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Biomedical subjects

M K Church

Publications and source records attributed to M K Church.

At least 37 records · Page 2Linked to original sources
PubMed1999

Measurement of interstitial cetirizine concentrations in human skin: correlation of drug levels with inhibition of histamine-induced skin responses.

BACKGROUND: The purpose of the present study was to measure the concentrations of cetirizine in the extracellular water compartment in intact human skin and assess simultaneously inhibition of histamine-induced wheal and flare reactions. METHODS: Skin cetirizine levels were collected by the microdialysis technique and analyzed by high-pressure liquid chromatography with mass spectrometry detection. Skin levels in 20 subjects were compared to plasma levels for 4 h after a single oral dose of 10 or 20 mg of cetirizine. Skin prick tests were performed with histamine 100 mg/ml. RESULTS: Plasma cetirizine levels increased within 30 min to reach peak values of 315+/-10 and 786+/-45 ng/ml 90-120 min after administration of 10 and 20 mg of cetirizine. This was followed by a slow decline. In the skin, dialysate cetirizine levels (non-protein-bound fraction only) peaked at 1.6+/-0.1 and 2.4+/-0.3 ng/ml at 120-180 min. In vivo recovery of cetirizine was 14.4+/-4.3%. It was estimated that the non-protein-bound concentration of cetirizine in the skin was 50-70% of corresponding plasma values. Both 10- and 20-mg doses of cetirizine inhibited wheal and flare reactions over 240 min. The time vs concentration profile of cetirizine in skin dialysate paralleled the inhibition of skin reactions, but no significant correlations were found between individual cetirizine levels in skin or plasma with wheal and flare reactions. CONCLUSIONS: Cetirizine concentrations in the skin could be monitored by the microdialysis technique. The results indicate no simple linear correlation between cetirizine skin levels and inhibition of skin reactions.

Adult↗
PubMed1999

Histamine release upon adenosine 5'-monophosphate (AMP) nasal provocation in allergic subjects.

BACKGROUND: Nasal provocation with adenosine 5'-monophosphate (AMP) elicits nasal symptoms in subjects with rhinitis. Histamine released from mast cells may play a part in AMP induced nasal responses. METHODS: Symptoms of rhinitis were recorded and histamine release in the fluid obtained by nasal lavage after AMP, guanosine 5'-monophosphate (GMP), and placebo instillations was measured in nine subjects with allergic rhinitis and nine non-allergic controls in a double blind, randomised, placebo controlled study. RESULTS: No symptoms or significant increases in histamine were observed after GMP and placebo challenge. Significantly higher levels of histamine were seen in the nasal lavage fluids of allergic subjects following AMP challenge than in nonallergic controls, the median (range) histamine concentration increasing from the baseline value of 1.62 (0.44-6.99) ng/ml to 6.45 (0.81-16.17) ng/ml at three minutes. No increase in histamine levels was seen in the non-allergic subjects in whom the median histamine concentration was 1.13 (0.29-4.25) ng/ml at baseline and 0.97 (0.31-5.89) ng/ml three minutes after AMP challenge. CONCLUSIONS: AMP elicits an immediate rise in histamine levels in the nasal lavage fluid of allergic subjects compared with non-allergic individuals. These findings indicate that the exaggerated nasal response to adenosine may reflect mast cell priming in vivo, thus supporting its application as a potential new marker of allergic inflammation.

Adenosine Monophosphate↗
PubMed1999

Non-H1-receptor effects of antihistamines.

Visit any international allergy meeting and you will soon learn that there are a plethora of very potent and effective histamine H1-receptor antagonists, or antihistamines as they are more often called. Thus, in order to make any particular antihistamine stand out therapeutically or commercially from the others, additional properties, e.g. anti-inflammatory properties, are often claimed. But are these claims valid and, if so, are the 'additional properties' clinically relevant? This document will review the evidence behind some of the 'additional properties' of antihistamines and lay the basis for discussion of their relevance.

Animals↗
PubMed1998

Human mast cells express stem cell factor.

