Sister chromatid exchange analysis in lung and peripheral blood lymphocytes of mice exposed to methyl isocyanate by inhalation

Mice were exposed to 1, 3, or 6 ppm methyl isocyanate (MIC) for 6 hr/day for four consecutive days. Lung cells and peripheral blood lymphocytes (PBLs) were removed and cultured for analysis of sister chromatid exchange (SCE) and cell cycle kinetics. MIC caused a small but significant increase in SCE frequency of cultured lung cells from mice exposed to 1, 3, or 6 ppm MIC. MIC did not significantly increase SCE levels in PBLs of mice exposed to concentrations as high as 6 ppm. In cultured PBLs, MIC had a stimulatory effect on cell cycling rates as measured by the replicative index, and it caused a significant reduction in mononuclear leucocyte counts and the mitotic indices.     

Induction of cytogenetic damage in rodents after short-term inhalation of benzene

Experiments were designed to investigate both the induction of sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs) and micronuclei (MN) in bone marrow polychromatic erythrocytes (PCEs) of mice and rats after inhalation of benzene (BZ). Male DBA/2 mice (17-19 weeks old) were exposed to target concentrations of either 0, 10, 100, or 1,000 ppm BZ for 6 hr. Male Sprague-Dawley rats (11-14 weeks old) were exposed to target concentrations of either 0, 0.1, 0.3, 1, 3, 10, or 30 ppm BZ for 6 hr. Blood was obtained by cardiac puncture 18 hr after exposure, and PBLs were cultured in the presence of lipopolysaccharide (mouse B cells, 60 micrograms/ml) or concanavalin A (rat T cells, 30 micrograms/ml) to stimulate blastogenesis for SCE analysis. Femoral bone marrow smears from both species were analyzed for MN in PCEs 18 hr after BZ exposure. Mouse PBLs revealed a significant concentration-related increase in the SCE frequency over controls at 10, 100, or 1,000 ppm BZ. Mouse bone marrow showed a significant concentration-dependent increase in MN over controls after exposure to 10, 100, or 1,000 ppm BZ. Rat PBLs showed a significant increase in the SCE frequency after exposure to 3, 10, or 30 ppm BZ. The statistical significance of the 1 ppm BZ result was borderline and dependent on the statistical test chosen. Rat cells revealed a significant concentration-related increase in MN after inhalation of either 1, 3, 10, or 30 ppm BZ. PBLs from treated mice showed significant concentration-dependent decreases in mitotic indices; however, cell cycle kinetics and leucocyte counts remained unaffected. Rat PBLs showed significant decreases in mitotic activity only after exposure to 3 and 30 ppm BZ, whereas cell cycle kinetics and leucocyte counts were unaffected. These results show that BZ can induce statistically significant cytogenetic effects in PBLs and PCEs of both mice and rats after a 6-hr inhalation of BZ at low concentrations.     

Induction of sister chromatid exchange in spleen and bone marrow cells of rats exposed by inhalation to different dose rates of ethylene oxide

We investigated the effects of dose rate on the frequency of sister chromatid exchange (SCE) in bone marrow and spleen cells of rats exposed to ethylene oxide (EtO). Four groups (18/group) of male Fischer 344 rats were exposed to EtO by inhalation. The exposures consisted of 100 ppm for 6 hr/day, 300 ppm for 2 hr/day, 600 ppm for 1 hr/day, and clean air control. All EtO treated rats were given a total exposure dose of 600 ppm-hr daily, 5 days/week for 3, 6, or 9 months. Six rats per group were sacrificed at each time point, and SCEs were measured in cultured spleen and bone marrow cells. A statistically significant increase was found in SCEs in both bone marrow and spleen cells for all treated groups and at each time point when compared to the control, except at the 3-month exposure for the middle and high dose-rate groups in bone marrow cells. In the spleen, the increases in SCEs were similar among the three experimental groups. In bone marrow, the lowest dose rate (100 ppm) resulted in higher SCE frequencies than the medium and high dose-rate group after 3 and 6 month exposures. The overall frequencies of SCEs in the spleen cells were higher than in the bone marrow cells. The increase in SCE frequencies and decrease in the replicative index in spleen cells were also dependent on the duration of exposure. These results indicate that (1) EtO, by inhalation, can cause SCEs both in spleen and bone marrow cells of Fischer 344 rats, (2) spleen cells are more sensitive to EtO than bone marrow cells, and (3) in bone marrow cells the lowest dose-rate (longest) exposure causes more SCEs than the highest dose-rate (shortest) exposures. © 1993 Wiley-Liss, Inc.     

