The complex biology and contribution of Staphylococcus aureus in atopic dermatitis, current and future therapies.

Atopic dermatitis (AD) is a complex, chronic inflammatory skin disorder affecting more than 10% of U.K. children and is a major cause of occupation-related disability. A subset of patients, particularly those with severe AD, are persistently colonized with Staphylococcus aureus and exacerbation of disease is commonly associated with this bacterium by virtue of increased inflammation and allergic sensitization, aggravated by skin barrier defects. Understanding the complex biology of S. aureus is an important factor when developing new drugs to combat infection. Staphylococcus aureus generates exoproteins that enable invasion and dissemination within the host skin but can also damage the skin and activate the host immune system. Antibiotics are often used by dermatologists to aid clearance of S. aureus; however, these are becoming less effective and chronic usage is discouraged with the emergence of multiple antibiotic-resistant strains. New ways to target S. aureus using monoclonal antibodies and vaccines are now being developed. This review will attempt to evaluate the key biology of S. aureus, current treatment of S. aureus infections in AD and recent advances in developing new anti-S. aureus therapies that have potential in severe AD.

Use of cell permeable NBD peptides for suppression of inflammation.

Nuclear factor (NF)-kappaB is a ubiquitous and essential transcription factor whose dysregulation has been linked to numerous diseases including arthritis and cancer. It is therefore not surprising that the NF-kappaB activation pathway has become a major target for development of novel therapies for inflammatory diseases and cancer. However, the indispensable role played by NF-kappaB in many biological processes has raised concern that a complete shutdown of this pathway would have significant detrimental effects on normal cellular function. Instead, drugs that selectively target the inflammation induced NF-kappaB activity, while sparing the protective functions of basal NF-kappaB activity, would be of greater therapeutic value and would likely display fewer undesired side effects. The recent identification and characterisation of the NF-kappaB essential modulator (NEMO)-binding domain (NBD) peptide that can block the activation of the IkappaB kinase (IKK) complex, have provided an opportunity to selectively abrogate the inflammation induced activation of NF-kappaB by targeting the NBD-NEMO interaction. This peptide is synthesised in tandem with a protein transduction domain sequence from Drosophila antennapedia which facilitates uptake of the inhibitory peptide into the cytosol of target cells.

Intravenous hyoscine as a premedication for colonoscopy: a randomized double-blind controlled trial.

BACKGROUND AND STUDY AIMS: There is wide variation in the use of antispasmodics to facilitate colonoscopy, both within and between countries, and its use before such procedures remains controversial. The aim of this study was to determine whether there was any objective benefit in using hyoscine as a premedication for colonoscopy in a district general hospital. PATIENTS AND METHODS: Consecutive day-case patients undergoing colonoscopy were included in the study. They were prospectively randomly allocated to receive either intravenous hyoscine (n = 61) or intravenous placebo (n = 56) as part of their premedication. RESULTS: Our analysis demonstrated no statistically significant difference between the two groups with respect to the median time from colonoscope insertion to caecal intubation (9.7 minutes in the hyoscine group vs. 8.3 minutes in the placebo group) or the median total procedure time (14.8 minutes in the hyoscine group vs. 13.8 minutes in the placebo group). There was also no statistically significant difference in success rates for caecal intubation between the two groups ( P < 0.06). However a type II error cannot be excluded because of the small sample size. CONCLUSION: This study demonstrated no obvious benefit in the routine use of hyoscine as a premedication for colonoscopy in a district general hospital setting.

Fibronectin and fibrinogen contribute to the enhanced binding of Staphylococcus aureus to atopic skin.

