The rate of wound healing is increased in psoriasis.

BACKGROUND: Psoriasis shares many features with wound healing, a process that involves switching keratinocytes from growth to differentiation. Ca2+ is known to regulate this process. The N-methyl-d-aspartate receptor (NMDAR), an ionotropic glutamate receptor found on keratinocytes, is expressed abnormally in psoriasis in vivo. OBJECTIVES: The goals of this study are to determine whether the rate of healing in the skin of psoriatic individuals differs from that observed in normal skin and whether the keratinocyte hyperproliferation found in psoriasis correlates with expression of specific NMDAR subunits. METHODS: Three mm punch biopsies were performed on the skin of normal, as well as, involved and uninvolved skin of subjects with psoriasis. On day 0, as well as, on day 6 after the biopsy, photographs were taken and the size of the wounds determined using ImageJ. Using immunohistochemistry, the biopsy material was stained for NMDAR and its subunits. RESULTS: Involved and uninvolved skin of individuals with psoriasis shows significantly more rapid healing than normal. The NR2C subunit of NMDAR is down-regulated in the basal cell layer of involved and uninvolved epidermis of psoriatic subjects compared to controls. By contrast, cells in the basal cell layer of the uninvolved epidermis showed a significantly greater percent strong staining for NR2D compared to those cells in normal epidermis. CONCLUSIONS: Wound healing is significantly accelerated in psoriasis compared to normal. Immunohistochemistry showed that the relative intensity of strong immunostaining for subunits of the NMDAR is altered in the basal cell layer in psoriatic skin compared to normal controls. We suggest that these alterations may contribute to the increased rate of wound healing in psoriasis.

Noninvasive genomic detection of melanoma.

BACKGROUND: Early detection and treatment of melanoma is important for optimal clinical outcome, leading to biopsy of pigmented lesions deemed suspicious for the disease. The vast majority of such lesions are benign. Thus, a more objective and accurate means for detection of melanoma is needed to identify lesions for excision. OBJECTIVES: To provide proof-of-principle that epidermal genetic information retrieval (EGIR™; DermTech International, La Jolla, CA, U.S.A.), a method that noninvasively samples cells from stratum corneum by means of adhesive tape stripping, can be used to discern melanomas from naevi. METHODS: Skin overlying pigmented lesions clinically suspicious for melanoma was harvested using EGIR. RNA isolated from the tapes was amplified and gene expression profiled. All lesions were removed for histopathological evaluation. RESULTS: Supervised analysis of the microarray data identified 312 genes differentially expressed between melanomas, naevi and normal skin specimens (P<0·001, false discovery rate q<0·05). Surprisingly, many of these genes are known to have a role in melanocyte development and physiology, melanoma, cancer, and cell growth control. Subsequent class prediction modelling of a training dataset, consisting of 37 melanomas and 37 naevi, discovered a 17-gene classifier that discriminates these skin lesions. Upon testing with an independent dataset, this classifier discerned in situ and invasive melanomas from naevi with 100% sensitivity and 88% specificity, with an area under the curve for the receiver operating characteristic of 0·955. CONCLUSIONS: These results demonstrate that EGIR-harvested specimens can be used to detect melanoma accurately by means of a 17-gene genomic biomarker.

Characterization of the expression and function of N-methyl-D-aspartate receptor in keratinocytes.

The N-methyl-D-aspartate (NMDA) receptor is expressed on neural tissue where it gates calcium ion entry upon stimulation. Using immunohistochemistry, it has been demonstrated in this study that the NMDAR1 receptor is also expressed on keratinocytes (KCs) in normal human skin and inflamed psoriatic skin in vivo. Furthermore, the NMDA receptor was functional as demonstrated by the ability of this receptor to trigger Ca++ influx in KCs. Incubation of cultured, human KCs with MK-801 decreases the cell growth and induces an increase in apoptosis. These findings demonstrate that the KC expression of NMDA receptor is a mechanism through which the influx of Ca++ into the cell can be regulated and suggest that the expression of this receptor may play a role in the regulation of KC growth and differentiation.

