Discovery and validation of novel biomarkers for detection of cervical cancer.

AIMS: To investigate novel biomarker for diagnosis of cervical cancer, we analyzed the datasets in Gene Expression Omnibus (GEO) and confirmed the candidate biomarker in patient sample. MATERIALS AND METHODS: We collected major datasets of cervical cancer in GEO, and analyzed the differential expression of normal and cancer samples online with GEO2R and tested the differences, then focus on the GSE63514 to screen the target genes in different histological grades by using the R-Bioconductor package and R-heatmap. Then human specimens from the cervix in different histological grades were used to confirm the top 8 genes expression by immunohistochemical staining using Ki67 as a standard control. RESULTS: We identified genes differentially expressed in normal and cervical cancer, 274 upregulated genes and 206 downregulated genes. After intersection with GSE63514, we found the obvious tendency in different histological grades. Then we screened the top 24 genes, and confirmed the top 8 genes in human cervix tissues. Immunohistochemical (IHC) results confirmed that keratin 17 (KRT17) was not expressed in normal cervical tissues and was over-expressed in cervical cancer. Cysteine-rich secretory protein-2 (CRISP2) was less expressed in high-grade squamous intraepithelial lesions (HSILs) than in other histological grades. CONCLUSION: For the good repeatability and consistency of KRT17 and CRISP2, they may be good candidate biomarkers. Combined analysis of KRT17, CRISP2 expression at both genetic and protein levels can determine different histological grades of cervical squamous cell carcinoma. Such combined analysis is capable of improving diagnostic accuracy of cervical cancer.

Correlation between salivary and serum anti-desmoglein 1 and 3 antibody titres using ELISA and between anti-desmoglein levels and disease severity in pemphigus vulgaris.

ELISA for anti-desmoglein antibodies (Dsg) is commonly used for diagnosis and assessment of treatment response in pemphigus vulgaris (PV). The present study was conducted to assess the relationship between salivary and serum Dsg1 and Dsg3 levels, and whether salivary Dsg1 and Dsg3 levels correlate with clinical disease severity of oral mucosal lesions in PV. In total 43, patients with PV with predominantly mucosal involvement were recruited. Both serum and salivary samples were collected from the cases, and salivary samples were also collected from five controls. There was a statistically significant correlation between serum and salivary Dsg1 levels and between serum and salivary Dsg3 levels. There was no correlation between serum or salivary Dsg1 and Dsg3 levels with the objective component of the oral mucosal Autoimmune Bullous Skin Disorder Intensity Score (ABSIS). Serum Dsg1 levels significantly correlated with cutaneous ABSIS, but there was no correlation between cutaneous ABSIS and either salivary Dsg1, salivary Dsg3 or serum Dsg3. As salivary Dsg titres correlate with serum levels, saliva can serve as a simple and noninvasive alternative to serum for Dsg ELISA.

Four cases of mucous membrane pemphigoid with clinical features of oral lichen planus.

BACKGROUND: Mucous membrane pemphigoid (MMP) and oral lichen planus (OLP) show similar clinical features on the oral mucosa. As clinical manifestations of oral mucosal lesions, MMP shows blisters and erosions, whereas OLP shows lace-like whitish lesions in an annular arrangement with erythema and erosions. Histopathologically, MMP shows subepithelial bullae with infiltrates of lymphocytes and neutrophils, whereas OLP shows band-like interface infiltration of lymphocytes with damage in basal cells. However, these two diseases are frequently difficult to distinguish both clinically and histopathologically. OBJECTIVES: We report four patients with oral MMP who showed OLP-like clinical and histopathological lesions. METHODS: We performed direct immunofluorescence, indirect immunofluorescence of normal human skin and 1 m NaCl-split skin, enzyme-linked immunosorbent assays for BP180, BP230, and desmogleins 1 and 3, and immunoblotting of normal human epidermal and dermal extracts, recombinant proteins of BP180-NC16a and -C-terminal domains, concentrated culture supernatant of HaCaT cells, and purified laminin-332. RESULTS: The results of various immunological studies suggested the diagnoses of various types of MMP for all four patients. CONCLUSIONS: Because MMP and OLP require different treatments, all dentists and dermatologists should have knowledge about the disease entity and the serological diagnostic methods for various types of MMP.