Stem cell factor (SCF) is a major cytokine regulator of mast cell growth and function. The present study demonstrates that human mast cells are able to produce SCF. Constitutive synthesis of SCF mRNA was seen in the mast cells isolated from human lung and skin by RT-PCR. This was confirmed by in situ hybridization in conjunctival mast cells of both tryptase-only (MCT) and tryptase/chymase (MCTC) subsets. SCF protein product was found in conjunctival MCT and MCTC mast cells by immunohistochemistry. Soluble SCF protein was detected in the culture supernatant of isolated lung mast cells by ELISA, and cross-linkage of IgE receptor (Fc epsilon-RI) on the lung mast cells in culture did not alter SCF mRNA expression, or the secreted soluble SCF protein. This was consistent with the finding that levels of SCF mRNA expression in conjunctival mast cells were similar between normal subjects and patients with seasonal allergic conjunctivitis (SAC). This study shows that human mast cells themselves are a cellular source of SCF, as well as being target cells for this growth factor. SCF may regulate mast cell growth and function via both paracrine and autocrine mechanisms. The production of SCF by mast cells may be regulated via mechanisms other than IgE receptor-mediated pathways.

Cell Culture Techniques↗
PubMed1998

Human lung mast cells are enriched in the capacity to produce granulocyte-macrophage colony-stimulating factor in response to IgE-dependent stimulation.

By using reverse transcription-PCR, in situ hybridization, enzyme-linked immunosorbent assay and immunocytochemistry, we have studied the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in human lung mast cells induced by cross-linkage of high-affinity Fc epsilon receptors (Fc epsilonRI). We have also confirmed the bioactivity of GM-CSF released from lung mast cells by investigating the effect of the supernatant from lung mast cells activated with anti-IgE on the release of eosinophil cationic protein (ECP) from eosinophils. Mast cells were purified using affinity magnetic selection with monoclonal antibody (mAb) YB5.B8 (93-99% pure). Purified mast cells were precultured with IgE for 16 h before challenge with 1 microg/ml anti-IgE with or without stem cell factor (SCF). Eosinophils were purified by immunomagnetic negative selection (> 98% pure). The activation of mast cells via Fc epsilonRI enhanced the intensity of the GM-CSF signal within 2 h and the cells produced GM-CSF protein 4 h after the activation. In the absence of recombinant human (rh) SCF, anti-IgE induced a median GM-CSF response of 202 (< 15 to approximately 681) pg/10(6) mast cells/24 h, whereas in the presence of rhSCF the median IgE-dependent GM-CSF release was 356 (152 to approximately 1216) pg/10(6) mast cells/24 h. This difference was statistically significant (p = 0.0029, n = 12). In contrast, mast cells produced only a small amount of GM-CSF in the absence of anti-IgE. The mast cell supernatant induced ECP release from eosinophils and the release was significantly inhibited by blocking mAb against GM-CSF. These findings indicate that human mast cells are an important cellular source of GM-CSF and as such may contribute to chronic eosinophil-mediated inflammation.

Blood Proteins↗
PubMed1998

Phosphodiesterase type 4 inhibitors, but not glucocorticoids, are more potent in suppression of cytokine secretion by mononuclear cells from atopic than nonatopic donors.

BACKGROUND: Both glucocorticosteroids and phosphodiesterase (PDE) type 4 inhibitors have modulatory effects on PBMC cytokine secretion. In this study we compared the effect of glucocorticoids and PDE inhibitors on IL-10 and TNF-alpha production by PBMCs from nonatopic versus atopic individuals. METHODS: PBMCs were incubated with glucocorticoids (beclomethasone dipropionate and mometasone furoate) or media alone for 24 hours. PDE type 4 inhibitors (Ro20-1724 and rolipram) were then added to the cells preincubated with media. After stimulation with PHA, incubation was continued for 48 hours. The cytokine content of the cell supernatants was determined by ELISA. RESULTS: PDE-4 inhibitors and glucocorticoids caused a concentration-dependent inhibition of the secretion of both TNF-alpha and IL-10. PDE-4 inhibitors were over 20 times more potent in suppressing cytokine secretion by PBMCs from atopic than nonatopic donors, and approximately 5 times more potent in preventing TNF-alpha than IL-10 secretion. In cells from nonatopic donors, glucocorticoids inhibited the production of TNF-alpha to a greater extent than IL-10, but these drugs were more potent in cells from nonatopic than atopic persons. CONCLUSION: In conclusion, both PDE-4 inhibitors and glucocorticoids suppress secretion of TNF-alpha and IL-10. However, because PDE-4 inhibitors are more potent in suppressing cytokine secretion by PBMCs from atopic individuals but less potent in inhibiting production of IL-10, PDE-4 inhibitors may have greater therapeutic potential than glucocorticoids in allergic diseases.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Glucocorticoids inhibit proliferation and interleukin-4 and interleukin-5 secretion by aeroallergen-specific T-helper type 2 cell lines.

