Biomedical subjects
Publications and source records attributed to M Cumberbatch.
The ingestion of the new sun-tan capsules 'Orobronze' containing the carotenoid derivative canthaxanthin discolours plasma orange. This is noticeable in the plasma of blood donations received 4 days after starting the capsules and continues for a further 4 weeks after finishing the course. This cause of plasma discolouration should be recognised as it was found to mask haemolysis within a plasma sample.
1. The erythrocyte content of sodium and of potassium were measured in 231 unselected patients with hypokalaemia, and together with net ouabain-sensitive sodium efflux in patients with severe hypokalaemia, before (20 patients) and during potassium repletion (14 patients). 2. The erythrocytes of the patients with hypokalaemia compared with control subjects had on average an increase in sodium content, a decrease in potassium content and a reduction in the rate constant of ouabain-sensitive sodium efflux. All three changes had a similar curvilinear relation to the concentration of potassium in plasma with relatively little change in the measured variable unless the plasma potassium was very low. 3. There was a similar curvilinear relation between the final sodium and potassium content of normal erythrocytes and the potassium concentration of the medium in which they were incubated for 48 h in vitro. 4. These results suggest that the changes in the sodium and potassium content of erythrocytes in hypokalaemia are due to a direct inhibiting effect of the hypokalaemia on the activity of the sodium pump. 5. In many patients with hypokalaemia of moderate degree the increase in erythrocyte sodium content was less than expected from the effect in vitro of a low extracellular potassium concentration. This finding suggests that a compensatory change, presumably an increase in the number of sodium pumps, is a common event even in moderate hypokalaemia.
1. The sodium content, the rate and rate constant of ouabain-sensitive sodium efflux and the number of sodium pumps (indicated by the ouabain-binding capacity) were measured in erythrocytes from patients with hyperthyroidism and compared with values obtained in euthyroid patients and healthy control subjects. Erythrocyte zinc content was measured as a simple estimate of the content of carbonic anhydrase. 2. In the hyperthyroid patients, erythrocyte sodium content was increased, whereas the rate and rate constant of ouabain-sensitive sodium efflux, the ouabain-binding capacity and erythrocyte zinc content were all decreased. 3. The sodium pumps in hyperthyroidism had the same affinity for ouabain and the same rate constant per pump as those in healthy controls. 4. The decrease in the efflux rate constant could be entirely accounted for by the decrease in the number of sodium pumps. 5. Although the sodium efflux was decreased in the hyperthyroid patients, the change was less than expected for the decrease in the efflux rate constant. This suggests that there is an increase in the ground permeability of the erythrocyte membrane in hyperthyroidism. 6. In the hyperthyroid patients the number of sodium pumps and erythrocyte zinc content were inversely related to the plasma levels of thyroxine and tri-iodothyronine, but more closely to the latter. 7. These results suggest that the thyroid hormones may influence the erythrocyte's content of a range of proteins which happens to include the sodium pump.
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1. We have examined the inter-relationships between erythrocyte sodium content and sodium transport in a group of healthy subjects and in groups of patients with disorders known to change the sodium content of erythrocytes. 2. In the healthy subjects the sodium content of erythrocytes was inversely related to both the permeability of the erythrocyte membrane to sodium (as measured by the unidirectional, ouabain-sensitive, sodium efflux) and the total activity of the sodium pumps (as measured by the rate constant of ouabain-sensitive sodium efflux). There was a correlation between the total activity of the sodium pumps and the membrane permeability to sodium. 3. Changes in the erythrocyte sodium content were due to a decrease in the activity of the sodium pumps (as in hypokalaemia and digoxin treatment), or a decrease in the permeability of the erythrocyte membrane to sodium (as in chronic renal failure) or a reduction of both the membrane permeability and the number of sodium pumps (as in hyperthyroidism or elderly patients). 4. One interpretation of the results in the healthy subjects is that there are two components of sodium influx; one associated with the sodium pumps in what we have called 'membrane-units' and the other determined by the ground permeability of the membrane. 5. On the basis of this model we suggest that in the geriatric and hyperthyroid patients there is a reduction in the number of 'membrane-units', that in hypokalaemia and during digoxin treatment there is inhibition of the sodium-pump component of the 'membrane-units' and that in chronic renal failure there is a decrease in the permeability of the membrane to sodium.
