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R H Clothier

Publications and source records attributed to R H Clothier.

At least 37 records · Page 2Linked to original sources

Thymus-replacing activity from the metamorphic spleen of Xenopus laevis.

Anuran metamorphosis offers an interesting vertebrate immunological paradigm, for adult cells that arise within the immunocompetent larval body are MHC Class I disparate from those of the larva. The animals, in order to avoid immune self-destruction during this transition period, are made unresponsive to these modified-self cells by an impairment of T-cell functions. However, it remains to be discovered how an animal with compromised T-cell functions can protect itself from those environmental pathogens protection from which is thymus dependent. During metamorphosis, larval and adult immunocytes capable of reacting to each other coexist within the animal. Their interaction might stimulate the secretion of a cytokine capable of circumventing T-cell functions by acting directly on B cells. Here, we report that such an activity is released in vitro by metamorphic and not by adult splenocyte suspensions. This activity will amplify in vitro anti-hapten responses by immunized, but not carrier-primed, adult splenocytes. The activity is unaffected by dexamethasone and, since it will amplify anti-hapten responses in T-cell-depleted immunized adult splenocyte suspensions, the antibody-producing (B) cell population may be affected directly. Two radiolabeled protein peaks of 65 kD and 40 kD were obtained by SDS-PAGE analysis from secreting, metamorphosing, but not from adult, splenocytes.

Aging↗

A monoclonal mouse anti-human IL-2 receptor antibody (anti-Tac) will recognize molecules on the surface of Xenopus laevis immunocytes which specifically bind rIL-2 and are only slightly larger than the human Tac protein.

We have made visible the binding of a mouse monoclonal anti-human interleukin 2 (IL-2) receptor (anti-Tac) antibody on the surface of phytohemagglutinin (PHA)-stimulated Xenopus thymocytes using a colloidal gold-conjugated goat anti-mouse antibody and transmission electron microscopy. No binding was found when a different mouse monoclonal antibody (mAb) of the same isotype and subclass was tested, or when the anti-Tac antibody was omitted from the procedure. After metabolic radiolabeling of the IL-2 receptors with [35S]methionine using PHA-stimulated thymocytes of Xenopus laevis, the South African clawed toad, we show that a concentrated preparation of the mouse anti-human Tac antibody will immunoprecipitate a radiolabeled molecule just slightly larger than 55 kDa. Phorbol dibutyrate (PDB), an effective T cell mitogen, and cyclosporin A, an inhibitor of T cell mitogenesis in this species, are both capable of regulating the expression of this IL-2-binding molecule on Xenopus immunocytes. Here, we use the calcium ionophore A23187 to show that the relationship between IL-2 receptor expression and mitogenesis, which was previously established in X. laevis, is associated with a calcium ion flux. Flow cytometry is used for assaying alterations in epitope expression after binding the lectin-stimulated cells under test with a fluorescence (Fl*) conjugate of the anti-Tac antibody or a control mAb, which is either anti-DNP or anti-keyhole limpet hemocyanin (KLH) in specificity, but of the same mouse isotype and subclass as the anti-IL-2 receptor antibody.

Animals↗

Amphibian metamorphosis: an immunologic opportunity!

Anuran amphibian metamorphosis is an immunologically interesting period. For the investigator, it provides an unusual opportunity for analyzing both humoral regulation of the immune response and the development and maintenance of self-tolerance. Some of the questions one can ask are: Why don't immunocompetent larvae destroy antigenically disparate adult cells as they differentiate within them during metamorphosis? Do the dramatic hormonal changes occurring during this period regulate immunological function? How do animals in metamorphosis protect themselves from their immunologically hostile environment?

Amphibians↗

Thyroid function and immune reactivity during metamorphosis in Xenopus laevis, the South African clawed toad.

