A murine model system for contact sensitization to poison oak or ivy urushiol components

A murine model of contact sensitization to components of poison oak or ivy urushiol oils was developed. Sensitization was effected by painting such compounds on abdominal skin, and was routinely assessed by challenging on the ears and monitoring increases in ear thickness. Sensitization to 3-heptadecylcatechol (HDC, a component of poison oak urushiol) was studied in detail. Contact sensitivity as indicated by ear swelling reactions was observed from 2 until around 25 days after primary abdominal painting with HDC. In all cases maximal ear swelling occurred 3–4 days after HDC challenge. Sensitivity could also be assessed by monitoring the uptake of radioiodinated deoxyuridine at the ear challenge site, and this correlated with the ear swelling assay in terms of kinetics. The sensitization effect induced by HDC had properties of delayed-type hypersensitivity, being antigen specific, and transferable with sensitized lymph node and spleen cells but not by serum. Also, T cells were required for activity as transfer with spleen cells was abrogated by treatment with anti-Thy-1.2 antibody and complement. In this system HDC and 3-pentadecylcatechol (PDC, a component of poison ivy urushiol oil) were completely cross-reactive both in sensitization and challenge, and both compounds also cross-reacted with native urushiol oil itself. Thus murine sensitization to HDC can be used as a model system for investigating mechanisms for the immunogenicity of such catechols.     

Relationship of In Vivo MR Parameters to Histopathological and Molecular Characteristics of Newly Diagnosed,Nonenhancing Lower-Grade Gliomas

The goal of this research was to elucidate the relationship between WHO 2016 molecular classifications of newly diagnosed, nonenhancing lower grade gliomas (LrGG), tissue sample histopathology, and magnetic resonance (MR) parameters derived from diffusion, perfusion, and ¹H spectroscopic imaging from the tissue sample locations and the entire tumor. A total of 135 patients were scanned prior to initial surgery, with tumor cellularity scores obtained from 88 image-guided tissue samples. MR parameters were obtained from corresponding sample locations, and histograms of normalized MR parameters within the T2 fluid-attenuated inversion recovery lesion were analyzed in order to evaluate differences between subgroups. For tissue samples, higher tumor scores were related to increased normalized apparent diffusion coefficient (nADC), lower fractional anisotropy (nFA), lower cerebral blood volume (nCBV), higher choline (nCho), and lower N-acetylaspartate (nNAA). Within the T2 lesion, higher tumor grade was associated with higher nADC, lower nFA, and higher Cho to NAA index. Pathological analysis confirmed that diffusion and metabolic parameters increased and perfusion decreased with tumor cellularity. This information can be used to select targets for tissue sampling and to aid in making decisions about treating residual disease.     

A direct, stimulating effect of cyclic GMP on purified phosphoribosyl pyrophosphate synthetase and its antagonism by cyclic AMP

The activity of phosphoribosyl pyrophosphate synthetase, purified from a line of rat hepatoma cells in continuous culture, is maximally stimulated (2–4 fold) by less than 10^?7M cyclic GMP. Half maximal stimulation occurs at 2 × 10^?9M. Cyclic GMP stimulates phosphoribosyl pyrophosphate synthetase by decreasing the Km of the enzyme for ATP from 50 μM to 10 μM without affecting the Vmax; it has no effect on the Km for ribose 5-phosphate, the other substrate. Cyclic AMP alone has no effect on the enzyme activity, but at micromolar concentrations it antagonizes the stimulation by cyclic GMP. GMP, GDP, and GTP do not stimulate enzyme activity; and AMP and ADP at micromolar concentrations do not antagonize the effect of cyclic GMP.There is no detectable cyclic nucleotide-activated protein kinase in the enzyme preparation. Cyclic GMP significantly stabilizes the enzyme to heat inactivation. We conclude that cyclic GMP binds directly to the enzyme in an allosteric fashion, causing it to have an increased affinity for one of its substrates, and that cyclic AMP directly antagonizes this effect.     

