Plasma and urine enrichments following infusion of L-[1-13C]phenylalanine and L-[ring-2H5]phenylalanine in humans: evidence for an isotope effect in renal tubular reabsorption.

Amino acids labeled with 13C or deuterium are commonly used in studies of amino acid metabolism. Traditionally, amino acid flux has been estimated by measurement of isotopic enrichment in the plasma pool; however, urine sampling as a noninvasive means of determining isotope enrichment has been increasing. The isotope enrichments and fluxes estimated from plasma and urine sampling were compared when two phenylalanine tracers (L-[1-13C]phenylalanine and L-[ring-2H5]phenylalanine) were intravenously infused for 4 hours in seven healthy men. This is the first evaluation of these isotopes as urinary tracers for assessing amino acid metabolism in adult humans. Before infusion, the mean ratio of plasma to urine (P:U) isotope enrichment was 0.99 +/- 0.03 (SD) and 0.99 +/- 0.02 for [13C]phenylalanine and [13C]tyrosine, respectively (isotope enrichment of [2H5]phenylalanine is zero at baseline). At isotopic steady state, the ratio was 1.06 +/- 0.05, 0.98 +/- 0.03, and 0.60 +/- 0.10 for [13C]phenylalanine, [13C]tyrosine, and [2H5]phenylalanine, respectively. The [13C]phenylalanine isotope showed a high correlation (R2 = .96) between enrichment in plasma and urine. However, use of [2H5]phenylalanine resulted in a significantly higher enrichment in urine than in plasma. Since amino acid flux is inversely related to enrichment, urine sampling would result in an underestimation of flux. The plasma to urine difference is probably due to discrimination of the [2H5]phenylalanine isotope in renal transport; therefore, this isotope may not be suitable for in vivo use where cellular transport mechanisms are involved.

Immune response to Schistosoma mansoni infections in inbred rats. VII. Resistance is contingent on OX-8(+)-regulated high affinity IL-2 receptor-bearing W3/25+ lymphocytes but not on IL-4-dependent cells.

These studies assess the roles of subpopulations of T lymphocytes in resistance to Schistosoma mansoni. CDF rats were depleted of the T cell subpopulation bearing the high affinity IL-2R by in vivo treatment with ART18+ mAb or of soluble IL-4 by treatment with 11B11 mAb. The development of parasites, the expression of resistance after sensitization, and the intensity of delayed type hypersensitivity (DTH), Ag-mediated blast transformation (AMBT), IgG2a, passive cutaneous anaphylaxis, and IgE-mediated antibody-dependent cell-mediated cytotoxicity responses against S. mansoni or control Ag were ascertained. Isolated T cell subpopulations were assessed in vivo and in vitro for effects upon the protective Ir. Depletion with ART18 mAb suppressed the development of W3/25+ helper-inducer cells and resulted in the initial survival of more worms, decreased resistance to challenge after initial sensitization, decreased IgG2a and IgE antibody, AMBT, and DTH reactivity against schistosome Ag. Depletion with ART18 did not prevent the development of OX8+ (T suppressor) cells. Depletion with 11B11 mAb led to insignificant changes in initial parasite survival and resistance to challenge; had no effect on IgG2a antibody, AMBT, or DTH; but profoundly suppressed the IgE responses against the parasite. Protective immunity to S. mansoni in rats is dependent upon IL-2R-bearing T lymphocytes and regulated by OX8+ cells but not absolutely contingent upon IL-4 or the IgE response.

Resistance in murine schistosomiasis is contingent on activated IL-2 receptor-bearing L3T4+ lymphocytes, negatively regulated by Lyt-2+ cells, and uninfluenced by the presence of IL-4.

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. C57BL/6 mice were depleted in vivo of L3T4+, Lyt-1+, Lyt-2+, IL-2R+ cells, or IL-4 by administration of appropriate mAb. Resistance and various correlative parameters of the immune response were studied in normal, depleted, and congenitally athymic mice. Depletion of T lymphocytes by anti-L3T4 or anti-IL-2R mAb reduced the development and expression of resistance, IgG2a and IgE antibody formation, and delayed type hypersensitivity reactivity against schistosome Ag. Depletion with anti-IL-4 antibody led to profound suppression of IgE-eosinophil-mediated antibody-dependent cell-mediated cytotoxicity and passive cutaneous anaphylaxis responses against the parasite and no effect on IgG2a antibody, Ag-mediated blast transformation, or resistance. Depletion of Lyt-2+ cells produced augmented development and expression of resistance and an increase in the immunological parameters of anti-schistosome reactivity. These studies suggest that protective immunity to S. mansoni in mice, induced by irradiated cercariae, is dependent on L3T4+, IL-2R+ lymphocytes and negatively regulated by Lyt-2+ cells. IL-4 does not appear to be essential for the development of resistance but is essential for the IgE response to the parasite.

IL-4 is required to generate and sustain in vivo IgE responses.

Antibodies of the IgE isotype play a predominant role in immediate hypersensitivity reactions. IL-4, a T cell-derived lymphokine that stimulates increased Ia expression by resting B cells and increased IgG1 secretion by LPS-activated B cells in vitro, has also been shown to regulate in vitro and in vivo polyclonal IgE responses. We report that large quantities of a purified anti-IL-4 mAb inhibit primary in vivo polyclonal IgE responses by 99% in mice infected with Nippostrongylus brasiliensis or injected with anti-IgD antibodies, and totally inhibit secondary Ag-specific IgE responses to TNP-keyhole limpet hemocyanin without effect on either IgG1 or IgG2a responses to these stimuli. The lack of effect of anti-IL-4 antibody on IgG1 secretion cannot be explained simply by inadequate neutralization of IL-4, inasmuch as the doses of anti-IL-4 antibody used blocked an N. brasiliensis-induced increase in B cell Ia expression by more than 85%, whereas in vitro studies indicate that enhancement of B cell Ia expression requires less IL-4 than induction of IgG1 secretion. In addition to demonstrating that IL-4 plays a necessary role in the generation of an in vivo IgE response, we show that IL-4 has an important role in sustaining established IgE responses, because anti-IL-4 antibody, when given at the peak of an N. brasiliensis- or TNP-keyhole limpet hemocyanin-induced IgE response, accelerates the declines in total serum IgE and in IgE anti-TNP antibody levels, respectively. These observations suggest that the effects of IL-4 on in vivo immune responses may be more specific than might have been predicted from in vitro observations, and that regulation of IL-4 production or action may be useful for the prevention or therapy of immediate hypersensitivity disorders.