Ichthyosis Linearis Circumflexa as the Only Clinical Manifestation of Netherton Syndrome.

Ichthyosis linearis circumflexa (ILC) presents as serpiginous and migratory erythematous patches with double-edged scales. ILC is rarely an isolated skin manifestation, but most commonly a part of Netherton syndrome (NS). NS is caused by SPINK5 mutations, which lead to absent or sometimes reduced expression of the serine protease inhibitor LEKTI. NS is characterised by congenital ichthyosiform erytroderma, trichorrhexis invaginata (TI) and atopy. We report 2 children who presented since the first months of life cheek erythema followed by the appearance of sparse ILC lesions on the face, trunk and proximal extremities. Erythroderma at birth, TI and atopy were absent. LEKTI immunoreactivity was reduced in patient epidermis, and serine protease activity was modestly increased, while desmoglein-1 expression remained unaffected. SPINK5 mutation and expression analysis in patient keratinocytes revealed compound heterozygous splicing variants, which allowed residual LEKTI secretion. Our results show that ILC can be the only clinical manifestation of NS.

The value of trichoscopy in the differential diagnosis of scalp lesions in pemphigus vulgaris and pemphigus foliaceus

BACKGROUND: Trichoscopy is becoming increasingly popular in diagnosing hair and scalp diseases. Scalp involvement in pemphigus is common. The scalp may be the first or only site of clinical manifestation of the disease. OBJECTIVE: The aim of this study was to analyze whether trichoscopy may be useful in aiding differential diagnosis of scalp lesions in patients with pemphigus vulgaris and pemphigus foliaceus. METHODS: Trichoscopy was performed in 19 patients with scalp lesions in the course of pemphigus (9 patients with pemphigus vulgaris and 10 with pemphigus foliaceus). In all patients, the diagnosis of scalp pemphigus was confirmed by histopathology. The working magnification was 20-fold and 70-fold. RESULTS: The most frequently observed trichoscopy features of pemphigus lesions were: extravasations (18/19; 94.7%) and yellow hemorrhagic crusts (11/19; 57.9%). Yellow dots with whitish halo were observed in 6/19 (31.6%) patients with pemphigus. White polygonal structures were observed in pemphigus foliaceus (6/10; 60%), but not in pemphigus vulgaris. Vascular abnormalities were more frequent in pemphigus vulgaris, when compared to pemphigus foliaceus, and were associated with a severe course of disease. Linear serpentine vessels were the most frequent vascular abnormality in patients with pemphigus vulgaris and pemphigus foliaceus (77.8% and 30%, respectively). CONCLUSION: Trichoscopy may serve as a useful supplementary method in the differential diagnosis of pemphigus, especially in cases of desquamative or exudative lesions limited to the scalp. Extravasations, yellow hemorrhagic crusts, yellow dots with whitish halo, white polygonal structures and linear serpentine vessels are trichoscopy features which may suggest the diagnosis of pemphigus. .

The value of trichoscopy in the differential diagnosis of scalp lesions in pemphigus vulgaris and pemphigus foliaceus.