Inhibition by somatostatin of the proliferation of T-lymphocytes and Molt-4 lymphoblasts

The proliferation of Molt-4 lymphoblasts and phytohemagglutinin-stimulated human T lymphocytes in vitro was inhibited significantly by 10(-12) to 10(-10) M to 10(-13) to 10(-9) M somatostatin, as assessed by the uptake of [3H]thymidine and [3H]leucine, respectively. The inhibitory effect of somatostatin was not attributable to cytotoxicity and was associated with a mean degradation of 52 and over 95% of immunoreactive somatostatin, respectively, after 3 and 24 hr of incubation at 37 degrees C. The specific suppression of Molt-4 lymphoblasts and T lymphocytes by somatostatin represents a distinct mechanism for the specific regulation of immunological responses by neuropeptides of the peripheral nervous system and gastrointestinal tract.     

Reversible inhibition by interferon of the maturation of human peripheral blood monocytes to macrophages

We demonstrated that interferon delays the maturation of human monocytes to macrophages in vitro as assessed by morphologic, histochemical, and biochemical parameters. After exposure to interferon, monocytes were slightly smaller, less stretched out, and had a delayed loss of granules with peroxidase positive reactivity, as compared with control, noninterferon-treated cells. Also, interferon prevented the increase of the specific activity of three lysosomal enzymes (β-galactosidase, β-glucuronidase, and β-N-acetylglucosaminidase) in the monocytes, and enzyme activities were 30–40% of that observed in untreated cells. Both human leukocyte and human fibroblast interferons were effective in delaying the maturation process. The effects of the interferon were species specific and reversible and were neutralized by antiinterferon serum. These studies describe a new nonantiviral effect of interferon, unique in that it involves the delay of maturation of cells in a system which is not associated with cell proliferation. Thus interferon could potentially effect host defense mechanisms and aspects of the immune response which are dependent on the maturation of monocytes to macrophages.     

Simultaneous isolation of major viral proteins in one step.

We have developed a rapid and simple technique for the simultaneous isolation of all the major viral proteins from RNA tumor viruses. The basis for this procedure is analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using dansylated virus as internal marker it is possible to follow the migration of unlabeled viral proteins since dansylation does not change the mobility of labeled proteins (8). The method results in approximately 80% recovery of starting protein and is very reproducible. Using radioimmunoassay no alteration of the purified proteins is detectable.     

Study of granule reappearance and histamine synthesis in rat mast cells maintained in short-term cultures

A single cutaneous application of components of poison oak or ivy urushiol oils to mice results in contact sensitivity with properties of delayed-type hypersensitivity. The compounds, 3-heptadecylcatechol (HDC, from poison oak urushiol) and 3-pentadecylcatechol (PDC, from poison ivy urushiol) are completely cross-reactive. Covalent bond formation between the o-quinone intermediate of PDC and nucleophilic functionalities such as those found on proteins is known to occur in a regiospecific manner. Amino nucleophiles preferentially attack the 5-position on the catechol ring while thiol nucleophiles attack the 6-position. The present paper describes the immunological properties of the three possible ring monomethylated analogs of PDC. When mice were treated with a single epicutaneous painting with these analogs, only the 5-methyl compound (5-Me-PDC) was found to be an ineffective sensitizer. The 5-Me-PDC analog, however, was capable of inducing cellular proliferation in draining lymph nodes. Furthermore, epicutaneous pretreatment with 5-Me-PDC suppressed the subsequent induction of contact sensitivity to PDC and HDC in an antigen-specific manner. Equivalent treatment with the 6-Me-PDC analog (a good sensitizing agent) resulted in a less consistent and weaker suppressive effect, while the 4-Me-PDC analog did not display any suppressive activity. The suppressive activity could be demonstrated up to 15 days following primary painting with 5-Me-PDC. Lymph node cells obtained from mice 10 days after a single painting with 5-Me-PDC could transfer the suppressive effect. Under certain circumstances 5-Me-PDC could also sensitize, indicating that the analog retains some sensitizing abilities. Hence, blocking the 5-position of the catechol ring results in a major alteration in the type of immune response elicited. Since 5-Me-PDC is specifically blocked in terms of nucleophilic attack by amino groups, it is suggested that attack by amino nucleophiles is of primary importance in the peripheral metabolic processing of these catechols, which in turn determines the outcome of the immune response.     

Deoxyguanosine toxicity in a mouse T lymphoma: relationship to purine nucleoside phosphorylase-associated immune dysfunction.

