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Biomedical subjects

G R Sharpe

Publications and source records attributed to G R Sharpe.

At least 37 records · Page 2Linked to original sources
PubMed1994

Intracellular free calcium and growth changes in single human keratinocytes in response to vitamin D and five 20-epi-analogues.

Vitamin D, 1,25(OH)2D3, decreases proliferation and promotes differentiation of keratinocytes, and other keratinocyte differentiation stimuli have been associated with an early rise in intracellular free calcium, [Ca2+]i. We therefore investigated the effect of 1,25(OH)2D3, its precursor D3 and five 20-epi-analogues (EB1089, KH1060, KH1139, MC1288, MC1301) on growth and [Ca2+]i levels of normal human keratinocytes. Cells were cultured in medium MCDB153 with an extracellular calcium concentration of 70 microM or 1 mM. All the analogues were more potent than 1,25(OH)2D3 at inducing the morphological changes of differentiation, but D3 was inactive. At concentrations down to 10(-8) M 1,25(OH)2D3, caused significant inhibition of growth, as assessed by counting cells and measurement of thymidine labelling. At 5 days 50% inhibition of growth occurred with 64 nM 1,25(OH)2D3 and 3330 nM D3. All the analogues were more potent than 1,25(OH)2D3, and KH1060 inhibited growth at 10(-10) M. In single keratinocytes [Ca2+]i was measured by microspectrofluorimetric techniques using the dye fura-2. No immediate rise in [Ca2+]i was observed following addition of 1,25(OH)2D3 or the analogues up to 10(-6) M. However 10(-7) M 1,25(OH)2D3 or the analogues induced a gradual increase in [Ca2+]i, significant at 4 h (P < 0.001), which increased further over 2-3 days. D3 had no effect on [Ca2+]i. Increases in [Ca2+]i following the differentiation stimuli of either 2 mM extracellular calcium or 1,25(OH)2D3 were similar at 48 h, increasing from 100 +/- 3 nM (mean +/- SEM) in control cells to 150 +/- 3 nM with 2 mM calcium and 144 +/- 6 nM with 10(-7) M 1,25(OH)2D3. The effect of extracellular calcium in raising [Ca2+]i within minutes was more rapid than 1,25(OH)2D3, but in combination the two were not additive.

Calcitriol↗
PubMed1994

Changes in oncogene mRNA expression during human keratinocyte differentiation.

The nuclear proto-oncogenes are involved in transcriptional regulation and control many cell processes. The role of changes in proto-oncogene expression in controlling the balance between proliferation and differentiation was studied in cultured keratinocytes. Normal human keratinocytes were grown in the serum-free medium MCDB153 with an extracellular calcium concentration of 70 microM. After treatment with different differentiation conditions, cellular RNA was size-fractionated on agarose gels and transferred to nylon membranes which were subsequently hybridized with c-myc, c-jun, and H-ras 32P-labelled probes. Relative RNA loading was assessed using probes for beta-actin and ribosomal 18s RNA. Inducing differentiation by increasing the calcium concentration of the medium from 70 microM to 1.5 mM resulted in a marked decrease in c-myc RNA levels to 26% of control levels within 8 h. After 48 h in 1.5 mM calcium, c-myc levels had recovered to approximately 50% of control levels. There was a gradual reduction in c-jun levels to 56% of control levels by 4 days. Treatment with 10 nM TPA, which also induces keratinocyte differentiation, reduced c-myc RNA levels to 70% of control levels during the first 4 h, but thereafter c-myc levels remained approximately constant for a further 20 h. TGF beta (2 ng/ml), which inhibits keratinocyte growth without inducing differentiation, did not alter c-myc RNA levels over a 4-day period. There were no changes in c-myc levels following the addition of retinoic acid and none of the conditions altered H-ras levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1994

Characterization and analysis of skin wealing by computerized non-linear regression.

Weal formation in human skin may be induced by histamine, histamine releasers and other inflammatory mediators. Weals occur spontaneously in chronic urticaria, and in response to frictional pressure in dermographic urticaria. We present an improved method for the analysis of wealing in human skin by the use of non-linear regression. The method has the advantage of speed, and by the use of non-linear regression permits the full characterization of the response curves. The time course of histamine-induced wealing is a double exponential function corresponding to the separate components of weal appearance and disappearance. Dermographic wealing corresponds to an increasing exponential function with increasing pressure. The method of computerized non-linear regression is a considerable advance on previous methods for the analysis of urticarial wealing, the effect of vasoactive agents, and their therapeutic action.

