Changes in the intracellular accumulation and distribution of metallothionein in rat liver and kidney during postnatal development

Metallothionein (MT) bound to zinc and copper was detected in high concentration in fetal and newborn rat livers by a cadmium saturation method. The levels of both hepatic zinc and MT remained high for the first 14 days after birth and decreased to adult levels by 24 days of age. There was a direct linear relationship between hepatic metallothionein and zinc concentrations during the first 31 days after birth. The ratio of MT to zinc levels also decreased with age suggesting a rapid degradation of MT during postnatal development. Immunohistochemical localization of MT by peroxidase-antiperoxidase technique, using a specific antibody to MT, showed intense intranuclear staining for MT in fetal and newborn rat liver which persisted until Day 9. The nuclear MT staining decreased with age; at 11 days it was equal both in nucleus and cytoplasm and at 14 days, MT was localized mainly in the cytoplasm, similar to adult rat liver pattern. The intranuclear localization of MT in neonates could be considered as a typical fetal-neonatal morphological pattern and its subsequent presence in the cytoplasm, an adult pattern.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Regulation of humoral immune responses by a bone marrow-derived glycolipid-like molecule

Bone marrow cells have been shown to nonspecifically suppress primary in vitro antibody responses. This suppression appears to be mediated by a low-molecular-weight soluble factor, B-SF which was released from a fraction of cells of similar size to the suppressor as obtained by velocity sedimentation. Like the suppressor cell, B-SF was also shown to be effective very early in the immune response. It was produced by all strains of mice tested and functioned across strain barriers. Characterization of the active suppressor molecule showed it to be a highly heat-stable, nonsialic acid-containing glycolipid of 1000 to 35000 daltons in molecular weight. Recovery of the purified suppressor from thin-layer chromatography plates was achieved indicating that the major glycolipid component visualized on TLC is likely the active suppressor molecule. The characteristics of this suppressor may show it to be a fundamental immune regulatory mechanism.     

Delayed hypersensitivity and migration inhibition in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin

Delayed-type hypersensitivity (DTH) and cell migration inhibition (MI) were studied in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) in vitro response of their lymphoid cells to phytochemagglutinin (PHA). A rapid photoelectric procedure for reading cell migrations enabled the study of MI over a wide range (10 log) of antigen concentrations in vitro. Hi/PHA mice required immunization with a 10 times higher dose of ovalbumin (OVA) in Freund's complete adjuvant (FCA) than Lo/PHA mice for a comparable response in DTH (footpad swelling) and MI of their induced peritoneal exudate cells (PEC). Lo/PHA spleen showed marked bizonal MI on Day 5 after immunization with low doses (0.1 and 0.5 micrograms) of OVA in FCA, one peak being obtained in presence of in vitro concentrations of 10(-3) or 10(-2) micrograms/ml OVA and another peak at 1 or 10 micrograms/ml, whereas Hi/PHA spleen showed stimulation of migration. In contrast, MI in Lo/PHA spleen failed to persist beyond Day 19, whereas it appeared progressively in Hi/PHA spleen, being maximal by Day 27. Low-zone inhibition in Hi/PHA spleen and PEC was lacking or poor even after immunization with higher doses of OVA in FCA. The implications of these findings are discussed.     

A link between helper and suppressor factors and the lymphokines migration inhibition factor and migration stimulation factor

The immunogenic and tolerogenic activities of solubilized T2 phage antigens in a primary in vitro response are augmented when these antigens are presented in cell-bound form. This enhancement is shown to be effected equally by an antigen adsorbed to autologous cells, to cells matched or mismatched with respect to major histocompatibility antigens, or to xenogeneic cells. Spleen or lymph node cells are more effective carriers of adsorbed T2 antigens than are thymocytes or erythrocytes. Among lymphocytes, T cells are more effective than B cells. Erythrocytes and B lymphocytes also are shown to bind significantly less solubilized T2 proteins than do T lymphocytes or thymocytes.     

Lymphocyte responses of human neonates to bacterial antigens

Human lymphoid lines derived from normal or neoplastic B cells were assayed for insulin binding. ¹²⁵I-Labeled insulin was allowed to bind to cells. Bound radioactivity which was inhibited with unlabeled insulin was regarded as specific binding. Among 46 lines tested, 43 bound more insulin than normal peripheral B lymphocytes. The majority of the lines resembled activated lymphocytes, with regard to their insulin binding. More mature cells represented by EBV-transformed lines of normal origin, bound more insulin than the less differentiated Burkitt lymphoma lines. However, even the latter bound significantly more insulin than peripheral blood lymphocytes.     