Sister chromatid exchange induction and persistence in peripheral blood and spleen lymphocytes of mice treated with ethylnitrosourea

The induction and persistence of sister chromatid exchanges (SCEs) were studied in peripheral blood and spleen lymphocytes of mice given a single i.p. injection of ethylnitrosourea (ENU) of 100, 350, or 600 muMoles ENU/kg. SCE frequencies were measured on days 1, 3, 5, and 7, and at seven additional times up to 172 days post-injection. SCEs were analyzed statistically by comparing the mean frequencies as well as the distribution of SCEs per cell at each time. The latter approach was based on a non-parametric method of identifying high frequency cells (HFCs). The SCE frequencies and proportion of HFCs in each dose and tissue remained elevated for up to 172 days following treatment, although the degree and statistical significance of the increase varied according to the tissue, dose, and statistical test employed. The SCE frequencies were found to oscillate during the first week. Following this, however, the return of the SCE frequencies to control levels was fit to a linear regression model with time as the only independent variable. The persistence of SCE-forming lesions was found to be dose-dependent for the spleen but not for blood. Within each dose the persistence of SCE-forming lesions was significantly greater for the blood relative to the spleen. The results emphasize that tissue, dose, and time since exposure are important factors to consider when quantifying SCEs in vivo; analysis of high frequency cells may be a more sensitive method of detecting exposure than the t-test; and a single determination of SCE frequencies may not be sufficient to quantitatively assess genotoxic damage in the first week following exposure.     

Sister chromatid exchanges induced in rabbit lymphocytes by ethylene oxide after inhalation exposure

Ethylene oxide, which is the simplest epoxide and an extremely important commercial compound, has been used by many investigators as a model compound to study mutagenicity by alkylation of DNA. Knowledge of in vivo dose-effect relations under experimental conditions may provide further insight into the dynamics of the sister chromatid exchange (SCE) response. It may also provide information on temporal aspects of sampling design for human worker populations. Groups of four male New Zealand white rabbits were exposed in inhalation chambers to 0, 10, 50, and 250 parts per million (ppm) ethylene oxide for 6 hr a day, 5 days a week, for 12 weeks. Peripheral blood samples were taken before the start of exposure, at intervals during exposure, and up to 15 weeks after the end of exposure to measure SCE rates in peripheral lymphocytes as well as standard hematological endpoints. Additionally, the level of reduced glutathione (GSH) in liver and blood was measured in a set of concurrently exposed animals at the end of the 12-week exposure. Results show that exposure to 10 ppm does not cause a detectable increase in SCE rates. However, exposure to 50 and 250 ppm does cause an increase in SCEs that decreases after exposure ends, but still remains above baseline levels 15 weeks after exposure. Hematological and GSH measurements did not differ between control and exposed groups. These results indicate that inhalation exposure to the mutagenic alkylating agent ethylene oxide results in a dose-related SCE effect, and that SCE is a more sensitive indicator of exposure than either standard hematological end points or GSH levels.     

Modulation of cellular immune responses in mice with disseminated histoplasmosis by recombinant interleukin-2.