Cetirizine↗
PubMed1994

Proliferating cell nuclear antigen decreases in normal human keratinocytes with differentiation stimuli but not in an HPV immortalised cell line.

Proliferating cell nuclear antigen (PCNA) is a co-factor for DNA polymerase delta, which replicates genomic DNA during cell growth and division. Using a monoclonal antibody to PCNA (PC10) and conventional immunofluorescent techniques, we have compared the effect of differentiation stimuli on PCNA expression in normal and HPV immortalised keratinocytes. Two positive nuclear staining patterns were observed, a strong speckled form characteristic of proliferating cells and a weaker diffuse form. Strong nuclear staining was present in 44 +/- 4% (mean +/- SEM) of normal keratinocytes proliferating as a monolayer in 70 microM calcium serum-free medium but decreased to 13 +/- 3% after the differentiation stimulus of 2 mM calcium medium for 2 days. An even greater reduction was observed following other differentiation agents, 1,25 dihydroxyvitamin D3, the phorbol ester TPA and the non-specific protein kinase C inhibitor staurosporine. Transforming growth factor-beta, which slows keratinocyte growth without inducing differentiation, reduced strong staining to 17 +/- 3% of cells, but with an increase in the diffuse pattern of staining from 39 +/- 4 to 57 +/- 3%. HPV immortalised cells were resistant to the above agents except staurosporine, which inhibited growth and reduced the strong nuclear staining from 44 +/- 5% to 15 +/- 2%.

Alkaloids↗
PubMed1993

Growth and differentiation stimuli induce different and distinct increases in intracellular free calcium in human keratinocytes.

The effect of growth and differentiation stimuli on intracellular free calcium ([Ca2+]i) in cultured human keratinocytes was investigated using micro-spectrofluorimetric techniques and the calcium-sensitive dye FURA-2. The mean [Ca2+]i of keratinocytes in 70 microM calcium medium was 104 +/- 3 nM (mean +/- SEM), significantly lower than the transformed keratinocyte line SVK14 (128 +/- 2 nM). When cultured in 2.0 mM calcium medium the [Ca2+]i increased in both normal and transformed keratinocytes to 135 +/- 4 nM and 180 +/- 4 nM, respectively. Keratinocytes grew more slowly in the absence of EGF, but [Ca2+]i was unaltered. Stimulation with EGF (10 ng/ml) induced, over 4 min, a large transient rise in [Ca2+]i up to 230 nm, due to an influx of extracellular calcium. Heterogeneity of keratinocytes was observed with 46% (n = 13) responding, but confluent or differentiated keratinocytes did not respond. TGF--beta (1 ng/ml) reduced cell growth without inducing differentiation and was not associated with any change in [Ca2+]i. The phorbol ester TPA (50 nM) induced irreversible growth arrest and terminal differentiation and increased the [Ca2+]i from 102 +/- 2 nM to 126 +/- 3 nM at 2 h, an effect similar to that of 2 mM extracellular calcium. Addition of 500 nM TPA was associated with a rise in [Ca2+]i, over several minutes to a plateau of 200-300 nM, due to release from internal stores and an influx of extracellular calcium. In normal human keratinocytes an increase in [Ca2+]i appears to be an early event in differentiation, whether induced by calcium or TPA, but not during growth inhibition without differentiation.

Calcium↗
PubMed1993

In dermographic urticaria H2 receptor antagonists have a small but therapeutically irrelevant additional effect compared with H1 antagonists alone.

Two studies of the additional effect of an H2 receptor antagonist when given in combination with an H1 antagonist were undertaken in dermographic urticaria. Using a randomized, double-blind, crossover design in 19 patients, a combination of cetirizine (10 mg at night) and ranitidine (150 mg twice daily) was compared with a combination of cetirizine (10 mg at night) and placebo. The addition of ranitidine did not produce any significant difference in linear analogue scores for weal, itch or sleep disturbance. There was a significant depression of the frictional force/wealing response curve with an increase in wealing threshold (P < 0.0001) following the addition of H2 blockade. The wealing threshold was 54.7 +/- 4.4 (mean +/- SEM) g/mm2 for the H1 antagonist alone, and 73.2 +/- 5.7 for the combination of H1 and H2 antagonists. In a second similar study involving nine different patients, comparing terfenadine (120 mg twice daily) with a combination of terfenadine and ranitidine (150 mg twice daily), the weal threshold was 59.8 +/- 6.6 for the H1 antagonist alone, and 73.0 +/- 6.4 for the combination of H1 and H2 antagonists. Thus, in dermographic urticaria, adding an H2 antagonist to treatment with a potent H1 antagonist gives a small, significant reduction in wealing response, but no symptomatic benefit. We conclude that involvement of the H2 receptor in this urticarial disease is minimal, and does not justify the use of H2 receptor antagonists.