The absence of either of the enzymes adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with an immunodeficiency disease. Because all four nucleoside substrates of the enzyme purine nucleoside phosphorylase accumulate in the urine of patients who lack this enzyme (Cohen et al., 1976), we examined the toxicity of each of the four substrates using a mouse T cell lymphoma (S49) in continuous culture. Of the four substrates (inosine, deoxyinosine, guanosine and deoxyguanosine), only deoxyguanosine is cytotoxic at concentrations lower than 100 μM; furthermore, only deoxyguanosine is directly phosphorylated in S49 cells. Mutant S49 cells lacking deoxycytidine kinase (EC 2.7.1.74) are resistant to the toxic effects of deoxyguanosine, and these same mutants do not phosphorylate deoxyguanosine. Thus the cytotoxicity of exogenous deoxyguanosine correlates with the intracellular concentration of accumulated deoxyGTP.The addition of deoxyguanosine results in the depletion of deoxyCTP in S49 cells, indicating that deoxyGTP is an inhibitor of ribonucleotide reductase. Furthermore, the addition of deoxycytidine prevents the toxic effects of deoxyguanosine. Thus a therapy for purine nucleoside phosphorylase-deficient patients might include deoxycytidine to alleviate the proposed deoxyCTP starvation in those tissues capable of phosphorylating deoxyguanosine.     

Timing and irreversibility of Müllerian duct inhibition in the embryonic reproductive tract of the human male

A study was undertaken to determine (1) the effects of endogenous Müllerian inhibiting substance (MIS) on the developing human fetal genital tract; (2) the time in fetal life when MIS is first capable of inhibiting the growth of the embryonic Müllerian ducts; and (3) the reversibility of the effects of MIS on the developing male Müllerian ducts. Human fetal reproductive tracts were transplanted and grown for sustained periods in vivo in athymic nude mice. The genital tracts from 12 male human fetuses, ages 51 to 68 days postovulation, were grafted without their associated gonads into castrated murine hosts and grown for 30 to 70 days. Controls consisted of genital tracts from 8 female human fetuses, ages day 53 to 70 that were grown under identical conditions. Male specimens grew to approximately one-half the size of female specimens and disclosed varying degrees of inhibition of the Müllerian duct system from absence of the Müllerian ducts in older specimens (after Day 63) to poorly segregated segments of stroma as the mildest defect (less than Day 61). It is concluded that (1) MIS secretion by the embryonic testes probably begins before Day 51 of gestation; (2) the effects of MIS are progressive during the so-called critical window; (3) the effects of MIS are permanent; and (4) the mesenchyme is an important target of MIS.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

Alkyl alkanethiolsulfonate sulfhydryl reagents: β-Sulfhydryl-modified derivatives of l-cysteine as substrates for trypsin and α-chymotrypsin

Derivatives of l-cysteine and the A chain of bovine insulin have been chemically modified at the cysteinyl β-sulfhydryl by certain sulfhydryl-specific alkyl alkanethiolsulfonate reagents. The alkanethiolation products possess mixed-disulfide side chains structurally similar to the side chains of lysine and phenylalanine and hence were studied here as substrates for trypsin and α-chymotrypsin, respectively. Kinetic parameters were obtained for the enzyme-catalyzed hydrolyses of the modified l-cysteine analogs and of specific reference amino acids which were derivatized analogously at both the α-amino and α-carboxyl groups and assayed identically. For both enzymes it was found that the specificity constants, $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, for analog esters compare favorably with those for specific reference esters, whereas specificity constants for analog amides compare much less favorably with those for specific reference amides. This discrepancy is largely a consequence of the k_cat values for the analog amides being relatively much lower than the corresponding values for the reference amides. Consistent with this trend, no detectable enzyme-catalyzed hydrolysis of the amide bonds at the sites of modified cysteine residues in the A chain of bovine insulin was observed. It is proposed that the predominant kinetic consequence of the mixed-disulfide side chains of the alkanethiolated cysteine moieties is a decrease in the acylation rate constants, k₂, arising from an increase in the transition-state free energies of acylation.     

