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Biomedical subjects

G R Sharpe

Publications and source records attributed to G R Sharpe.

At least 19 recordsLinked to original sources
PubMed2000

Inflammatory mediators in normal, sensitive and diseased skin types.

The role of inflammatory mediators in the pathogenesis and pathophysiology of skin diseases is now widely accepted. We analysed the profiles of inflammatory mediators in normal, sensitive (past history of eczema, but currently patch test negative) and diseased (psoriasis and eczema) skin types to identify the patterns associated with various degrees of inflammatory dermatoses. Compared with normal skin, prostaglandin E2 was increased approximately 3.8-fold (p<0.0002) and 4.7-fold (p<0.0001) in suction blister fluids from sensitive and diseased skin types, respectively. Leukotriene B4 and interleukin-1alpha showed no differences between normal and sensitive skin types. However, in lesional skin from psoriasis and eczema patients, leukotriene B4 was increased approximately 6.6-fold (p<0.0001), whereas interleukin-1alpha was decreased approximately 3.1-fold (p<0.001). Interleukin 6 and tumour necrosis factor-alpha could not discriminate between skin types. We conclude that only prostaglandin E2 showed a significant stepwise increase on progression from normal through sensitive and inflammatory skin diseases. Levels of leukotriene B4 and interleukin-1alpha were also indicative of disease state and may be important in the pathophysiology of these conditions.

Adult↗
PubMed1999

Regulation of epidermal homeostasis through P2Y2 receptors.

1. Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. 2. Reverse transcriptase-polymerase chain reaction (RT - PCR) revealed expression of mRNA for the G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes. 3. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgammaS were found to stimulate the proliferation of keratinocytes. 6. Using a real-time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Adenosine Triphosphate↗
PubMed1999

The antiproliferative effect of lectin from the edible mushroom (Agaricus bisporus) on human keratinocytes: preliminary studies on its use in psoriasis.

Lectins or agglutinins are proteins with affinity for specific sugar residues. Peanut agglutinin (PNA) and the lectin from the edible mushroom (Agaricus bisporus, ABL) both bind to the disaccharide galactosyl beta-1,3-N-acetyl galactosamine alpha-. This is expressed in keratinocytes as an O-linked chain on CD44, a polymorphic membrane glycoprotein. Many lectins are mitogens and PNA is a mitogen for colonic epithelial cells. However, ABL reversibly inhibits proliferation of colonic cancer cell lines without cytotoxicity and thus has therapeutic potential in situations such as psoriasis where proliferation is increased. We have therefore investigated the effect of ABL on the growth of normal human cultured keratinocytes and a human papilloma virus (HPV)-transformed cell line. In a 5-day dose-response study, keratinocyte growth was greatly reduced by 1.0 microg/mL ABL and completely inhibited by 3.0 microg/mL ABL (ANOVA, P < 0.0001). Exposure to 1.0 microg/mL ABL for only 8 h gave the same growth inhibition as did continued exposure for 3 days. No cytotoxic or morphological changes were observed. An HPV-immortalized cell line was relatively resistant to ABL: in a 5-day dose-response study, exposure to 30 microg/mL was required to inhibit cell growth completely. Topical application of ABL 0.01% or 0.1% to normal human skin caused no change in skin erythema, blood flow or thickness compared with vehicle or baseline (n = 6). ABL 0. 1% in white soft paraffin was compared with vehicle in 11 psoriatic patients, using comparative contralateral plaques. Twice daily application for 2 weeks showed no significant difference from vehicle-treated sites, although the skin thickness of plaques fell from 5.3 +/- 0.4 (n = 11, mean +/- SEM) to 4.1 +/- 0.3 mm. In view of the in vitro results further studies are warranted, particularly if means can be found to improve the epidermal penetration of the relatively large ABL molecule (60 kDa).

Adult↗
PubMed1998

Human keratinocytes express transcripts for three isoforms of parathyroid hormone-related protein (PTHrP), but not for the parathyroid hormone/PTHrP receptor: effects of 1,25(OH)2 vitamin D3.