Is psoriasis a T-cell disease?

The etiology and pathogenesis of psoriasis--one of the most common chronic, inflammatory, hyperproliferative skin disorders of man--have long fascinated dermatologists, pathologists and biologists alike. Here, we have a model disease that offers to study neuroectodermal-mesenchymal interactions in the widest sense possible. Epithelial, endothelial, and hematopoietic cells as well as neurons projecting into the skin apparently all interact with each other to generate the characteristic psoriatic lesion. For decades, the ongoing controversy on the molecular nature, choreography and hierarchy of these complex interactions e.g. between epidermal keratinocytes, T cells, neurotrophils, endothelial cells and sensory nerves has served as a driving force propelling investigative dermatology to ever new horizons. This debate has not only been at the heart of our quest to develop more effective forms of therapy for this socially crippling disease, but it also has profoundly influenced how we view the skin as a whole: the numerous competing theories on the pathogenesis of psoriasis published so far also are reflections on the evolution of mainstream thought in skin biology over the last decades. These days, conventional wisdom infatuated with a T-cell-centered approach to inflammatory skin diseases-- portrays psoriasis as an autoimmune disease, where misguided T lymphocyte activities cause secondary epithelial abnormalities. And yet, as this CONTROVERSIES feature reminds us, some authoritative "pockets of academic resistance" are still quite alive, and interpret psoriasis e.g. as a genetically determined, abnormal epithelial response pattern to infectious and/or physicochemical skin insults. Weighing the corresponding lines of argumentation is not only an intriguing, clinically relevant intellectual exercise, but also serves as a wonderful instrument for questioning our own views of the skin universe and its patterns of deviation from a state of homeostasis.

Firm stroking of human skin leads to vasodilatation possibly due to the release of substance P.

Many animal species invest a large amount of time in grooming behavior without deriving any apparent benefit. In order for this behavior to have survived, however, it must confer some survival advantage. In seven of eight humans tested, an elevation in the skin's temperature was documented after massaging of the cheeks of the face. The elevation of the skin's temperature reached a plateau after about 40 min of massaging and was correlated to visible erythema. This effect could be inhibited by repeated pretreatment of the skin with topical capsaicin, a chemical that results in the release of substance P from peripheral nerve endings. Thus, it appears that the temperature elevation induced by stroking of human skin is controlled, at least in part, by release of the neurotransmitter, substance P. In conclusion, it appears that the release of neurotransmitter(s) may be the survival advantage that grooming confers to animals.

A noninvasive method for quantifying and distinguishing inflammatory skin reactions.

BACKGROUND: The ribonuclease protection assay (RPA) represents a technology that allows detection of small amounts of intact RNA. Recent progress in understanding cytokine networks in the skin suggests that measurements of cytokine mRNA levels could provide a method to distinguish various reactions such as irritant contact dermatitis and allergic contact dermatitis that can occur in the skin. OBJECTIVE: We attempted to differentiate and quantitate irritant and immunologic skin reactions by measuring mRNA levels. METHODS: We have used the technique of tape stripping human skin to remove superficial cell layers and have extracted RNA from these skin samples. This RNA was used for RPA analysis. RESULTS: By means of RPA analysis, we have demonstrated distinct cytokine profiles that appear to discriminate, for example, irritant from immunologic skin reactions. CONCLUSION: We have shown that multiple cytokine mRNA levels can be defined in these RNA samples obtained from the skin. This approach assesses not only the cytokine gene profiles, but at the same time may quantify the severity of common irritant versus allergic skin reactions.

Changes in the skin's capacitance after damage to the stratum corneum in humans.