Adolescent↗
PubMed1993

The effect of cetirizine on symptoms and wealing in dermographic urticaria.

The effect of cetirizine, 10 mg at night, on dermographic urticaria, was studied in 19 patients. The study design was a randomized, double-blind, crossover comparison with placebo, each treatment being given for 7 days. Patients kept a daily diary of itch and weal severity (100-mm linear analogue scale), and recorded sleep disturbance. The dermographic weal response was measured objectively with a spring-loaded stylus, and the weal threshold calculated from the force/response curve. There was a small, insignificant subjective response to placebo, but no objective response. On cetirizine, the subjective assessment of wealing was reduced from 34.3 +/- 6.7 (mean +/- SEM, 0-100 scale) to 16.8 +/- 4.1 (P = 0.02), itch was reduced from 43.2 +/- 6.6 to 19.4 +/- 4.1 (P = 0.001), and nights disturbed from 46.2 to 8.8% (P = 0.03). There was a shift to the right in the position of the force/response curve, and the wealing threshold increased from 24.6 +/- 3.2 to 54.7 +/- 4.4 g/mm2 (P = 0.00001), but there was no correlation between change in itch scores and wealing threshold. Cetirizine 10 mg daily is an effective treatment in dermographic urticaria, and its usefulness will depend on the prevalence of unwanted effects.

Adolescent↗
PubMed1993

Response of psoriasis to twice weekly PUVA.

In the U.K., PUVA treatment for psoriasis is usually given three times weekly, with the starting dose of UVA chosen according to the skin type of the patient. Observations on the time-course and dose-response characteristics of PUVA erythema suggest that larger doses of UVA could be used safely, provided that the frequency of PUVA treatment is reduced. We have examined this by treating 100 patients with chronic plaque-type psoriasis with a PUVA protocol in which treatment using oral 8-methoxypsoralen was given twice weekly, with the starting dose of UVA based on each patient's minimal phototoxic dose, and with weekly UVA dose increments calculated as a percentage of the dose used in the previous treatment. Clearance of psoriasis was achieved in 92% of patients. The median number of treatments required for clearance was 12, and the median cumulative UVA dose for clearance was 52J/cm2. Although erythema occurred at some stage during the course of PUVA in 48% of patients, in only 16% of cases was the erythema of sufficient intensity to result in more than one treatment being missed. These results compare favourably with previous studies in which treatment was given three or four times weekly. Thus, twice weekly PUVA treatment for psoriasis is at least as effective as treatment given more frequently, and may be safer, as lower cumulative UVA doses are required for clearance. It also allows for more efficient operation of a PUVA unit and is more convenient for patients.

Adolescent↗
PubMed1993

Prevalence of skin and other cancers in patients with psoriasis.

The prevalence of skin and internal malignancies was estimated from the general practitioners' notes of 2247 patients with psoriasis and 4494 age- and sex-matched controls. The prevalence of skin cancer in the psoriatics was 155% that of controls, but this was not significant at the 5% level. Subgroup analysis showed an increase in skin cancers in women (P < 0.05). There was no difference in the age of onset of skin cancers between psoriatics and controls and there was no evidence of a cumulative therapeutic risk. There was no difference in the prevalence of non-skin cancers between psoriatics and controls.

Adult↗
PubMed1993

Topical indomethacin aggravates the weal and flare response in chronic dermographic urticaria: evidence for a new class of histamine receptors.

The effect of topical indomethacin on the weal and flare response was measured in 9 patients with chronic dermographic urticaria. An augmentation of dermographic wealing by topical indomethacin was shown with lowering of the weal threshold from 22.3 +/- 4.7 g/mm2 (mean +/- SEM, n = 9) to 16.4 +/- 3.8 (p < 0.005), but without a change in the shape of the force/response curve. Flare was increased by indomethacin particularly in patients with a greater lowering of the weal threshold. The augmentation of weal and flare by indomethacin in individual patients was not related to the degree of inhibition of UVB erythema in individual patients. These findings indicate that in chronic dermographic urticaria there is an abnormality involving eicosanoid production by a non-cyclo-oxygenase pathway. It is suggested that this acts by augmenting the effect of histamine on a new class of histamine receptor, the definition and antagonism of which should lead to better control of urticarial disease.

