Skin stem cell hypotheses and long term clone survival--explored using agent-based modelling.

Epithelial renewal in skin is achieved by the constant turnover and differentiation of keratinocytes. Three popular hypotheses have been proposed to explain basal keratinocyte regeneration and epidermal homeostasis: 1) asymmetric division (stem-transit amplifying cell); 2) populational asymmetry (progenitor cell with stochastic fate); and 3) populational asymmetry with stem cells. In this study, we investigated lineage dynamics using these hypotheses with a 3D agent-based model of the epidermis. The model simulated the growth and maintenance of the epidermis over three years. The offspring of each proliferative cell was traced. While all lineages were preserved in asymmetric division, the vast majority were lost when assuming populational asymmetry. The third hypothesis provided the most reliable mechanism for self-renewal by preserving genetic heterogeneity in quiescent stem cells, and also inherent mechanisms for skin ageing and the accumulation of genetic mutation.

Skin differences based on age and chronicity of ultraviolet exposure: results from a gene expression profiling study.

BACKGROUND: Skin ageing represents an inevitable physiological consequence of getting older but the impact on personal health and wellbeing can be significant, and therefore considerable efforts have been made to understand the biology and pathophysiology of skin ageing to try to identify new targets that might offer therapeutic intervention and prevention. OBJECTIVES: This study was designed to identify differences at the gene expression level between young and old, sun-exposed and sun-protected skin. METHODS: We generated transcriptomic data from young and old skin from sun-exposed and sun-protected sites (10 samples of each) using HG-U133 Plus 2.0 Affymetrix GeneChips. The data were analysed using hierarchical clustering, theme analysis and interaction mapping to identify regulated pathways, processes and potential targets for therapy. RESULTS: With 54,613 probe sets on the GeneChip, 2731 significant differences would be expected by chance (at P = 0·05), but we noted that 13,640 probe sets were significantly different comparing young arm skin vs. older arm skin (photoageing), and 7215 probe sets were significant for the young buttock vs. older buttock comparison (intrinsic ageing). In both types of ageing there was reduced expression of many genes implicated in lipid biosynthesis and epidermal differentiation with functional relevance to skin barrier integrity and maintenance. Increased expression of genes contributing to oxidative stress and decreased expression of antioxidant defences were also common to both types of ageing. Differences between intrinsic ageing and photoageing were mainly noted in extracellular matrix gene expression with reduced expression of interstitial collagen genes in intrinsic ageing and increased expression of elastic tissue genes in photoageing. CONCLUSIONS: Collectively, the data identified new biomarkers of aged skin, particularly involving abnormalities of proteases, matrix proteins and inflammation. These findings offer the prospect of new and more specific targets for therapeutic development based on an improved understanding of the biology of skin ageing.

Sclerostin antibody treatment enhances bone strength but does not prevent growth retardation in young mice treated with dexamethasone.

OBJECTIVE: Exposure to supraphysiologic levels of glucocorticoid drugs is known to have detrimental effects on bone formation and linear growth. Patients with sclerosteosis lack the bone regulatory protein sclerostin, have excessive bone formation, and are typically above average in height. This study was undertaken to characterize the effects of a monoclonal antibody to sclerostin (Scl-AbI) in mice exposed to dexamethasone (DEX). METHODS: Young mice were concomitantly treated with DEX (or vehicle control) and Scl-AbI antibody (or isotype-matched control antibody [Ctrl-Ab]) in 2 independent studies. Linear growth, the volume and strength of the bones, and the levels of bone turnover markers were analyzed. RESULTS: In DEX-treated mice, Scl-AbI had no significant effect on linear growth when compared to control treatment (Ctrl-Ab). However, in mice treated with DEX and Scl-ABI, a significant increase in trabecular bone at the femoral metaphysis (bone volume/total volume +117% versus Ctrl-Ab-treated mice) and in the width and volume of the cortical bone at the femoral diaphysis (+24% and +20%, respectively, versus Ctrl-Ab-treated mice) was noted. Scl-AbI treatment also improved mechanical strength (as assessed by 4-point bending studies) at the femoral diaphysis in DEX-treated mice (maximum load +60% and ultimate strength +47% in Scl-AbI-treated mice versus Ctrl-Ab-treated mice). Elevated osteocalcin levels were not detected in DEX-treated mice that received Scl-AbI, although levels of type 5b tartrate-resistant acid phosphatase were significantly lower than those observed in mice receiving DEX and Ctrl-Ab. CONCLUSION: Scl-AbI treatment does not prevent the detrimental effects of DEX on linear growth, but the antibody does increase both cortical and trabecular bone and improves bone mechanical properties in DEX-treated mice.

