Multicenter prospective study on multivariant diagnostics of autoimmune bullous dermatoses using the BIOCHIP technology.

BACKGROUND: The current standard in the serologic diagnosis of autoimmune bullous diseases (AIBD) is a multistep procedure sequentially applying different assays. In contrast, the BIOCHIP Mosaic technology combines multiple substrates for parallel analysis by indirect immunofluorescence. METHODS: Sera from 749 consecutive, prospectively recruited patients with direct immunofluorescence-positive AIBD from 13 international study centers were analyzed independently and blinded by using (1) a BIOCHIP Mosaic including primate esophagus, salt-split skin, rat bladder, monkey liver, monkey liver with serosa, recombinant BP180 NC16A, and gliadin GAF3X, as well as HEK293 cells expressing recombinant desmoglein 1, desmoglein 3, type VII collagen, and BP230 C-terminus and (2) the conventional multistep approach of the Department of Dermatology, University of Lübeck. RESULTS: In 731 of 749 sera (97.6%), specific autoantibodies could be detected with the BIOCHIP Mosaic, similar to the conventional procedure (725 cases, 96.8%). The Cohen κ for both serologic approaches ranged from 0.84 to 1.00. In 6.5% of sera, differences between the 2 approaches occurred and were mainly attributed to autoantigen fragments not present on the BIOCHIP Mosaic. LIMITATIONS: Laminin 332 and laminin γ1 are not represented on the BIOCHIP Mosaic. CONCLUSIONS: The BIOCHIP Mosaic is a standardized time- and serum-saving approach that further facilitates the serologic diagnosis of AIBD.

Prospective studies on the routine use of a novel multivariant enzyme-linked immunosorbent assay for the diagnosis of autoimmune bullous diseases.

BACKGROUND: Serologic diagnosis of autoimmune blistering disease (AIBD) usually follows a sophisticated multistep algorithm. OBJECTIVE: We sought validation of a multivariant enzyme-linked immunosorbent assay (ELISA) in the routine diagnosis of AIBD. METHODS: The multivariant ELISA comprising 6 recombinant immunodominant forms of major AIBD target antigens, ie, desmoglein 1, desmoglein 3, envoplakin, BP180, BP230, and type VII collagen was applied in: (1) a cohort of well-characterized AIBD (n = 173) and control sera (n = 130), (2) a prospective multicenter study with 204 sera from patients with newly diagnosed AIBD with positive direct immunofluorescence microscopy, and (3) a prospective monocenter study with 292 consecutive sera from patients with clinical suspicion of AIBD in comparison with the conventional multistep diagnostic algorithm. RESULTS: Concordant results in the multivariant ELISA compared with direct immunofluorescence microscopy were seen in 94% of patients with pemphigus and 71% of patients with pemphigoid (Cohen κ value, 0.95 and 0.66) and with the conventional multistep diagnostic approach in 91% of patients with pemphigus and 88% of patients with bullous pemphigoid and 93% of autoantibody-negative sera (Cohen κ, 0.95, 0.84, and 0.78). LIMITATIONS: IgA autoantibodies and less common target antigens were not analyzed. CONCLUSIONS: The multivariant ELISA is a practical, highly standardized, and widely available novel diagnostic tool for the routine diagnosis of AIBD.

Inzidenz von bullösen Autoimmunerkrankungen in Serbien: eine retrospektive Studie über 20 Jahre.