BACKGROUND: Staphylococcus aureus colonizes the skin lesions of more than 90% of patients with atopic dermatitis (AD). The mechanism for increased S aureus colonization in AD is unknown. However, the initial event in colonization requires adherence of S aureus to the skin. OBJECTIVE: The purpose of this study was to examine the roles of various bacterial adhesins on S aureus binding to AD skin. METHODS: In an attempt to delineate the mechanism behind this adherence process, an in vitro bacterial binding assay was developed to quantitate the adherence of various S aureus strains to AD, psoriatic, and normal skin sections. S aureus strains used in this study were obtained either from cultures of AD skin lesions or from genetically manipulated strains of S aureus that lacked specific microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)--namely, fibronectin-binding protein (Fnbp), fibrinogen-binding protein (Clf), collagen-binding protein (Cna), and their parent strains. In addition, S aureus strains from patients with AD were pretreated with fibronectin or fibrinogen to block MSCRAMM receptors and interfere with binding. RESULTS: Under all experimental conditions, binding of S aureus was localized primarily to the stratum corneum. Immunocytochemical staining of AD skin sections showed a redistribution of fibronectin to the cornified layer, an observation not seen in normal skin. S aureus binding to uninvolved AD skin was significantly greater than the binding to uninvolved psoriatic skin (P <.0001) and normal skin (P <.0005). The Fnbp-negative S aureus showed a significant reduction in binding to the AD skin (P <.0001) but not to the psoriatic and normal skin. In the AD skin, a significant reduction in the binding of S aureus was also observed in the Clf-negative strain (P <.0001) but not in the Cna-negative S aureus. Preincubation of S aureus with either fibronectin or fibrinogen also inhibited bacterial binding to AD skin (P <.0001). CONCLUSION: These data suggest that fibronectin and fibrinogen--but not collagen--play a major role in the enhanced binding of S aureus to the skin of patients with AD.

Preferential binding of Staphylococcus aureus to skin sites of Th2-mediated inflammation in a murine model.

Staphylococcus aureus is found on over 90% of atopic dermatitis skin lesions and is thought to contribute to skin inflammation via the production of potent exotoxins. In contrast, less than 5% of normal subjects harbor S. aureus. This suggests that an atopic immune response itself may play a role in preferential binding of S. aureus to the skin. To examine this issue more directly, we analyzed the S. aureus binding characteristics of skin in mice undergoing different T helper type 1 cell versus T helper type 2 cell inflammatory responses using a novel in vitro bacterial binding assay. BALB/C female mice were first sensitized to ovalbumin with alum or ovalbumin with complete Freund's adjuvant to induce T helper type 2 or T helper type 1 responses, respectively. Mice were then challenged intradermally with either saline (control) or ovalbumin. Forty-eight hours later, skin specimens were obtained from the challenge sites, and the number of S. aureus binding to each skin section was quantitated. Bacterial binding was found to be significantly greater at skin sites of BALB/C mice that had been ovalbumin/alum sensitized compared with ovalbumin/complete Freund's adjuvant sensitized (p < or = 0.01). When compared to the ovalbumin sensitized/challenged skin of wild type BALB/C mice or interferon-gamma gene knockout mice, interleukin-4, but not interferon-gamma, gene knockout mice had significantly less S. aureus binding at their ovalbumin sensitized/challenged skin sites. Mutant S. aureus strains that lacked either fibronectin- or fibrinogen-binding protein expression showed significantly reduced S. aureus binding compared with the parent wild type strain (p < 0.005). Moreover, preincubation of the wild type bacteria with fibronectin or fibrinogen, but not collagen, resulted in significantly less skin binding of S. aureus (p < 0.01). Incubation of skin with interleukin-4, and less so with interferon-gamma, led to more binding of wild type S. aureus but not of an S. aureus mutant deficient in fibronectin binding protein expression. After interleukin-4 incubation, but not interferon-gamma, epidermal immunoreactivity for fibronectin was observed in murine skin explants. These results show that a T helper type 2 inflammatory environment can promote skin binding by S. aureus and that this binding is mediated by fibronectin and fibrinogen.

High constitutive glucocorticoid receptor beta in human neutrophils enables them to reduce their spontaneous rate of cell death in response to corticosteroids.