High plasma titers of thyroid and adrenocorticoid hormones are present during the metamorphosis of Xenopus laevis. Here we examine the influence of thyroid hormones on several features of immune reactivity during this period, e.g., the capacity of thymus-derived immunocytes to reduce (immune suppression) or amplify (helper function) antibody production. Further, we test whether thyroid hormone is able to modulate the expression of putative interleukin 2 (IL-2) receptors on lectin-activated adult Xenopus splenocytes, an aspect of helper function. Finally, we have tested the ability of thyroid hormones to affect larval antibody-producing cells directly. Our data suggest that all three functions (suppressor, helper, and antibody producing) are independent of thyroid function during metamorphosis. However, the anatomical distribution of two features of immune suppression, as well as the numbers of lectin-activated splenocytes able to bind anti-IL-2 receptor antibody, were changed by thyroid function. In vivo thyroid blockade by thiourea prevented the transition from the premetamorphic to the adult pattern of distribution of the two suppressor functions; triiodothyronine in vitro stimulated an increase in the numbers of cells able to bind an IL-2 receptor antibody.

Adrenal Cortex Hormones↗

Differential cyclophosphamide sensitivity of suppressor function in Xenopus, the clawed toad.

Thymocyte and Splenocyte cultures from in vivo immunised Xenopus were assayed to test their suppressive capacity. Immunisation with TNP-Polyvinylpyrrolidone induced suppression. Suppression induced by the haptenated antigens, TNP-Red blood cells, TNP-Lipopolysaccharide, and TNP-Ficoll affected the anti-TNP antibody response of splenocytes from TNP-PVP immunised animals. Pretreatment with cyclophosphamide revealed both a sensitive and insensitive suppression capacity in Xenopus laevis.

Animals↗

Responses in Xenopus to the thymus independent antigen polyvinylpyrrolidone (PVP) and its haptenated derivative, trinitrophenylated PVP (TNP-PVP).

Xenopus laevis (the South African clawed toad) can respond to thymus dependent (TD) and thymus independent (TI) antigens. However, the response to trinitrophenylated Ficoll (TNP-Ficoll), a TI-2 antigen in mammals, is thymus dependent in Xenopus. Polyvinylpyrrolidone (PVP), classed as a TI antigen in mammals, is also a TI antigen in Xenopus, but responses to PVP and TNP-PVP are thymus regulated. As with TNP-Ficoll, capacity to respond to TNP-PVP diminishes during metamorphosis, and tolerance can be induced via the stimulation of TD suppression with trinitrobenzene sulphonic acid. Animals treated with N-methyl-N-nitrosourea and adult-thymectomised Xenopus, which lack certain TD responses, can nevertheless respond to TNP-PVP. Based on this and other information, it is concluded that TNP-PVP should be classed as a TI-2 antigen in Xenopus.

Aging↗

The characterization of the toad splenocytes which bind mouse anti-human IL-2 receptor antibody.

We have utilized two depletion protocols to characterize the spleen cells of the South African clawed toad, which bind specifically mouse-anti-human IL-2 receptor (anti-Tac) antibody and recombinant DNA produced IL-2. When the selectively lymphotoxic reagent, N-CH3-N-nitrosourea (NMU), is injected into adult toads, it removes the thymic cortex and lymphocytes throughout the body required for helper and cytotoxic cell functions. We have also used a monoclonal mouse anti-Xenopus IgM antibody to deplete toad splenocyte populations of surface (s) Ig+ cells. Freshly biopsied and cultured spleen cells were compared with respect to their capacity to bind fluorescent (Fl*-) anti-Tac antibody and its ligand, rIL-2, after a depletion protocol. The results clearly show that a phytohemagglutinin (PHA)/IL-2 sensitive splenocyte population is removed by NMU injection. While many of the remaining NMU insensitive, previously cultured cells are Tac+, they fail to bind rIL-2. Freshly biopsied spleen cells with constitutive IL-2 receptors are found in both the sIg- (T cell enriched) and sIg+ (B cell enriched) populations following panning. Moreover, both populations are able to bind the ligand with equal efficiency. Thus, constitutive IL-2 receptor bearing cells are not restricted to either the T or B cell populations. The predominant PHA activatable, rIL-2 binding cell populations of the toad appear to be T cells which are involved with helper and cytotoxic functions.

Animals↗

The detection of cytotoxicity produced by short-lived reactive intermediates: a study with bromobenzene.

