Biomedical subjects

R H Clothier

Publications and source records attributed to R H Clothier.

64 records · Page 4Linked to original sources

PubMedDate not supplied

Phosphatidylserine expression on apoptotic lymphocytes of Xenopus laevis, the South African clawed toad, as a signal for macrophage recognition.

Inflammation is avoided in apoptosis by early removal of dying cells by macrophages (MOs). In mammalian cells, an early aspect of apoptosis is the translocation of phosphatidylserine (PS) from the inner leaflet of the cell membrane to the surface. PS recognition can serve as a signal for triggering removal of dying cells. PS expression on splenocytes and thymocytes of Xenopus laevis was quantified using FITC-Annexin and flow cytometry following exposure in vitro to several known apoptogens for this species. All apoptogens used induced PS expression. Dose dependency and the kinetics of PS expression following exposure to the calcium ionophore, A23187, were also examined. Peritoneal exudate cells (PEC's) were cultured with A23187-treated thymocytes to test MO capacity for recognition of PS. MO binding to apoptotic thymocytes was reduced following exposure of PEC's to a water soluble analogue of PS, phospho-L-serine. The presence of a phagocytic PS-dependent recognition system in amphibia is supportive of the evolutionary conservation of this function in mammals that is crucial in limiting inflammation induced by dying cells.

Animals↗

PubMedDate not supplied

Apoptosis in the thymus of developing Xenopus laevis.

Metamorphosis in Xenopus laevis is a time when thyroxine and glucocorticoid levels rise, dramatic morphological and physiological changes take place, and tolerance is established to newly expressed adult antigens. In vitro exposure of thymocytes tested at different metamorphic stages, to the T-cell lectin, phytohemagglutinin (PHA), stimulates increased apoptosis, but incubation with the synthetic glucocorticoid, dexamethasone (DEX), fails in this regard. Altered-self antigenicity, following trinitrobenzene sulfonic acid (TNBS) treatment, increases apoptosis only in the late stages of metamorphosis. Developmentally blocked metamorphosing larvae demonstrate low thymic apoptotic rates that are also unaffected by in vitro exposure to DEX or by in vivo exposure to thyroxine, but are increased by PHA and in some individuals by TNBS. When released from blockade, their thymic apoptotic rates rise as progress through metamorphosis is renewed. Larval thymic apoptosis is glucocorticocoid- and thyroxine insensitive, but is lectin and altered-self antigen activated, particularly during postclimax stages.

Animals↗

PubMedDate not supplied

In situ lymphocyte apoptosis in larval Xenopus laevis, the South African clawed toad.

During Anuran metamorphosis larval structures regress, adult structures form anew and impaired T cell immune functions are noted, as are alterations in endogenous glucocorticoid titers. In situ histological data, after staining for DNA fragmentation, reveal patterns of lymphocyte suicide in the thymus and spleen of non-antigenically challenged, laboratory bred, developing larvae, that do not correlate with either impaired immune functions or plasma glucocorticoid titers. Apoptotic levels in the thymus are high in premetamorphic stages, low during prometamorphosis and high again, after metamorphic climax, reflecting a periodic removal of thymocytes. Lymphocytic apoptosis in the spleen is low during premetamorphosis, rises in prometamorphic stages, principally within the red pulp, reaching a peak at climax, before declining as metamorphosis is completed.

Animals↗

PubMedDate not supplied

The use of an in vitro submerged keratinocyte model to predict induction of squamous metaplasia.

An in vitro submerged keratinocyte model of squamous metaplasia (SQ) in epithelia is being developed to assess the risk associated with exposure to certain environmental agents. Tracheobronchial epithelium (TBE) in vivo can respond to airborne environmental insult by becoming squamous. Epidemiological evidence suggests that cigarette smoke is capable of inducing this change. Retinoic acid has been shown to maintain cells in the mucociliary state. SQ is considered protective and adaptive but potentially preneoplastic if unrelenting and is used histologically in the diagnosis of squamous cell carcinoma. SQ is characterised by upregulation of the expression of transglutaminase I (TGI), TGI activity leading to the formation of isopeptide cross-linked envelopes and replacement of the mucociliary cell type with non-polar squamous cells out of contact with the basal lamina. The ability of the in vitro keratinocyte submerged model to predict the squamous metaplastic response in vivo has been investigated in vitro using TG catalysed fluorescein cadaverine incorporation as a measure of cross-linked envelope formation, Alamar blue conversion to measure viability and Coomassie blue incorporation to measure total cellular protein. The modulation of the squamous condition by retinoic acid (RA), cigarette smoke condensate (CSC) and nicotine has been assessed in keratinocytes cultured in Green's medium. RA inhibited FC incorporation by 95% at 1 x 10(-5) M and simultaneously increased cell viability providing evidence to support its role in the regulation of the non-differentiated state. Nicotine (0-1 mg/ml) induced a dose-dependent increase in viability at 6 days, a response that was accompanied by an increase in FC incorporation at 12 days. CSC (0-5 microg/ml) increased FC incorporation after 12 days. Hence, nicotine modulated the squamous condition by up-regulating TGI activity following a period of hyperactivity. CSC induced a gradual change to the differentiated state and RA served to maintain the cells in an undifferentiated state.

Animal Testing Alternatives↗

PubMedDate not supplied

Hydrogen peroxide induced stress in human keratinocytes and its effect on bithionol toxicity.