BACKGROUND: Wound healing is a complex process to study, especially in humans, because the endpoint(s) of wound-induction and healing are subjective and, therefore, difficult to quantitate. Experiments were performed to establish quantifiable endpoints during the wound-induction process in 15 humans (11 women, 4 men). OBJECTIVE: Measurement of the skin's capacitance was used as a marker for the three stages of wound healing: 1) establishment of a wound, 2) the healing process, and 3) complete re-epithelialization. METHODS: Superficial wounds in the epidermis were generated by tape stripping the skin on the arm of 15 healthy volunteers. The skin's capacitance was measured before tape stripping, at various time points during the induction of the wound, immediately after glistening of the epidermis was achieved, and when the wound had healed according to clinical assessment. RESULTS: The values of the skin's capacitance at the point of removal of the stratum corneum varied from one individual to the next. Furthermore, the number of adhesive cellophane-tape strips needed to remove the stratum corneum and achieve glistening, due to accumulation of moisture, differed greatly from one volunteer to the next. These experiments suggest that the number of tape strips needed to remove the stratum corneum varies with age, sex, and possibly ethnicity of the subject. By contrast, during the wound healing process when a scab could be visualized, the skin's capacitance measured -122 picofarads (pF). Moreover, when the scab had disappeared, the skin's capacitance returned to baseline values (0 pF). CONCLUSION: Measurement of the skin's capacitance is a useful tool to determine two endpoints in the wounding and healing processes. The presence of a scab and the re-establishment of an intact stratum corneum can be quantitated easily. Less amenable to quantitation is the exact pF value that correlates to a specific type of wound.

Immunophenotyping of a sarcoidal granuloma in a scar, a manifestation of a possible autoimmune process.

BACKGROUND: Old scars rarely develop sarcoid lesions. To determine whether the immunophenotype of the cellular infiltrate found in sarcoid in scarred skin resembled that seen in sardoidosis, we performed routine staining with hematoxylin and eosin, as well as immunophenotyping of a sarcoid lesion in a scar. Currently, there is controversy about the etiology of sarcoidosis in general and about sarcoidal granulomas in scars in particular. A new hypothesis is suggested in this case report. OBJECTIVE: The purpose was to determine whether the cellular infiltrate in a sarcoidal granuloma in an old scar was similar to that in sarcoidosis. METHODS: Staining with hematoxylin and eosin as well as immunophenotyping was performed using standard techniques. RESULTS: The dermis of the sarcoid lesion demonstrated predominantly macrophages, followed by CD-4+ T-helper cells. CD-8+ cytotoxic suppressor cells were rare. CONCLUSIONS: The lymphoid cell infiltrate in a sarcoidal granuloma found in a scar is similar to that found in sarcoidosis. Furthermore, the staining pattern of sarcoid described in this paper and a review of the data in the literature suggest that sarcoidosis shares many characteristics of diseases with an autoimmune origin. Thus, we suggest that sarcoid isolated to scars represents a more benign variant of sarcoidosis, a possible systemic autoimmune disease.

Skin2--an in vitro human skin model: the correlation between in vivo and in vitro testing of surfactants.

The availability of an in vitro test system to replace animal testing of potential irritants is becoming more and more urgent especially in Europe as a consequence of the European Community Cosmetics Directive. To evaluate the ability of Advanced Tissue Sciences' (ATS) ZK1301 skin model to predict the skin irritation potential of surfactants, we performed a pilot validation study utilizing four different laboratories. The in vitro protocol was designed as a quantitative pre-screen for the clinical patch studies. Sixteen substances, representing various surfactant categories and ranges of irritation potential, were tested. The 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to quantitate viability in vitro. We documented the viability of tissues exposed to unknown substances for specific periods. The in vitro results were calculated as percent distilled water controls (DWC). The time required to reduce the viability of each tissue to 50% of the distilled water controls (T50) was compared to mean erythema and edema scores from the clinical studies by Pearson's correlation. The individual laboratories demonstrated coefficients of 0.72. The results indicated that the 30 min percent untreated control values best predicted the 24 h clinical patch scores. No statistically significant interlab variability was found. Only one false negative was seen when non/mild and moderate/severe irritant categories were assigned according to the in vitro scores. These results demonstrate that the skin2 in vitro test system may serve as a good screening method prior to clinical patch studies.