Heterogeneity in the response of T cells to antigens presented by B lymphoma cells

Proliferation of antigen-specific T-cell populations was induced in cultures stimulated with antigen and a suitable source of antigen-presenting cells. Soluble (keyhole limpet hemocyanin) and particulate (horse red blood cells) antigens were presented by irradiated spleen cells and by a variety of B-lymphoma-cell lines, providing support for antigen-specific H-2-restricted T-cell responses. A marked heterogeneity was demonstrated, however, in the capacity of T-cell lines to proliferate in response to antigen presented by the B-lymphoma cells. T-cell populations were prepared from the lymph nodes of antigen-primed mice and restimulated in vitro in the presence of antigen and irradiated spleen cells. During the first six in vitro restimulations, these T-cell populations maintained the capacity to respond to antigen presented either by irradiated spleen cells or by B-lymphoma cells. Continued growth of these T-cell populations, again in the presence of antigen and irradiated spleen cells, resulted in the generation of T-cell lines which had lost the ability to respond to antigen presented by B-lymphoma cells. These lines however, fully retained the capacity to proliferate in the presence of antigen and irradiated spleen cells. T-cell clones derived from one of these lines were also unable to respond to antigen presented by B-lymphoma cells but again proliferated in the presence of antigen and irradiated spleen cells. Supernatants containing high levels of IL-1, IL-2, or IL-3 activity failed to reconstituted the antigen-specific response of T-cell lines which had lost the capacity to respond to antigen presented by B-lymphoma cells. Furthermore, titrated numbers of irradiated spleen cells, while having the capacity to support T-cell proliferation themselves, failed to synergize with B-lymphoma cells in the support of antigen-specific T-cell proliferation. Thus we have defined populations of antigen-specific, H-2-restricted T cells which do not recognize antigen presented by B-lymphoma cells and can therefore discriminate between different antigen-presenting cell types.     

Detection of lipopolysaccharides in polyacrylamide gels by transfer to nitrocellulose followed by immunoautoradiography with antibody and 125I-protein A: "LPS blotting"

Lipopolysaccharides (LPS), which constitute the somatic (O) antigen of gram-negative bacteria, were used to demonstrate the procedure of LPS blotting involving the electrophoretic transfer of electrophoretically resolved LPS from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose filters. Immobilized LPS could then be immunoautoradiographically visualized in situ by reaction with specific anti-LPS antibody and subsequent binding of radioiodinated Staphylococcus protein A. LPS blotting is expected to provide an efficient and specific means of investigating the LPS (O) antigens of gram-negative bacteria.     

Neonatally induced tolerance to soluble protein antigens. I. Analysis of B-cell and T helper cell function in mice tolerant to bovine serum albumin or fowl gamma-globulin

High dose tolerance to either bovine serum albumin (BSA) or fowl γ-globulin (FGG) was induced in CBA mice by neonatal injection. Tolerance to BSA lasted about 9 weeks, and that to FGG, about 18 weeks. Splenic B-cell function was analyzed using quantitative in vivo assays and in vitro limiting dilution analysis. Tolerogen-specific IgM- and non-IgM-producing B cells are depleted at least threefold in the spleens of tolerant mice. Tolerogen-specific T-helper-cell function was examined by immunization with haptenated antigens. Analysis of the recovery from tolerance indicates that the return to normal function in the tolerogen-specific B-cell and T helper fractions coincides with the return to normal responsiveness by the whole animal.     

Correlation of inhibition of fructose 1,6-bisphosphatase by AMP and the presence of the nucleotide-binding domain

Chicken liver fructose 1,6-bisphosphatase binds to blue dextran-Sepharose affinity columns and is eluted by AMP, an allosteric inhibitor of the enzyme. On the other hand, bumblebee fructose 1,6-bisphosphatase, which is not inhibited by AMP, does not bind to blue dextran-Sepharose. Chicken liver 1,6-bisphosphatase binds 3.6 mol of AMP/mol of enzyme, while the bumblebee enzyme binds no AMP. However, bumblebee fructose 1,6-bisphosphatase can be activated by subtilisin, indicating that it possesses a protease-sensitive region similar to that present in mammalian fructose 1,6-bisphosphatase.     

Receptors for the complement C3d component and the Epstein-Barr virus are quantitatively coexpressed on a series of B-cell lines and their derived somatic cell hybrids

Various human Burkitt lymphoma and LCL lines established in vitro and their derived somatic cell hybrids were tested for their comparative EBV receptor levels in a virus binding assay. Their graded C3b and C3d complement receptor expression was estimated simultaneously by means of isotope labeled rosette marker cells. The receptor concentration of each cell line was related to Raji as the standard of comparison, K 562, P3HR-1, and YACUT were used as negative controls. In general, the charging curves for EBV and C3d receptors parallelled each other (r = 0.97) while C3b receptor charging showed no correlation (r < 0.60). In the Raji hybrids between the C3b receptor positive Raji parent and various patents that were negative for this receptor, C3b receptor expression was low or negative. In contrast, the C3d negative P3HR-1 line gave rise to hybrids, after fusion with receptor-positive cells, that were intermediate with regard to their C3d receptor expression. The host range restriction of the Epstein-Barr virus is determined at the receptor level. The close relationship between the EBV receptor and the C3d receptor, a B-lymphocyte-specific moiety, suggests that the moderate interaction with EBV with the B lymphocytes may have had a selective advantage, favoring the presence of EBV. Since EBV causes lytic infections after artificial introduction into nonnatural host cells, it may represent a B-lymphocyte-specific host range mutant, derived from an originally lytic herpesvirus with a much broader target cell range.     