BACKGROUND: Glucocorticoids play an important role in the treatment of allergic disease. The atopic process, itself, may reduce the response of peripheral blood mononuclear cells (PBMC) to these drugs. OBJECTIVE: In this study we compared the effect of hydrocortisone (HC), beclomethasone (BDP), and mometasone (MF) on interleukin (IL)-4 and IL-5 secretion by aeroallergen-specific T-helper type 2 cells (Th2) and proliferation of PBMC from atopic donors. METHODS: Cells were incubated with drug before stimulating with phytohemagglutinin and assessing proliferation (PBMC) and cytokine secretion (Th2). RESULTS: The glucocorticoids concentration dependently inhibited proliferation and cytokine secretion, but had less effect on proliferation of cells from severe atopics than on cells from those whose symptoms required little treatment. The rank order of potency was MF (average IC50 0.01 nM) > BDP (4.0 nM) > HC (250 nM). CONCLUSIONS: These experiments demonstrate glucocorticoid inhibition of IL-4 and IL-5 secretion by human Th2-like cells and proliferation of PBMC from severely and mildly allergic donors.

Allergens↗
PubMed1998

Effects of H1 antagonists on the cutaneous vascular response to histamine and bradykinin: a study using scanning laser Doppler imaging.

Histamine plays an important part in the cutaneous weal and flare response which underlies many allergic skin conditions. It has a direct effect on the local vasculature to promote vasodilatation and increase microvascular permeability and may also initiate the more widely spread neurogenic flare. Quantification of these responses and studies of the mediator mechanisms underlying them have been limited by the lack of appropriate techniques to investigate them. To address this we have used two relatively new techniques, scanning laser Doppler imaging (LDI) and dermal microdialysis to measure changes in skin blood flow and the release of histamine within the weal and flare, following intradermal injection of histamine or bradykinin. These measurements have been made both in the absence and presence of the H1 receptor blockers cetirizine and loratadine. Scanning LDI of the inflammatory response revealed marked differences in both the development and steady state responses to the intradermal injection of histamine (1-3 mumol/L) and bradykinin (1 mumol/L). The development of the flare and the weal response to both histamine and bradykinin was significantly reduced by cetirizine but not by loratadine. The histamine-induced flare area fell by 57 +/- 4% (mean +/- SEM, n = 10, P < 0.001) after cetirizine and the area of the weal fell by 73 +/- 11% (P < 0.009). Bradykinin-induced inflammatory responses were similarly reduced by cetirizine, the weal by 60 +/- 16% (P < 0.02) and the flare by 61 +/- 4% (P < 0.005). Measurement of histamine concentration in skin using microdialysis, in six subjects, confirmed that histamine levels rose in the dialysate collected from the weal to 310 +/- 16 nmol/L following injection of histamine. Histamine levels also rose following bradykinin injection in some subjects (mean 147 +/- 46 nmol/L, range 18-336). Little increase in histamine concentration was seen in the dialysate from the flare following injection of either histamine or bradykinin. The histamine concentration in dialysate from unprovoked skin was 4.19 +/- 0.75 nmol/L. These data reveal differences in the dermal responses to different mediators when assessed using scanning LDI. They confirm that histamine is released within the weal but not the flare response to the intradermal injection of both histamine and bradykinin and that its effects on the local vasculature to cause the oedematous weal and the axon reflex-mediated flare are significantly attenuated by the H1 antagonist cetirizine and to a lesser extent by the H1 antagonist loratadine.

