Variation in the diagnosis and clinical management of lentigo maligna across Europe: a survey study among European Association of Dermatologists and Venereologists members.

BACKGROUND: Lentigo maligna (LM), a form of melanoma in situ, is treated to prevent progression to lentigo maligna melanoma (LMM). Surgical treatment is the gold standard. However, treatment guidelines are based on expert opinion, and comparative studies are lacking. OBJECTIVE: The objective of this study was to assess the diagnostic methods and clinical management of LM patients among European dermatologists and residents. METHODS: A survey consisting of 29 questions about diagnostic methods and treatment options used for LM patients was sent to 3308 members of the European Association of Dermatologists and Venereologists (EADV). RESULTS: Most questions were multiple choice, and multiple answers could be ticked per question. A total of N = 415 (12.5%) completed surveys were included in the analyses. A combination of clinical diagnosis (65.7%), dermatoscopy (83.4%) and histopathology (88.2%) is used by most respondents to diagnose LM. Tissue for histopathological evaluation was collected most often using a single punch biopsy in 61.0%. The most common treatment for LM patients <60 years of age is surgery (97.6%). For LM patients >70 years of age, 66.8% of the respondents preferred surgical treatment. Non-surgical options such as radiotherapy (17.0%), topical imiquimod (30.6%), watchful waiting (19.6%) or cryotherapy (20.4%) were used in this elderly group. Subanalysis showed that respondents who take into account patient preference used topical imiquimod, radiotherapy and watchful waiting more often. CONCLUSION: In conclusion, the results of this survey show that there is a variance in the diagnostic methods and treatment modalities used for LM across Europe. Surgery remains the most utilized option. However, non-surgical options, such as topical imiquimod and radiotherapy, are most often used for elderly patients. We recommend that future studies focus on patient preference and compare surgical to non-surgical therapy.

A systematic review on the role of imiquimod in lentigo maligna and lentigo maligna melanoma: need for standardization of treatment schedule and outcome measures.

Lentigo maligna (LM) is an in situ variant of melanoma. Our objective was to systematically review clinical and histological clearance and recurrence rates of imiquimod treatment of LM with emphasis on progression to lentigo maligna melanoma (LMM). PubMed, EMBASE and the Cochrane library were searched from inception to May 2015. Articles were included if they described histologically proven LM treated with imiquimod 5% monotherapy or combined with another topical therapy. Analysed outcomes were clinical and histological clearance, recurrence rates and number of LMM. The quality was assessed using the GRADE-like checklist, and results were reported according to the PRISMA Statement. Twenty-six case reports, 11 retrospective studies, three prospective studies and one randomized controlled trial were included. One case report of poor quality was excluded. Complete clinical clearance was seen in 369 of 471 patients (78.3%). Histological clearance was present in 285 of 370 (77%) patients. LMM was diagnosed in nine (1.8%) patients 3.9 months (range 0-11 months) post-treatment. Univariate multinominal logistic regression showed that 6-7 applications/week had a 6.47 greater odds (P = 0.017) of resulting in complete clinical clearance compared to 1-4 applications/week. An intensity of 6-7 applications/week showed a 8.85 greater odds (P = 0.003) of resulting in histological clearance compared to 1-4 applications. Applying imiquimod >60 times during a treatment period of 12 weeks (range 4-36) showed a 7.75 greater odds (P = 0.001) of resulting in histological clearance compared to <60 total applications. In conclusion, a treatment schedule using imiquimod 6-7 applications per week, with at least 60 applications, shows the greatest odds of complete clinical and histological clearance of LM. Imiquimod is an option for patients unfit for or not willing to undergo surgery or radiotherapy. Nine cases of LM progressed to LMM shortly after treatment. Our hypothesis is that these LMM may have been present before starting imiquimod.

[A baby with progressive skin lesions on the scalp]. / Een baby met toenemende huidafwijkingen op de scalp.

An eight-month-old girl was referred to the dermatologist with a progressive desquamative and purulent eruption on the scalp. Laboratory tests confirmed the diagnosis kerion celsi and the girl was successfully treated with an antimycoticum. Kerion celsi is a deep inflammatory fungal infection on the scalp. Early diagnosis is essential to prevent irreversible hair loss.

The distortive mechanism for the activation of complement component C1 supported by studies with a monoclonal antibody against the "arms" of C1q.