Langerhans cells may trigger the psoriatic disease process via production of nitric oxide.

Psoriasis is a skin disease that appears to result from a dysfunction in the normal mechanism(s) that regulates wound healing. The Langerhans cell is a specialized epidermal macrophage that may instigate wound healing via production of nitric oxide and epidermal growth factor. Here, Vera Morhenn suggests that, whereas precise coordination of the synthesis of these two substances regulates normal wound healing, a disturbance of this regulation could lead to psoriasis.

Cell-mediated autoimmune diseases of the skin: some hypotheses.

The propensity of most autoimmune diseases to manifest themselves in the skin may result from faulty teaching of what is 'self' by epithelial cells in the thymus. Whether T cells learn what is 'self' in the thymus or not until later, peripheral to the thymus, may explain the wide range of severity of autoimmune diseases. Also, I suggest that lichen planus, alopecia areata, vitiligo and psoriasis, as well as sarcoidosis and granuloma annulare are autoimmune in nature.

Allelic instability in the mitosis model and the inheritance of psoriasis.

BACKGROUND: Allelic instability in mitosis has been proposed as a model for dominantly inherited diseases. OBJECTIVE: Our purpose was to analyze our own and published data on the inheritance of psoriasis according to the predictions of the allelic instability-in-mitosis model. METHODS: Frequency of psoriasis in father, mother, and siblings was extracted or calculated from data in the literature. Our case records were reviewed and supplemented by telephone or personal interviews when records were incomplete. Inheritance rates from each sex of parent were compared. Correlation between ages at onset in affected siblings was determined. RESULTS: From our own data and the literature summary, the preponderance of inheritance from the father over the mother is statistically significant. A direct correlation exists between ages at onset in affected siblings. Age at onset in parent and child is also positively correlated. Average age at onset in parents is greater than in offspring. CONCLUSION: Both data from literature and our own data are in agreement with the predictions of the allelic instability in mitosis model. This provides evidence that an unstable gene may contribute to the genetics of psoriasis.

The response of human dermal microvascular endothelial cells to hypoxia.

Dermal microvascular endothelial cells (DMEC) exposed to hypoxic conditions show a rapid induction of several proteins that do not increase in other cell types placed in a similar environment. These DMEC proteins differ from the well-characterized stress proteins that have been observed in a wide variety of cultured cell types. The DMEC proteins are induced rapidly, within 2-4 h, and are expressed transiently. They include a group of acidic proteins (pI approximately 5-5.2) with molecular weights in the range 100,000-120,000 and at least one glycoprotein (pI 5.1, M(r) 57,000) that is probably expressed on the cell surface. In some primary DMEC cell strains, this response is accompanied by a transient overall increase in protein synthesis. The oxygen-regulated proteins (ORP) that are induced in most other cell types under hypoxic conditions show little variation in their rate of synthesis in DMEC within the first 24 h. The response of DMEC differs from that of umbilical vein endothelial cells (UVEC) and from spindle-shaped cells derived from DMEC, that show a response to hypoxia that is similar to most other cell types. The changes seen in DMEC proteins take place in the same time scale as ischemia-reperfusion injury and may reflect the specialized change of functions of the microvasculature observed under conditions of hypoxic stress in vivo.

Evidence for an NMDA receptor subunit in human keratinocytes and rat cardiocytes.

Receptor binding studies have demonstrated the presence of an [3H]MK-801 ([3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate) binding site in human keratinocytes. The affinity found in keratinocytes was lower than that found in brain membranes. Northern blots identified mRNA in human keratinocytes and rat cardiocytes, as well as rat brain, that hybridized with high stringency to a probe for NMDAR1, an NMDA receptor subunit. In each tissue, mRNA that hybridized to another glutamate binding protein that might be part of an NMDA receptor complex, was also present. The presence of NMDA or NMDA-like receptors in keratinocytes and rat cardiocytes together with the low affinity [3H]MK-801 binding suggests that this protein may be a general channel forming protein that is present in many tissues, and forms specific receptors by interacting with additional subunits.

