mg/kg body weight/day (0, 6.25, 25 and 100 ppm, respectively) in a subchronic toxicity study. With regards to the serum immunoglobulin levels, a slight increase of IgM was observed only at 6.9 mg/kg (26% increase), while levels of IgG and IgA did not fluctuate at any dose. Flow cytometric analysis of splenic cells revealed a dose-dependent decrease of T lymphocyte/B lymphocyte (CD3(+)/B220(+)) ratio from 1.5mg/kg and an elevated CD4(+)helper/CD8(+)cytotoxic T lymphocyte ratio at 6.9 mg/kg. Furthermore, increases of natural killer (NK) activity of splenic lymphocytes against YAC-1 target cells were observed at all doses, while the magnitude of changes was similar between 1.5 and 6.9 mg/kg. At 6.9 mg/kg, the reduction of the ratio of NKR-P1A(+) splenic cells, which is an indicator of NK cells in the spleen, was apparent. As with other previous studies of NIV, decreased body weight was observed from 1.5 mg/kg during the experiment in the present study. In summary, NIV affected immune function in rats after 90-day dietary exposure, the effects being apparent from 0.4 mg/kg judging from the increase of NK activity, although nutritional suppression might have influenced the immunological changes appeared from 1.5mg/kg.     

Genistein modulates splenic natural killer cell activity,antibody-forming cell response,and phenotypic marker expression in F(0) and F(1) generations of Sprague-Dawley rats

The potential effects of the phytoestrogen genistein (GEN) on the immune system were evaluated in both F(0) (dams) and F(1) generations of Sprague-Dawley rats exposed to a soy-free diet containing low (L: 25 ppm), middle (M: 250 ppm), and high (H: 1250 ppm) levels of GEN. In dams, exposure to GEN from Gestation Day 7 to Postpartum Day 51 (totally 65 days) produced a significant increase in NK cell activity (M and H), while a decrease in the percentage of helper T cells (H). In F(1) males, exposure to GEN gestationally, lactationally, and through feed from Postnatal Days 22 to 64 (total 78 days) produced an increase in the relative weights (% body) of spleen (L and H) and thymus (L). Furthermore, exposure to GEN increased the number of splenic B cells (H), T cells (L, M, and H), and T-cell subsets (L, M, and H). Although GEN decreased the percentages of splenic NK cells (L, M, and H), no effect on the activity of NK cells was observed. In F(1) females, exposure to GEN produced a decrease in terminal body weight (H), with an increase in the relative weight of spleen (L, M, and H). Exposure to GEN also increased the number of splenic B cells (L), macrophages (L and M), T cells (H), helper T cells (L and H), and cytotoxic T cells (M and H). Additionally, exposure to GEN increased the percentages of T cells (M and H), helper T cells (H), and cytotoxic T cells (M and H). Moreover, the spleen IgM antibody-forming cell response to sheep red blood cells was enhanced (H), although the percentages of B cells were decreased (M and H). No effect on the activity of NK cells was observed; however, the percentages of splenic NK cells were decreased by GEN (L and H). In conclusion, these results demonstrate that exposure to GEN can modulate the immune responses in Sprague-Dawley rats. Furthermore, the sexual dimorphic effects of GEN in F(1) male and female rats suggest that there may be interactions between GEN and the responses modulated by sex hormones.     

Murine retroviral disease-enhancing effects of a pyrimidinone immunomodulator.