Alpha melanocyte stimulating hormone inhibits prolactin secretion in fetal and infant lambs

The intravenous administration of αMSH (25 μg/kg) to 11 lambs (3 to 29 days of age) suppressed plasma PRL by 15 minutes. The mean basal concentration was 15.3 ± 2.9 ng/ml and the mean nadir was 4.9 ± 0.8 ng/ml (p<0.01). In chronically catheterized fetuses (128–140 days), intravenous administration of αMSH (25 μg/kg) decreased basal PRL levels (89.6 ± 12.4 ng/ml) significantly at 15–30 minutes to levels of 74.3 ± 11.4 ng/ml (p<.01). The degree of suppression of basal PRL levels was less in fetusus (76.9 ± 4.1%) than that induced in the neonates (40.5 ± 7.1%). In younger fetuses <120 days in whom basal PRL levels are low (3.0 ± 2.1 ng/ml), administration of αMSH was without effect. Plasma GH concentrations were not altered by administration of αMSH. The suppression of PRL secretion by αMSH administration could result from increased release of hypothalamic dopamine or be a direct effect on secretion of prolactin by the pituitary.     

T-lymphocyte function and sensitivity to interferon in trisomy 21

Previous studies have shown that cells from subjects with trisomy 21 have enhanced sensitivity to the antiviral effects of interferon, presumably because of the location of the gene, IfRec, coding for the species-specific response to interferon on chromosome 21. Interferon is also known to have many other effects including the ability to inhibit the proliferation of many types of cells. To determine whether proliferating trisomic lymphocytes are more sensitive to the antiproliferative effect of interferon we have investigated, using healthy noninstitutionalized subjects with trisomy 21, the ability of interferon to inhibit the proliferation of lymphocytes stimulated with phytohemagglutinin P(PHA), concanavalin A (Con A), and tetanus toxoid. The trisomic subjects had normal numbers of peripheral blood leukocytes, and normal numbers and proportions of T and B lymphocytes. The production of interferon by PHA-stimulated trisomic T lymphocytes was normal. Trisomic lymphocytes also had normal proliferative responses to PHA and Con A. There were no differences between the inhibitory effects of interferon on the proliferation of PHA-stimulated trisomic and normal lymphocytes. However, trisomic lymphocytes stimulated with low doses of Con A did display significantly enhanced sensitivity to the antiproliferative effects of interferon. In contrast to normal lymphocytes, trisomic lymphocytes were not stimulated to proliferate by tetanus toxoid, and exposure to interferon resulted in enhancement, rather than inhibition, of DNA synthesis.     

Involvement of the neurointermediate lobe of the pituitary gland in the secretion of prolactin and luteinizing hormone in the rat

The relationship between prolactin (PRL) secretion and the neurointermediate lobe (NIL) of the pituitary gland was investigated. Plasma PRL concentrations in rats bearing anterior pituitaries autografted with or without the NIL to the renal capsule were elevated to equal extents at 1 through 6 weeks after surgery (p > 0.10). PRL levels in ovariectomized rats in which the NIL had been removed surgically (NIL-X) or only visualized (NIL-C) were 3–7 ng/ml 4, 7, and 28 days after surgery (p > 0.10); however, they were slightly higher in NIL-X $\text{vs.}$ NIL-C rats 14 days after surgery (p < 0.05). Plasma luteinizing hormone (LH) concentrations in NIL-C rats increased by 36% from 2 to 4 weeks after surgery (p < 0.05); this increase was not detected in NIL-X rats. PRL and LH surges were induced by estradiol implants in ovariectomized NIL-X and NIL-C rats; the profiles of the PRL surges were superimposable, although the magnitude of the LH surge was only 50% that in NIL-C rats (p < 0.05). These results cast doubt on the importance of the NIL in the regulation of PRL secretion either $\text{via}$ secreting hypophysiotropic hormones or $\text{via}$ conducting anterior pituitary hormones directly to the median eminence. However, the NIL may have a physiologically important role in the regulation of LH secretion.     