Activated human Langerhans cells express mRNA for IL-1 alpha and IL-1 beta and produce these cytokines but do not secrete them.

Human Langerhans cells (LC) were isolated from epidermal cell preparations by panning with mouse anti-CD1 monoclonal antibody. RNA was prepared and probed for the presence of mRNAs for various cytokines using radiolabeled cDNAs. After stimulation with phorbol myristate acetate LC express RNA for interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta) and produce proteins but do not secrete them at detectable levels. LC-associated IL-1, particularly IL-1 alpha, may play a role in antigen presentation. PMA did not induce IL-6 expression in LC. The addition of lipopolysaccharide, a muramyl dipeptide analog, ionomycin, IL-1 alpha, tumor necrosis factor-alpha, insulin-like growth factor-1 or IL-6 did not induce IL-1 mRNA in LC. UVB augmented IL-1 beta mRNA expression. Glucocorticoids did not detectably affect IL-1 alpha or IL-1 beta mRNA levels following PMA induction, however, staurosporin inhibited IL-1 beta mRNA synthesis. Thus the inducers and regulators of IL-1 formation in human LC and monocytes are not identical.

Comparison of second messenger formation in human keratinocytes following stimulation with epidermal growth factor and bradykinin.

We have examined the ability of recombinant human epidermal growth factor (EGF) and bradykinin (BK) to stimulate formation of inositol polyphosphates and sn-1,2-diacylglycerol (DAG), and mobilize intracellular Ca2+ ([Ca2+]i) in adult human keratinocytes (KC). Inositol polyphosphates were resolved by high performance liquid chromatography coupled with flow detector spectroscopy. Free intracellular calcium was quantitated using digital ratio imaging fluorescence microscopy of fura-2 loaded KC. The mass amount of DAG was quantitated using the DAG kinase reaction. When comparing maximal doses of BK (0.1 microM) and EGF (200 ng/ml), BK stimulated larger increases in all second messengers measured. The majority of cells responded rapidly to BK with global increases in [Ca2+]i. Cells responding to EGF were fewer in number and slower to respond with the Ca2+ signal being less pronounced. Treatment of cells with pertussis toxin (PTX) for 24 h significantly attenuated the BK-stimulated inositol polyphosphate formation and [Ca2+]i while the EGF response remained unaffected in both parameters. BK (10(-9) to 10(-6) M) did not stimulate DNA synthesis in KC as measured by [3H]-thymidine incorporation when cultures were treated for 5 days. These results demonstrate that the coupling and biochemical signals produced by stimulation of BK and EGF receptors in human KC are different and suggests that stimulation of second messenger formation from inositol lipid hydrolysis may not be an absolute requirement for the initiation of cell proliferation.

The effect of recombinant human growth hormone on wound healing in normal individuals.

Growth hormone therapy has been suggested to speed wound healing in postoperative patients and in patients with severe burns. This study was undertaken to determine the effect of recombinant human growth hormone on the rate of wound healing in normal individuals. Twenty-three healthy males were evaluated in a randomized, double-blind placebo-controlled study. Each subject received a split-thickness wound (Davol-Keratome) on one buttock and a full-thickness wound (3-mm punch biopsy) on the other. The full-thickness wound healed significantly more slowly in the recombinant human growth hormone-treated group as compared with the placebo control group (t-test, P = .001). No statistically significant difference was noted in the healing of the split-thickness wounds. It is concluded that recombinant human growth hormone may impede healing in normal patients with full-thickness wounds as compared with treatment with placebo. We cannot rule out, however, that the recombinant human growth hormone affected the quality of the scab in full-thickness wounds and thereby only appeared to alter the wound-healing process.

Autocrine stimulation of interleukin-1 alpha and transforming growth factor alpha production in human keratinocytes and its antagonism by glucocorticoids.