Xenobiotic metabolism gene expression in the EpiDermin vitro 3D human epidermis model compared to human skin.

There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm model appears to be representative of human skin. Models such as EpiDerm provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics.

Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro.

Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2'+'/ErbB3'+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2'+'/ErbB3'+' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.

Detection of molecular diversity in Bacillus atrophaeus by amplified fragment length polymorphism analysis.

Phenotypically, Bacillus atrophaeus is indistinguishable from the type strain of Bacillus subtilis except by virtue of pigment production on certain media. Several pigmented variants of B. subtilis have been reclassified as B. atrophaeus, but several remain ambiguous in regard to their taxonomic placement. In this study, we examined strains within the American Type Culture Collection originally deposited as Bacillus globigii, B. subtilis var. niger, or Bacillus niger using 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis to determine the level of molecular diversity among these strains and their relationship with closely related taxa. The 16S rRNA gene sequences revealed little variation with one base substitution between the B. atrophaeus type strain ATCC 49337 and the other pigmented bacilli. AFLP analysis produced high-quality DNA fingerprints with sufficient polymorphism to reveal strain-level variation. Cluster analysis of Dice similarity coefficients revealed that three strains, ATCC 31028, ATCC 49760, and ATCC 49822, are much more closely related to B. atrophaeus than to B. subtilis and should be reclassified as B. atrophaeus. A very closely related cluster of B. atrophaeus strains was also observed; this cluster was genetically distinct from the type strain. The level of variation between the two groups was approximately the same as the level of variation observed between members of the two B. subtilis subspecies, subtilis and spizizenii. It is proposed that the cluster of strains typified by ATCC 9372 be designated a new subspecies, B. atrophaeus subsp. globigii.

A non-invasive tape absorption method for recovery of inflammatory mediators to differentiate normal from compromised scalp conditions.

BACKGROUND/AIMS: A simple non-invasive tape (Sebutape) adsorption method was used to recover inflammatory proteins from normal and compromised human scalp (i.e. dandruff and seborrheic dermatitis) in order to assess the inflammatory and immunologic changes relevant to these clinical conditions. METHODS: The scalps of subjects identified by a dermatologist as having either dandruff (n = 18), seborrheic dermatitis (n = 19) or normal scalp (n = 16) were visually graded to obtain total adherent scalp flaking scores (TASFS). Sebutape samples were then collected from both the high and low TASFS scalp sites using a one-minute tape application. To recover inflammatory molecules, tapes were extracted in buffered saline with sonication and the tape extracts analysed using commercial immunoassay methods for pro-inflammatory cytokines [i.e. interleukin-1alpha (IL-1alpha), IL-1beta, IL-1 receptor antagonist (IL-1ra), IL-8 and tumor necrosis factor-alpha (TNF-alpha)] and the immunologic cytokines [i.e. IL-2, IL-4, IL-10, IL-12, IL-15 and interferon gamma (IFN-gamma)]. Nitric oxide (NO) was also assayed on tape extracts using the Greiss reaction. To account for differences in protein loading on the tapes all cytokine and NO results were normalized using the total protein (TP) amounts recovered in tape extracts. RESULTS: The IL-1alpha/TP levels recovered from dandruff and seborrheic scalps were significantly decreased (P = 0.03) compared to normal appearing scalp levels. The scalp levels of IL-1ra/TP and the ratio of IL-ra to IL-1alpha were significantly (P = 0.002) or directionally (P = 0.07) higher in seborrheic dermatitis scalps and dandruff scalps, respectively, compared to normal scalps. The IL-1ra and the IL-1ra/IL-1alpha ratio values correlated well with the TASFS. The TNF-alpha/TP levels recovered from dandruff scalps were significantly higher (P = 0.02) than levels recovered from seborrheic dermatitis and normal scalp subjects. IL-2/TP was significantly increased (P = 0.01) and IFN-gamma and NO significantly decreased (P = 0.05) in the seborrheic dermatitis scalp samples compared to normal controls. CONCLUSION: The Sebutape method has proven useful for distinguishing normal from diseased scalp conditions. The cytokines recovered from the scalp tape samples showed distinct patterns that differentiated dandruff, seborrheic dermatitis and normal scalp populations. These methods may also prove useful for monitoring the clinical efficacy of therapeutic actives for treating dandruff and seborrhea.