Neutrophils are markedly less sensitive to glucocorticoids than T cells, making it difficult to control inflammation in neutrophil-mediated diseases. Development of new antiinflammatory strategies for such diseases would be aided by an understanding of mechanisms underlying differential steroid responsiveness. Two protein isoforms of the human glucocorticoid receptor (GR) exist, GRalpha and GRbeta, which arise from alternative splicing of the GR pre-mRNA primary transcripts. GRbeta does not bind glucocorticoids and is an inhibitor of GRalpha activity. Relative amounts of these two GRs can therefore determine the level of glucocorticoid sensitivity. In this study, human neutrophils and peripheral blood mononuclear cells (PBMCs) were studied to determine the relative amounts of each GR isoform. The mean fluorescence intensity (MFI) using immunofluorescence analysis for GRalpha was 475 +/- 62 and 985 +/- 107 for PBMCs and neutrophils, respectively. For GRbeta, the MFI was 350 +/- 60 and 1,389 +/- 143 for PBMCs and neutrophils, respectively (P < 0.05). After interleukin (IL)-8 stimulation of neutrophils, there was a statistically significant increase in intensity of GRbeta staining to 2,497 +/- 140 (P < 0.05). No change in GRalpha expression was observed. This inversion of the GRalpha/GRbeta ratio in human neutrophils compared with PBMCs was confirmed by quantitative Western analysis. Increased GRbeta mRNA expression in neutrophils at baseline, and after IL-8 exposure, was observed using RNA dot blot analysis. Increased levels of GRalpha/GRbeta heterodimers were found in neutrophils as compared with PBMCs using coimmunoprecipitation/Western analysis. Transfection of mouse neutrophils, which do not contain GRbeta, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRbeta by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death. Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

Evidence for superantigen involvement in skin homing of T cells in atopic dermatitis.

The environmental factors that contribute to the homing of T cells in skin disease is unknown. The skin lesions of atopic dermatitis (AD) are frequently colonized with superantigen (SAg), producing strains of Staphylococcus aureus. In vitro, these superantigens have the capacity to activate and expand T cells expressing specific T cell receptor BV gene segments, and also to increase their skin homing capacity via upregulation of the skin homing receptor, cutaneous lymphocyte-associated antigen (CLA). These activities have been proposed to enhance the chronic cutaneous inflammation of AD, but an in vivo role for SAg has not been conclusively demonstrated. In this study, we sought direct evidence for in vivo SAg activity by comparing the SAg profiles of S. aureus cultured from the skin of AD subjects to the T cell receptor Vbeta repertoire of their skin homing (CLA+) T cells in peripheral blood. SAg secreting S. aureus strains were identified in six of 12 AD patients, and all of these subjects manifested significant SAg-appropriate Vbeta skewing within the CLA+ subsets of both their CD4+ and their CD8+ T cells. T cell receptor Vbeta skewing was not detectable among the overall CD4+ or CD8+ T cell subsets of these subjects, and was not present within the CLA+ T cell subsets of five patients with plaque psoriasis and 10 normal controls. T cell receptor BV genes from the presumptively SAg-expanded populations of skin homing T cells were cloned and sequenced from three subjects and, consistent with a SAg-driven effect, were found to be polyclonal. We conclude that SAg can contribute to AD pathogenesis by increasing the frequency of memory T cells able to migrate to and be activated within AD lesions.

The role of superantigens in human diseases: therapeutic implications for the treatment of skin diseases.

Although it is well established that immune mechanisms contribute to the pathogenesis of chronic inflammatory skin diseases such as atopic dermatitis (AD) and psoriasis, the actual events that trigger the immunological pathways resulting in these skin diseases are not well understood. Colonization and infection with Staphylococcus aureus and streptococci has been reported to exacerbate AD and psoriasis. Recent studies demonstrating that bacterial toxins can act as superantigens provide mechanism(s) by which S. aureus and streptococci could mediate an inflammatory skin lesion that consists predominantly of activated T-cells and monocytes. This review will explore the diverse mechanisms by which bacterial superantigens can induce skin inflammation following systemic or local infection. These observations provide a new direction for the development of novel approaches for the treatment of skin inflammation.

TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression.

The in vivo response to ultraviolet B (UVB) radiation in skin is characterized by the accumulation of both mononuclear and polymorphonuclear cells within the dermis and an induction of vascular endothelial adhesion molecules. Epidermal production of cytokines (IL-8 and TNF-alpha) has been strongly implicated in the development of UVB-induced inflammation. In the current study, we examined the time course of IL-8 and TNF-alpha mRNA and protein expression in the epidermis over a 24-h period after in vivo UVB irradiation. Also, the induction of adhesion molecule expression and the accumulation of neutrophils within the dermis were followed. We found constitutive expression of both cytokines (mRNA and protein) in the epidermis of unirradiated skin. IL-8 was rapidly upregulated after irradiation and mRNA and protein increased at 4 h, reaching a maximum between 8 and 24 h. TNF-alpha mRNA and protein was minimally increased by 8 h after UVB irradiation and reached a maximum by 24 h. No significant alteration in ICAM-1 or VCAM-1 expression was observed. E-selectin expression, which was absent from control samples, was increased from 4 h onward and also reached a maximum at 24 h, coinciding with peak neutrophil accumulation. A strong correlation (r = 0.96) was found between number of E-selectin-positive vessels and numbers of infiltrating neutrophils at this time. Moreover, because E-selectin expression was increased before any apparent increase in TNF-alpha protein (4 h), TNF-alpha does not appear to be involved in the early induction of the adhesion molecule, but cytokines such as TNF-alpha and IL-8 may act subsequently to augment the inflammatory response.

Comparison of changes in endothelial adhesion molecule expression following UVB irradiation of skin and a human dermal microvascular cell line (HMEC-1).

We have assessed the pattern of dermal endothelial adhesion molecule expression following broadband UVB irradiation in vivo and in vitro. Skin biopsies were taken from 4 human volunteers at baseline and at 4, 8 and 24 h post-irradiation with 2.5 minimal erythema doses of UVB. Sections were stained immunohistochemically for E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1). CD31 and neutrophil elastase. The effect of direct UVB irradiation on E-selectin, ICAM-1 and VCAM-1 was examined in a human dermal microvascular endothelial cell line, HMEC-1. Cultured HMEC-1 were irradiated with 2.5-40 mJ/cm2 of UVB, and assessed for adhesion molecule expression by immunofluorescence microscopy and fluorescence-activated cell sorter analysis. In vivo, E-selectin was minimally expressed on EC at baseline and was induced by 4 h following irradiation, P < 0.01. ICAM-1 was moderately expressed at baseline and appeared mildly induced at 24 h, although this did not reach statistical significance. VCAM-1 was weakly expressed in unirradiated skin while CD31 was moderately expressed, but neither was induced by UVB irradiation. A significant neutrophilic infiltrate appeared by 8 h and was maximal at 24 h, P < 0.05. Neutrophil infiltration correlated with E-selectin expression, r = 0.96. In HMEC-1, ICAM-1 was upregulated at 24 h post-irradiation, with an increase in mean channel fluorescence from 100% at baseline to 145 (SD12)% at 24 h, P < 0.05. No change was seen in expression of E-selectin, VCAM-1 or CD31. These studies support the involvement of endothelial adhesion molecules E-selectin and ICAM-1 in UVB-induced inflammation. Whereas ICAM-1 is upregulated by direct irradiation of endothelial cells, E-selectin stimulation appears to be an indirect effect.

Effect of biomaterial surface charge on the inflammatory response: evaluation of cellular infiltration and TNF alpha production.