(B10.A x A/WySn)F1 mice, infected with the Friend virus (FV) complex, were used as a predictive therapeutic model for AIDS. These infected mice exhibit many of the viral and immunologic manifestations of AIDS. Bropirimine (2-amino-5-bromo-6-phenyl-4[3H]pyrimidinone, ABPP) is an immunomodulating compound which has been shown to inhibit other viral infections. Oral (per os treatment) dosages of ABPP ranging from 50 to 400 mg/kg/day for 3 days resulted in increased numbers of infectious centers in the infected mice and increased splenomegaly and percentage of Ig+ (B cells) in spleens of infected and uninfected mice. Decreased percentages of total Thy-1.2+ (total T) cells and L3T4+ (T-helper) cells were seen in both uninfected and infected mice and a slightly decreased percentage of Ly-2+ (T-suppressor/cytotoxic) cells was observed in spleens of the infected mice. No effect on Ly2+ cells in spleens of uninfected mice was found. Intraperitoneal injection, single or multiple, of 20-200 mg/kg ABPP prior to FV injection resulted in increased spleen weights but had no effect on numbers of infectious centers in the spleens or on FV antibody titers in the plasma. Intraperitoneal treatment of uninfected mice with ABPP resulted in slight or no changes in percentages of Thy-1.2+, L3T4+ and Ly-2+ cells. Mice receiving multiple exposures of ABPP had an increase in percentage of splenic B cells and a depressed response to the T cell mitogen PHA. Treatment with ABPP induced the production of interferon (IFN); however, a state of hyporesponsive IFN production was seen following multiple administrations of ABPP. These data suggest that the immunomodulator ABPP may have an enhancing effect on this retroviral disease.     

Comparative immunotoxicity evaluation of amphotericin B and ABELCET, an amphotericin B lipid complex (ABLC)

ABELCET, an amphotericin B lipid complex formulation (ABLC) and an aqueous, non-lipid-containing formulation with sodium deoxycholate (AmBd), were evaluated for their potential to induce immunotoxicity in B6C3F1 female mice. ABLC was administered intravenously at doses of 1, 3, and 10 mg/kg daily for 28 days, while AmBd at 1 mg/kg was administered by the same route and duration. The effect of ABLC and AmBd on clinical signs, body weight, and spleen weight was determined. Peritoneal macrophage function was measured by phagocytosis of 51Cr-labeled chicken red blood cells and generation of hydrogen peroxide during respiratory burst. The ability of natural killer cells to lyse radiolabeled tumor target cells was evaluated in a short-term chromium-release assay. The ability of splenic T and B cells to undergo blastogenesis and of splenic T cells to recognize alloantigens present on foreign cells was assessed in a splenic lymphocyte assay and the ability of mice to generate antibody-forming cells following immunization with sheep red blood cells was measured. Neither ABLC nor AmBd affected the metabolic or functional activity of murine phagocytic cells. These agents also did not cause any biologically significant or dose-related changes in B- or T-cell responses to mitogens, T-cell responses to allogeneic cells in the mixed lymphocyte culture assay, or natural killer cell function. The ability to generate a primary antibody response to a T cell-dependent antigen was also unimpaired. Based on the results of this study, it was concluded that neither ABLC at dose up to 10 mg/kg nor AmBd at dose up to 1.0 mg/kg produce biologically significant immunologic changes in B6C3F1 mice.     

Methamphetamine administration produces immunomodulation in mice

The abuse of methamphetamine (MA) is an increasingly growing problem globally and produces serious side effects. In the present study, the immunomodulating effects of MA were examined on the immune system after MA (5 mg/kg body weight) was administered daily orally for 14 d. The immune system was evaluated by the antibody response to sheep red blood cells (SRBC; plaque assay and serum immunoglobulin [Ig] G), natural killer (NK) activity, lymphocyte subpopulations in the spleen and thymus, and concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated lymphocyte proliferation using splenocytes. Body weight, spleen weight, and thymus weight generally decreased in MA-treated mice. MA treatment induced an increase in the percentage of CD4(+) cells with simultaneous decrease in the percentages of CD8(+) and double-positive CD4(+)CD8(+) in thymus. MA inhibited the IgM plaque-forming cell number, and lowered the level of IgG, the proliferation of mitogen-stimulated B and T cells, and the growth of granulocyte-macrophage colony-forming units (CFU-GM). Exposure to MA also decreased interleukin-2 production by splenocytes. In contrast, splenic NK activity in exposed mice was significantly enhanced. Taken together, data indicate that the immune system was suppressed by oral MA exposure.     