Depression of the cellular immune responses in mice with disseminated histoplasmosis is associated with deficient production of interleukin-2 (IL-2) by splenocytes. Therefore, we examined whether a highly purified preparation of IL-2, recombinant human IL-2 (rIL-2), could modify the cellular immune responses in infected mice and whether this lymphokine could alter the severity of histoplasmosis in animals. Exogenous rIL-2, at concentrations of up to 1,000 U/ml, failed to augment the proliferative responses to concanavalin A by unfractionated splenocytes or splenic T cells from mice infected for 1 week. In addition, rIL-2 did not modulate the plaque-forming cell response to sheep erythrocytes by splenocytes from these same mice. However, at week 3, rIL-2 in concentrations ranging from 10 to 1,000 U/ml considerably augmented the proliferative response to concanavalin A and plaque-forming cell response to sheep erythrocytes by splenocytes from infected mice. Kinetics studies demonstrated that rIL-2 exerted maximal immunoregulatory activity when added on day 0 or 1 to cultures of splenocytes. In vivo administration of rIL-2, 200 to 20,000 U/day, for 10 days to normal and 3-week-infected mice did not alter the proliferative activity of splenocytes to concanavalin A; 200,000 U of rIL-2 per day actually depressed the proliferative responses of splenocytes from normal and infected mice. In vivo, rIL-2 did not modify delayed-type hypersensitivity responses to sheep erythrocytes or to histoplasmin by normal and infected mice. Moreover, treatment with rIL-2 in vivo did not reduce the number of Histoplasma CFU in spleens of mice. Thus, despite the immunoenhancing effect of rIL-2 in vitro, this lymphokine failed to exert similar effects in vivo.     

Cytogenetic effects of short- and long-term exposure of chick embryos to the phenoxyherbicide 2,4-D

Before incubation, chick embryos were treated with the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) by injecting onto the inner shell membrane solutions of 0, 0.5, 1, 2, or 4 mg 2,4-D. A commercial formulation containing 37% 2,4-D iso-octyl ester as active ingredient and pure 2,4-D were tested. Sister chromatid exchange (SCE) and cell cycle kinetics were examined at days 4, 7, and 10 from 22 to 30 embryos per group. After 4 days of exposure to commercial 2,4-D, a small (P < 0.05) dose-related increase of SCE was seen for the 4-mg group. An enhanced SCE response upon long-term exposure to 2,4-D was apparent. After 10 days of exposure, SCE frequencies for the 2- and 4-mg commercial 2,4-D, and 4-mg pure 2,4-D groups were significantly higher than for the controls. A significant slowing of cell cycle at concentrations at and above 1 mg was seen. Also observed was a slight, not statistically significant proliferative effect at the lowest dose of 0.5 mg/embryo. Consistent with the results from other test systems, the present findings indicate that 2,4-D has a low to moderate genotoxic activity.     

Persistent suppression of humoral immunity produced by 7,12-dimethylbenz(A)anthracene (DMBA) in B6C3F1 mice: correlation with changes in spleen cell surface markers detected by flow cytometry

The purpose of these studies was to examine the effects of DMBA on subpopulations of splenocytes obtained from B6C3F1 mice, using cell surface markers defined by monoclonal antibodies and multiparameter flow cytometry. Changes were correlated with alterations in humoral immune function assessed in vitro. Mice were treated for 10 days during a 2 week period by subcutaneous (s.c.) injections of DMBA in corn oil at doses of 0.5, 5 and 10 micrograms/g/day (5-100 micrograms/g total dose). Four mice from each exposure group and an additional corn oil control group of mice were studied at 4 and 8 weeks following the last injection with DMBA. These studies demonstrated a dose-dependent decrease in the total number and percentage of spleen cells expressing B-cell markers (mu heavy chain, kappa light chain and 14.8 antigen) as well as T-cell markers (Thy 1.2, Lyt-1 and Lyt-2). The percentage of splenocytes expressing Mac-1 was increased by DMBA. Helper T-cells appeared to be a very sensitive population of spleen cells to DMBA exposure, as suggested by a decrease in the number and percentage of Lyt-1 positive cells recovered from the spleen 4 weeks after exposure to DMBA. DMBA produced a dose-dependent suppression of the in vitro primary humoral immune responses to SRBC, TNP-Ficoll and TNP-LPS. The fact that a functional suppression of humoral immunity was accompanied by a decrease in the number of mature B-cells and T-cells in the spleen suggests that cell surface markers may be useful indicators of immunotoxicity in animals receiving DMBA in sub-chronic studies.     