A mouse monoclonal antibody (IgG1 isotype) against human C1q (MAb 130) is presented that activates C1 in serum through its antigen-binding sites at an optimal molar ratio of 3 MAbs:1 C1q. The antibody does not inhibit binding of C1q to IgG. Experiments with pepsin- and collagenase-digested C1q showed that MAb 130 binds to the fibril-like strands (arms) of C1q, close to the globular heads. Bivalency of MAb 130 was a requirement for C1-activation, but not for binding to C1q. Increasing the segmental flexibility of the intact antibody by reduction and alkylation destroyed its capacity to activate C1. A MAb against the globular heads of C1q completely inhibited C1-activation by aggregated IgG (AHG), but did not prevent activation by MAb 130. C1, reconstituted by adding C1q-stalks that lack the globular heads to C1q-depleted serum was not activated by AHG, whereas activation by MAb 130 was not affected. Activation of serum-C1 by AHG and MAb 130 was inhibited by addition of excess purified C1-inhibitor in a comparable and dose-dependent manner. Sucrose-gradient analysis indicated a predominance of stable complexes of a single C1q-molecule with three MAbs at the optimal activating ratio. When isolated and added to C1q-depleted serum, these complexes activated C1 efficiently. A mechanism for activation by MAb 130 is proposed that supports the "distortive" model of C1-activation.

Rat polymeric IgA binds C1q, but does not activate C1.

Immune complexes, prepared with monoclonal rat IgA antibodies directed against DNP, activate the alternative pathway of the complement system in rat serum. In this study, the interaction of these monoclonal IgA antibodies with the classical pathway of complement was investigated. Monoclonal polymeric IgA (p-IgA) was shown to inhibit the IgG2b-mediated classical pathway-dependent lysis of TNP-coated sheep red blood cells. In addition, the binding of C3 to solid phase IgG2b immune complexes was inhibited by p-IgA. Monoclonal monomeric IgA (m-IgA) was much less efficient in this respect. To further analyse the effect of p-IgA on the activation of the classical pathway by IgG2b immune complexes, the interaction of p-IgA with C1 was studied. It was found that p-IgA antibodies bind C1q. No species-specificity was observed, since both rat and human C1q were bound. Whereas binding of C1q in C1 to IgG2b resulted in activation of C1, binding to p-IgA did not. The binding of C1q to both p-IgA and IgG2b could be inhibited by monoclonal antibodies directed against the globular heads of C1q, but not by monoclonal antibodies directed against the collagen tail. The formation of insoluble p-IgA immune complexes was inhibited in the presence of rat serum or C1. These studies indicate that C1q binds to p-IgA by its globular heads, and thereby may modulate classical pathway-mediated reactions such as the inhibition of immune precipitate formation.

Indomethacin increases the sensitivity of the monocyte migration inhibition assay.

In ophthalmo-immunological investigations only small samples of ocular tissues and fluid are available and assays which are feasible with very small volumes or cell numbers are mandatory. Indomethacin, which is known to augment the immune response both in vivo and in vitro was therefore tested for its effect on the monocyte migration inhibition (MIF) assay using low cell or antigen doses. The sensitivity of the MIF assay may be greatly increased by adding indomethaci during the first step of the assay. Titration of either the antigen dose, the mononuclear cells number or both per assay, resulted in a 10-50-fold increase in sensitivity of the assay, with a broad inter-individual variability. Increasing the sensitivity of the MIF assay with indomethacin has clear advantages with regard to the number of cells required but also confronts us with a new problem: activation of specific cells that circulate at very low frequencies in non-immunized individuals. The enhanced response could be reversed to some extent by adding prostaglandin E2 together with indomethacin to the first step of the assay. Moreover, adding leukotriene B4 to the first step of the assay had an enhancing effect over a limited concentration range. We conclude that in the presence of indomethacin, the MIF assay provides a highly sensitive technique for the demonstration of cellular immune responses in small samples of biological fluids containing very small numbers of antigen-specific lymphocytes.

Human corneal extract enhances serum complement activity.

PURPOSE: The concomitant presence of complement-activation products in the cornea during inflammation is well described. The present study was undertaken to analyze the complement activity of normal human corneal tissue and to assess the presence of complement-modulating activities. METHODS: Human corneal tissue was extracted in veronal-buffered saline. The overall complement (C) activity of the human corneal extract (HCE) and the effect of HCE on serum C-activity were investigated using a hemolytic assay. Anion-exchange chromatography and gel filtration were applied for biochemical analysis of HCE. Monoclonal anti-human C3 was used to detect corneal C3 and to remove C3 from HCE by immunoadsorption. RESULTS: It was found that C-activity of HCE was less than 200 U/g tissue. Experiments to test whether HCE exhibited inhibitory activity led to an unexpected result: When added to human serum dilutions, HCE caused a significant, dose-dependent increase of C-activity. Pretreatment of HCE at 56 degrees C abolished the effect. Analysis of HCE by anion-exchange chromatography revealed two C-enhancing peaks. One peak was identified as C3 whereas the identity of the other protein peak remained unknown. CONCLUSIONS: Results indicate that the human cornea contains an as yet unidentified heat sensitive factor(s) able to enhance complement activity of serum. It is postulated that this factor(s) may play an important role in corneal physiology and pathology.

