Biomedical subjects
Publications and source records attributed to K W Johnson.
A survey of blood lead concentrations is reported in a group of 199 young Walsall children (3-6 years of age). The geometric mean blood lead concentration was 0.47 mumol/l (range 0.2-1.6 mumol/l). There were no significant differences in blood lead concentrations between groups of children with different ages, sex, ethnic origin, or environment.
Treatment of murine spleen cells with 30 nM TCDD resulted in an approximately 3 fold increase in unstimulated antibody production after 3 days in culture. This response was not accompanied by increased cellular proliferation and may represent an effect of TCDD on B cell activation or differentiation. Since PMA is capable of activating B cells, presumably via PKC, we have compared the effects of PMA and TCDD on protein kinase activation and phosphorylation of endogenous proteins in a highly purified preparation of B cells. In contrast to a reduction of cytosolic PKC activity, the expected effect of PMA, TCDD caused an increase in basal kinase activity with no effect on PKC activity. Addition of either PMA or TCDD resulted in enhanced phosphorylation of a similar profile of proteins, including proteins of Mr 12.2, 14.6, 29.2, 52.3 and 62.7 KDa. Addition of TCDD also resulted in the increased phosphorylation of a protein of Mr 45.2, which was unaffected by PMA. Combined treatment with PMA and TCDD resulted in additive responses. The additive effects of PMA and TCDD suggest an interaction at the level of protein phosphorylation which is mediated by different kinases. Therefore, TCDD may be stimulating B cells via an early effect on an unidentified protein kinase.
The suppression of in vitro antibody responses by dimethylnitrosamine (DMN) was produced in a mouse hepatocyte and splenocyte co-culture system. Mouse hepatocytes were isolated from female B6C3F1 mice and cultured for 20-24 hr to allow the formation of a monolayer on collagen-coated plastic petri dishes. Spleen cells were isolated from the same hybrid and were co-cultured with the hepatocytes along with DMN. Cyclophosphamide (CP), an immunosuppressive agent requiring metabolic activation that was included as an initial positive control, produced a marked suppression of the in vitro antibody responses to LPS, DNP-Ficoll, and SRBCs in 4 hr in the co-culture system. Under comparable conditions DMN markedly suppressed the response to SRBCs, marginally suppressed the response to DNP-Ficoll, and did not suppress the polyclonal response to LPS. The suppression by DMN was related to the rocking speed during the 4-hr co-culture period and was optimally produced when the cultures were not rocked. Addition of serum into the medium (10% fetal calf serum) during the co-culture period did not change the effects of DMN on the antibody response. However, the addition of extracellular DNA (1 mg calf thymus DNA/ml) prevented the suppression of the antibody response by DMN. These results suggest that DNA represents the primary macromolecular target for the reactive intermediate of DMN, and indicate that a syngeneic co-culture system can be used to characterize the in vitro immunosuppression
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.
The in vitro activation of dimethylnitrosamine (DMN) to an immunosuppressive form was studied utilizing liver-enzyme fractions and intact hepatocytes. The N-demethylation of DMN by mouse S9 and microsome preparations was confirmed by determination of formaldehyde generation. S9 fractions from both phenobarbital(PB)- and isopropanol(iso)-pretreated mice displayed significantly greater demethylase activity than uninduced S9 fractions. However, when incubated with spleen cells, neither S9 preparation was capable of activating DMN to a form capable of suppressing antibody responses by recovered spleen cells. In contrast, the positive control, cyclophosphamide, was activated to a markedly immunosuppressive form. S9 fractions failed to activate DMN to an immunosuppressive form regardless of S9 concentration, time of preincubation, or rocking speed. Liver microsomes from PB-pretreated mice displayed significantly greater N-demethylase activity than S9 fractions yet were unable to activate DMN to an immunosuppressive form. In contrast, the addition of DMN to mixed cultures of mouse hepatocytes and mouse spleen cells resulted in activation of DMN and marked suppression of antibody responses. The separation of spleen cells from the hepatocyte monolayer by an agar layer less than 1 mm thick resulted in complete reversal of the immunosuppressive effect of DMN. Unlike the metabolism of DMN to a mutagenic form, the in vitro activation of DMN to an immunosuppressive form was therefore dependent on intact cells. Furthermore, the activation by intact hepatocytes was shown to be dependent on cell-cell contact or close proximity of activating and target cells.
Immunosuppression by gamma-chlordane was examined by the direct addition of chlordane to cultured spleen cells from untreated B6C3F1 mice. Both cell-mediated and humoral immune responses were markedly suppressed upon in vitro exposure. The mixed lymphocyte response and the proliferative response to both B- and T-cell mitogens were significantly suppressed at micromolar concentrations of chlordane. The antibody response to sheep erythrocytes (SRBC) was suppressed 90% at 10 microM chlordane. The kinetics of the SRBC response were not altered by chlordane. Addition of chlordane to the antibody cultures on various days indicated an effect at the early stages of the response. Previous studies with chlordane failed to demonstrate immunosuppression following in vivo exposure. The possibility that chlordane was metabolized in vivo to a less immunosuppressive form was studied by examining the effect of the major metabolite, oxychlordane, on the in vitro antibody response and by incubating splenocytes with chlordane and a liver S9 preparation prior to culture with SRBC. Oxychlordane was immunosuppressive by itself, and the activity of chlordane was unaltered in the co-culture experiments. The association of chlordane with serum components was evaluated in vitro in cultures of mouse bone-marrow cells (BMC). The chlordane-induced suppression of [3H]thymidine incorporation by BMC was reversed by the addition of mouse or human serum. In summary, chlordane produces marked suppression of in vitro immune responses via an apparent antiproliferative action. The failure of chlordane to produce in vivo immunosuppression may be related to extensive association of chlordane with serum components.