Induction of sister chromatid exchange in multiple murine tissues in vivo by various methylating agents

In order to study the reliability of in vivo sister chromatid exchange (SCE) assays for predicting carcinogenicity, several known animal carcinogens were tested in a multicellular in vivo SCE assay and an in vivo/in vitro murine lymphocyte assay. The methylating agents 1,2-dimethylhydrazine.2 HCl (DMH), dimethylnitrosamine (DMN), methylnitrosourea (MNU), methyl methane-sulphonate (MMS), and methylazoxymethanol acetate (MAM) were tested for SCE induction in several murine tissues in vivo, including bone marrow, alveolar macrophages, regenerating and intact liver, and kidney from B6D2F1 mice. In all cell types, clear dose-responses were observed following exposure of mice to subcytotoxic fractions of the LD50 dose of DMH, MNU, or MMS. DMN (0.03-0.27 mmol/kg) produced small, although not dose-related, increases in SCE in all cell types. At the doses tested (0.06 and 0.08 mmol/kg), MAM did not induce elevated SCE in the various cell types. Following a series of multiple i.p. injections of low, non-toxic doses of DMH (0.15 mmol/kg, once a week, for 10 weeks), significant differences were observed in intact vs. regenerating liver and in single vs. multiple injections in regenerating liver. Following exposure of B6D2F1 mice to a single i.p. injection of 0.25 mmol/kg DMN, DMH, or MMS or 0.19 mmol/kg MNU, SCE responses were evaluated in Concanavalin A (Con A)- and LPS-stimulated blood and spleen lymphocytes. Considerable cytotoxicity was observed in blood lymphocytes. In Con A- and LPS-stimulated spleen lymphocytes, DMH-, and DMN- and MMS-induced SCE frequencies were approximately 1.5-2 x baseline levels and MNU-induced SCE were approximately three- to fourfold higher than baseline values in cultures initiated at 1 and 24 h postexposure. At 48 and 72 h after an i.p. injection of 0.131 mmol/kg MNU, SCE responses in lymphocytes were approximately 2 x baseline levels. At 24 h following one, two, or four injections (one/week) of 0.075 mmol/kg MNU dose-related increases in SCE were observed in spleen lymphocytes. These studies illustrate that carefully designed in vivo SCE assays may have the capacity to predict the tumorigenic potential of chemical agents.     

Cytogenetic studies of ethyl acrylate using C57BL/6 mice

The clastogenicity of ethyl acrylate (EA) was examined in vivo by injecting i.p. five male C57BL/6 mice per dose group with either 125, 250, 500 or 1000 mg/kg EA dissolved in saline. Controls received solvent only. Acrylamide (100 mg/kg), because of its similarity in structure and mode of action to EA, was used as a positive control. Twenty-four hours after injection, the animals were anesthetized and the spleens aseptically removed. Splenocytes were isolated on density gradients and cultured with concanavalin A to stimulate cell division. In half the cultures bromodeoxyuridine was added at 21 h for analysis of chromosome aberrations (CAs) in first division cells and sister chromatid exchange (SCE) in second division cells. In the remaining cultures cytochalasin B was added to produce binucleated cells for scoring of micronuclei (MN). There was no significant increase in SCE or CAs at any of the doses of EA examined. At the highest dose examined (1000 mg/kg), EA did cause a small but significant increase in binucleated cell MN. Acrylamide caused an increase in MN and SCEs in splenocytes. Because others have found EA to be clastogenic in vitro, isolated splenocytes were exposed to a wide range of concentrations of EA during the G0 stage of the cell cycle or 23 h after mitogen stimulation during the late G1 or early S phase of the cell cycle. Although EA was toxic for both exposure regimens, significant increases in chromatid-type aberrations were found only when the target cells were treated 23 h after mitogenic stimulation. No statistically significant increase in SCE frequency was found after either treatment regimen. These data suggest that EA is only clastogenic at near toxic concentrations during a specific stage of the cell cycle.     