Parathyroid hormone-related protein (PTHrP) is strongly expressed in the epidermis and has been implicated in the regulation of growth and differentiation of keratinocytes. PTHrP has N-terminal sequence homology with parathyroid hormone (PTH) and binds to the type I PTH/PTHrP receptor, but earlier reports suggest that keratinocytes do not possess this cell surface receptor. In order to determine which PTHrP mRNA isoforms are expressed by keratinocytes and whether the type I receptor mRNA is present, we designed specific primers for reverse transcriptase-polymerase chain reaction. The interaction of PTHrP with other promoters of keratinocyte differentiation is unclear. In particular, 1,25(OH)2D3 is also fundamental in calcium homeostasis and induces changes in intracellular calcium. We therefore investigated the effect of 1,25(OH)2D3 on PTHrP mRNA expression and protein production in cultured human keratinocytes. Cells were incubated for 3 days at concentrations of 1.25(OH)2D3 of 10(-10)-10(-6) mol/L. PTHrP in culture supernatant, measured by two site immunoradiometric assay, was 915 +/- 98 PTHrP fmol/mg of cell layer protein in untreated cultures decreasing to 570 +/- 113 with 10(-8) mol/L and 402 +/- 24 with 10(-6) mol/L 1,25(OH)2D3 (mean +/- SEM, P < 0.01, n = 6). Transcripts for all three PTHrP isoforms (139, 141 and 173 amino acids) were detectable in keratinocyte mRNA. Corresponding to the decrease in PTHrP protein we demonstrated a reduction in all three PTHrP mRNA transcripts after 3 days' incubation with 1,25(OH)2D3 over a concentration range 10(-10)-10(-6) mol/L. Repeated studies failed to detect type I PTH/PTHrP receptor mRNA in human keratinocytes, either in control cultures or in the presence of 1,25(OH)2D3. We have shown that keratinocytes produce abundant PTHrP and that this is modulated by 1,25(OH)2D3, suggesting a physiological role. Further studies are required to investigate the relative expression of PTHrP isoforms, their role in keratinocyte signalling and the receptors involved.

Calcifediol↗
PubMed1998

Seborrhoeic dermatitis is not caused by an altered immune response to Malassezia yeast.

The immune response of patients with seborrhoeic dermatitis and healthy age- and sex-matched controls was examined to test the hypothesis that an inadequate or inappropriate immune response to Malassezia yeast leads to seborrhoeic dermatitis. Antibody responses were examined using enzyme-linked immunosorbent assays (ELISAs) and Western blots and lymphocyte responses using lymphocyte proliferation assays. The level of IgG and IgM specific for whole yeast cells or extracted proteins of two isolates of M. furfur was tested in ELISA. A wide range of antibody levels was found but the patient and control groups were indistinguishable (n = 19), and the groups could not be distinguished by the pattern of Malassezia proteins recognized by their sera in Western blots. The average affinity of the subjects' antibodies specific for Malassezia cells or proteins was measured using ammonium thiocyanate dissociation. Most of the sera had moderate affinities corresponding to 50% dissociation at thiocyanate concentrations of 0.5-1.0 mol/L. There was no difference between patients and matched controls. The proliferation of the patients' lymphocytes in response to a number of M. furfur cell preparations was measured: whole cells, cytoplasmic proteins, cell walls, soluble molecules extracted from the cell walls using sonication, and a commercial preparation. There was a wide range of responses between individuals, but there was no difference between the three groups: patients with seborrhoeic dermatitis (n = 16), healthy controls (n = 16) and a group suffering from other inflammatory skin conditions (n = 15). The results do not support the hypothesis that an inadequate immune response to Malassezia yeast could lead to seborrhoeic dermatitis. Other possible pathological mechanisms include toxin production or lipase activity.

Adult↗
PubMed1997

Inspiratory muscle fatigue following running to volitional fatigue: the influence of baseline strength.