Black Americans continue to have large numbers of premature and excess deaths, measured against white experience, from seven major causes. Major differences in chronic disease, disability and case fatality rates persist despite similarities in the amount of health care received; the nature and quality of care is likely to be dissimilar. Epidemiological and clinical evidence suggests various strategies to reduce these differentials through well-designed public health efforts at prevention. These will have to deal with harsh realities of sociocultural, economic, and political contexts.
The hypothesis that dimethylnitrosamine (DMN)-induced humoral immune suppression is mediated by reactive intermediates was tested in female B6C3F1 mice using three approaches. First, distribution of reactive species to known target organs (liver and kidney) and to immune organs (peripheral blood components, bone marrow cells, thymus and spleen) was demonstrated by monitoring acid-insoluble radioactivity after single or multiple doses of 6 mg/kg and 2 microCi of [methyl-14C] DMN. Most of the radioactivity resided in acid-insoluble material, which increased with the number of exposures and correlated with suppression of the in vivo antibody response to sheep erythrocytes as determined by enumerating immunoglobulin M antibody-forming cells. Fractionation of spleens from exposed animals into various cell types indicated greater association of acid-insoluble radioactivity with T- and B-lymphocytes as compared to macrophages. Fractionation of spleen cell lysates into protein, DNA and RNA indicated that most radioactivity was associated with the nucleic acid component. Pretreatment of mice with aminoacetonitrile reduced acid-insoluble disintegrations per minute in the spleen. The second approach was to demonstrate the immunosuppressive nature of compounds forming the same toxic intermediate as DMN. The addition of N-nitroso(acetoxymethyl)methylamine to spleen cell cultures resulted in completely suppressed in vitro antibody responses to sheep erythrocytes and dinitrophenylated Ficoll at micromolar concentrations. The third approach involved activating DMN to an immunosuppressive form in vitro. Preincubation of splenocytes with Aroclor-induced mouse hepatocytes resulted in activation of DMN to intermediates capable of markedly suppressing the response of antibody-forming cells to sheep erythrocytes.
Various toxicological and immunological parameters were assessed after exposure of female B6C3F1 mice to 0.1, 1.0, 4.0, and 8.0 mg/kg gamma-chlordane for 14 days via oral gavage. Variables evaluated included periodic body weights, terminal organ weights, hematology including leukocyte differentials, antibody response to sheep red blood cells (SRBC), lymphoproliferative responses to the mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and lipopolysaccharide (LPS) and allogeneic cells, and the delayed hypersensitivity response (DHR) to keyhole limpet hemocyanin (KLH). When compared to the corn oil (vehicle) controls, chlordane produced a significant, dose-related increase in liver weight. Total leukocytes were significantly increased in chlordane-treated groups and seemed to be due to a significant increase in the lymphocyte population. Humoral immunity (HI), as assessed by enumeration of SRBC-specific immunoglobulin M (IgM) antibody forming cells (AFC) and proliferation of LPS-stimulated spleen cells, was not significantly altered in mice exposed to chlordane. In vitro evaluation of cell-mediated immunity (CMI), as measured by proliferation of Con A and PHA-stimulated spleen cells from chlordane-treated animals, indicated a significant and dose-related increase. The response to allogeneic cells was also enhanced. Results from an in vivo indicator of cell-mediated immune status, the delayed hypersensitivity response to KLH, did not support chlordane-enhanced T cell function suggested by mitogen and mixed lymphocyte responses. Therefore, a potent cyclodiene insecticide of environmental concern produced an enhancement of certain indicators of CMI. The expression of a DHR in vivo and the antibody response to SRBC was unaltered in mice exposed to the chemical. These results suggest that gamma-chlordane, at the concentrations utilized, does not produce a biologically significant alteration of the immunocompetence of intact animals.
Gallstone formation complicating cardiac valve replacement has been amply documented in adults. Two children who developed gallstones shortly after cardiac valve surgery are reported.
A microtiter hemolytic assay was utilized to determine sodium azide (NaN3) modulation of B6C3F1 and C3H mouse serum complement levels in vivo and in vitro. Functional complement was expressed in CH50 units per milliliter. Experiments were performed to determine the in vitro effect of NaN3 on complement mediated lysis of IgM sensitized rabbit erythrocytes. Concentrations of 5, 10, 20, 30, 40, 60, and 80 mM NaN3 were added to microtiter wells containing Tris buffer, IgM sensitized rabbit erythrocytes, and serum complement from naive female C3H mice. Although NaCl and KCl controls had an inhibitory effect, NaN3 demonstrated a significant dose-dependent inhibition of complement-mediated lysis. In the three in vivo experiments, female B6C3F1 mice were exposed to NaN3 and physiological saline (vehicle control). Complement hemolytic ability was evaluated after a 1-day, single iv injection of 0.2, 2.0, and 20.0 mg/kg NaN3; at Days 1, 2, 3, 4, and 6 of a 6-day time course study after ip administration of 20 mg/kg NaN3; and at the end of an 11-day study involving daily injections of 10, 15, and 20 mg/kg NaN3 given ip. No significant changes in complement-mediated hemolysis were observed in the in vivo experiments. These studies indicate that NaN3 does not affect mouse complement levels in vivo. However, NaN3 suppresses in vitro complement hemolytic ability.
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