BACKGROUND: Trichoscopy is becoming increasingly popular in diagnosing hair and scalp diseases. Scalp involvement in pemphigus is common. The scalp may be the first or only site of clinical manifestation of the disease. OBJECTIVE: The aim of this study was to analyze whether trichoscopy may be useful in aiding differential diagnosis of scalp lesions in patients with pemphigus vulgaris and pemphigus foliaceus. METHODS: Trichoscopy was performed in 19 patients with scalp lesions in the course of pemphigus (9 patients with pemphigus vulgaris and 10 with pemphigus foliaceus). In all patients, the diagnosis of scalp pemphigus was confirmed by histopathology. The working magnification was 20-fold and 70-fold. RESULTS: The most frequently observed trichoscopy features of pemphigus lesions were: extravasations (18/19; 94.7%) and yellow hemorrhagic crusts (11/19; 57.9%). Yellow dots with whitish halo were observed in 6/19 (31.6%) patients with pemphigus. White polygonal structures were observed in pemphigus foliaceus (6/10; 60%), but not in pemphigus vulgaris. Vascular abnormalities were more frequent in pemphigus vulgaris, when compared to pemphigus foliaceus, and were associated with a severe course of disease. Linear serpentine vessels were the most frequent vascular abnormality in patients with pemphigus vulgaris and pemphigus foliaceus (77.8% and 30%, respectively). CONCLUSION: Trichoscopy may serve as a useful supplementary method in the differential diagnosis of pemphigus, especially in cases of desquamative or exudative lesions limited to the scalp. Extravasations, yellow hemorrhagic crusts, yellow dots with whitish halo, white polygonal structures and linear serpentine vessels are trichoscopy features which may suggest the diagnosis of pemphigus.

The value of anti-desmoglein enzyme-linked immunosorbent assay in the immunological follow-up of pemphigus.

BACKGROUND: The value of anti-desmoglein 1 and 3 (Dsg1, Dsg3) enzyme-linked immunosorbent assay (ELISA) is controversial in the follow-up of pemphigus. OBJECTIVE: To evaluate anti-desmoglein ELISA (Dsg ELISA) in the follow-up of pemphigus and compare ELISA with direct and indirect immunofluorescence in complete remission (CR). METHODS: We performed a retrospective monocenter study of patients with pemphigus and consecutive sera samples collected at baseline (M0), 12 months (M12) and 24 or 36 months after M0 (M24/36). Tests were compared in CR and in active disease. Direct immunofluorescence and circulating autoantibodies were compared for patients with stable CR. RESULTS: We included 36 patients. At M12, ELISA values did not differ between CR and active disease. At M24/36, Dsg3 but not Dsg1 ELISA values were lower in CR (p = 0.07). For 5/8 patients with stable CR, direct immunofluorescence and ELISA findings remained positive. CONCLUSION: In routine practice, Dsg ELISA seems to be of little interest for immunological follow-up of pemphigus.

[D-penicillamine-induced pemphigus: changes in anti-32-2B immunostaining patterns]. / Autonomisation d'un pemphigus induit par la D-pénicillamine: évolution de l'immunomarquage par l'anticorps anti-32-2B.

BACKGROUND: It has been reported that D-penicillamine causes pemphigus that is typically superficial. Immunostaining with monoclonal anti-32-2B antibody targeting desmoglein 1 and 3 can help differentiate between drug-induced and classical auto-immune pemphigus. Absence of specific staining militates in favour of drug-induced pemphigus whilst positive staining suggests an auto-immune aetiology that is ongoing despite discontinuation of drug therapy. PATIENTS AND METHODS: A 59-year-old male patient was referred for management of superficial pemphigus 1 year after starting D-penicillamine treatment for scleroderma. The diagnosis of pemphigus was confirmed histologically (intra-epidermal cleavage, acantholysis and perikeratinocytes, deposition of IgG and complement C3). Immunochemical staining with anti-32-2B antibody was initially normal, in keeping with drug-induced pemphigus. Despite discontinuation of D-penicillamine, pemphigus recurred in 2008. A further skin biopsy was undertaken and anti-32-2B staining was abnormal, which is consistent with auto-immune pemphigus. DISCUSSION: Numerous cases of drug-induced pemphigus have been described in the literature. In approximately half of all cases, the pemphigus recedes after cessation of the causative drug. However, there have been no previous reports that changes over time in the immunostaining with anti-32-2B antibodies can mirror a change in form of pemphigus from a drug-induced type to an idiopathic type as well as the associated clinical feature of persistence after drug withdrawal. CONCLUSION: Normal staining with anti-32-2B antibody is associated with a favourable prognosis as regards resolution of drug-induced pemphigus. When, as in this case, status changes to abnormal staining, there is a risk that the pemphigus may become chronic despite discontinuation of therapy.