Interleukin-1 (IL-1) and transforming growth factor alpha (TGF alpha) mRNA expression was analyzed in cultured normal human keratinocytes. Keratinocytes constititively express IL-1 mRNA when cultured in keratinocyte growth medium but not in Dulbecco's minimal essential medium containing fetal bovine serum, in which the cells differentiate. The predominant form of IL-1 expressed by keratinocytes is IL-1 alpha. Addition of IL-1 alpha to keratinocytes increased IL-1 alpha and TGF alpha mRNA expression in a dose-dependent manner. TGF alpha induced a similar increase in IL-1 alpha and TGF alpha mRNA in keratinocytes. Hydrocortisone decreased the expression of both IL-1 alpha and TGF alpha mRNA in keratinocytes. These findings document an autocrine mechanism by which IL-1 alpha and TGF alpha can stimulate the proliferation of keratinocytes in the skin. It is proposed that this autocrine loop may be hyperactive in psoriasis. Antagonism of the effects of this autocrine loop may be one of the mechanisms by which glucocorticoids exert clinically useful effects in psoriasis and other diseases of the skin.

Immunophenotyping of skin cells during healing of suction blister injury.

The time course of appearance and the dynamic changes of immunocompetent cells were assessed in human skin following sterile suction blister would healing. During epidermal regeneration, a local increase in Langerhans cells (LC) and the appearance of a mononuclear cell infiltrate were observed. Initially, T cells were exclusively of the T helper/inducer subtype, whereas during the later stages of the healing process an increasing number of cytotoxic/suppressor T cells (up to 30%) was observed. Keratinocytes of neither the adjacent nor the newly formed epidermis expressed HLA-DR over the course of the wound-healing process. Our results suggest that immune-competent cells such as T cells and LC may play an important role in the regulation of the wound-healing process.

Insulin-like growth factors are mitogenic for human keratinocytes and a squamous cell carcinoma.

Normal adult human keratinocytes in monolayer culture and SCL-1, a skin-derived squamous-cell carcinoma cell line, were investigated for the expression of receptors for insulin-like growth factors (IGF) and insulin. As demonstrated by affinity crosslinking, radiolabeled IGF-1, IGF-2, and insulin bound specifically to both cell types. Each cell expressed type I IGF receptors, with affinity for IGF-1 greater than IGF-2 much greater than insulin. Insulin receptors, with highest affinity for insulin, were also present on both cells. However, keratinocytes and SCL-1 cells differed in 125I-IGF-2 binding. 125I-IGF-2-bound to both type I and type II IGF receptors in normal keratinocytes, but bound predominantly to membrane-associated IGF binding proteins in SCL-1. IGF-1 was slightly more potent than IGF-2 in stimulating growth of both keratinocytes and SCL-1 cells. In keratinocytes, concentrations of IGF-1 ranging from 5-100 ng/ml, and of IGF-2 from 50-100 ng/ml, resulted in a significant increase in cell number. At the maximum dose of 100 ng/ml, either IGF-1 or IGF-2 caused a 2.3-times increase in cell number. In SCL-1 cells, IGF-1 was more potent than IGF-2 or insulin at lower concentrations, but either IGF-1 or IGF-2 at the maximal concentration of 333 ng/ml stimulated a 4.7-times increase in thymidine incorporation. The stimulatory effect of insulin in SCL-1 was 10-50 times less potent than that of the IGF. The effect of either IGF on SCL-1 was completely inhibited by the type I IGF receptor antibody alpha IR-3, suggesting that both IGFs are mitogenic through the type I IGF receptor. Insulin action was partially blocked by alpha IR-3, suggesting that insulin can act through both the insulin and type I IGF receptors. It thus appears that IGF-1 and IGF-2 are mitogens for normal and transformed human keratinocytes and that their actions are primarily mediated through the type I IGF receptor, whereas insulin is a mitogen through both the IGF-1 receptor and the insulin receptor.