Adult↗
PubMed1998

Measurement of nitric oxide concentration in human skin in vivo using dermal microdialysis.

Using microdialysis, we measured nitric oxide (NO) levels in healthy human skin, in vivo, before and during the local inflammatory response to histamine. Basal dialysate NO concentration, assayed using an amperometric technique, was 0.49+/-0.06 microM (mean+/-S.E.M., 21 probes, 14 subjects). Histamine injection produced transient increases in NO concentration within both the weal and flare which was blocked by the NO synthase inhibitor, NG-nitro-L-arginine-methyl ester (L-NAME). Dialysate NO concentration also increased following transdermal delivery of the nitrosovasodilator, glyceryl trinitrate. Thus, using microdialysis, it is possible to quantify NO production in human skin in vivo and study its modulation during the acute inflammatory response.

Adult↗
PubMed1998

Human skin mast cells produce TNF-alpha by substance P.

Using in situ hybridization and the reverse transcriptase polymerase chain reaction (RT-PCR) we show that messenger RNA for IL-4, IL-5 and tumor necrosis factor-alpha (TNF-alpha) is induced by cross-linkage of high-affinity Fc(epsilon) receptors (Fc(epsilon)RI) on human skin mast cells, but that only TNF-alpha mRNA is selectively induced by substance P. Skin mast cells were purified using the Percoll density technique. T cells were removed by serial negative selection using a CD2 monoclonal antibody (mAb) to achieve a final mast cell purity >95%. Purified mast cells were precultured with recombinant human stem cell factor (rhSCF; 10 ng/ml) and myeloma IgE (3 microg/ml) for 16 h before challenge with sheep polyclonal antihuman IgE antibody (anti-IgE; 1 or 10 microg/ml) in the presence of rhSCF (50 ng/ml). Using in situ hybridization, we demonstrated that IgE-dependent stimulation induces the expression of IL-4, IL-5 and TNF-alpha mRNA in skin mast cells. We have investigated the expression of IL-4, IL-5 and TNF-alpha mRNA by substance P, with the result that substance P, 0.003-30 microM, selectively induced TNF-alpha mRNA. However, substance P did not induce IL-4 mRNA and did not enhance IL-5 mRNA. Furthermore, we confirmed the release of TNF-alpha by substance P from skin mast cells using an ELISA technique. These findings demonstrate the capacity of human skin mast cells to transcribe IL-4, IL-5 and TNF-alpha by immunological activation and to transcribe and release TNF-alpha by substance P.

Base Sequence↗
PubMed1998

Asthma, adenosine, mast cells and theophylline.

Many clinical trials have suggested that theophylline has anti-inflammatory properties in the treatment of bronchial asthma. Proposed mechanisms of theophylline inhibition include phosphodiesterase inhibition, an adenosine receptor antagonist, the increase of circulating adrenaline, mediator antagonist and inhibition of calcium ion influx. Further to these observations we report on the inhibition by theophylline of NF-kappaB, a key transcription factor found in human purified mast cells, which plays a role in the transcription of TNF alpha, GM-CSF, and IL-8 within this cell. The suppression of NF-kappaB activation, indicates that theophylline, in addition to its bronchodilator activities, has the potential for anti-inflammatory activity.

Adenosine↗
PubMed1998

Theophylline inhibits the release of eosinophil survival cytokines--is Raf-1 the protein kinase A target?

Increased numbers of activated eosinophils in bronchial tissue is a feature of asthma and may, in part, be attributed to the prolonged cytokine-dependent survival of eosinophils within the inflamed microenvironment. Low-dose oral theophylline was previously shown to reduce the number of activated eosinophils within the sub-mucosa following allergen exposure. A number of inhibitory actions of theophylline have been described which relate to eosinophil recruitment and activation, including inhibition of cell migration and release of granule basic proteins. In this study we investigated the ability of theophylline to inhibit the release of preformed GM-CSF and IL-8 from eosinophils in vitro, as these cytokines may serve an autocrine function in eosinophil survival in vivo. Eosinophils rapidly released GM-CSF and IL-8 spontaneously, and release was further enhanced in response to sIgA-coated beads. Theophylline inhibited the stimulated, but not the spontaneous, release of both cytokines. We previously reported the role of protein kinase A in inhibition of arachidonic acid mobilization and LTC4 synthesis. Therefore we speculate that cAMP-dependent activation of protein kinase A following theophylline treatment of eosinophils resulted in inhibition of Raf-1 and MAPK/MAPKK dependent activation of phospholipase A2 and consequently inhibition of degranulation and cytokine release.

