Hapten-specific carrier-dependent tolerance induction in man in vitro

We sought to determine whether hapten-specific tolerance can be induced in cultured human lymphocytes in vitro. Unfractionated as well as T and B cells from peripheral blood lymphocytes of healthy human volunteers were cultured with different hapten-carrier conjugates before in vitro challenge with dinitrophenyl (DNP) linked to keyhole limpet hemocyanin. Hapten-specific antibody was detected in the supernatant by solid-phase radioimmunoassay. Both hapten specificity and carrier dependence in addition to the cellular basis of tolerance induction were examined. The results show that hapten-specific tolerance of antibody production was induced by human gamma-globulin (HGG) conjugated to DNP but not by other conjugates of DNP nonhuman gamma-globulin, as well as human serum albumin. Moreover, both T and B cells are involved in tolerance induction to DNP-HGG in vitro. The significance of tolerance in human in vitro for the specific therapy of autoimmune disease is discussed.     

Characterization of an immune suppressor from transformed human trophoblastic JEG-3 cells

Cultured human choriocarcinoma JEG-3 cells secrete an immunosuppressor that inhibits lymphocyte proliferation stimulated by either an antigen or a mitogen. In this study, the immunosuppressive factor was characterized by three methods: ion-exchange and exclusion chromatography, partition in organic solvents, and thin-layer chromatography on silicic acid. This JEG-3 cell factor appeared to be a protein complex of about 150,000–200,000 Da that contained an immunologically active polar lipid. The structural and functional characteristics of JEG-3 cell immunosuppressor are similar if not identical to those of SIF, a suppressor lymphokine derived from T cells. These secretions from transformed trophoblastic cells may correspond to normal placental products or represent a function of malignant cells.     

Nucleoside-specific suppression in MRL/MP +/+ mice

The induction of nucleoside-specific nonresponsiveness was further studied in the autoimmune strain MRL/MP +/+ (MRL/n). Experiments were undertaken to determine (i) whether nucleoside-conjugated spleen cells are able to induce specific nonresponsiveness to T-dependent nucleoside antigens in MRL/n mice, and (ii) whether periodic treatment with nucleoside-conjugated spleen cells would retard the development of spontaneous anti-DNA antibodies and associated indicators of autoimmunity. The results show that nonresponsiveness to nucleoside antigens is inducable in male, but not in female, MRL/n mice. Nonresponsiveness in male MRL/n was transferable and mediated by T cells. Treatment of male MRL/n mice with nucleoside-conjugated spleen cells (NSC) appeared to attenuate the progress of autoimmune symptoms in experimental animals. These results are discussed in the context of recent studies exploring the etiology of autoantibody production and the loss of self-tolerance in murine models of autoimmunity.     

Influence of cortisone on immunologic tolerance in vivo: Cortisone prolongs the duration of unresponsiveness but fails to affect its induction

The influence of cortisone administration on either the induction or the duration of immunologic tolerance was examined in vivo. Tolerance induced by isologous IgG coupled to fluorescein was chosen because the hapten-bearing cell can be directly visualized and the hapten-specific immune response to either a TD antigen or a TI₂ antigen can be tested. It was found that cortisone facilitates the maintenance of tolerance, but fails to affect its induction to either class of antigen. Fluorescein-IgG-bearing cells are cortisone resistant. They are seen for a longer period of time in animals treated with cortisone and tolerogen than in animals treated with tolerogen, and fluorescent cells are either T or B cells. We propose that cortisone facilitates the maintenance of tolerance by maintaining a receptor blockade in vivo. This finding might have clinical implications for the treatment of autoimmunity.     

Dexamethasone-resistant cystic fibrosis fibroblasts show cross-resistance to sex steroids