Respiratory exposure to diesel exhaust particles decreases the spleen IgM response to a T cell-dependent antigen in female B6C3F1 mice.

We investigated the systemic immunotoxic potential of respiratory exposure to diesel exhaust particles (DEP) in this study. Female B6C3F1 mice (approximately 8 weeks old) were exposed to increasing concentrations of DEP intratracheally, 3 times every two weeks, and sacrificed 2 or 4 weeks after the first exposure. The systemic toxicity and immune status in mice were evaluated. Mice exposed to DEP (1 to 15 mg/kg) showed no significant changes in body, spleen, or liver weights. Lung weights were increased in the mice exposed to 15 mg/kg DEP for 2 or 4 weeks. Except for a decreased platelet count, no significant alterations occurred in hematological parameters following DEP exposure. The number of splenic anti-sheep red blood cell (sRBC) IgM antibody-forming cells (AFC) decreased following DEP exposure for 2 weeks. This effect was less severe following 4 weeks of exposure and was only evident in the high dose group. Exposure to DEP also resulted in a significant decrease in the absolute numbers and the percentages of total spleen cells for total, CD4(+), and CD8(+) T cells, while the numbers of B cells and total nucleated cells in spleen were not significantly changed. The proliferative response of splenocytes to the T-cell mitogen, concanavalin A (ConA), as well as their production of IL-2 and IFN-gamma, was decreased dose-dependently following exposure of mice to DEP for 2 weeks, whereas proliferation was not changed in response to anti-CD3 monoclonal antibody. In summary, short-term respiratory exposure of mice to DEP resulted in systemic immunosuppression with evidence of T cell-mediated and possibly macrophage-mediated mechanisms.     

Effects of subchronic inhalation exposure to ethyl tertiary butyl ether on splenocytes in mice

Ethyl tertiary-butyl ether (ETBE) is a motor fuel oxygenate used in reformulated gasoline. The current use of ETBE in gasoline or petrol is modest but increasing. To investigate the effects of ETBE on splenocytes, mice were exposed to 0 (control), 500 ppm, 1750 ppm, or 5000 ppm of ETBE by inhalation for 6 h/day for 5 days/wk over a 6- or 13-week period. Splenocytes were harvested from the control and exposed mice, and the following cell phenotypes were quantified by flow cytometry: (1) B cells (PerCP-Cy5.5-CD45R/B220), (2) T cells (PerCP-Cy5-CD3e), (3) T cell subsets (FITC-CD4 and PE-CD8a), (4) natural killer (NK) cells (PE-NK1.1), and (5) macrophages (FITC-CD11b). Body weight and the weight of the spleen were also examined. ETBE-exposure did not affect the weight of the spleen or body weight, while it transiently increased the number of RBC and the Hb concentration. The numbers of splenic CD3+, CD4+, and CD8+ T cells, the percentage of CD4+ T cells and the CD4+/CD8+ T cell ratio in the ETBE-exposed groups were significantly decreased in a dose-dependent manner. However, ETBE exposure did not affect the numbers of splenic NK cells, B cells, or macrophages or the total number of splenocytes. The above findings indicate that ETBE selectively affects the number of splenic T cells in mice.     

Alleviation of cyclosporin-induced immune suppression by the cytokine inducer ADA-202-718