Evaluation of sister chromatid exchange as an indicator of sensitivity to N-ethyl-N-nitrosourea-induced carcinogenesis in rats

Sister chromatid exchange (SCE) frequencies in peripheral lymphocytes are a frequently used endpoint to indicate exposures to genotoxins in groups of humans. The aim of this study was to ascertain, in an experimental design, whether or not SCE rates have any association with the risk of cancer at the individual level in rats exposed to a known carcinogen. Individual SCE rates were determined in three consecutive analyses in cultured blood lymphocytes of 50 adult male Wistar rats. Analyses were done before as well as 24 hr and 7 days after a single intraperitoneal administration of 0, 25, 50, or 75 mg/kg of N-ethyl-N-nitrosourea (ENU). The animals were followed until death; also, the relationship between SCEs and carcinogenic outcome, i.e., the presence or absence of tumors, and their latency period were examined. ENU significantly decreased the life expectancy of the rats. The tumor types most clearly associated with ENU treatment were various gliomas and thyroid-gland and testicular tumors. ENU induced a moderate (maximally 1.6-fold) increase in the mean frequency of SCEs/cell at both sampling times after treatment. The effect was somewhat more pronounced 1 day rather than 1 week after treatment. The mean SCE rates in rats with ENU-specific cancers or in animals with early or multiple tumors did not differ from those in animals that survived no less than 65 weeks or longer without developing tumors. In ENU-treated animals with tumors, no relationship was found between the mean SCE rate and survival time. It is concluded that in outbred Wistar rats the SCE response in cultured lymphocytes does not indicate individual susceptibility to the carcinogenic action of ENU. On a group basis, however, animals with high SCE rates were shown to have increased risk of cancer.     

Effect of cosensitization with buckwheat flour extract on the production of house dust mite-specific IgE

There are studies reporting food sensitization in infancy increases the risk of sensitization to inhalants later in life. We performed a study to evaluate whether cosensitization with buckwheat (BW) has an effect on the production of house dust mite-IgE. C3H/HeJ mice (4 weeks, female) were sensitized with house dust mite (HDM)/Al (OH)(3), intraperitoneally on day 0, followed by 4 intranasal sensitizations (on days 14, 15, 16, and 21). Group 1 was cosensitized intragastrically with BW/cholera toxin (CT) (on days 0, 1, 2, 7, and 18) during sensitization with HDM, group 2 was cosensitized intragastrically with CT only (on days 0, 1, 2, 7, and 18), and group 3 was used as controls. HDM- and BW-IgE and antigen-specific T-cell proliferation and cytokine production were evaluated. In Group 1, BW-IgE levels were highest at week 4, and the HDM-IgE at week 3 (98.45+/-64.37 ng/mL and 169.86+/-55.54 ng/mL, respectively). In Group 2, HDM-IgE levels reached a peak at week 3, remarkably higher (810.52+/-233.29 ng/mL) compared to those of Group 1 (169.86+/-55.54 ng/mL). The interleukin (IL)-4 and interferon (IFN)-beta in the HDM-stimulated culture supernatants of splenocytes were not significantly different among groups. We postulate that the cosensitization with BW may down-regulate the specific IgE response to HDM.     

Intravenous administration of splenocytes in early pregnancy changes the implantation window in mice

To clarify the role of immune cells in the reproductive phenomenon during early pregnancy, we investigated the effect of splenocytes obtained from mice in early pregnancy on embryo implantation. We prepared splenocytes from 5 week old pregnant ICR mice on day 4 (pregnancy day 4 splenocytes; P4-spl) and day 8 (pregnancy day 8 splenocytes; P8-spl), from 5 week old pseudopregnant ICR mice on day 4 (pseudopregnancy day 4 splenocytes; PP4-spl) and from 5 week old di- oestrous virgin mice (di-oestrous splenocytes; Di-spl). The supernatants (P4-sup, P8-sup, PP4-sup and Di-sup) derived from the suspensions of P4-spl, P8-spl, PP4-spl and Di-spl respectively, were used in the control groups. The recipient pseudopregnant mice (6 weeks of age) were injected i.v. with splenocytes (2 x 10(7) cells) or supernatants as controls, and embryo transfer was performed on day 2. Embryo implantation was evaluated 7 days later under laparotomy. The successful implantation rate per embryo was markedly higher compared with the control groups in the P4-spl (30.5 +/- 8.55 versus 1.0 +/- 1.37%, n = 200, P < 0.01) and P8-spl (20.1 +/- 10.3 versus 1.17 +/- 1.62%, n = 200, P < 0.05) groups. On the other hand, a slight but not significant increase was observed in the PP4-spl (8.33 +/- 8.80% versus 1.5 +/- 1.37%, n = 200) and Di-spl (9.0 +/- 5.76% versus 2.3 +/- 2.28%, n = 200) groups. Among the splenocyte administration groups, the implantation rate in the P4-spl group was significantly higher than in the PP4-spl and Di-spl groups (P < 0.01). These findings indicate that i.v. administration of splenocytes can change the murine implantation window. Since the splenocytes during early pregnancy are most effective on embryo implantation, peripheral immune cells in early pregnancy may be involved in embryo implantation.      