1. A V79 cell incubation incorporating rat liver 9000 g supernatant (S9) fractions, used previously to detect the toxicity due to long-lived, stable metabolites of cyclophosphamide, has been used to study the toxicity of short-lived, reactive metabolites generated from bromobenzene. 2. Cytotoxicity was observed in the presence of S9 fractions from rats treated with phenobarbitone but not in the presence of S9 fractions from untreated or beta-naphthoflavone-treated animals. This toxicity was enhanced by depletion of the glutathione in the S9 fraction by prior treatment of the animals with diethyl maleate and was reduced by SKF 525 A, in agreement with results in vivo on the mechanism of bromobenzene-induced hepatotoxicity. 3. This study demonstrates that cytotoxicity due to the generation of short-lived, reactive metabolites can be detected in this system in vitro provided that procedures are used to modify the activating and detoxifying enzyme systems within the S9 fraction.

7-Alkoxycoumarin O-Dealkylase↗

Signals provided in vivo by human rIL-2 and Con A can switch hapten-specific tolerance from unresponsiveness to responsiveness in the South African clawed toad.

Injection in vivo of trinitrobenzene sulphonic acid (TNBS) will conjugate trinitrophenyl (TNP) to the cells and proteins of rodents, and induce hapten-specific tolerance to this epitope. However, the induction of hapten-specific tolerance by this method in the South African clawed toad, Xenopus laevis, is restricted to its subsequent presentation on Ficoll or polyvinylpyrrolidone (PVP). This restricted tolerance depends on the stimulation of an N-methyl-N-nitrosourea (NMU)-insensitive, cyclophosphamide (CyP)-sensitive hapten-specific (suppressor) population that is functionally demonstrable in vitro. Unresponsiveness to TNP-Ficoll can be switched to responsiveness by injection in vivo of recombinant DNA-produced human IL-2 (rIL-2) or the plant-derived lectin, concanavalin A (Con A). Responsiveness to TNP-Ficoll requires thymic presence, although thymic extracts from rIL-2- or Con A-injected toads will suffice. Unresponsiveness to TNP-PVP can also be broken by these reagents, but thymic presence is not required with this immunogen. TNP-Ficoll responses are thymus requiring, while those to TNP-PVP are not, in the toad. Since TNBS failed to stimulate hapten-specific tolerance to a secondary challenge to TNP-Ficoll, we suggest that the suppressor function involved in the establishment of the unresponsive state may act on the differentiation, rather than on the function, of the relevant B cells.

Animals↗

Transfer of antibiotic resistance genes between yeast and mammalian cells under conditions favoring cell fusion.

Antibiotic resistance to G418 has been transferred into Chinese hamster cell lines via a plasmid vector. The same plasmid, which also contained the Leu2 gene, has been used to transform Leu2- yeast (strain MC16) to leucine prototrophy. Subsequent fusion between transformed yeast and untransformed hamster cells demonstrated that plasmid DNA could be transferred and its genes expressed within the mammalian cell genome. The fusion of transformed hamster cells with untransformed MC16 yeast cells demonstrated that DNA integrated within the mammalian cell genome could be transferred to correct the Leu2 deficiency and also confer G418 resistance on some yeast colonies.

Animals↗

Two cellular pathways regulate the response to TNP-Ficoll in Xenopus laevis.

The initiation of the anti-TNP response to TNP-Ficoll in the amphibian Xenopus laevis has been studied. Although the response to this antigen is thymus independent in mammals, it is thymus dependent for the first three days following immunization in Xenopus. This thymus regulation is not MHC restricted, since it can be substituted for by thymus xenografts, and by prior or co-injection of heterologous red blood cells or Concanavalin A. The pathway which is activated by the Con A to substitute for the thymus is NMU sensitive, unlike the thymic pathway. The peripheralised alternative pathway is activated by particulate but not soluble TNP-Ficoll. The thymus-dependent and alternative pathways are discussed in terms of their possible nature, regulation and evolutionary significance.

Animals↗

Thymic involvement in memory responses after primary challenge with TNP-Ficoll in Xenopus laevis, the South African clawed toad.

Memory to TNP-Ficoll in Xenopus laevis is demonstrable as a more rapid secondary response rather than an augmented response. While adult thymectomy abrogates ability to respond to both primary and secondary challenge with soluble TNP-Ficoll, memory to this antigen can be revealed in thymectomized animals by challenge with TNP-Ficoll on bentonite beads. Memory to TNP-Ficoll cannot be revealed by secondary challenge with TNP-LPS or TNP-RBC, but memory to TNP-LPS is revealed by TNP-Ficoll challenge. These results are consistent with the hypothesis that, whereas B1 cells (capable of responding to TI-2 antigens) can develop from a pool of B1 and B2 memory cells (generated in response to TD or TI-1 antigens), initial challenge with TI-2 antigen does not produce a pool containing both B-cell subsets.