Anti-E-selectin is ineffective in the treatment of psoriasis: a randomized trial.

BACKGROUND: Skin-homing, memory T lymphocytes play an important role in the pathogenesis of psoriasis by interacting with the vascular addressin, E-selectin and trafficking into lesional skin. Thus an attractive option for targeted therapy of the disease would be blockade of skin-homing T cells with an antibody directed at E-selectin. OBJECTIVE: We performed a multicentre, randomized, placebo-controlled trial to investigate the clinical efficacy and side-effect profile of a humanized monoclonal antibody to E-selectin, CDP850, in the treatment of moderate to severe chronic plaque psoriasis. METHODS: Patients with moderate/severe chronic plaque psoriasis were selected for study. Nine male subjects (mean age 37 years, range 25-47) were given 20 mg kg-1 CDP850 intravenously as a single dose and four subjects (three males, one female; mean age 40 years, range 23-50) received placebo infusion. Clinical response to treatment was assessed using the psoriasis area and severity index (PASI). Skin biopsies were taken for immunohistochemical analysis at the baseline, pretreatment, visit and also at day 2 and weeks 1 and 4 postinfusion. RESULTS: The treatment was well-tolerated with a minimal side-effect profile. Plasma E-selectin levels were significantly decreased in those subjects who received CDP850 compared with those who had placebo for the entire study period. At the end of study (8 weeks postinfusion), there was no significant reduction in PASI from baseline for either the CDP850 or placebo-treated groups. Immunohistochemical analysis of biopsies taken from lesional psoriatic skin showed that 2 days after dosing with CDP850, staining for E-selectin was decreased, although not absent, on dermal vascular endothelial cells when compared with baseline (P < 0.01). This decrease in E-selectin expression was maintained 4 weeks after infusion (P < 0.05). It was not, however, accompanied by a significant reduction in numbers of neutrophils or lymphocytes in the dermis. There was a statistically significant increase in CD1a-positive epidermal Langerhans cells compared with pre-dose levels at week 1 (P < 0.05). CONCLUSIONS: This clinicopathological study shows that anti-E-selectin (CDP850), although a well-tolerated, logical and safe therapy, does not appear to possess a therapeutic role in the treatment of chronic plaque psoriasis.

Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products.

The dermatotoxicologist today is faced with a dilemma. Protection of workers and consumers from skin toxicities (irritation and allergy) associated with exposure to products, and the ingredients they contain, requires toxicological skin testing prior to manufacture, transport, or marketing. Testing for skin corrosion or irritation has traditionally been conducted in animals, particularly in rabbits via the long established Draize test method. However, this procedure, among others, has been subject to criticism, both for its limited predictive capacity for human toxicity, as well as for its use of animals. In fact, legislation is pending in the European Union which would ban the sale of cosmetic products, the ingredients of which have been tested in animals. These considerations, and advancements in both in vitro skin biology and clinical testing, have helped drive an intensive effort among skin scientists to develop alternative test methods based either on in vitro test systems (e.g. using rat, pig or human skin ex vivo, or reconstructed human skin models) or ethical clinical approaches (human volunteer studies). Tools are now in place today to enable a thorough skin corrosion and irritation assessment of new ingredients and products without the need to test in animals. Herein, we describe general testing strategies and new test methods for the assessment of skin corrosion and irritation. The methods described, and utilized within industry today, provide a framework for the practicing toxicologist to support new product development initiatives through the use of reliable skin safety testing and risk assessment tools and strategies.

A noninvasive method to assess skin irritation and compromised skin conditions using simple tape adsorption of molecular markers of inflammation.