Diploid skin fibroblasts derived from individuals with the autosomal recessive disease, cystic fibrosis (CF), were shown previously to be significantly more resistant to the cytotoxicity of dexamethasone, a glucocorticoid hormone, than were normal human fibroblasts. Here cystic fibrosis fibroblasts are also shown to be more resistant than normal human fibroblasts to the cytotoxic effects of the sex hormones, 17 beta-estradiol, dihydrotestosterone and progesterone. Since cells are believed to contain different receptors for each of the steroid hormones, it is not probable than the resistance of CF cells to these hormones results from a receptor deficiency. This was shown by the fact that CF cells were found to exhibit the same receptor activity as normal cells for 3-H-dexamethasone. Furthermore, neither normal human nor CF fibroblasts could be demonstrated to contain detectable receptor activity for 3H-17 beta-estradiol. In addition, the studies of fibroblast killing by hormones led to the further interesting observation that normal human diploid fibroblasts, regardless of the sex of the tissue donor, are sensitive to killing by each of the sex hormones. These findings suggest that the cytotoxic effects of the steroid hormones may be observed independently of the specific hormone receptors. The studies reported here thus suggest that the resistance of CF cells to the different steroid hormones is probably the result of a defect in a pathway in cellular steroid hormone metabolism other than that involving receptors.     

Characterization of cross-reacting antigens on the Epstein-Barr virus envelope and plasma membranes of producer cells.

A rabbit antiserum has been prepared against the B95-8 transforming strain of EBV. The antiserum has a high virus neutralizing titer (approximately 1:1000) against both the marmoset B95-8 EBV and the human P3HR-1 EBV. The neutralizing antibodies may be absorbed completely with EBV producer cell lines, but not with nonproducer cell lines or producer cell lines treated with phosphonoacetic acid (PAA) so as to be nonproducer. After repeated absorption with PAA-treated B95-8, the serum remains reactive with the membranes of producer cell lines as judged by immunofluorescence or the 125I--Staphylococcal protein A radioimmunoassay. Thus the neutralizing antigens are expressed on the membranes of producer cell lines and may be purified from this source using the serum and 125I--Staph A binding as an assay. The ability of the serum to differentiate between producer and nonproducer cells by means of cell surface determinants has been exploited to achieve a separation of these two populations from the same culture. Immunoprecipitation by the protein A technique shows that the serum recognizes two polypeptides from producer cells of approximate molecular weights 150,000 and 75,000.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

Mouse bone collagenase inhibitor: purification and partial characterization of the inhibitor from mouse calvaria cultures

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.     

Prevention of overoxidation in periodate oxidation of reducing oligosaccharides by their conversion into 1,5-anhydroalditol derivatives

A procedure for the conversion of reducing oligosaccharides into their 1,5-anhydroalditol derivatives was devised to prevent overoxidation during periodate oxidation. Gas-chromatographic analysis of the aldehydes in the products of complete oxidation of the resultant 1,5-anhydroalditol derivatives by the dithioacetal method^1b indicated that new types of dialdehyde characteristic of the linkage-types were formed, together with ordinary simple aldehydes. A number of d-gluco-oligosaccharides having various types of interglycosidic linkage were examined by this method. The results were consistent with expectations.     

High-performance liquid chromatographic determination of indoleamines, dopamine, and norepinephrine in rat brain with fluorometric detection

A high-performance liquid chromatography-fluorescence procedure for the determination of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan, dopamine, and norepinephrine has been developed. The method uses an ion-pairing system on an Ultrasphere ODS (5-microns) column with detector wavelength settings of excitation at 290 nm and emission at 330 nm. The procedure has been used to quantitate these indoleamines and catecholamines in rat brain tissue after homogenization in a perchloric acid solution; an aliquot of this solution is injected directly onto the HPLC column. Column sensitivities range from 6.1 pmol for tryptophan to 1.1 pmol for 5-hydroxytryptamine.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

The catalytic effect of tyrosinase upon oxidation of 2-hydroxyestradiol in presence of catechol

The hydroxylating activity of mushroom tyrosinase has been utilized for over a decade in the preparation of 2-hydroxyestradiol from estradiol, yet this same enzyme is known to function as an oxidant of o-dihydric compounds to the corresponding o-quinones. It was questioned why catechol estrogens do not react further, particularly since the tyrosinase activity (hydroxylating) is exceeded many fold by the diphenol oxidase activity of the enzyme. This report describes that the catechol estrogen will react in presence of enzyme but only if catechol is also present. Diphenol oxidase activity was measured either by the polarographic oxygen-utilization technique or by changes in the absorption spectrum at 206 and 256 nm. The enzyme activity was standardized with catechol (Km = 5.2 X 10(-4) M). The steroid did not react with the enzyme if catechol was absent. With catechol, the steroid reacted rapidly and completely (Km = 4.2 X 10(-4) M). The consumption of oxygen with catechol and 2-hydroxyestradiol was additive and stoichiometric, 1 g-atom oxygen/mol of either substrate. Kinetic analysis shows that catechol functions as an activator of the tyrosinase.     