Animals↗

Murine and human interleukin 2 can substitute for the thymus in immune responses to TNP-Ficoll in Xenopus laevis, the South African clawed toad.

Extirpation of the thymuses of Xenopus laevis abrogates the capacity to respond to trinitrophenol (TNP)-Ficoll regardless of the age of the animal. This thymus requirement can be substituted for by a variety of treatments which stimulate thymus-derived (T)cell activity in the periphery, such as the rejection of allogeneic skin grafts, immunologic challenge with thymus-dependent immunogens, (e.g., heterologous erythrocytes), or plant-derived lectins (e.g., concanavalin A). Here we report that interleukin 2 (IL-2), a T-cell-produced hormone of mammalian origin also substitutes for this thymus requirement in thymectomized toads.

Animals↗

Lectin effects on thymic suppression of hemagglutinin production in vitro by spleen fragments from Xenopus laevis, the South African clawed toad.

Suppression of hemagglutinin (HA) production was studied in co-cultures of thymus and spleen fragments from the South African clawed toad, Xenopus laevis. Concanavalin (Con)A and peanut (PNA) and wheatgerm (WGA)agglutinins were tested in conjunction with, or as a substitute for antigenic induction of thymus suppression. While thymuses from animals injected with PNA or WGA and antigen expressed suppressor function, those treated with Con A and antigen did not. Spleen fragments from animals challenged with PNA or WGA and antigen were resistant to suppression by normal thymus in reciprocal co-cultures; those receiving Con A and antigen alone were equally suppressible. All three lectins can substitute for antigen in the induction of thymus suppression. However, only PNA and WGA induce suppressor function in Xenopus spleen. Since WGA can induce splenic suppression in adult thymectomized animals, it is now possible to distinguish thymic and peripheral aspects of suppression of HA in this primitive vertebrate.

Animals↗

Immunologic reactivity to TNP-Ficoll during development and ageing in Xenopus laevis, the South African clawed toad.

The immunological response of Xenopus laevis to haptenated Ficoll was examined throughout development. The capacity to respond to TNP-Ficoll originated with the first appearance (at stage 48, 8 days post-fertilisation) of mature thymic lymphocytes, and preceded the ability to respond to heterologous erythrocytes. During premetamorphosis the response increased, while during prometamorphosis , particularly at stages 57-58, there was a marked reduction. The response recovered during the metamorphic climax and immature adult stages, increasing further at the onset of sexual maturity. The reduced response during prometamorphosis parallels the reported reduction in other thymus-dependent responses.

Aging↗

Genetic limits of thymic immunosuppression of anti-hapten antibody production in Xenopus laevis laevis, the South African clawed toad.

Thymic immunosuppression in adult Xenopus laevis laevis, the South African clawed toad, is antigen-dependent and antigen-specific, but it is not genetically restricted. In this report, we show that combination in vitro with a thymus from an immunized Xenopus laevis laevis can suppress antibody production to a hapten from spleen fragments, if the spleen fragments are derived from a subspecies of Xenopus laevis or a species of Xenopus which shares the same diploid chromosome number (2N = 36). Some aspects of the evolution of thymic immunosuppression are considered in the light of these results.

Animals↗

Lectins and substitution for helper function in anti-hapten responses in Xenopus laevis.

Substitution by lectins for the carrier-priming requirement in thymus-dependent, antigen-binding responses in Xenopus laevis has been examined. Concanavalin A (Con A) was found to substitute for carrier priming in control, early-thymectomized and adult-thymectomized animals, but not in animals given a single, high dose of N-methyl-N-nitrosourea, which has a permanent effect on certain thymus-dependent functions in this species. Lipopolysaccharide and other lectins, such as peanut agglutinin and wheat germ agglutinin, were unable to substitute for carrier priming. These effects of Con A are discussed in terms of substitution via amplifier T cells or a helper T cell subset.

Age Factors↗