ADA-202-718 (ADA) has been shown to augment the production of various cytokines (IL-1, IL-2 and gamma-interferon) by murine T lymphocytes. Administration of 1 mg/kg/day to mice from the time of immunization significantly enhanced delayed-type hypersensitivity responses to sheep red blood cells (SRBC). ADA also inhibited immune suppression in mice given 25 but not 50 mg/kg/day cyclosporin A (CsA). There were, however, few changes compared with vehicle-treated controls in the numbers of splenic regulatory T cell subsets (L3T4+ and Ly2+) following treatment with CsA, ADA or both drugs. On the other hand, splenic lymphocytes from ADA-treated animals exhibited augmented proliferative responses to polyclonal T and B cell mitogens and antigen. ADA (1 mg/kg) also stimulated the splenic IgM plaque-forming cell response to SRBC and prevented CsA (25 mg/kg)-induced suppression of humoral immunity. In vitro, ADA (2 micrograms/ml) inhibited CsA-induced suppression of T cell proliferation, the effect being most marked at lower inhibitory concentrations of CsA. These data illustrate the capacity of ADA to augment or restore T cell responses in experimental animals and are consistent with the view that CsA acts in these experimental in vivo systems by inhibition of cytokine production.     

Ganoderma tsugae mycelium enhances splenic natural killer cell activity and serum interferon production in mice.

Effects of the water-soluble extract of Ganoderma tsugae mycelium (GT), its alcohol-insoluble subfraction (GTI), and its alcohol-soluble subfraction (GTS) on splenic natural killer (NK) cell activity and serum interferon (IFN) production were assessed in mice. Intraperitoneal administration of GT (4-200 mg/kg) or GTI (1-50 mg/kg), but not GTS, augmented the NK cytotoxic activity in a dose-dependent manner in C3H/HeN mice. This augmentation of splenic NK cytolytic activity was not mouse-strain-dependent. The serum IFN titers of mice were also elevated after i.p.-doses of GTI. The GTI-induced serum IFN was reduced by either IFN-(alpha+beta) antiserum or IFN-gamma monoclonal antibody in vitro. The treatment with antiserum neutralizing IFN-(alpha+beta) resulted in a 70% reduction of GTI-induced IFN, while monoclonal antibody against mouse IFN-gamma, moderately neutralized the GTI-induced IFN (50%). These results demonstrated that both the splenic NK activity and serum IFN [IFN-(alpha+beta) and IFN-gamma] titers are elevated by Ganoderma tsugae mycelium extracts in mice.     

Enhancement of natural killer cell function by titanocenes in mice bearing Ehrlich ascites tumour

In the present work, we studied the effects of two titanocenes, biscyclopentadienyldichlorotitanium IV, (DDCT) and its derivative, biscyclopentadienylditiocianatetitanium IV (BCDT), on the activity of natural killer (NK) cells in Ehrlich ascites tumour (EAT)-bearing BALB/c mice. In order to investigate a more direct effect of these compounds on NK cell function, we performed experiments with severe combined immunodeficiency (SCID) mice, which exhibit a normal NK cell response in the absence of T and B cells. The treatment consisted of intraperitoneal (i.p.) administration of 15 mg/kg/day of DDCT for 2 days or 10 mg/kg/day of BCDT for 3 days. In addition, to verify whether the effects produced by the titanocenes were compound specific or related to a direct antitumour effect, we also investigated the effects of a 3-day treatment with 100 mg/kg of cyclophosphamide cyclophosphamide on NK cell activity. Our results demonstrated that, in BALB/c and SCID mice, NK cell function declined to subnormal levels after inoculation of the tumour. In these animals, although treatment with DDCT and BCDT significantly enhanced NK cell function, only DDCT restored NK cell activity to normal values in all stages studied. Conversely, treatment with cyclophosphamide reduced NK cell function in nontumour bearing SCID mice and was also unable to restore the decreased NK activity of tumour-bearing SCID mice, thus demonstrating that the enhancement of NK cell function by titanocenes is compound specific. The same effect of cyclophosphamide was observed with BALB/c mice. In the present study, the up-modulatory effects of these two compounds on NK cell function reveal a new aspect of the mechanism of antitumoural action of titanocenes.     