Induction kinetics of human suppressor cells in the mixed lymphocyte reaction and influence of prednisolone on their genesis

The induction kinetics of human suppressor cells in mixed lymphocyte cultures (MLC) and the influence of prednisolone on the genesis of these suppressor cells is reported. We induced over 1 to 6 days suppressor cells in one-way MLC (MLC-1), the inhibitory activity of which was tested on a secondary MLC (MLC-2), and on responder cells alone, where lymphocytes were obtained from the same lymphocyte donors as for the MLC-1. In four experiments the degree of inhibition ($\text{x}\text{?}\text{±}\text{SE}$) when suppressor cells were induced for 2, 4, or 6 days was 38.5 ± 11.8, 79.5 ± 7, and 85 ± 6%, respectively, compared to 50.5 ± 9.4, 83.3 ± 7.8, and 85.3 ± 9.8% when 500 ng/ml prednisolone was added to the MLC-1. A similar inhibition pattern was observed when the generated suppressor cells were incubated with responder cells only. The inhibitory activity of these MLC-induced suppressor cells was abrogated by irradiation with 3000 R. Suppressor cells apparently are generated in MLCs between Days 1 and 4; furthermore, their genesis is not affected by usual therapeutic concentrations of prednisolone.     

Further studies on the effect of aldosterone on electrical resistance of toad bladder

The fall in transepithelial electrical resistance which accompanies aldosterone stimulation of short-circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) in toad urinary bladder has been studied further to evaluate the possible causal role of this response in hormonal stimulation of Na⁺ transport. A steady-state change in tissue conductance was found to depend upon both the simultaneous stimulation of transport by the steroid and the metabolic state of the tissue. Changes in metabolic state alone did not alter resistance. A sustained increase in Na⁺ transport, dependent on pretreatment with aldosterone and elicited by addition of glucose, could be obtained without a sustained decrease in resistance. Amiloride, an inhibitor of Na⁺ uptake, produced changes in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ that were linearly correlated with its effects on tissue conductance. On the basis of the $\text{conductance}\text{-I}{}_{\mathrm{<mtext>sc</mtext>}}$ relationship with amiloride, the $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ response to aldosterone was about two-fold higher than would be predicted from its effects on conductance alone. Despite the apparent lack of a simple quantitative dependence of the change in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ on the change in conductance when the response is fully developed, the results suggest that conductance changes may mediate the initial or early stage of the response.     

Intercellular adhesion in the cellular slime mold Polysphondylium pallidum inhibited by interaction of asialofetuin or specific univalent antibody with endogenous cell surface lectin.

Previous work has shown that, as amoebae of the cellular slime mold Polysphondylium pallidum become aggregation competent, they accumulate on their cell surface a carbohydrate-binding protein (lectin) named pallidin. These amoebae also possess cell surface receptors, presumed to contain complex oligosaccharides with a high affinity for the endogenous lectin. If lectin-receptor interactions mediate cell-cell contact, then appropriate concentrations of pallidin inhibitors should block cell cohesion. Two potent macromolecular antagonists of the lectin were employed: the desialylated form of the glycoprotein fetuin and the univalent antibody (Fab) prepared against pallidin. We studied the effects of these inhibitors on rotation-mediated aggregation of P. pallidum amoebae under a variety of assay conditions. Amoebae exposed to hypertonic conditions or to antimetabolites (“Permissive conditions”) were selectively blocked from associating by microgram quantities of the lectin inhibitors, whereas cells in isotonic buffer (“nonpermissive condition”) were only slightly affected. A comparison of the morphology of agglutinates formed under the various conditions allows several explanations for the different susceptibilities to inhibition by antipallidin reagents. Although not conclusive, the work supports a model of cell adhesion in this simple eukaryotic system based at least in part on specific interactions between carbohydrate-binding proteins and receptors on adjoining cells.     

The effect of cyclic nucleotides on purine biosynthesis and the induction of PRPP synthetase during lymphocyte activation

The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.     

Aldosterone plasma radioimmunoassay interference by a spirolactone metabolite

The plasma aldosterone radioimmunoassay developed by Ito et al. was found to be non-specific for aldosterone following administration of the spirolactones, spironolactone and canrenoate-K, in rabbits, dogs and humans. The assay interfering principle was identified as a hydroxylated derivative (M_B) of canrenone, which itself is a metabolite common to both spironolactone and canrenoate-K. The metabolite M_B possessed a high cross-reactivity to the 21-hemisuccinate aldosterone antibody relative to other spirolactones. A modified procedure was developed specific for plasma aldosterone in the presence of MB. Following single doses of spironolactone and canrenoate-K, aldosterone plasma levels were unchanged in humans and in dogs and decreased in rabbits.