BACKGROUND/AIMS: We have developed a simple noninvasive method to assess inflammatory changes in human skin, even in the absence of visible clinical irritation. Our approach is based on a simple tape (Sebutape) adsorption method to recover molecular mediators of skin inflammation (e.g., cytokines). This procedure has been used to investigate baseline cytokine levels on skin, to assess normal skin condition and to evaluate changes due to chemical insult, existing dermatitis, or sun exposure. METHODS: In clinical studies, Sebutape was applied to normal appearing uncompromised skin, as well as to compromised (diaper or heat rash), chemically treated (sodium laurel sulfate), or sun-exposed skin. Sebutape was applied to the skin for a 1 min collection interval. Tapes were extracted in saline using a 10 min sonication, and the extracts were analyzed for human interleukin-1alpha (IL-1alpha), IL-1 receptor antagonist (IL-1RA) and IL-8 using commercial immunoassay test kits. The cytokine levels recovered from each tape extract were normalized to total protein (TP) levels. In infant product use tests, the severity of skin irritation (diaper and heat rash or erythema) was also assessed using a visual grading scale. RESULTS: The method itself caused minimal, if any, skin damage. Additionally, Sebutape was shown to quantitatively adsorb detectable levels of cytokine from normal-appearing (control) or compromised (e.g., rashed or chemically treated) skin. In infant studies, significant increases in IL-1alpha levels were found in skin exhibiting diaper rash, heat rash and erythema compared with normal appearing control skin sites. When these results were normalized to total protein levels recovered from each tape, the significance was maintained. A positive correlation (r2=0.82) existed between IL-1RA levels and diaper rash severity. Significant increases in IL-8 levels were recovered from diaper rash versus control skin sites. There were differences in baseline cytokine levels in normal skin related to body site and sun exposure. The IL-1 RA/IL-1alpha ratios for sun-exposed skin of the face and lower leg were significantly (P<0.05) higher (3-6-fold) than those for skin sites that typically receive minimal sun exposure (i.e., underarm, upper leg and upper back). There was a significant increase in IL-1alpha and a directional increase in IL-8 levels in adult skin sites treated with the irritant, sodium lauryl sulfate, even in the absence of visible skin irritation (erythema). CONCLUSION: Our results demonstrate that this method is a useful noninvasive technique for assessing skin inflammatory events. In addition, the method is simple and easily applied in a clinical setting, whether on infants or adults.

Leukocyte integrin Mac-1 recruits toll/interleukin-1 receptor superfamily signaling intermediates to modulate NF-kappaB activity.

The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) regulates important cell functions in inflammation, including adhesion, phagocytosis, and oxidative burst. Deficiency of Mac-1 reduces vessel wall inflammation and neointimal thickening after murine carotid artery injury. Although Mac-1 has been implicated in modulating AP-1 and NF-kappaB activity, the signal transduction pathways involved are undefined. cDNA array analysis of Mac-1-clustered compared with -nonclustered monocytic THP-1 cells showed increased expression of the signal transducer TRAF6 (TNF receptor-associated factor 6), leading us to consider the possibility that Mac-1 used a Toll/IL-1 receptor family-like signaling pathway. Mac-1-dependent activation of NF-kappaB was potentiated by wild-type, and attenuated by dominant negative, TRAF6- and TGF-beta-activated kinase (TAK1) constructs. IRAK1 (IL-1 receptor associated kinase), a kinase immediately upstream of TRAF6, coimmunoprecipitated with Mac-1. Taken together, these observations indicate that Mac-1 recruits a Toll/IL-1 receptor family-like cascade to modulate NF-kappaB activity. This represents a new pathway for integrin-dependent modulation of gene expression.

The Caenorhabditis elegans EGL-15 signaling pathway implicates a DOS-like multisubstrate adaptor protein in fibroblast growth factor signal transduction.

EGL-15 is a fibroblast growth factor receptor in the nematode Caenorhabditis elegans. Components that mediate EGL-15 signaling have been identified via mutations that confer a Clear (Clr) phenotype, indicative of hyperactivity of this pathway, or a suppressor-of-Clr (Soc) phenotype, indicative of reduced pathway activity. We have isolated a gain-of-function allele of let-60 ras that confers a Clr phenotype and implicated both let-60 ras and components of a mitogen-activated protein kinase cascade in EGL-15 signaling by their Soc phenotype. Epistasis analysis indicates that the gene soc-1 functions in EGL-15 signaling by acting either upstream of or independently of LET-60 RAS. soc-1 encodes a multisubstrate adaptor protein with an amino-terminal pleckstrin homology domain that is structurally similar to the DOS protein in Drosophila and mammalian GAB1. DOS is known to act with the cytoplasmic tyrosine phosphatase Corkscrew (CSW) in signaling pathways in Drosophila. Similarly, the C. elegans CSW ortholog PTP-2 was found to be involved in EGL-15 signaling. Structure-function analysis of SOC-1 and phenotypic analysis of single and double mutants are consistent with a model in which SOC-1 and PTP-2 act together in a pathway downstream of EGL-15 and the Src homology domain 2 (SH2)/SH3-adaptor protein SEM-5/GRB2 contributes to SOC-1-independent activities of EGL-15.