A rat model was used to investigate the effect of net surface charge on polymer biocompatibility and its potential to modify and stimulate the inflammatory response. Poly(ether)urethane was taken as the base material and the net charge altered by introducing sulphonate ionic groups to the polymer backbone. Three differently charged poly(ether)urethanes were made with 10, 20, and 30% sulphonate substitution, giving a range of negative charge, with unmodified poly(ether)urethane used as a control. The polymers were implanted intramuscularly into rats for 2 days, and for 1, 2, and 12 weeks. After explantation, the cellular infiltration in the tissue surrounding the implants was evaluated using immunohistochemistry to stain for specific cell types: macrophages, neutrophils, lymphocytes, and the cytokine TNF alpha. In situ hybridization was used to detect expression of mRNA encoding TNF alpha. Stained sections were analyzed and the cellular response quantified using image analysis. Initially macrophages and neutrophils were observed around all the materials, but neutrophils were absent in all samples at 12 weeks. The 2-day time point had significantly more macrophages than the later time points. By 2 weeks the 20%-charged polymer elicited significantly less neutrophil infiltration than the other three polymers. In all samples where macrophages were observed, cells staining positive fore TNF alpha protein and message also were observed. No T or B lymphocytes were observed in the infiltrates around the materials at any time point. The results indicate that surface charge can influence the early phase acute inflammatory response to an implanted material.

Double-blind controlled trial of effect of housedust-mite allergen avoidance on atopic dermatitis.

BACKGROUND: The role of housedust-mite (HDM) allergen (Der p1) in the pathogenesis of atopic dermatitis is controversial. We tested the hypothesis that atopic dermatitis improves if amounts of HDM allergen in the home are reduced. METHODS: Active treatment comprised Goretex bedcovers (placebo, cotton covers), benzyltannate spray (placebo, water), and a high-filtration vacuum cleaner (placebo, a conventional domestic vacuum cleaner). Dust was sampled monthly from the mattress covers and bedroom and living-room carpets. 48 patients (24 adults [mean age 30] and 24 children [mean age 10]) completed the 6-month study. 28 were in the active treatment group and 20 in the placebo group. FINDINGS: The weight of dust collected from Goretex-covered mattresses had fallen by 98% at 1 month (from 386 to 9 mg/m2) with no change thereafter. Placebo covers caused a smaller reduction in dust load (361 to 269 mg/m2); the difference between active and placebo covers at 6 months was highly significant (p = 0.002). Both active and placebo treatments caused significant reductions in Der p1 concentrations in bedroom and living-room carpets and the differences between the treatments were not significant. The severity of eczema decreased in both groups, but the active group showed significantly greater improvements in severity score (difference between mean final scores 4.3 units, p = 0.006) and area affected (difference between mean final areas 10%, p = 0.006) in analysis of covariance with initial mattress dust weights and bedroom carpet Der p1 load as covariates. Reported analysis with final values for the covariates showed that most of the treatment effect was due to the reduction in mattress dust and carpet Der p1. INTERPRETATION: The activity of atopic dermatitis can be greatly reduced by effective HDM avoidance. Methods to identify individuals who will benefit most from such measures are needed.

Investigation of mechanisms in toxic epidermal necrolysis induced by carbamazepine.

BACKGROUND: Erythema multiforme and toxic epidermal necrolysis can occur as serious and even life-threatening adverse drug reactions. The underlying mechanisms are unknown, but evidence suggests that affected individuals may have impaired capacity to detoxify reactive intermediate drug metabolites. Such intermediates may be directly toxic or may react with host tissues to form antigens, evoking an immune response. We describe our investigation of a patient with carbamazepine-induced erythema multiforme and toxic epidermal necrolysis. The inflammatory infiltrate was examined immunocytochemically in lesional skin specimens from the patient, in the patient's patch test response to carbamazepine, and in lesional skin specimens from five other patients with drug-induced erythema multiforme. The patient's lymphocytes were examined both for susceptibility to cytotoxic damage by liver microsome-induced carbamazepine metabolites and for proliferative responses to native carbamazepine, which might indicate cell-mediated immune sensitization. OBSERVATIONS: Lesions of toxic epidermal necrolysis were more florid, but findings were essentially similar in all the skin samples examined. In the dermis there were CD14+ macrophages, CD1a+ Langerhans cells, and CD3+ CD45RO+ T cells. The CD4-CD8 T-cell ratio was 2:1, and 10% of the T cells were CD25+, suggesting activation by recent encounter with antigen. The epidermis contained CD14+ macrophages and T cells, but the CD8+ cells out-numbered the CD4+ cells. Up to 25% of the T cells were CD25+. Lymphocyte proliferation was not induced by native carbamazepine, but the patient's lymphocytes were significantly more susceptible to cytotoxic killing by liver microsome-induced carbamazepine intermediates. CONCLUSIONS: The inflammatory reaction in skin affected by erythema multiforme and toxic epidermal necrolysis was rich in CD8+ T cells, suggesting an immune cytotoxic reaction. The patient appeared to have a reduced capacity to detoxify reactive intermediates. This, together with the lack of lymphocyte response to native drug but a positive patch test response, suggests that the immune response may be directed at drug-modified epidermal cells.