Reduced lysis by CD8+ cytotoxic T cells in mixed lymphocyte reactions induced via CD4+ T cells exposed to chemically modified antigen presenting cells.

The resistance by T lymphocytes to activation by antigen (anergy) is well documented for CD4+ T-helper (Th) cells, although less is known about CD8+ cytotoxic T lymphocytes (CTL). One widely used method of inducing anergy of CD4+Th is presentation of antigen by ECDI (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide)-fixed antigen-presenting cells (APCs). We report here that in murine mixed lymphocyte reactions (MLRs), a marked reduction in detected cytotoxicity (which is mediated predominantly by CD8+ CTL) occurs on day 7 if the bulk cultures are restimulated 2 days previously with ECDI-fixed allogeneic splenocytes. No differences were seen between untreated cultures on days 5 and 7, or on day 7 of cultures to which were added unfixed allogeneic splenocytes, fixed or unfixed syngeneic splenocytes, or 'third-party' allogeneic splenocytes, 2 days previously. The effect is not mediated directly on CD8+ cells, since MLRs depleted of CD4+ cells immediately prior to exposure to fixed allogeneic splenocytes fail to show reduced lysis. On the other hand, reduced lysis did occur if CD4+ cells, purified from the MLRs on day 4, were exposed to ECDI-fixed allogeneic splenocytes and then returned to MLRs previously depleted of CD4+ cells. Moreover the effect is overcome using exogenous interleukin-2 (IL-2). We propose that CD4+ cells, restimulated by a regimen shown previously to induce their anergy, can cause a reduction in CD(8+)-mediated cytotoxicity in MLRs.     

Effects of 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene and cyclosporin A on murine cytomegalovirus infection: studies of resistance mechanisms

Susceptibility to murine cytomegalovirus (MCMV) was enhanced by treating B6C3F1 and CD-1 mice subcutaneously with 100 mg 7,12-dimethyl-benz[a]anthracene (DMBA)/kg fractionated over a 2 week period prior to sub-lethal infection. Virus-augmented natural killer cell (NKC) activity was depressed in B6C3F1 mice treated with 100 mg DMBA/kg, while serum interferon (IFN) levels were unaffected. Treatment with 50 mg DMBA/kg had no effect on susceptibility to virus or virus-augmented NKC activity. Susceptibility to MCMV was not affected by treating mice with 400 mg benzo[a]pyrene (B[a]P)/kg using the same exposure regimen. Virus-augmented NKC activity was suppressed in B[a]P-treated mice, but the magnitude of the suppression (18%) was much less than that for DMBA-treated mice (39%). Susceptibility to MCMV, virus-augmented NKC and IFN induction were not affected in mice treated intraperitoneally with 50 mg cyclosporin A (CSA)/kg/day for 5 days and infected on the 5th day of treatment. In contrast, enhanced susceptibility to MCMV and depressed NKC activity were observed in mice treated by the same exposure regimen on days 1-5 post infection. Susceptibility was not affected by CSA given on days 5-9 post infection. The data are useful not only because they show that DMBA and appropriately-timed CSA treatments suppress virus augmented NKC and enhance susceptibility to MCMV, but also because they help to define the relative importance of certain immune responses in defending against the infection, thus improving the usefulness of MCMV as a host resistance model for immunotoxicity testing. The data suggest that chemicals which depress NKC are likely to enhance susceptibility to MCMV, and conversely that effects on NKC should be suspected when chemical exposure enhances susceptibility to MCMV.     