Assessment of desmosomal components (desmoglein 1-3, plakoglobin) in cardia mucosa in relation to gastroesophageal reflux disease and Helicobacter pylori infection.

Gastroesophageal reflux disease is associated with impaired epithelial barrier function and abnormal expression of proteins forming cell-cell contacts by tight junctions and desmosomes in distal esophageal squamous mucosa. Although gastroesophageal reflux disease and Helicobacter pylori are both associated with chronic inflammation of the adjacent cardia mucosa, it is not known whether these lead to derangements of the desmosomal complexes. Here, we assessed the expression of 4 proteins (plakoglobin and desmoglein 1, 2, and 3) forming epithelial desmosomal complexes by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in biopsies from 67 patients with gastroesophageal reflux disease and 23 gastroesophageal reflux disease-negative controls. Plakoglobin and desmoglein 2 were ubiquitously expressed in all samples, whereas desmoglein 1 and 3 were not expressed in cardia mucosa. Gastroesophageal reflux disease was specifically associated with elevated transcript levels of desmoglein 2 and plakoglobin. These were significantly increased from 2.0- to 2.7-fold in patients with gastroesophageal reflux disease compared with controls (P < .01), and significantly increased immunohistochemical scores for both proteins were observed (P < .05) as well. The combined presence of gastroesophageal reflux disease and Helicobacter pylori infection had no additional effect on desmosomal gene expression. Taken together, the up-regulation of plakoglobin and desmoglein 2 in cardia mucosa of patients with gastroesophageal reflux disease supports the concept that the "transition zone" between distal esophagus and proximal stomach is affected by gastroesophageal reflux disease as well, and architectural and molecular changes in the desmosomal compartment contribute to the pathogenesis of gastroesophageal reflux disease in the cardia mucosa.

Tetracycline and niacinamide control bullous pemphigoid but not pemphigus foliaceus when these conditions coexist.

Pemphigus and pemphigoid are different types of autoimmune bullous disease and can occur in the same patient. We report a female patient with this condition. At first, we diagnosed her with bullous pemphigoid, and we treated her with tetracycline, niacinamide and a topical steroid. Tense bullas disappeared shortly after that, but crusted erythemas mainly on her head and trunk persisted. We examined BP180 and desmoglein 1 enzyme-linked immunosorbent assays, and also histological features, which showed coexistence of bullous pemphigoid and pemphigus foliaceus concurrently. Therefore, we tried prednisolone, which could control both conditions. This case showed that tetracycline and niacinamide could control bullous pemphigoid, but could not control pemphigus foliaceus, and that prednisolone was effective for both conditions.

Nikolsky's sign on the gingival mucosa: a clinical tool for oral health practitioners.

BACKGROUND: Nikolsky's sign is a clinical sign which is elicited by a horizontal, tangential pressure to the mucosa and/or skin resulting in blisters extending and separating or peeling away. Few data are currently available in the literature about its usefulness, specificity, and sensitivity in the diagnosis of either oropharyngeal or cutaneous bullous diseases. The purpose of this study was to determine the sensitivity and specificity of the gingival Nikolsky's sign in the identification of an autoimmune blistering disease. METHODS: Over a period of 13 years, we recruited 566 patients with autoimmune oral bullous and non-bullous diseases who possessed either maxillary or mandibular gingival mucosal lesions. All patients were subjected to a test causing a gingival Nikolsky's sign at their first visit during the diagnostic algorithm and in the active disease phase before commencing treatment. RESULTS: A total of 566 patients (184 with and 382 without bullous lesions) had at least gingival involvement. A positive gingival Nikolsky's sign resulted in 100 (17.7%) of 566 patients: 86 patients with bullous lesions (53 with pemphigus vulgaris, eight with mucous membrane pemphigoid, 22 with bullous/mixed lichenoid lesions, and three with erythema multiforme) and 14 with non-bullous lesions (12 with non-bullous lichenoid lesions and two with systemic lupus erythematous/mixed connective tissue disease). Thus, the specificity of Nikolsky's sign was higher (96.3%) than the sensitivity (46.7%). CONCLUSION: The results of this study support the use of Nikolsky's sign of the gingival mucosa as a viable test to establish the presence of oral bullous diseases.