1 Erythrocyte sodium content, sodium transport (ouabain sensitive sodium flux Eos, and ouabain sensitive efflux rate constant ERCos) sodium, potassium activated ouabain sensitive adenosine triphosphatase (Na+K+ATPase) and plasma digoxin were measured in patients during acute digitalisation and in patients who were on long-term digoxin treatment. 2 In the six patients who were studied during digitalisation, the ERCos and Na+K+ATPase activity decreased and erythrocyte sodium content increased during days 2-4 treatment, but there was no change in Eos. 3 In 39 patients on long term digoxin therapy (2-119 months) the erythrocyte sodium content was normal, but the erythrocyte Na+K+ATPase activity was higher than the control group. When the results from these 39 patients were divided according to the duration of treatment it was found that the erythrocyte sodium content was higher in patients treated for 2-4 months than in patients treated for longer periods and the erythrocyte Na+K+ATPase activity increased with duration of treatment. In eight patients (duration of treatment greater than 29 months) in whom ERCos and Eos were measured, ERCos and Eos were higher than the control group. 4 The results suggest that the effects of digoxin on erythrocytes which occur during acute digoxin treatment do not persist in the long term. 5 The possible explanation for the higher ERCos, Eos and Na+K+ATPase activity in patients treated with digoxin for more than 2 months is discussed.
1 Four healthy persons and ten patients with heart failure were studied for 5 to 20 days after they started taking digoxin. The sodium content of their erythrocytes increased and there was an equimolar decrease in potassium content. 2 The increase in erythrocyte sodium for a given increase in plasma digoxin during this acute digitalization was less on average and varied more in the patients than in the healthy persons, that is the patients' erythrocytes were less responsive to digoxin. 3 The average erythrocyte sodium was greater in 183 patients who had been taking digoxin for at least 2 months than in 100 healthy persons not taking digoxin but there was no significant correlation between the plasma digoxin concentrations and erythrocyte sodium concentration in the patients. Indeed, there was no apparent change in the erythrocyte sodium in many of the patients taking digoxin. 4 If the erythrocyte sodium concentration is a reliable guide to the tissue effects of digoxin then the results suggest that there is a wide variation in the response to digoxin between patients both during acute digitalization and during chronic treatment with digoxin.
A new, simple and non-radioactive technique is described for the measurement of ouabain-sensitive sodium transport in red cells, which is an alternative to the more complex standard technique using radioactive sodium. The new technique is based on the observation that when excess ouabain is added to blood, the sodium content of the red cells rises, due to complete inhibition of ouabain-sensitive sodium efflux while influx remains unchanged. The rate of increase of red cell sodium is therefore a measure of ouabain-sensitive sodium efflux (Eos). This estimate of Eos, when expressed as a fraction of the original red cell sodium concentration, gives a measure of the ouabain-sensitive, efflux rate constant (ERCos). In healthy persons, ERCos obtained with this new technique agrees closely with that obtained with the standard radioactive technique. In hypokalaemic patients the ERCos estimated by the new technique was less than that obtained with the standard technique, but this difference disappeared as the patients became normokalaemic. We suggest that this difference arises because the new technique measures only net ouabain-sensitive sodium transport whereas the standard technique also measures the ouabain-sensitive Na : Na exchange which is present in patients with hypokalaemia.