Respiratory muscle fatigue has been demonstrated following short-term exercise to volitional fatigue, as well as following prolonged submaximal exercise. There is some suggestion that the respiratory muscles of 'athletic' individuals have superior strength and greater fatigue resistance but it is not known whether inspiratory muscle strength influences fatigueability of the inspiratory muscles. The present study examined this question in 24 moderately trained young men. Inspiratory muscle strength was measured at residual volume using a hand held Mouth Pressure Meter before and after an incremental, multistage shuttle run to volitional fatigue. Following the run, there was a significant fall in inspiratory mouth pressures (-10.5 +/- SD 8.2%; p < 0.001 Pre- vs Post Pipeak). The subjects with the weakest inspiratory muscles exhibited significantly greater fatigue than those with the strongest (-17.0 +/- SD 7.8% c.f. 6.8 +/- SD 4.4% for the 25th and 75th percentiles respectively p < 0.01). These data support existing evidence that the respiratory muscles fatigue following high intensity exercise. In addition, they provide new evidence that this phenomenon occurs in moderately trained young men and that the severity of the fatigue is related to the baseline strength of the inspiratory muscles.

Adult↗
PubMed1997

Kaposi's sarcoma associated with immunosuppression for bullous pemphigoid.

Kaposi's sarcoma may occur in transplant recipients on immunosuppressive regimens, but is not well recognized in association with treatment for dermatological disease. We report two cases where multifocal Kaposi's sarcoma developed following iatrogenic immunosuppression with prednisolone and azathioprine for bullous pemphigoid. Both patients were HIV negative and, in one case, lesions regressed both clinically and histologically when immunosuppressive therapy was withdrawn.

Aged↗
PubMed1995

Intracellular calcium modulates the responses of human melanocytes to melanogenic stimuli.

Ultraviolet radiation (UVR), the synthetic diacyglycerol (DAG), 1-oleoyl-2-acetylglycerol (OAG), and cyclic AMP (cAMP) stimulants, including cholera toxin (CT) have all been shown to increase melanogenesis in cultured human melanocytes. Indirect evidence suggests that an increase in intracellular free Ca2+ ([Ca2+]i) may be important in stimulated melanogenesis. Therefore, to determine whether melanogenic responses are modulated by [Ca2+]i, the Ca2+ in the culture medium of melanocytes ([Ca2+]o) was raised from 70 microM to 1 mM. This switch in [Ca2+]o was associated with a biphasic increase in [Ca2+]i, with an early transient rise, over minutes, and a delayed sustained rise in [Ca2+]i, over hours. The early increase was blocked by nickel chloride (NiCl2), but not affected by depletion of [Ca2+]i stores by thapsigargin, suggesting that this [Ca2+]i rise was due to Ca2+ entry across the plasma membrane. Melanocytes cultured in the absence of CT had a reduced basal melanin content following the switch to 1 mM [Ca2+]o, but in the presence of CT, which acts by stimulating cAMP synthesis, the basal level was increased. Raising [Ca2+]o resulted in enhanced melanogenic responses to UVR and OAG, in the presence or absence of CT, suggesting that Ca(2+)-dependent mechanisms are important. UVR also stimulated a delayed rise in [Ca2+]i, over 24 h, but OAG did not. These results indicate that while [Ca2+]i is not essential for melanogenesis, it plays an important role in modulating the responses of melanocytes to melanogenic stimuli.

Calcium↗
PubMed1995

Intracellular calcium as a second messenger following growth stimulation of human keratinocytes.

The mitogenic effect of the neuropeptide substance P and bombesin was investigated in normal human keratinocytes in serum-free culture, both with and without the presence of epidermal growth factor (EGF). Although both neuropeptides induced a small increase in cell numbers in the presence of EGF, the response was much greater in its absence, and cell numbers increased to 200% of controls at 5 days. Changes in intracellular free calcium are frequently seen following mitogenic stimulation of cells, and this phenomenon was studied in individual keratinocytes. Epidermal growth factor (10 ng/ml) induced calcium transients in 57% (n = 21) of cells. The mean intracellular free calcium was 97 +/- 11 nM (mean +/- SEM) in quiescent cells, and the calcium transients reached approximately 250 nM for 3-4 min. In the presence of EGF, calcium transients were never observed with the addition of either substance P or bombesin. For EGF-deprived cultures, 20% of keratinocytes (n = 10) showed a large calcium transient following the addition of 500 nM bombesin, and 63% (n = 12) of cells gave calcium transients following the addition of 700 nM of substance P. Studies in calcium-free medium, and following depletion of intracellular calcium stores with thapsigargin, showed that all of the calcium transients were dependent on the presence of intracellular stores, but also partially mediated by an influx of extracellular calcium. These studies demonstrate the mitogenic effect of substance P and bombesin on human keratinocytes in the absence of EGF. The ability of the neuropeptides to increase keratinocyte growth in culture suggests a possible in wound healing.