HINTERGRUND UND ZIELE: Die meisten früheren Arbeiten zu den klinisch-epidemiologischen Merkmalen von bullösen Autoimmunerkrankungen (AIBD) konzentrierten sich vor allem auf eine einzige Krankheitsentität oder nur eine Krankheitsgruppe; nur in wenigen Studien wurde die Inzidenz verschiedener AIBD untersucht. Bei der vorliegenden Studie war es unser Ziel, das gesamte Spektrum der AIBD zu betrachten, die Inzidenz der häufigsten AIBD zu ermitteln und die zeitlichen Trends ihres Auftretens in Zentralserbien über einen Zeitraum von 20 Jahren zu untersuchen. METHODEN: Wir rekrutierten retrospektiv 1161 AIBD-Fälle, die in Zentralserbien von Januar 1991 bis Dezember 2010 neu diagnostiziert wurden. Die Diagnose stützte sich auf eine strikte klinische, histologische und immunhistologische Beurteilung. ERGEBNISSE: Folgende Inzidenzraten wurden für die einzelnen Erkrankungen ermittelt: 4,35 pro eine Million Einwohner/Jahr (pME/Jahr) für Pemphigus, 4,47 pME/Jahr für Pemphigoid, 1,42 pME/Jahr für Dermatitis herpetiformis (DH), 0,25 pME/Jahr IgA-Dermatose und 0,08 pME/Jahr für Epidermolysis bullosa acquisita. Im betrachteten Zeitraum stieg die altersbereinigte Inzidenzrate für Pemphigus und insbesondere für Pemphigoid signifikant an, während sie für DH, allerdings nicht signifikant, abnahm. SCHLUSSFOLGERUNGEN: Unsere Studie befasst sich zum ersten Mal mit den Inzidenzraten des gesamten Spektrums der AIBD in Serbien und untersucht die zeitlichen Trends ihres Auftretens über einen Zeitraum von 20 Jahren. Nach unserem besten Wissen wurde ein ähnlicher Befund wie der unsere, dass nämlich die Inzidenzraten von Pemphigus und Pemphigoid vergleichbar sind, bisher noch nicht publiziert.

Incidence of autoimmune bullous diseases in Serbia: a 20-year retrospective study.

BACKGROUND AND OBJECTIVES: While most previous surveys on the clinico-epidemiological features of autoimmune bullous diseases (AIBDs) have predominantly focused on a single disease entity or just one disease group, there have been only few studies examining the incidence of various AIBDs. In the present study, we set out to determine the spectrum of AIBDs, to estimate the incidence of the most common AIBDs, and to examine their temporal trends in Central Serbia over a period of 20 years. METHODS: We retrospectively recruited 1,161 new AIBD cases diagnosed in Central Serbia during the period from January 1991 to December 2010. The diagnosis was based on strict clinical, histological, and immunohistological evaluation. RESULTS: The incidence rates were: 4.35 per million population/year (pmp/year) for pemphigus, 4.47 pmp/year for pemphigoid, 1.42 pmp/year for dermatitis herpetiformis (DH), 0.25 pmp/year for linear IgA disease, and 0.08 pmp/year for epidermolysis bullosa acquisita. In the period observed, age-adjusted incidence rates significantly increased for pemphigus and particularly for pemphigoid, whereas they decreased, albeit not significantly, for DH. CONCLUSIONS: For the first time, our study evaluates the incidence rates of the entire spectrum of AIBDs in Serbia, and examines their temporal trends over a 20-year period. To the best of our knowledge, our finding of similar incidence rates for pemphigus and pemphigoid has previously not been reported.

Direct immunofluorescence of the outer root sheath in anagen and telogen hair in pemphigus vulgaris and pemphigus foliaceus.

Direct immunofluorescence of peri-lesional skin is the gold standard in the diagnosis of pemphigus. A specific immunofluorescence pattern may also be demonstrated in the outer root sheath of anagen and telogen hair. We demonstrated an intercellular reticular deposition of immunoglobulin G in the outer root sheath of plucked anagen and telogen hair in all pemphigus vulgaris patients with active disease and for the first time in all patients with active pemphigus foliaceus. Moreover, we demonstrated for the first time that plucked hair samples may be kept at -20°C for at least 2 weeks before immunofluorescent staining and analysis.

Aloe-emodin inhibits proliferation of adult human keratinocytes in vitro.

Aloe-emodin (AE) is a plant-derived hydroxyanthraquinone with several biological activities. It is present in a variety of skin-conditioning agents containing aloe extracts, but its influence on keratinocyte growth was not examined so far. We investigated the influence of AE on human keratinocyte proliferation and apoptosis in vitro. AE significantly inhibited proliferation of cultivated human keratinocytes at 5 µM concentration, as revealed by incorporation of radioactive thymidine. The antiproliferative effect of AE was accompanied with induction of apoptosis, but not necrosis, as demonstrated by flow cytometric analysis and lactate dehydrogenase release assay. Based on the half maximal inhibitory concentration values, we demonstrated that AE may impair proliferation of keratinocytes at concentrations far below the industry standards for commercial products containing aloe extracts. Therefore, further research of AE effects on the human skin and proper labeling of products are necessary for maximizing benefits from aloe extracts and to avoid undesired responses.