Microfluorometric detection of asymmetry in the centromeric region of mouse chromosomes

A lateral asymmetry in the centromeric region of mouse chromosomes is revealed in studies involving the BUdR quenching of 33258 Hoechst fluorescence. This cytologically detected asymmetry may reflect the unequal distribution of thymidine between the two chains of mouse satellite DNA.     

The dissociation of the extracellular hemoglobin of Tubifex tubifex at extremes of pH and its reassociation upon return to neutrality

The dissociation of the extracellular hemoglobin of Tubifex tubifex at alkaline and acid pH, and its reassociation upon return to neutral pH, was investigated using gel filtration, ultracentrifugation, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). Tubifex hemoglobin dissociated at pH above 8 and below 6; both dissociations appeared to be equilibrium processes. The extent of dissociation increased as the pH moved away from neutrality; although dissociation was virtually complete at pH 11, its extent at acid pH did not exceed 50–60% at pH 4. Ca(II), Mg(II), and Sr(II) cations over the range 1–100 mm decreased the extent of the dissociation only at alkaline pH. The visible absorption spectrum of the oxyhemoglobin remained unaltered in the pH range 4–9. At more extreme pH, it changed with time, altering irreversibly to that of the aquo ferri form. Gel filtration of the hemoglobin at both extremes of pH showed that it dissociated into two heme-containing fragments; one consisting of subunit 1 (M_r ~ 17,000) and the other containing subunits 2, 3, and 4 of the hemoglobin (M_r ~ 60,000). Upon return to neutral pH, the dissociated fragment reassociated to the extent of 50 to 80% to whole hemoglobin molecules. The reassociation decreased with increase in alkaline pH, and with decrease in acid pH to which the hemoglobin had been exposed; it increased in the presence of Ca(II), Sr(II), and Mg(II) only subsequent to dissociation at alkaline pH. The SDS-PAGE patterns, gel-filtration elution volumes, and α-helical contents, determined from circular dichroism at 222 nm, of the reassociated whole molecules were identical to those of the native hemoglobin.     

Two-dimensional, computer-controlled film scanner: quantitation of fluorescence from ethidium bromide-stained DNA gels

A two-dimensional scanner based on a digital plotter is described. The device is used to analyze photographic negatives of ethidium bromide-stained DNA-agarose gels. Scanning is controlled by and photometric data transferred to a computer for processing, storage, display, and analysis such as integration of the areas under bands and determination of the mean distances of migration of polydisperse samples. An integral light source and detector module designed for reading optical "bar-codes" is mounted in place of the pen of the plotter. Spatial resolution and reproducibility are about 0.2 and 0.005 mm, respectively. Photometric precision as good as one part per thousand is achieved by sinusoidal modulation of the intensity of the light source and synchronous, phase-sensitive detection of the signal from the detector by a lock-in amplifier. No part of the sensor assembly touches the surface of the negative. In contrast to a densitometer, the computer transforms photometric data to values directly proportional to the amount of DNA at given points on the original gel. The ability to move the sensor in two dimensions over the negative allows for the integration across the width of a lane correctly allowing for the nonuniform distribution of the DNA.     

Determination of picogram quantities of zinc in zinc metalloproteins by atomic absorption spectrometry using a graphite furnace atomizer

Optimum operating conditions have been determined for the atomization of zinc from metalloproteins in a graphite furnace. Addition of 50 mm ammonium dihydrogen phosphate to to protein and measurement of the integrated absorbance suppresses or eliminates matrix interference effects. Using a 5-μl sample both the sensitivity and the detection limit are 0.3 ng of Zn/ml, i.e., 1.5 pg of zinc on an absolute basis. For 10 ng/ml of zinc in 5-μl samples of a zinc metalloenzyme, the coefficient of variation is 1.5%. Accuracy has been established by analysis of zinc metalloenzymes of known zinc stoichiometry. The method has been applied successfully to the determination of zinc in several proteins for which zinc stoichiometry had been unknown.