Analysis of aqueous humor immunoglobulin G in uveitis by enzyme-linked immunosorbent assay, isoelectric focusing, and immunoblotting.

Immunoglobulin G (IgG) in aqueous humor from patients with various uveitis syndromes was analyzed using a number of immunologic techniques. Sixty-five percent of patients with Fuchs' heterochromic cyclitis (FHC), 70% of patients with other forms of uveitis, and 44% of controls showed local synthesis of IgG, as demonstrated by an elevated IgG:albumin relative concentration ratio. Using an enzyme-linked immunosorbent assay to measure the concentration of IgG subclasses 1-4, a relative excess of IgG1 was found in the aqueous compared with the serum in FHC. Isoelectric focusing and immunoblotting studies revealed oligoclonal IgG bands in the aqueous of 13 of 23 (57%) patients with FHC, most being of the IgG1 subclass. Oligoclonal bands were not found in 18 patients with other types of uveitis or 13 patients undergoing surgery for senile cataract. These findings indicate intraocular production of IgG of restricted specificity in FHC, providing further evidence for local immune dysfunction in this condition. As yet the antigenic stimulus for this oligoclonal B-cell response has not been identified.

Endotoxin-induced uveitis in the rat. The significance of intraocular interleukin-6.

The potential role of interleukin-6 (IL-6) was studied as an inflammatory mediator of endotoxin (or lipopolysaccharide [LPS])-induced uveitis (EIU) in the rat. In young Lewis rats, levels of intraocular IL-6, but not serum IL-6, correlated with the severity of uveitis and with aqueous humor protein levels in response to foot pad injections of LPS (P less than 0.001). Adult Lewis rats did not develop uveitis and had no intraocular IL-6, although IL-6 was released systemically. Resistance to EIU and absence of IL-6 levels in the aqueous humor, despite the ability to release serum IL-6, also were observed in brown Norway rats, irrespective of age and weight. Intravitreal injection of as little as 1 ng of human recombinant IL-6 induced uveitis in young Lewis rats. In adult Lewis rats, and in young animals made tolerant to LPS, intravitreal IL-6 still caused substantial leakage of plasma proteins into the anterior chamber but no influx of inflammatory cells. As early as 2 hr after intravitreal injection of IL-6, immunohistochemical analysis showed invasion of the iris, corneal stroma, and anterior chamber by polymorphonuclear leukocytes (PMN) and expression of major histocompatibility complex (MHC) class II antigen in the retina by large cells that were macrophage-marker ED2 negative. This was followed by massive PMN infiltration of the retinal layers and vitreous. The MHC class II antigen expression of ciliary and iris epithelium occurred at a later stage (greater than 8 hr).(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-mediated immunity against human retinal extract, S-antigen, and interphotoreceptor retinoid binding protein in onchocercal chorioretinopathy.

Autoimmune mechanisms are thought to be involved in the pathogenesis of onchocercal chorioretinopathy. Cell-mediated immune responses to human retinal S-antigen, interphotoreceptor retinoid binding protein (IRBP), and crude retinal extract were investigated in patients with onchocerciasis from Sierra Leone, West Africa using a two-step migration-inhibition factor assay. Patients were subdivided into three groups: (1) without ocular involvement (n = 10), (2) with ocular onchocerciasis limited to the anterior segment (n = 19), and (3) with onchocercal chorioretinopathy (n = 21). A group of endemic controls (n = 25) from Sierra Leone were also studied. The cellular immune response to concanavalin A (Con A) was measured to assess the general capacity of lymphocytes to respond to a mitogen. Four of 50 (8%) patients with onchocerciasis and four of 25 (16%) endemic controls reacted with at least one retinal antigen. From the patients with onchocercal chorioretinopathy two of 21 (10%) showed a positive cellular response. The general mitogen response tested with Con A was positive in all these individuals. A role for an antiretinal autoimmune mechanism in the pathogenesis of onchocercal chorioretinopathy, as studied with human S-antigen, IRBP, or crude retinal extract, could not be shown because the cellular response to these antigens did not differ in patients with or without onchocercal chorioretinopathy or in endemic controls.