The development of in vitro methods for the identification of skin sensitizers based upon analysis of Langerhans cell (LC) function has been constrained by the fact that these cells represent only a minority population in the skin that, once isolated, alter their phenotype spontaneously and rapidly. Methods have been developed recently that allow the expansion in culture using appropriate cytokine conditions of LC-like dendritic cells (DCs) from certain tissues, including human peripheral blood. It has been demonstrated that culture of human blood-derived LC-like cells with selected potent contact allergens such as 2,4-dinitrofluorobenzene (DNFB) stimulates selective phenotypic changes, including the up-regulation of interleukin 1 beta (IL-1 beta) mRNA expression, under conditions where skin irritants are without effect. However, in our own previous investigations, we have observed that there appear to be differences between blood donors with respect to the responsiveness of DCs to DNFB-induced changes in IL-1 beta expression, differences that could compromise the utility of this approach as a screening method for contact allergens. We have therefore investigated donor variability in DC responsiveness to a panel of known human contact allergens (DNFB; paraphenylene diamine, PPD; methyl- chloroisothiazolinone/methylisothiazolinone, CMIT), to the skin irritant benzalkonium chloride and to the mitogen phorbol myristate acetate (PMA). Dendritic cells derived from all donors expressed IL-1 beta mRNA constitutively. Treatment of DCs isolated from donors with a responder phenotype to DNFB with PPD or CMIT resulted also in up-regulation of IL-1 beta mRNA expression, although such changes were always comparatively modest, generally resulting in a twofold induction compared with vehicle-treated controls. Dendritic cells derived from donors with a non-responder phenotype to DNFB failed also to respond to these additional contact allergens under conditions where the mitogen PMA caused similar increases in IL-1 beta expression to those observed for allergen-responsive donors. Benzalkonium chloride failed to provoke changes in the expression of this cytokine in any donor examined, irrespective of their responder phenotype. The temporal stability of the responder/non-responder DC phenotype was confirmed, with stable phenotypes with respect to DNFB-induced changes in IL-1 beta mRNA expression observed over a period of some 18 months. Fifty per cent (6/12) of donors tested over this period displayed a responder phenotype. These data demonstrate that chemical allergens do stimulate consistent changes in IL-1 beta mRNA expression in the proportion of donors who have a responsive phenotype, and that such responses are apparently selective for allergen using the relatively narrow range of materials assessed to date. However, the modest response to very strong contact allergens, coupled with the difficulties of responder/non-responder phenotypes, means that in its present form this approach does not lend itself to the routine assessment of skin sensitizing activity.
It has been demonstrated previously that tumour necrosis factor alpha (TNF-alpha) provides an important signal for the migration of epidermal Langerhans cells (LC) from the skin. Intradermal administration to mice of homologous recombinant TNF-alpha induces both a rapid reduction in the frequency of LC local to the site of exposure and, somewhat later, an accumulation of dendritic cells in draining lymph nodes. It has been proposed recently, however, that the influence of TNF-alpha on LC function may be dose-dependent in nature with lower concentrations inducing migration, but higher concentrations immobilizing LC in the epidermis. To investigate this proposal we examined the kinetics and dose-response relationships of TNF-alpha-induced LC migration in mice. At all concentrations tested (50, 150 or 300 ng/ear), intradermal exposure to TNF-alpha caused within 30 min a significant reduction in the frequency of MHC class II (Ia)+ LC within epidermal sheets. With the lower concentrations of TNF-alpha this effect was still apparent when LC were enumerated in the epidermis up to 4 h following cytokine treatment. In contrast, however, exposure of mice to 300 ng of TNF-alpha was consistently associated with a considerably less marked, and statistically insignificant, reduction in LC frequency by 4 h. These data indicate that at all concentrations of the cytokine examined here, TNF-alpha was able to stimulate a rapid (within 30 min) reduction in epidermal LC numbers, but that the rapidity with which the epidermis was repopulated following the initiation of LC migration was influenced by the concentration of TNF-alpha administered. It is suggested that TNF-alpha may influence not only the tempo of LC migration, but also the kinetics of epidermal repopulation.
Chemical allergy can take a variety of forms, those of greatest importance in an occupational setting being skin sensitization resulting in allergic contact dermatitis and sensitization of the respiratory tract associated with asthma and other symptoms. In both cases there is a need for predictive test methods that allow the accurate identification of sensitizing chemicals. Well characterized methods are available for skin sensitization testing, and although to date no tests for respiratory sensitization have been formally validated, progress has been made in defining suitable animal models. In recent years there have been significant advances in our understanding of the cellular and molecular mechanisms through which allergic sensitization to chemicals is induced and regulated. Such progress provides us now with new opportunities to consider alternative approaches to sensitization testing, including the design of in vitro test methods. The greatest investment has been in exploring novel methods for the identification of contact sensitizers and it is upon this aspect of chemical allergy that this article is focused. Described here are some of the general requirements of in vitro test methods for skin sensitization, and progress that has been made in developing suitable approaches with particular emphasis on the utility of dendritic cell culture systems.