Tumor-specific immunity induced by somatic hybrids: IV. Relationship between immunogenicity and expression of surface tumor-associated antigens

Semi-allogeneic hybrid clones were derived by fusion of the TEPC-15 plasmacytoma (H-2^d) and mouse L cells of C3H (H-2^k) origin. Three representative clones were chosen to study the relationship between the expression of different membrane antigens and their immunogenicities leading to protection of recipient mice from the parent TEPC-15 plasmacytoma. The level of surface tumor-specific transplantation antigens (TSTA) was measured by radioimmunoprecipitation with syngeneic anti-TSTA and by inhibition of anti-TSTA binding to the TEPC-15 tumor cells. The most immunogenic hybrid clone (LTC-1) expressed the highest level of TSTA and the weakly immunogenic (LTC-2) and nonimmunogenic (LTC-4) hybrid clones exhibited relatively low levels of TSTA on the surface. Moreover, the strongly immunogenic LTC-1 hybrid cells, but not the parent tumor cells, were effective in priming recipient spleen cells to generate TEPC-15 tumor-specific cytotoxic cells upon subsequent in vitro exposure to the TSTA-bearing cells. Therefore, the level of TSTA on the semi-allogeneic hybrid clones may play an important role in enhancing the immunogenicity of TSTA.     

Distribution of actin and myosin in mouse trophoblast: Correlation with changes in invasiveness during development in vitro

Mouse trophoblast is an invasive tissue that undergoes conversion to a noninvasive state during normal development. We examined the distribution of actin and myosin during trophoblast development in vitro with double label fluorescence microscopy using fluoresceinated subfragment-1 of myosin to identify actin and indirect immunofluorescence with rhodamine-conjugated antibody to detect myosin. During the outgrowth stage trophoblast spread as a sheet by active movement of the marginal cells. These cells exhibited different patterns of actin and myosin distribution in connection with lamellar extension and fiber formation. Marginal and submarginal cells were packed with overlapping layers of actin fibers, some of which were organized into a lattice that extended throughout the trophoblast. The cytoskeletal function of the fibers appeared to involve maintenance of the cells in a coherent sheet. Cessation of trophoblast spreading was associated with conversion of the cell sheet into a cell network. Cells stained more densely for actin and myosin and contained distinctive actomyosin condensations in the cortex and the cytoplasm. At the same time there was disorganization and then loss of the actin fiber system. These changes in actin and myosin distribution may be associated with mechanisms that control invasiveness by limiting trophoblast expansion.     

Differential sensitivity to 2'-deoxyguanosine of antigen-specific and nonspecific suppressor T cells in delayed hypersensitivity

The cell wall components involved in the adherence of Bacillus globigii to lymphocytes were studied. Lipoteichoic acid inhibited B. globigii-lymphocyte interaction. The interaction was not affected by Escherichia coli outer membranes or lipopolysaccharides.     

Feedback suppression of the immune response in vivo. I. Immune B cells induce antigen-specific suppressor T cells

Stimulated lymphocytes are capable of synthesizing and secreting a variety of lymphokines which can affect the functions of several types of target cells. We report here the existence of a soluble factor released by activated human mononuclear leukocytes which produces a selective inhibition of human pulmonary fibroblast migration. This fibroblast migration inhibitory factor (FIF) was produced by antigen- or mitogen-stimulated human peripheral blood mononuclear leukocytes (PBML) and purified T cells. It inhibited the migration of ⁵¹Cr-labeled fibroblasts in a dose-dependent fashion with optimal effect (65–70% inhibition) obtained at 1:10 dilution and 8–20 hr of incubation. Sephadex G-100 fractionation revealed most activity to be found between 28,000 and 34,000 daltons. FIF was stable at 56 °C for 15 min, but destroyed at 80 °C or at low pH. This factor may play an important role in the modulation of fibrogenesis and healing processes by the immune system.     