Aqueous humor interleukin-6 levels in uveitis.

The level of Interleukin-6 (IL-6) in the aqueous humor of 24 patients with 2 types of uveitis was measured with a specific bioassay using the murine hybridoma cell line B9. Sixteen patients had Fuchs' heterochromic cyclitis (FHC) and 8 had toxoplasma uveitis (TU). Sixty-three percent of each of the FHC and TU groups had raised levels of IL-6 in their aqueous (mean: 543 and 19,228 units/ml respectively). Thirteen control aqueous samples, obtained at surgery for senile cataract, showed IL-6 levels of less than 10 units/ml. Serum obtained at the same time as each aqueous humor sample also showed IL-6 levels of less than 10 units/ml, indicating that the raised levels of IL-6 found in the aqueous of uveitis patients did not result from serum leakage, but from local production. This is the first report on intraocular IL-6 levels, and indicates that IL-6 may play a role as an inflammatory mediator in uveitis.

Analysis of interleukin-6 in endotoxin-induced uveitis.

The mechanisms underlying the induction of intraocular inflammation in the rat model of endotoxin-induced uveitis (EIU) and the subsequent development of tolerance after repeated endotoxin injections are poorly understood. Interleukin-6 (IL-6) was measured in the aqueous humor and serum of Lewis rats after single and repeated injections of endotoxin into the footpad. After a single injection, a rise in serum and aqueous-humor levels of IL-6 was seen after 2 and 16 hr, respectively. The highest aqueous-humor level of IL-6 was seen 20 hr postinjection and was tenfold that seen in the serum sample taken at the same time, suggesting intraocular synthesis of this cytokine. Four hours later the most active uveitis and the highest total aqueous-humor protein level were observed. Repeated injection of endotoxin still resulted in a moderate but significant systemic release of IL-6 but no detectable IL-6 in the aqueous humor and the absence of uveitis. Intravitreal injection of endotoxin-free human recombinant IL-6 (10-10(5) U) in rats resulted in uveitis, resembling the ocular response to endotoxin. There appeared to be a prozone effect regarding the total aqueous-humor protein concentration. The largest amount of aqueous-humor protein was seen in the eyes injected with 10(2) U of IL-6, but increasing concentrations of intravitreal IL-6 showed a corresponding decrease in protein levels. In the fellow saline-injected eyes, a clear consensual response was observed with regard to the extravasation of protein, although the uveitic grade in these eyes was low or zero. Repeated intravitreal injection of IL-6 resulted in ocular unresponsiveness in nine of 11 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of intraocular tumor necrosis factor and interleukin-6 in endotoxin-induced uveitis in the rat.

PURPOSE: To determine the kinetics of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in serum and aqueous humor of rats with different susceptibilities to endotoxin-induced uveitis (EIU), after footpad injection of lipopolysaccharide (LPS). METHODS: Samples were collected from EIU-susceptible Lewis rats and EIU-resistant Brown Norway (BN) rats for up to 72 hours after LPS injection. Specific bioassays were used to measure TNF and IL-6 activity. Northern blot analysis was used to assess intraocular IL-6 mRNA expression. RESULTS: High levels of TNF and IL-6 were detected in serum of both rat strains early after LPS injection. A second rise in serum TNF was observed at 18 to 20 hours in Lewis rats only. In aqueous humor of Lewis rats, high levels of TNF and IL-6 were observed early after LPS injection (2 to 8 hours) and concomitant with maximal uveitis (18 to 24 hours). Low levels of TNF and IL-6 were found in aqueous humor of BN rats. Ocular IL-6 mRNA was detected at the same time as IL-6 activity was measured in aqueous humor. CONCLUSIONS: The results of this study indicate that both TNF and IL-6 may play a role in the pathogenesis of EIU. The early release of TNF in aqueous humor during EIU suggests that this cytokine may serve as an initial mediator of intraocular inflammation. Furthermore, Northern blot analysis indicates that IL-6 is produced locally during EIU.

Adhesion molecule expression in basal cell carcinoma.