Administration, Topical↗
PubMed1992

A case of Sweet's syndrome and myelodysplasia: response to cyclosporin.

A case of severe Sweet's syndrome associated with myelodysplasia is reported. The skin lesions responded to high doses of systemic steroids, but recurred rapidly on dose reduction. Treatment with a low dose of cyclosporin resulted in sustained clearance of the skin lesions, and was associated with a stable haemoglobin level, without the need for further blood transfusions.

Cyclosporins↗
PubMed1990

Time-dependent inhibition of growth of human keratinocytes and fibroblasts by cyclosporin A: effect on keratinocytes at therapeutic blood levels.

The effect of cyclosporin A (CyA) on the growth of cultured normal human keratinocytes and fibroblasts was studied. Tritiated thymidine incorporation per DNA content was used for short-term studies (1-2 days) and cell counting for longer term studies (up to 14 days). CyA caused a dose- and time-dependent inhibition of growth of both cell types. Keratinocytes (serum free, 70 microM Ca2+) were more sensitive to CyA than fibroblasts (10% serum). For keratinocytes, at 2 days growth was 61% and 6% of controls with 3 and 10 micrograms/ml CyA, respectively, at 4 days growth was 51% with 1 microgram/ml with complete inhibition at 10 micrograms/ml, and at 11 days growth was 58 and 39% with 0.1 and 1 micrograms/ml. For fibroblasts, at 4 days growth was 72 and 58% of controls with 10 and 30 micrograms/ml CyA, respectively, at 10 days growth was 76, 43 and 10% with 3, 10 and 30 micrograms/ml, and at 14 days growth was 41 and 11% with 3 and 10 micrograms/ml with complete inhibition with 30 micrograms/ml. For keratinocytes the effect of 24 h of CyA on growth was reversible. Growth of keratinocytes was inhibited by 10% delipidized serum, and 1% serum had a small stimulatory effect, but the action of CyA was unaltered. We conclude that increasing the duration of exposure to CyA increases the concentration-dependent growth inhibition. At therapeutic blood levels of CyA the growth of cultured keratinocytes but not fibroblasts is inhibited. The benefit of CyA in reducing the hyperproliferation of psoriasis may include a direct effect on keratinocytes.

Cell Count↗
PubMed1990

Unilateral naevoid basal-cell-carcinoma syndrome--an individually controlled study of fibroblast sensitivity to radiation.

A patient with unilateral naevoid basal-cell-carcinoma syndrome (NBCCS, Gorlin's syndrome) provided a unique opportunity to compare the radiation sensitivity of fibroblasts from the affected and unaffected sides of the same individual. Although the growth rate of the fibroblast strain established from the affected side was faster than that from the unaffected side there was no difference in X-ray, UV-B or UV-C radiation sensitivity between fibroblasts from the two sides. These results are discussed in the context of previous studies of radiation sensitivity in NBCCS.

Adult↗
PubMed1989

An increase in intracellular free calcium is an early event during differentiation of cultured human keratinocytes.

The effect of [Ca2+]o on [Ca2+]i in cultured human keratinocytes was studied using dual-wavelength microspectrofluorometric techniques. The results show that increasing [Ca2+]o from 70 microM to 1 mM causes an early rise in [Ca2+]i complete by 2 h. Heterogeneity within cultures was demonstrated. The [Ca2+]i in spontaneously differentiated cells of low Ca2+ cultures was similar to that of Ca2+ induced differentiated cells. The increase in [Ca2+]i preceded the morphological changes and growth inhibition induced by increasing [Ca2+]o. These observations are consistent with an increase in [Ca2+]i mediating differentiation of human keratinocytes.

Calcium↗
PubMed1987

Effect of naloxone on the antral motor response to solid food in man.

Antroduodenal motor activity was recorded in eight healthy subjects using perfused tubes connected to external strain gauge transducers. Each subject was studied over a 2.5-h period following ingestion of a solid meal, on 2 separate days. Intravenous saline was administered on one day and saline plus naloxone (40 micrograms kg-1 h-1) on the other, in randomized order. Naloxone markedly inhibited the antral motor response to food and this effect was due to decreased amplitude and contractile frequency. The duodenal motor response to solid food and the postprandial rise in serum gastrin and plasma pancreatic polypeptide were not altered by naloxone. These observations suggest that peripheral or central opiate receptors play a role in regulating the antral motor response to food.

Adult↗