Novel and recurrent FERMT1 gene mutations in Kindler syndrome.

Kindler syndrome (OMIM 173650) is an autosomal recessive condition characterized by skin blistering, skin atrophy, photosensitivity, colonic inflammation and mucosal stenosis. Fewer than 100 cases have been described in the literature. First reported in 1954, the molecular basis of Kindler syndrome was elucidated in 2003 with the discovery of FERMT1 (KIND1) loss-of-function mutations in affected individuals. The FERMT1 gene encodes kindlin-1 (also known as fermitin family homologue 1), a 77 kDa protein that localizes at focal adhesions, where it plays an important role in integrin signalling. In the current study, we describe five novel and three recurrent loss-of-function FERMT1 mutations in eight individuals with Kindler syndrome, and provide an overview of genotype-phenotype correlation in this disorder.

Pemphigus vulgaris in three adolescents: the course of the disease.

We report three adolescents with pemphigus vulgaris whose disease started at the age of 13, 15 and 14 years, respectively. The course of the disease and the treatment approaches were reviewed. Pemphigus vulgaris during childhood and adolescence is a very rare disease in this part of Europe. Among 410 pemphigus vulgaris patients that we treated during the 20-year period, only three patients (0.73%) were under the age of 18 years. According to our experience, the course of pemphigus vulgaris in patients before the age of 18 years is comparable with the course of pemphigus vulgaris in adults.

Dexamethasone-cyclophosphamide pulse therapy in pemphigus: a review of 72 cases.

BACKGROUND: Autoimmune pemphigus is a group of severe blistering diseases. Although corticosteroids have dramatically altered the prognosis of pemphigus, morbidity and mortality resulting from the adverse effects of systemic corticosteroids remain high. Dexamethasone-cyclophosphamide pulse (DCP) therapy was introduced to diminish the adverse effects of prolonged conventional daily dose regimens. OBJECTIVE: To report our experience with the use of the DCP regimen in patients with autoimmune pemphigus. METHODS: In the period 1998-2002, 72 patients with various forms of autoimmune pemphigus treated with DCP therapy were included, of whom 36 patients were previously treated with conventional corticosteroid therapy, and 36 were newly diagnosed patients. RESULTS: Of the 72 patients, 43 completed treatment, while 13 patients did not respond adequately to the treatment and continued with the conventional daily regimen, nine patients were lost to follow-up, and seven patients died. Two of these deaths were probably a consequence of DCP therapy. CONCLUSION: DCP regimen is a beneficial treatment for patients with pemphigus, sparing the adverse effects of conventional regimens.

Bullous pemphigoid induced by penicillamine in a patient with Wilson disease.

We report a 47-year-old man with Wilson disease who developed bullous lesions on the trunk and extremities after 20 years of penicillamine treatment. The histologic and immunofluorescence findings were diagnostic of bullous pemphigoid. When penicillamine was replaced by zinc sulfate, the patient's bullous skin lesions improved rapidly. However, after 2 months of zinc sulfate treatment, the patient's skin condition remained improved but his neurologic disease became worse and penicillamine was reinstituted. Bullous lesions recurred within 1 week and the diagnosis of penicillamine-induced bullous pemphigoid was confirmed. This is the first report of penicillamine-induced bullous pemphigoid in a patient with Wilson disease.

Patients' reported quality of life in chronic venous disease in an outpatient service in Belgrade, Serbia.