Bronchodilator Agents↗
PubMed1997

Platelet-activating factor induces histamine release from human skin mast cells in vivo, which is reduced by local nerve blockade.

BACKGROUND: Intradermal injection of platelet-activating factor (PAF) causes wheal and flare reactions, which are inhibited by antihistamines. However, PAF does not release histamine from human dispersed skin mast cells in vitro. The purpose of this study was to investigate the extent and possible mechanisms of PAF-induced histamine release in human skin in vivo with the use of dermal microdialysis. METHODS: Hollow dialysis fibers were inserted into the upper dermis in forearm skin and each fiber was perfused with Krebs-Ringer bicarbonate solution at a rate of 3.0 microliters/min. PAF (4.5 to 36 mumol/L), lyso-PAF (36 mumol/L), vehicle (negative control), and codeine 750 or 250 mumol/L (positive control) were injected intradermally above separate fibers. Dialysate was collected in 2-minute fractions for 20 minutes and histamine analyzed spectrofluorometrically. RESULTS: PAF, but not lyso-PAF, caused statistically significant dose-related histamine release and wheal and flare reactions. Intradermal mepivacaine administration significantly abrogated flare reactions by PAF and codeine and inhibited histamine release and wheal reactions by PAF but not by codeine. Long-term topical capsaicin administration inhibited histamine release and wheal reactions by PAF but not by codeine. It inhibited flare reactions induced by both compounds. PAF did not release histamine from blood basophils. CONCLUSIONS: These data suggest that PAF induced histamine release from mast cells in intact human skin indirectly via neurogenic activation. Further, on the intradermal injection of PAF histamine release and the skin responses, the wheal and the flare, are differentially regulated by neurogenic components.

Adult↗
PubMed1997

Histamine is released in the wheal but not the flare following challenge of human skin in vivo: a microdialysis study.

BACKGROUND: The mediator mechanisms of the cutaneous wheal and flare response, which underlies allergic skin and urticarial conditions, are controversial. The wheal results primarily from a direct effect of histamine on the local vascular bed but to what extent does histamine diffuse within the wheal? The flare is neurogenic, in origin, being disseminated within the dermis by axon reflexes, but do the neuropeptides released from the nerve endings cause the vasodilatation directly or do they induce the further release of histamine which then transduces the flare? OBJECTIVE: We have addressed these questions by inserting 216 microns diameter microdialysis fibres into the dermis within the different areas of the wheal and flare to monitor changes in histamine levels provoked by intradermal injections of histamine, allergen, codeine and substance P. Twenty-one subjects participated in the investigations. RESULTS: The histamine concentration in unprovoked skin was 10.5 +/- 0.6 nM. As the dialysis efficacy was approximately 50%, this equates to tissue concentrations of 20 nM. All provicants released large amounts of histamine at the injection site, maximum histamine levels being 337-1293 nM. Diffusion of histamine within the wheal was poor, levels at 2.3 mm and 3.7 mm from the site of injection being 4-22% and 0.2-3.7% respectively of those 1 mm from the injection site. No increased histamine levels were detected in the flare with any provicant. Atraumatic delivery to the skin of histamine in infusion concentrations of 30-10,000 nM caused concentration-related effects, at least 100 nM being necessary to induce a significant increase in skin blood flow, a threshold of 300-1000 being required to stimulate a visible flare and a measurable erythema, and 3000-10,000 nM being the minimum for induction of a wheal. Thus the skin blood vessels and nerves are responsive to histamine, but at relatively high concentrations. CONCLUSIONS: These data support the theory that the flare reaction to focal histamine injection or release is a neurogenic reflex not involving histamine release at its effector end.

Adult↗