Basal cell carcinomas (BCCs) are frequently associated with a peritumoral mononuclear infiltrate. Until now, the function of this inflammatory infiltrate and its possible role in the control of tumor growth is unclear. Mechanisms controlling endothelial and target cell adhesiveness for leukocytes are important features in the development of a specific local immune response. The expression and distribution of the adhesion molecules ICAM-1, VCAM-1 and E-selectin by microvascular endothelial cells and tumor cells, together with their leukocyte receptors LFA-1, VLA-4 and CLA respectively, were studied in 33 BCCs of different histological subtypes. In normal skin, ICAM-1 is expressed by resting endothelial cells, whereas VCAM-1 and E-selectin expression correlates with endothelial activation. The epidermis in normal conditions displays no ICAM-1, VCAM-1, or E-selectin expression. In BCC, endothelial ICAM-1 expression was only slightly increased compared to normal skin, whereas expression of endothelial VCAM-1 and E-selectin was low or absent in all BCCs examined. Peritumoral infiltrates contained mostly LFA-1-expressing lymphocytes, with minimal VLA-4 and CLA positivity. In none of the cases studied was adhesion molecule expression by BCC tumor cells identified. The lack of significant expression of adhesion molecules on peritumoral vascular endothelial cells and BCC tumor cells does not support the idea of specific, cell-mediated immunity being an important mechanism in limiting BCC tumor spread.

Cytokines and intraocular inflammation.

Although new endogenous mediators of inflammatory and immune responses are reported almost on a monthly basis, the cytokines IL-1, TNF, and IL-6 have emerged as the primary regulators of local inflammation in man. In this paper, uveitogenic and other properties of these particular cytokines are discussed and attention is payed to the possible involvement of a cytokine-network in the development of uveitis.

Cytokines and uveitis, a review.

Although the exact pathogenic mechanisms underlying uveitis are unknown, cytokines appear to be involved in this inflammatory disorder. This review describes the studies in which the uveitogenic properties of several cytokines, including tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, IL-8 and interferon gamma (IFN-gamma), were investigated and the reports on intraocular expression of cytokines, such as TNF, IL-2, IL-6 and IFN-gamma, during uveitis. The exact contribution of these mediators to uveitis remains to be determined. This may provide new clues in the treatment of uveitis.

Humoral and cellular immune reactions against retinal antigens in clinical disease.

Autoimmune reactions against retinal antigens have been suggested to play an important role in clinical uveitis in man. As yet the evidence for this assertion is very weak. Sympathetic Ophthalmia is a disease entity which comes closest to acceptance as an autoimmune disease although the autoantigen involved has not been identified. Both cellular and humoral autoreactivity against retinal antigens have been found both in uveitis patients as well as in healthy controls. Very high levels of retinal antibodies were found in onchocerciasis patients but no relation was observed with the occurrence of chorioretinitis. Differences were observed when testing patient sera against human or bovine retinal antigens (S-antigen or IRBP) emphasizing the need for using human tissue when investigating autoimmune responses. Circumstantial evidence in favor of an autoimmune etiology of uveitis include the morphology of the inflammatory infiltrate, effect of immuno-suppressive therapy and especially the establishment of experimental animal models. The experimental models of S-antigen or IRBP induced uveitis are primarily T cell mediated and also show pineal gland involvement. As yet no "established" human autoimmune disease has been described with a dominant role for T cells. Furthermore there is no evidence for pineal gland involvement in clinical uveitis. Analysis of the specificity of the T cell infiltrate or deposited immunoglobulins obtained from the diseased tissues may provide conclusive evidence for a possible autoimmune character of certain clinical uveitis entities.

Detection of aldehyde dehydrogenase activity in human corneal extracts.

The major soluble protein in bovine corneal epithelial extracts is a 54 kD protein (BCP 54) which has recently been identified as the corneal aldehyde dehydrogenase. Although ALDH activity has been reported in human corneal extracts it was not yet clear whether this was identical with the 54 kD protein described in bovine corneas. To investigate this question, we studied human corneal extracts for the presence of ALDH using enzyme analysis, SDS-PAGE, native electrophoresis, isoelectric focusing and immunoblotting techniques. The corneal epithelium was the most active layer (8.46 +/- 1.9 IU/mg protein) followed by the stroma (2.83 +/- 0.56 IU/mg protein) and endothelium (0.06-3.6 IU/mg protein). When comparing substrate specificity between human and bovine corneal ALDH, using NADP as coenzyme, it was shown that the human enzyme preferred benzaldehyde whereas the bovine enzyme revealed the strongest enzymatic activity with hexanal. Human corneal ALDH was partly inhibited by disulfiram. Bovine and human cornea ALDH lost their enzymatic activity after heating at temperatures above 56 degrees C. Both human and bovine corneal extracts contained a prominent 54 kD protein which reacted with a rabbit anti BCP 54 antibody. Isoelectric focusing followed by enzyme staining in the gel revealed 5 human corneal isozyme species and 4 in bovine corneal extracts, migrating at a pH between 6.5 and 7.0. All isozymes could also be detected after immunoblotting with a rabbit anti BCP 54 antibody.