Chronic venous disease (CVD) has been reported to substantially affect patients' quality of life (QoL). To evaluate the impact of CVD on patient-reported QoL in a patient series in Serbia, a cross-sectional study of 570 CVD patients, classified according to the CEAP clinical classification into classes (C) 1-6, was performed in a Belgrade outpatient clinic. QoL was assessed by the general short-form (SF)-36 questionnaire, and additionally by a brief CVD-specific questionnaire. The SF-36 scores for all QoL dimensions showed a progressive reduction from C1 to C6. Class 5 and 6 patients scored the lowest across all dimensions, with significant (p < 0.05) reductions in physical functioning, role-physical, general health, vitality and mental health. The score for bodily pain decreased from C1 to C4, but increased in C5 and C6 as compared to C4 patients. Interestingly, despite an increasing rate of aesthetic concerns as the disease progresses, no variations were found in the social functioning and emotional role scores across the groups. There were no age or gender differences in any QoL item across the classes. The data presented show that QoL of CVD patients decreases, particularly after the appearance of skin changes, and suggest that even patients in the early stages consider CVD a disease and not merely a cosmetic problem.

Unilateral multiple lichen striatus treated with tacrolimus ointment: a case report.

A previously healthy 11-year-old boy with multiple, unilateral lichen striatus (LS) on the left side of the body is described. The distribution of the lesions corresponded to the lines of Blaschko. Histology of the lesional skin was compatible with LS. Two years after the onset of the linear eruption, treatment with tacrolimus ointment resulted in significant improvement over a short period of time. To our knowledge, this is the first time that the use of tacrolimus for successful treatment of LS with multiple lesions has been reported.

Antioxidative capacity of C(60) (buckminsterfullerene) and newly synthesized fulleropyrrolidine derivatives encapsulated in liposomes.

C(60) (buckminsterfullerene or fullerene C(60)) has been recognized an efficient free-radical scavenger. Owing to its high antioxidative capacity, C(60) and its derivatives have a great potential for application in biological systems where prevention of oxidative cell damage is desirable. However, poor solubility of native C(60) in water represents a major drawback for its use in biological systems. In order to increase the efficiency of delivery of fullerenes to target tissues it is of great interest to enhance their water solubility by developing hydrophilic organoderivatives of C(60) with retained antioxidative properties, and/or by encapsulating fullerenes in biocompatible liposomes. In the present study, using EPR spin-trapping and spin-labelling techniques, we investigated the antioxidative capacity of C(60) and newly synthesized fulleropyrrolidine derivatives Q-C(60) [N-methyl-(2-quinolyl)fulleropyrrolidine(60)] and I-C(60) [N-methyl-(2-indolyl)fulleropyrrolidine(60)] encapsulated in multilamellar phospholipid liposomes. The capacity for removal of (*)OH (hydroxyl radical) and O2(*-) (superoxide radical) and for the prevention of lipid peroxidation should be stressed as the most relevant biological antioxidative parameters. When these parameters for novel organofullerenes were compared with the the performance of C(60), Q-C(60) and I-C(60) showed similar, or even better, antioxidative characteristics. However, further research is required to establish the toxicity of these derivatives and their antioxidant efficacy in vivo.

Cytokine concentrations in basal cell carcinomas of different histological types and localization.

BACKGROUND: Basal cell carcinoma (BCC) is the most common malignant skin tumor. Cytokines as major mediators of the immune system have been shown to play an important role in biology of the neoplasm with the general predomination of Th2 cytokines, whereas IFN-? and other Th1 cytokines are prevalent in spontaneously regressing tumors. OBJECTIVE: We were interested in comparing cytokine levels in BCC and cutaneous squamous cell tumors with BCC of different localization and histological subtypes. MATERIAL AND METHODS: Explants from freshly excised BCC from 18 patients, and cutaneous squamous cell tumors (solar keratoses and Bowen's disease) from 9 patients were cultivated for 24 h. Cytokine (IL- 2, IFN-gamma, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNFalpha, IL-1beta) concentrations in culture supernatants were determined by a sandwich immunoassay. RESULTS: Tissue explants of BCC contained significantly higher concentrations of IL-1beta, IL-4, IL-5, and IL-6 compared to those of squamous cell tumors. Higher levels of TNF-alpha (p = 0.042), IL-4 (p = 0.028), and IL-5 (p = 0.012) were found in tumors localized to the head and neck compared to those on the trunk or extremities. Interleukin-6 concentrations were higher in aggressive BCC variants (infiltrative and micronodular), but the difference was not statistically significant (p = 0.068). CONCLUSIONS: Confirming the earlier findings that BCC is a tumor with a Th2 cytokine microenvironment, this study further shows that BCC situated on the head and neck produce even more of certain Th2 cytokines (IL-4 and IL-5) and TNF-alpha, a crucial immunosuppressive cytokine released upon UVB irradiation.