Human brain monoamine oxidase: solubilization and kinetics of inhibition by octylglucoside

Complete solubilization of both the A and B forms of human brain monoamine oxidase (MAO) occurred when crude mitochondria were incubated in the presence of 50 mM octylglucoside (OG). Upon removal of this nonionic detergent by dialysis, approximately 100% of the starting activity was present in the dialysate. The effects of solubilization were examined by comparison of several properties of the membrane-bound and OG-treated oxidases. The percentage inhibition of phenylethylamine (PEA) and the 5-hydroxytryptamine (5-HT) deamination by deprenyl and clorgyline were identical. The Km values obtained for the deamination of PEA, a B-selective substrate, 5-HT, an A-selective substrate, and tyramine (TYR), a nonselective substrate, were also comparable. OG was found to inhibit type A (I50 = 8.1 mM) and B (I50 = 4.7 mM) MAO activities at concentrations at least 10-fold below those used to solubilize the oxidases. Kinetic studies revealed that OG was an apparent competitive inhibitor of PEA deamination whereas OG produced a mixed-type pattern of inhibition when 5-HT was the variable substrate. Inhibition of TYR deamination by either the A or B form of MAO produced a mixed pattern of inhibition. The findings herein suggest that solubilization of the A and B forms of MAO by OG does not significantly alter the substrate and inhibitor specificity of the oxidases following removal of detergent. However, in the presence of concentrations of OG 50 times less than the critical micellar concentration of this detergent, marked inhibition of deamination by both forms of human brain MAO is observed. Accordingly, the usefulness of OG is limited to situations where the detergent is completely removed before quantitation of MAO activity.     

Determination of levels of glycolytic intermediates and nucleotides in platelets by pulse-labeling with [32P]orthophosphate

A method is presented for the separation and quantification of ³²P-labeled carbohydrates and nucleotides in blood platelets which have been pulse-labeled with [³²P]orthophosphate. The procedure is based on two-dimensional paper chromatography, identification of the spots by radioautography and enzymatic methods, and quantitation of ³²P radioactivity by liquid scintillation counting. The data show that ³²P is homogeneously distributed among the compounds studied so that the total radioactivity is proportional to the levels of these compounds in the metabolic compartment of the cells. Thus, this method provides a sensitive and accurate means to evaluate phosphorylated intermediates in glycolysis and nucleotide metabolism and to assess the transfer of energy-rich phosphate groups between these pathways in particular.     

Preparation and properties of biologically active radioiodinated parathyroid hormone

A constant-current microelectrolytic radioiodination method was used to label bovine parathyroid hormone (BPTH) with ¹²⁵I to an overall iodination ratio of 1:1 iodide atoms per PTH molecule. Such iodinated preparations were shown to be fully active in several bioassay systems: in vitro adenylate cyclase activation in rat renal and skeletal membranes, in vitro calcium release from rat calvaria, and the in vivo hypercalcemic response in chickens. Analysis by Sephadex G-15 chromatography after enzymatic digestion showed the radioiodine to be incorporated predominantly as monoiodotyrosine. Bioassay of iodinated preparations from which uniodinated hormone had been removed by isoelectric focusing showed essentially full hormonal activity. Such methods can be used to consistently produce radioiodinated biologically active preparations of BPTH 1–84 with high specific activity (2000 Ci/mmol).     

The suppression of experimental allergic encephalomyelitis in Lewis rats by treatment with myelin basic protein-cell conjugates

Pretreatment of Lewis rats with guinea pig (GP) myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) suppressed the subsequent induction of experimental allegic encephalomyelitis (EAE) with GP-MBP in Freund's complete adjuvant. The degree of suppression correlated positively with the amount of antigen coupled to the SL. GP-MBP coupled to syngeneic red blood cells (RBC) also resulted in suppression of EAE and the extent of the suppression was related to the dose of cells. These regimens of pretreatment also resulted in a decrease in the in vitro lymphocyte proliferative response to GP-MBP and in the extent of perivascular cuffing in the spinal cord. No decrease in the anti-MBP antibody response was detected in rats pretreated with either GP-MBP-SL or GP-MBP-RBC conjugates. Transfer of lymph node cells from rats pretreated with GP-MBP-RBC resulted in a decrease in disease severity in recipients. It is concluded that prior administration of MBP-cell conjugates is an effective way of suppressing the symptoms of EAE.     

Functional evaluation of murine allogeneic T lymphoblasts separated by Vicia villosa lectin positivity

Mixed-lymphocyte culture-stimulated cells have been fractionated by their ability to bind the lectin Vicia villosa (Vv) and assessed for their cytolytic and suppressor activity in vitro. Vv positive and negative cells were separated either by cell affinity chromatography using Vv-Sepharose 6MB chromatography or by electronic cell sorting with FITC-Vv. Both populations expressed marked cytolytic and suppressor cell activity. Thus this lectin cannot be used to discriminate between these and other functional lymphoid cell population of blastoid cells binding the FITC-Vv appears following allogeneic stimulation; treatment with the immunosuppressive drug cyclosporin A, which affects cytotoxic cells preferentially, results in a considerable reduction of the Vv positive blastoid cells.