Immune responses of mice after conjunctival exposure toChlamydia trachomatis serovar A

CBA/J mice were inoculated in the lower conjunctival sac with live elementary bodies (EBs) ofChlamydia trachomatis serovar A. Recovery of chlamydia after exposure was done by culture of conjunctival swabs and draining lymph node (D-LN) cells in McCoy cells grown on coverslips in isolation vials. Cellular immune responsiveness was measured by lymphocyte proliferation assay of D-LN cells stimulated with irradiated EBs of serovars A, C, or L2. Humoral immunity was measured by enzyme-linked immunosorbant assay (ELISA) and immunoblot analysis. Chlamydia were consistently isolated from the conjunctiva and from the D-LN at 1 and 7 days after exposure respectively. Intermittent isolations were obtained from the conjunctiva up to day 4 and from the D-LN up to day 14 after a single exposure. Serovar A EB-stimulated lymphocyte proliferation was strong by 1 week after conjunctival exposure, but by 4 and 5 weeks, blastogenic responsiveness was very low. This lack of responsiveness may reflect a state of immunosuppression. Responses to serovars C and L2 EBs were consistantly lower than to serovar A EBs. Serum IgG antichlamydia antibodies were not detected by ELISA until 2 weeks after exposure, peaked by 4–5 weeks, and decreased between 5 and 7 weeks after exposure. The IgM response was minimal at all times tested. There was, however, a modest increase in IgM antibodies at 3 and 5–7 weeks after exposure. Immunoblot analysis showed reactivity of mouse serum antibodies with polypeptide bands of 30, 41, and 52 kD at 3 and 4 weeks post exposure and predominantly with the 52 kD moiety at 5 weeks post exposure. These studies along with the effect of multiple exposures on the immune response may provide a model system which will assist in the understanding of the immune responses and immunomodulation that occur in trachoma.     

Alterations in interleukin-6 production by LPS- and Con A-stimulated mixed splenocytes,spleen macrophages and lymphocytes in prenatally diazepam-exposed rats

Prenatal exposure to diazepam leads to a suppression of mitogen or allogen-induced lymphocyte proliferation as well as to a reduced production of tumour necrosis factor (TNF)- from rat splenocytes during postnatal development of rats. We analysed the secretion of interleukin (IL)-6 which occurs at a later stage of the cytokine cascade. Splenocytes of male offspring from Long Evans rats, treated with a daily dose of diazepam (1.25 mg/kg) from gestational day 14 to 20, were stimulated with lipopolysaccaride (LPS) and concanavalin A (Con A). In response to LPS, IL-6 liberation was significantly lower in mixed splenocytes and spleen macrophages of 2 and 8 week old prenatally diazepam-treated rats than in controls. Spleen lymphocyte preparations of prenatally treated animals exhibited a reduction of IL-6 release at 12 h and an increase at 24 h of incubation. At 2 weeks of age, Con A-induced IL-6 production could only be detected in mixed splenocytes; prenatally treated rats were releasing significantly less IL-6 than controls. In 8 week old rats, IL-6 liberation from mixed splenocytes and spleen macrophages was significantly lower in prenatally treated animals than in controls. Spleen lymphocytes presented a complex response picture depending upon incubation conditions. Our data indicate that in prenatally diazepam-exposed rats, the disturbance of cytokine release also extends to cytokines which play an important role in the later phases of immune responses.     