Early time course of recruitment of immune surveillance in human skin after chemical provocation.

As part of the defence function of skin it seems probable that mechanisms exist for the rapid recruitment of immune surveillance to 'inspect' any foreign substance that penetrates the skin. In the present study, evidence of such mechanisms was sought by following the time course of early changes in distribution of immune cells, expression of cell adhesion molecules and cytokines after epicutaneous challenge with provoking chemicals to which subjects were known to be either specifically 'sensitive' or 'non-sensitive'; anthralin, an irritant chemical, was used as control. Fifty-seven individuals were studied and there were at least five biopsy samples at each time point. Regardless of whether individuals were sensitive or not, or of the type of chemical, dermal microvascular endothelial cells showed increased expression of the adhesion molecules ELAM-1 and VCAM-1 within 2 h, and ICAM-1 within 8 h. The intensity of immunohistochemical staining increased progressively up to 24 h. More vessels stained for ICAM-1 than for VCAM-1 or ELAM-1, implying that not every vessel expressed all three cell adhesion molecules. Another early change, observed 2 h after irritant challenge, was a significant increase in numbers of CD1a+ dendritic cells in the superficial dermis from a median of 3/high power field (hpf) to 9.5/hpf (P < 0.03). This was not observed with 'weak' provoking substances, such as nickel, but did occur with the potent provoking agent dinitrochlorobenzene (DNCB). Thus, as little as 2 h after contact with provoking chemicals, the skin activates cellular mechanisms to increase T cell infiltration for the presumed purpose of immune surveillance. These mechanisms are not dependent upon specific immune sensitivity and reflect a capacity of skin cells to respond to chemical provocation.

The therapeutic equivalence of oral and intravenous iron in renal dialysis patients.

Oral and intravenous iron were compared in patients treated with renal dialysis by a cross-over trial. Intravenous iron was given over 2 weeks as an iron dextran (equivalent to 100 mg elemental iron). Oral iron was given daily as ferrous sulphate (equivalent to 100 mg elemental iron) in a wax matrix tablet. Each treatment period lasted 26 weeks. There was no significant difference in therapeutic or unwanted effects between the treatments.

A double-blind controlled trial of 'Gaviscon' in patients with symptomatic gastro-oesophageal reflux.

In a double-blind trial in 26 patients with symptomatic gastro-oesophageal reflux, the alginate/effervescent/antacid compound 'Gaviscon' was compared with antacid containing placebo similarly formulated. Retrosternal pain after meals and at night was significantly reduced whilst patients were taking 'Gaviscon' and the beneficial effects lasted for many weeks after discontinuation of the active preparation.

A controlled trial of practolol in mild hypertension.

1 Fifteen patients with diastolic blood pressures between 90 and 120 mmHg were admitted to a trial comparing placebo with practolol at doses up to 500 mg 12 hourly. 2 The trial was a double blind cross over study. Treatments were allocated at random and each treatment block lasted 12 weeks. 3 Ten patients completed the trial, and none of those who withdrew experienced serious unwanted effects. 4 Practolol caused significant falls in standing systolic and diastolic pressures and the supine diastolic pressure.