Two cases of probable endogenous extensive cutaneous larva migrans in Serbia.

Cutaneous larva migrans (CLM) is a skin infestation clinically characterized by erythematous serpiginous lesions caused by nematode larvae, usually of animal hookworms. It is most commonly seen in tropical and subtropical geographic areas. It is occasionally seen in Europe and other temperate climates, most often in patients that have previously traveled to tropical areas. We present two male patients that did not travel abroad with clinical features of extensive CLM located on the trunk acquired in an unusual way. CLM is not characteristic of Serbia, which is located in southeast Europe Unusually hot and sunny weather with heavy rainfall and high humidity during the summers of 2005 and 2006 were favorable conditions for the development of parasites in the soil and infestation with large numbers of larvae. To the best of our knowledge, this is the first report of extensive CLM in Serbia.

Recessive dystrophic epidermolysis bullosa: presentation of two forms.

Dystrophic epidermolysis bullosa (DEB) and aplasia cutis congenita (ACC), also known as congenital localized absence of skin (CLAS) are rare clinical entities. Aplasia cutis congenita presented in conjunction with simplex, junctional, or dystrophic types of epidermolysis bullosa (EB) is classified as type-6 ACC. This association was initially described and referred in the literature as Bart syndrome. We describe two cases of recessive DEB (RDEB), one with the major Hallopeau-Siemens (RDEB-HS) subtype and one case with the minor RDEB inversa (RDEB-I) subtype associated with ACC localized on the lower extremities. Full clinical history and transmission electron microscopic findings are presented for both cases. To date, only five cases of RDEB presenting with ACC have been reported in the literature. Detailed descriptions of the association of RDEB and ACC in the literature are scarce. It seems that this condition is probably more common in clinical practice than described in the literature. Our findings confirm that the term, Bart syndrome, should not be considered as a separate entity or clinical variant of dominant dystrophic EB as it was initially described. Congenital localized absence of skin may be associated with any of the three major types of EB (simplex, junctional, or dystrophic).

Pagetoid reticulosis of Woringer-Kolopp.

Pagetoid reticulosis of Woringer-Kolopp is a rare form of cutaneous T-cell lymphoma that primarily affects middle-aged males. It is characterized by the presence of one or several scaly patches and plaques with an acral distribution. We present a case of a 58-year-old woman, otherwise healthy, with a 5-month history of asymptomatic, hyperkeratotic plaques on the hands and feet. Histological and immunohistochemical analysis confirmed the diagnosis. Nearly complete spontaneous regression was noted 7 months after the initial examination. After 5 years no evidence of the disease remained.

Male urethritis with and without discharge: relation to microbiological findings and polymorphonuclear counts.

OBJECTIVE: We studied the relation between the presence or absence of urethral discharge, urethral pathogens, and polymorphonuclear (PMN) counts on Gram stained urethral smears in men with symptomatic urethritis. METHODS: The study population was composed of 630 sexually active heterosexual men (aged 18-45 years) who had urethral symptoms and signs (discharge, dysuria or urethral discomfort). Participants were divided into two groups: the first (n=320) was comprised of men with urethral discharge confirmed on examination, while the other (n=310) was composed of patients with urethral symptoms but without discharge. Urethral swabs for Gram stained smears and microbiological analyses (N. gonorrhoeae, C. trachomatis, T. vaginalis and U. urealyticum) were taken from all study participants. Polymorphonuclear leukocytes (PMN) on Gram-stained urethral smears were counted in 5 oil immersion x1000 PMN per high power fields (phpf). Urethritis was defined as the presence of > or =5 PMN/hpf. RESULTS: N. gonorrhoeae was isolated only in men with urethritis accompanied by discharge. The prevalence of T. vaginalis, C. trachomatis and U. urealyticum was significantly higher (F=8.854, P<0.01) in urethral swabs of urethritis patients with discharge compared to patients with no discharge. The most common urethral pathogen in both groups of patients was T. vaginalis (31.56% and 26.45%, respectively). One or more microorganisms were isolated in 258 (81%) subjects with urethritis with discharge, and in 166 (53.5%) urethritis patients without discharge. There was a positive correlation between the significant number of PMN in Gram stained urethral smears and positive microbiological findings in men with urethritis both with and without urethral discharge (Spearmanns coefficients rho=0.986 and rho=0.993, respectively; P<0.01). CONCLUSIONS: The study found a relatively high prevalence of T. vaginalis among our men with urethritis irrespective of the presence or absence of urethral discharge, and showed that taking into account both discharge found on examination, and relevant PMN counts on Gram stained urethral smears fails to detect only 4.2% of oligosymptomatic urethritis patients who are infected with one of the strict urethral pathogens.