Evaluation of a quantitative clinical method for assessment of sensory skin irritation.

Sensory skin irritation refers to the myriad of symptomatic complaints (e.g., sting and burn) frequently associated with inflammatory skin conditions or skin intolerance to various chemicals or finished products. Sensory irritation is an important factor in consumer acceptance of the products that they buy and use; however, from a safety testing and risk assessment standpoint, it has been difficult to evaluate. Recently, methods have been developed to more quantitatively assess sensory irritation using a semantically-labeled scale of sensation intensity, the labeled magnitude (LM) scale. Using this device, studies were conducted to determine if test subjects' perceptions of recalled or imagined sensory responses (from a series of survey questions) were related to their actual sensory reactivity to chemical challenge. Subjects were presented with 15 skin sensation scenarios of varying intensities and asked to record their self-perceived recalled or imagined responses using the LM scale. Individual and mean responses to each of the 15 survey questions were compared within and across studies. Considerable variation was seen between subjects' responses to the questions, particularly for questions pertaining to stronger stimuli (e.g., scalding water or skin lacerations). There was also little consistency seen in the pattern of individual responses across the questions. However, among 4 different study populations, the group mean scores for each of the 15 survey questions showed a high degree of consistency. Also, in spite of the variability in perceived responses to the recalled/imagined skin sensations, statistically significant dose-response and time-response patterns were observed in chemical (lactic acid and capsaicin) challenge studies. In one capsaicin study, a direct relationship was observed, among 83% of the study subjects, between the mean recall intensity scores and actual responses to subsequent capsaicin challenge. This pattern was not seen in a lactic acid challenge study. However, a similar relationship was seen in this study if only recall stimuli related to sting-type responses were included in the analysis. Hence, use of recall/imagined skin sensation perception data for prediction of actual reactivity to chemical probes may have screening utility depending on the survey questions used. On the whole, the LM scale is of practical use for quantifying subjective sensory irritation responses. Combined with evolving noninvasive instrumental and bioassay procedures for identifying biophysical or inflammatory markers of sensory irritation, better methods are on the horizon for improving our sensory skin irritation testing and risk assessment capabilities.

Defining the repeating elements in the cysteine-rich region (CRR) of the CD18 integrin beta 2 subunit.

The cysteine-rich region (CRR) of the integrin beta subunits is organised into four repeating elements. By expression of a panel of truncated beta 2 subunits, and CRR segments fused to the C-terminal end of a CD4 soluble fragment, the segment required for the expression of two monoclonal antibody conformational epitopes was determined. This segment, E482-Q574, contains 16 cysteines representing two repeating units. We have thus defined the CRR unit motif of 'xC---C---C---CxCxxCxC---Cx', where 'x' represents a single residue, and '---' represents a stretch of four to 14 residues.

Intra-individual variations in acute and cumulative skin irritation responses.

It is well-known that humans show a wide range of variation in skin reactivity to irritant chemicals. This has been established through population studies, through the examination of inter-subject variability, and (to a limited extent) through studies of skin site variation in response within subjects. However, simple response variability within individual test subjects has not been examined as carefully, and this has implications for our ability to predict irritant reactivity. Some key questions are: (i) how consistently do human beings respond, even within a given study, to different equally irritating chemicals, or to the same chemical when comparing different concentrations or durations of exposure, and, (ii) Do individual test subjects' responses to one chemical (or exposure scenario) correlate with their responses to another? To examine these questions in some detail, we reexamined individual study subjects' responses from earlier published studies involving both acute and cumulative irritation patch test protocols. Acute irritation responses were compared across chemicals with similar irritation profiles. Cumulative irritation responses were compared across different concentrations of the anionic surfactant, sodium dodecyl sulfate (SDS). Acute (high concentration) and cumulative (low concentration) patch test responses to SDS were also compared. The analysis showed that, as might be expected, response correlations were greatest within test types, either when comparing chemicals of similar overall irritancy, or when comparing similar concentrations of a single chemical. However, individually divergent responses were also frequent, reinforcing the conclusion that a given individual's response to one chemical or exposure condition does not always predict their response to another. This has important ramifications for other questions related to population differences in skin reactivity.

Contact allergenic potency: correlation of human and local lymph node assay data.