Exposure to hydrogen peroxide causes oxidative stress in keratinocytes. Previous work has shown that the antiparasitic drug bithionol has an EC(50) of 0.7 microg/ml (2 microM) with primary human keratinocytes, but that these cells do not respond to photoactivated bithionol. Bithionol is known to be photoactivated by UV-A visible light, therefore this study aims to investigate the effects of inducing oxidative stress in the cells prior to bithionol treatment alone and in the presence of UV-A visible light. Oxidative stress, by hydrogen peroxide treatment, caused the cells to become sensitive to photoactivated bithionol. Bithionol alone reduced the amount of oxidative stress, while following photoactivation, an augmentation in the amount of oxidative stress and cell cytotoxicity was observed. The hydrogen peroxide treatment did not alter the sensitivity of the keratinocytes to 5 J/cm(2) UV-A visible light.

Antiplatyhelmintic Agents↗

PubMedDate not supplied

Adrenergic modulation of apoptosis in splenocytes of Xenopus laevis in vitro.

Impaired and healthy cells undergo suicide using an intrinsic genetic program. Exposure to stress-related alpha2- or beta-adrenergics for 4 or 20 h in vitro had no effect on apoptosis in splenocytes of adult Xenopus laevis, while a 4-hour coincubation of clonidine, an alpha2-agonist, with a calcium ionophore (A23187) or a phorbol diester (PMA), enhanced apoptosis induced by each apoptogen alone. Clonidine did not affect apoptosis stimulated with dexamethasone (DEX), however. Comparable in vitro exposures to isoproterenol, a beta-agonist, reduced apoptotic levels stimulated by all three apoptogens alone. Following 20 h coexposure, clonidine no longer affected A23187-induced apoptosis, but reduced PMA-induced apoptosis, while isoproterenol enhanced apoptosis stimulated with both. Neither agonist modulated apoptosis induced by 20 h of exposure to DEX. Thus, adrenergic agonists modulated apoptosis in cells coexposed to A23187 and PMA, in a time-dependent and adrenoceptor class-dependent fashion. These stress-induced products can affect concurrent apoptosis reversibly over time in vitro, and thus possibly in vivo.

Adrenergic alpha-2 Receptor Agonists↗

PubMedDate not supplied

Electrical stimulation of transforming growth factor-beta 1 secretion by human dermal fibroblasts and the U937 human monocytic cell line.

The in vitro effects on human dermal fibroblasts and the U937 human monocytic cell line of three phases of electrical microcurrents generated by the ACE Stimulator were investigated. The growth and viability of growing and confluent dermal fibroblasts were not directly influenced by the separate microcurrent phases. One form of microcurrent (designated phase 1) stimulated both dermal fibroblasts and U937 cells to secrete transforming growth factor-beta 1 (TGF-beta 1), which is an important regulator of cell-mediated inflammation and tissue regeneration, but none of the three phases stimulated secretion of the pro-inflammatory cytokine interleukin-6 by U937 cells. The stimulation of TGF-beta 1 secretion in these experiments was not dramatic (a median increase over control levels of 20-30%), although it could be biologically significant.

Animal Testing Alternatives↗

PubMedDate not supplied

A comparison of two cytotoxicity assays for the detection of metabolism-mediated toxicity in vitro: a study with cyclophosphamide.

The cytotoxicity to V79 Chinese hamster fibroblasts of cyclophosphamide (CPA) metabolites, generated by rat-liver S9 fractions, has been compared in two assay systems with different endpoints of toxicity, namely, reduction of cloning efficiency and inhibition of cell growth. The two assay systems were found to be equally sensitive in detecting the metabolism-mediated cytotoxicity of CPA and gave similar ID50 values. Further studies confirmed the cytochrome P-450 enzyme requirement for the bioactivation of CPA to cytotoxic metabolites. CPA activation was mediated by phenobarbitone-inducible forms of cytochrome P-450, but not by beta-naphthoflavone-inducible forms.

7-Alkoxycoumarin O-Dealkylase↗

PubMedDate not supplied

Comparison of the in vitro cytotoxicities and acute in vivo toxicities of 59 chemicals.

The in vitro cytotoxicities of 59 chemicals, expressed as ID50 values (i.e., concentrations of test chemicals that reduced the final cellular protein content of test cultures by 50% in comparison with appropriate solvent control cultures) and obtained using murine 3T3-L1 cells and the FRAME kenacid blue method, have been compared with rat oral and mouse intraperitoneal (ip) LD50 values. A better in vivo/in vitro correlation was obtained for the 59 chemicals with mouse ip LD50 values (r = .80) than with rat oral LD50 values (r = .76), but the best in vivo/in vitro correlation was found when the most toxic of the rat and mouse values were used in the comparison (r = .81).

Administration, Oral↗

PubMedDate not supplied

Assessment of two alternative methods for predicting the in vivo toxicities of metallic compounds.

The FRAME in vitro cytotoxicity assay and a physicochemical parameter for metal ions (i.e., "softness," sigma p) were assessed for their ability to predict the in vivo acute toxicities of 52 metallic compounds. The in vitro assay was found to be more useful, since it measures the toxicity of the whole compound, as does the in vivo method. The softness parameter applies to the metal ion only, so it cannot be used to predict the toxicity of compounds containing relatively nontoxic metal ions and toxic anions (e.g., potassium fluoride). The in vitro toxicity values (expressed as ID50 values, i.e., concentrations of test chemicals that reduced the final cellular protein content of test cultures by 50% in comparison with appropriate solvent control cultures) correlated better with mouse ip LD50 values than with rat oral LD50 values.

Animals↗