A kinetic study of the murine mixed lymphocyte reaction by 5,6-carboxyfluorescein diacetate succinimidyl ester labeling

Alternatives to the use of radioisotopes to measure cell proliferation in mixed lymphocyte reactions (MLR) are desirable to avoid the hazards and costs associated with radioisotope use. The versatile fluorochrome 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to measure MLR and provides the opportunity to measure several different growth parameters. This study was aimed at determining which growth parameter is most practical and suitable for measuring murine MLR. The parameters measured were: the relative number of daughter T-cells, the relative number and frequency of reactive T-cell precursors and the relative number of mitotic events. Responder cells were CFSE-labeled unfractionated splenocytes from C57BL/6 mice. Stimulator cells included irradiated splenocytes from C57BL/6 (control), B6D2F(1) (haplo-allogeneic) or FVB/N (allogeneic) mice. Cultures were harvested daily for 1 week. Stimulator T-cells rapidly declined to less than 0.2-0.3% of the mixed population by day 2 of culture. Experimental groups had a significantly higher number of daughter T-cells and mitotic events after 2 days of culture with the number of daughter T-cells climbing exponentially after 5 days of culture. The number and frequency of reactive T-cell precursors were significantly higher in experimental groups on days 2-3, but this difference became insignificant by day 4. Among all the parameters, the relative number of daughter T-cells was the most practical for measuring MLR, after 5 days of culture, based upon the growth kinetics of responder T-cells and the survival of the stimulator cells.     

Flow microfluorometry analysis of alterations in T-lymphocyte subsets during murine listeriosis

C57BL/6 mice were infected intravenously with 6 X 10(3) Listeria monocytogenes organisms. As early as day 3 of infection, there was a marked reduction in the number of lymphocytes recovered from the peripheral blood, bone marrow, and thymuses of infected animals. Concomitantly, there was an increase in the number of splenic lymphocytes. By day 14, both the total and differential cell counts were similar in both infected and normal animals. Flow microfluorometric studies comparing the Thy-1.2, Lyt-1, Lyt-2, and surface immunoglobulin (SIg) phenotypes of lymphocytes from normal and infected mice were performed. Between days 3 and 5, there was a decrease in the percentage of Thy-1.2+ cells in the spleens of L. monocytogenes-infected animals. Conversely, the percentages of Lyt-1+, Lyt-2+, and SIg+ cells remained constant. At day 7 of infection, the percentage of Thy-1.2+ splenocytes was within normal limits, and at day 10, the percentage of Thy-1.2+ cells was elevated slightly. The absolute numbers of Thy-1.2+ cells were comparable in both infected and normal animals at early stages (days 3 to 5) of L. monocytogenes infection, but there was a marked elevation of Thy-1.2+ splenocytes at days 7 to 14 of infection. Lyt-1+, Lyt-2+, and SIg+ splenocytes increased in absolute numbers as early as day 3 of infection and were still elevated at day 14. Adrenalectomy before infection had no effect on the results obtained, suggesting that these changes were not mediated by endogenous steroids.     

Vinclozolin modulates splenic natural killer cell activity, antibody-forming cell response and phenotypic marker expression in sprague dawley rats: a two-generation feeding study

The potential effects of the fungicide vinclozolin (VCZ) on the immune system were evaluated in F(0) (dams) and F(1) generations of Sprague Dawley rats exposed to a soy-free diet containing VCZ at 10, 150 and 750 ppm. In dams, exposure to VCZ at the highest concentration from gestation day 7 to postpartum day 51 (65 days total exposure) produced a significant increase in the numbers of splenocytes, B cells, T cells, helper T cells and cytotoxic T cells and a decrease in the percentage of NK cells. In F(1) males, exposure to VCZ gestationally, lactationally and through feed from postnatal day 22 to 64 (78 days total exposure) produced no effect on spleen or thymus weights or splenocyte subsets. However, increases in the spleen IgM antibody-forming cell response to sheep red blood cells (150 and 750 ppm) and the activity of NK cells (150 ppm) were observed. In F(1) females, exposure to VCZ produced a decrease in the activity of NK cells in all the treatment groups. Although decreases in the number of cytotoxic T cells (150 ppm) and the percentages of NK cells (10 ppm) and cytotoxic T cells (150 ppm) were also observed, the lack of a dose-related response suggested that these findings might not be biologically meaningful. In conclusion, these results demonstrate that exposure to VCZ at the concentrations tested modulates the immune responses in Sprague Dawley rats. Furthermore, the differential effect of VCZ in F(1) male and female rats is consistent with the reported anti-androgenic properties of VCZ.