In vivo and in vitro inhibition of human histidine decarboxylase by (S)-alpha-fluoromethylhistidine.

Histidine decarboxylase (HDC) activity in Ficoll-Hypaque purified human peripheral blood leukocytes (PBL) was determined by measuring the formation of [3H]histamine from L-[3H]histidine. HDC activity was inhibited in vitro to more than 90% by (S)-alpha-fluoromethylhistidine (alpha-FMH) at concentrations of 10(-5) M and above. Both polymorphonuclear and mononuclear cells possessed HDC activity, but on a per cell basis the former had several-fold higher enzyme activity than the latter. In safety and tolerability studies, alpha-FMH was administered orally to healthy human subjects twice daily for 7 days at doses of 2.5, 10, 50 and 100 mg per person. A dose-dependent inhibition of HDC activity was observed in PBL that were isolated both at 12 hr after administration of the first dose of alpha-FMH and after treatment for 1 week. At the 50 and 100 mg doses of alpha-FMH, there was complete inhibition of HDC activity and partial inhibition at the 10 mg dose. Twenty-four hours after the last dose, HDC activity had recovered to 64-100%, 44-46%, and 30-52% of control values in subjects that received 10, 50 and 100 mg alpha-FMH respectively.

A monoclonal antibody (100C5) to the Lyt-2-T cell population expanding in MRL/Mp-lpr/lpr mice detects a surface antigen normally expressed on Lyt-2+ cells and B cells.

MRL/Mp-lpr/lpr (MRL-lpr) mice develop a generalized lymphadenopathy reflecting the expansion of a Lyt-2- T cell population. The present report describes the pattern of reactivity of a xenogeneic monoclonal antibody (mAb 100C5) directed against this T cell population. By single- and two-color flow cytofluorometry analysis this mAb was found to stain brightly 80-90% of T cells in enlarged MRL-lpr lymph nodes. In contrast lymph node T cells from congenic MRL-MP-+/+ (MRL-+) mice were either unstained (70%) or weakly stained (30%), these latter cells being mostly Lyt-2+. Unexpectedly, all lymph node B cells from MRL-+ or MRL-lpr mice were as strongly 100C5+ as MRL-lpr T cells. Similar observations were made in C57BL/6-lpr/lpr and C57BL/6-+/+ mice. Molecular weight determination suggested that the 100C5 mAb binds to the same molecule (Mr = 220 000) on MRL-lpr T cells and normal B cells.

In vivo evidence for multiple opiate receptors mediating analgesia in the rat spinal cord.

In rats implanted with chronic catheters in the spinal subarachnoid space, intrathecal injections of SKF 10047 and dynorphin did not produce any elevation of the nociceptive threshold as defined by hot-plate and tail-flick tests. In contrast, intrathecal ethylketocyclazocine (EKC) and (D-Ala2,D-Leu5)-enkephalin (DADL) administration resulted in a dose-dependent antinociceptive effect which was reversible with intraperitoneal naloxone. Calculation of the Schild dose-ratio plots for the data derived from systemically administered naloxone reveals a slope of--1 and a calculated pA2 value of 6.8 for EKC and 6.2 for DADL. Also, animals made tolerant to systemic morphine showed a diminished analgesic response to intrathecal morphine and EKC when compared to naive animals. There was, however, no significant change in the dose response curve of intrathecal DADL. Thus, these experiments suggest that in addition to mu receptors a separate subpopulation of delta but not kappa or sigma receptors are involved with spinally mediated analgesia.

Regulation of IgE antibody production by serum molecules. VI: Preliminary biochemical and immunological characterization of serum molecules active in suppressing IgE antibody production.

Molecules present in the serum and ascites fluids of low IgE responder mice previously inoculated with complete Freund's adjuvant have been analyzed in terms of certain biochemical and immunological characteristics. These studies demonstrate that the active molecules, termed "suppressive factors of allergy" (SFA), are (1) nondialyzable, (2) not associated with low-density or high-density lipoproteins, (3) heat stable, (4) precipitable by ammonium sulfate, and (5) approximately 150,000 daltons in molecular size. Studies with immunoadsorbents prepared from various antisera indicate that the suppressive molecules are (1) not immunoglobulin in nature, (2) not reactive with specific anti-H-2 alloantibodies, but (3) reactive with anti-beta 2m antibodies as well as (4) heterologous antisera raised against CFA-immune mouse serum.

Structural studies on induced antibodies with defined idiotypic specificities. V. The complete amino acid sequence of the light chain variable regions of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

The complete amino acid sequence of the variable regions of light chains derived from anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype is reported. At least two and probably more than three distinct light chains are associated with this idiotypically characterized antibody. The antibodies have several differences in their "framework" structures but evidence is presented indicating that all three light chain hypervariable regions have a homogeneous sequence. The data are discussed in relation to the various theories of antibody diversity. In addition, the findings support the view that hypervariable regions, idiotypic determinants, and the antibody-combining site involve, to a large extent, the same molecular structures.