Bombesin↗
PubMed1994

Staurosporine, a non-specific PKC inhibitor, induces keratinocyte differentiation and raises intracellular calcium, but Ro31-8220, a specific inhibitor, does not.

The responsiveness of normal human keratinocytes to different modulators of protein kinase C (PKC) was investigated. The PKC agonist TPA, staurosporine (a non-specific inhibitor), and Ro31-8220 (a specific inhibitor) were studied for effect on cell morphology, growth rate, involucrin expression, and intracellular calcium levels. Surprisingly the response to nanomolar concentrations of staurosporine was similar to TPA and induced a fusiform morphology, inhibited growth, increased involucrin levels, and raised intracellular calcium. Staurosporine also increased the number of cornified envelopes, and its action therefore appeared identical to TPA. In contrast, Ro31-8220 had little effect on morphology or growth and blocked both the TPA-induced growth inhibition and calcium rise. Ro31-8220 had no effect on staurosporine-induced growth inhibition but partially reduced its associated calcium rise. These results suggest PKC activation is required for keratinocyte differentiation and that staurosporine acts like a PKC agonist to give a similar effect as TPA. Specific inhibition of PKC by Ro31-8220 inhibits TPA-induced differentiation.

Alkaloids↗
PubMed1994

Thapsigargin raises intracellular free calcium levels in human keratinocytes and inhibits the coordinated expression of differentiation markers.

Thapsigargin raises intracellular free calcium ([Ca2+]i) by potently inhibiting the endoplasmic reticulum Ca-ATPase, which sequesters calcium from the cytosol. In human keratinocytes a rise in [Ca2+]i has been associated with differentiation and therefore we investigated the action of thapsigargin on this process. At concentrations above 3 nM thapsigargin inhibited keratinocyte proliferation. Thapsigargin induced an immediate transient [Ca2+]i rise in calcium-free or 70 microM calcium medium but a more prolonged rise in 2 mM calcium. For keratinocytes cultured in 70 microM calcium medium a late [Ca2+]i rise was also observed, after 6 h, similar to the effect of known differentiation stimuli. However, immunohistochemical techniques did not show any expression of the differentiation-specific protein involucrin, a component of the cornified envelope. When keratinocyte differentiation was induced by an increase in the extracellular calcium from 70 microM to 2 mM abundant involucrin and desmoplakin, a component of desmosomes, were synthesised. Both proteins gave staining patterns which suggested incorporation into structural proteins, but thapsigargin disrupted the calcium-induced pattern of involucrin and desmoplakin synthesis. Thapsigargin did not induce differentiation, possibly due to its inability to activate protein kinase C and raise inositol trisphosphate levels. We conclude that a rise in [Ca2+]i does not alone induce keratinocyte differentiation but may act with other intracellular signals to promote differentiation.

Biomarkers↗
PubMed1994

Ni2+ blocks the Ca2+ influx in human keratinocytes following a rise in extracellular Ca2+.

In keratinocytes a rise in extracellular Ca2+ induces differentiation and is associated with a sustained increase in intracellular Ca2+, due to Ca2+ entry across the plasma membrane. We have investigated the mechanism of Ca2+ entry in human keratinocytes following this rise, using Fura-2-loaded cells and the cations Ni2+, Co2+, Mn2+, and La3+ and Ca2+ channel blocker verapamil. Keratinocytes were permeable to La3+, Mn2+, and Co2+; Fura-2 fluorescence was quenched by Mn2+ and Co2+. Verapamil was unable to block Ca2+ entry. Ni2+ did not enter keratinocytes, but blocked the influx of extracellular Ca2+ and Mn2+. Thapsigargin depleted Ca2+ stores, inducing a large transient intracellular rise, and the efflux of this Ca2+ was not blocked by Ni2+. We conclude that keratinocytes are permeable to a number of cations, but not Ni2+, which may be used to block the entry of the other cations during the study of cation flux into cells. The data presented are consistent with calcium entry by a nonspecific cation channel recently described on keratinocytes.

Calcium↗