Primary cellular target responsible for dimethylnitrosamine-induced immunosuppression in the mouse

The present studies were undertaken to identify the cellular targets responsible for immunosuppression by dimethylnitrosamine (DMN). The in vitro antibody responses of splenocytes from B6C3F1 mice exposed to 6 mg/kg DMN for 7 days to the T cell-independent antigen dinitrophenyl-Ficoll and the T cell-dependent antigen sheep erythrocytes were used for separation and reconstitution studies. The antibody-forming cell response of spleen cells from DMN-treated mice to the T-independent and T-dependent antigens was suppressed by 72% and 61%, respectively, when compared to vehicle controls. Whole spleen suspensions were fractionated into nonadherent populations by plastic adherence and Sephadex G-10 depletion of macrophages. Adherent antigen-presenting cells were obtained by incubating spleen suspensions in culture wells and removing nonadherent cells after 3 h. By combining vehicle and DMN nonadherent and adherent populations it was demonstrated that the population most affected by DMN exposure in both the sheep erythrocyte and dinitrophenyl-Ficoll responses was the nonadherent population. The lack of T cell dependence of the dinitrophenyl-Ficoll response was verified by elimination of T cells from whole spleen suspensions by monoclonal anti-thy 1.2 antibody plus complement treatment. These results indicated the B cell from DMN-treated mice as the splenic cell type responsible for suppressed antibody-forming cell responses to dinitrophenyl-Ficoll. B cells, T cells (prepared by cytotoxic elimination of B cells using anti-immunoglobulins) and adherent antigen-presenting cells (macrophages) from vehicle- and DMN-treated mice were fractionated and recombined and immunized with sheep erythrocytes (requires B cell, T cell, and macrophage cooperation). While splenic macrophages from DMN-treated mice supported control responses, T-helper activity was slightly reduced and B cell function was especially impaired. The conclusion that T cell function was less suppressed was supported by control responses of DMN-treated mice to concanavalin A but reduced responsiveness to lipopolysaccharide under conditions of limiting cell densities. These studies indicate that the primary cellular target of DMN exposure resulting in suppressed antibody responses is the B lymphocyte.     

Suppression of murine retroviral disease by 2',3'-didehydro-2',3'-dideoxythymidine (D4T).

The thymidine analog, 2',3'-didehydro-2',3'-dideoxythymidine (D4T), and 3'-azido-3'-deoxythymidine (AZT) were evaluated for activity against Friend virus complex (FV) in Mus dunni cells using a focal immunoenzyme assay. The 50% effective doses were, respectively, 1.2 and 0.1 microM for the two compounds; the 50% cytotoxic doses using trypan blue dye exclusion were 25.4 and > 100 microM. Four FV inhibition experiments with D4T were run in F1 hybrid mice containing the Rfv-3r/s genotype. This mouse strain allows the study of treatment effects on development of specific neutralizing antibodies and on splenomegaly, splenic and plasma virus titers, and splenic viral RNA. In the first experiment, D4T was given by oral gavage (p.o.) three times daily (t.i.d.) for 14 days beginning 4 h post-virus inoculation. All dosages used (187.5, 375, 750 mg/kg/day) significantly inhibited all viral parameters. Other experiments used D4T p.o. twice daily, with dosages of 46.9, 93.8, 187.5 and 375 mg/kg/day or four times daily with a dose of 375 mg/kg/day. No significant disease inhibition was seen using the twice daily treatment schedule, but efficacy was apparent using the four times daily treatment. The final experiment repeated the initial study, extending the t.i.d. treatments to 25 days and using dosages of 46.9, 93.8, 187.5 and 375 mg/kg/day. All but the lowest dose reduced each virus parameter. None of the D4T treatment regimens caused death in toxicity controls, although moderate host weight loss or less weight gain was seen, and variable hematocrit decreases occurred, particularly in mice receiving the highest drug dosage. Inhibition of natural killer (NK) cell activity also was seen in these same animals, but in infected mice, FV-induced decrease in NK cell activity was prevented by D4T treatment. Virus-specific neutralizing antibodies developed in all infected, treated animals. These data indicate D4T has potential as a possible candidate for anti-human immunodeficiency virus evaluations in the clinic.