BACKGROUND: Effective toxicologic evaluation of skin sensitization requires that potential contact allergens are identified and that the likely risks of sensitization among exposed populations are assessed. By definition, chemicals that are classified as contact sensitizers have the capacity to cause allergic contact dermatitis (ACD) in humans. However, this hazard is not an all-or-nothing phenomenon; clear dose-response relationships can be discerned and thresholds identified for both the induction of sensitization and the elicitation of ACD. Commonly, these parameters are grouped under the heading of potency, the determination of which is vital for risk assessment. Preclinical testing for sensitization potential is critically important for hazard assessment before human exposure. The murine local lymph node assay (LLNA) is the most recently accepted test method for sensitization hazard assessment. OBJECTIVE: The aim was to compare potency estimations derived from LLNA data with clinical determinations of relative potency based on human data. METHODS: No-effect levels (NOELs) for a range of 21 chemicals were determined from nondiagnostic human repeat patch test studies as reported in the literature. These levels were compared with LLNA EC(3) values, the estimated concentration required to produce a 3-fold increase (positive response) in draining lymph node cell (LNC) proliferative activity. RESULTS: Using available human repeat patch test data, together with expert judgment, the compounds were classified as strong, moderate, weak, extremely weak, or nonsensitizing. Additionally, the potency of each chemical was classified independently based on its LLNA EC(3) value. The results show clearly that LLNA EC(3) values are very comparable with the NOELs calculated from the literature. Moreover, the potency rankings based upon LLNA EC(3) data support their human classification. CONCLUSION: The present investigations show that the LLNA can be used to provide quantitative estimates of relative skin sensitizing potency EC(3) values that correlate closely with NOELs established from human repeat patch testing and from our clinical experience.

The N-terminal region and the mid-region complex of the integrin beta 2 subunit.

In the primary sequence of the integrin beta subunit, the N-terminal region (NTR) and mid-region are separated by the I-like domain. To determine the spatial relationship and functional properties of the integrin beta(2) NTR and mid-region, we constructed beta(2)/beta(7) chimeras in which the NTR, I-like domain, and the mid-region of the beta(2) subunit were replaced by those of beta(7). Changing either the beta(2) NTR or mid-region, but not the I-like domain to that of beta(7) did not affect LFA-1 (alpha(L)beta(2)) formation and surface expression. Thus, the specificity of alpha(L)beta(2) pairing is conferred by the I-like domain but not the NTR or mid-region. Using these chimeras, the epitopes of six anti-beta(2) mAbs (H52, 7E4, AZN-L18, AZN-L27, KIM202, and MEM-148) were mapped. All except H52 require both the NTR and mid-region for epitope expression. Since these mAbs have distinct properties in terms of epitope expression and effect on LFA-1 binding to ICAM-1, we conclude that the beta(2) NTR and mid-region interact extensively. Although the I-like domain is located between the NTR and mid-region, its removal does not affect the folding of the beta(2) NTR/mid-region complex because this complex alone can be expressed as a soluble protein and precipitated by the appropriate mAbs. Finally, the mAbs H52 and 7E4, abrogated KIM185- but not Mg/EGTAinduced LFA-1/ICAM-1 binding and the epitope of MEM-148 is expressed on Mg/EGTA-activated but not resting LFA-1. These results suggest that the NTR/mid-region complex is involved in the regulation of LFA-1 function.

Labelling of skin sensitizers: the new European Dangerous Preparations Directive.

The new Dangerous Preparations Directive (DPD, 1999/45/EC) introduces a special labelling requirement for skin sensitizers in products that are regulated under this Directive. The packaging of products containing 0.1% of a sensitizer must bear the inscription "Contains 'name of sensitizer'. May produce an allergic reaction." The aim is to protect individuals already sensitized by providing information which enables them to avoid products containing ingredients which may elicit their allergy. However, this is only of benefit where such sensitized individuals do exist in the population. Moreover, this labelling requirement does not take into account the potency of the skin sensitizer. For each sensitizer and type of skin exposure, there will be levels below which it will not elicit allergic contact dermatitis reactions in individuals who are sensitized to that chemical. We therefore propose that within the new DPD, it should be possible to override this labelling requirement with well-documented data, to ensure that information provided to the consumer on the product label is not misleading. The current implementation in the DPD of what is in principle a good idea means that further action (legislative changes; scope for derogation) is needed if the potential benefits are not to be lost.