Pemphigus vulgaris and pemphigus foliaceus determined by CD86 and CTLA4 polymorphisms.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are rare autoimmune blistering diseases with presumed T-cell-dependent pathology. Activation of naïve T cells is dependent on antigen recognition, subsequent signaling through the T-cell receptor complex (signal 1), and various other interactions of T cells with antigen presenting cells that may be collectively designated as signal 2, which is unconditionally required for T-cell activation both in response to infection and to autoantigens. Among the best described interactions contributing to signal 2 are those mediated by B7 family molecules, such as CD80 and CD86 with their ligands; CD28, providing activation signals; and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), conferring inhibition. Single nucleotide polymorphisms (SNPs) within genes encoding those molecules may alter the signaling process. It is not known whether functional genetic polymorphisms within genes encoding the aforementioned proteins may increase risk for developing PV and PF and, if so, whether they might serve as biomarkers for susceptibility to these diseases. To address those questions, we examined functional single nucleotide polymorphisms within CD86 (rs1129055) and CTLA4 (rs733618 and rs5742909) genes in 61 pemphigus patients and 486 healthy controls. We found statistically significant differences in allele and genotype frequencies between PV patients and controls for rs1129055, as well as for rs5742909 among PV and PF patients. Namely, the rs1129055 A allele was significantly more common in PV patients compared with controls (35.4% versus 25.7%, respectively; P = .040), whereas the rs5742909 T allele was significantly more common in PF compared with PV patients (19.2% versus 5.2%, respectively; P = .035). The frequency of the rs5742909 T allele did not, however, differ significantly in PF or in PV compared with controls (10.5%; P = .187 and P = .100, respectively). We report a novel association of SNPs within CD86 and CTLA4 genes with pemphigus. The CD86 rs1129055 A allele appears to confer susceptibility to PV but not to PF. © 2016 Elsevier Inc. All rights reserved.

Correlation of Antibodies against Desmogleins 1 and 3 with Indirect Immunofluorescence and Disease Activity in 72 Patients with Pemphigus Vulgaris.

The enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) have both been used for testing of antibodies to desmogleins 1 and 3 (anti-Dsg1 and anti-Dsg3) and for the serologic diagnosis of pemphigus. IIF values and antibody concentrations and profile do not always correlate with a specific clinical phenotype and with the disease activity. The purpose of the present study was to correlate the clinical phenotype of patients with pemphigus vulgaris (PV) and the disease activity with anti-Dsg1 and anti-Dsg3 antibodies and IIF titers. A total of 72 patients with PV underwent ELISA serum testing for the presence and titers of anti-Dsg1 and anti-Dsg3 and IIF which were correlated with the severity of the disease (evaluated using the Pemphigus Disease Area Index, PDAI), clinical phenotype, and clinical course. In 79.2% patients there was a perfect correlation between the clinical phenotype and antibody profiles; in 20.8% patients, clinical features and antigenic findings were discordant. A statistically significant correlation was found between disease activity and a) anti-Dsg3 and anti-Dsg1 concentrations (Rho=0.679, P<0.001 and Rho=0.363, P=0.02, respectively) and b) IIF titers (Rho=0.426, P<0.01), as well between IIF titers and anti-Dsg3 and anti-Dsg1 antibodies (Rho=0.742, P<0.01 and Rho=0.372, P=0.02, respectively). This study supports the previous observations that the disease severity in most patients with pemphigus correlates with IIF titers, which in turn is determined by the quantities of Dsg1 and Dsg3 antibodies, as well as the previous observation that the clinical phenotype and antibody profile are not always in correlation.