P120-catenin is a novel desmoglein 3 interacting partner: identification of the p120-catenin association site of desmoglein 3.

P120-catenin (p120ctn) is an armadillo-repeat protein that directly binds to the intracytoplasmic domains of classical cadherins. p120ctn binding promotes the stabilization of cadherin complexes on the plasma membrane and thus positively regulates the adhesive activity of cadherins. Using co-immunoprecipitation, we show here that p120ctn associates to desmogleins (Dsg) 1 and 3. To determine which region is involved in the association between Dsg3 and p120ctn, we constructed mutant Dsg3 proteins, in which various cytoplasmic subdomains were removed. The tailless Dsg3 constructs Delta IA:AA1-641Dsg3 and Delta 641-714Dsg3, which do not contain the intracellular anchor (IA) region, did not coprecipitate with p120cn, nor did they colocalize at the plasma membrane. Immunocytochemical analysis revealed that p120ctn does not localize to desmosomes, but colocalizes with Dsg3 at the cell surface. A biotinylation assay for Dsg3 showed that biotinylated Delta 641-714Dsg3 was turned over more rapidly than wild-type Dsg3. These results indicate that the membrane proximal region (corresponding to residues 641-714) in the IA region of Dsg3 is necessary for complex formation with p120ctn, and to maintain free Dsg3 at the cell surface before it is integrated into desmosomes. In summary, we show that p120ctn is a novel interactor of the Dsg proteins, and may play a role in desmosome remodeling.

Isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantibodies.

Pemphigus foliaceus (PF) is a blistering disease caused by autoantibodies to desmoglein 1 (Dsg1) that cause loss of epidermal cell adhesion. To better understand PF pathophysiology, we used phage display to isolate anti-Dsg1 mAbs as single-chain variable fragments (scFvs) from a PF patient. Initial panning of the library isolated only non-pathogenic scFvs. We then used these scFvs to block non-pathogenic epitopes and were able to isolate two unique scFvs, each of which caused typical PF blisters in mice or human epidermis models, showing that a single mAb can disrupt Dsg1 function to cause disease. Both pathogenic scFvs bound conformational epitopes in the N terminus of Dsg1. Other PF sera showed a major antibody response against the same or nearby epitopes defined by these pathogenic scFvs. Finally, we showed restriction of the heavy-chain gene usage of all anti-Dsg1 clones to only five genes, which determined their immunological properties despite promiscuous light-chain gene usage. These mAbs will be useful for studying Dsg1 function and mechanisms of blister formation in PF and for developing targeted therapies and tools to monitor disease activity.

Paraneoplastic pemphigus mimicking erosive mucosal lichen planus associated with primary hepatocellular carcinoma.

A 58-year-old Japanese male visited us with painful lesions on the lower lip, oral mucosa and genital region of an 8-month duration. Histological features of the genital lesion were almost consistent with lichenoid tissue reaction. A few intraepidermal acantholytic keratinocytes were also seen in the suprabasal clefts. Direct immunofluorescence exhibited cell surface immunoglobulin (Ig)G deposition and linear deposition of fibrinogen at the dermoepidermal junction. IgG anti-desmoglein (Dsg)3 antibody, but not anti-Dsg1 antibody, was detected in the patient's serum by enzyme-linked immunosorbent assay. Immunoblotting using normal human epidermal extract detected the 210-kD envoplakin, 190-kD periplakin and 130-kD Dsg3. The diagnosis of paraneoplastic pemphigus (PNP) was made. Subsequent investigation revealed a large space-occupying lesion in the liver. Histological findings from liver biopsy specimen were consistent with hepatocellular carcinoma. The patient has been alive 38 months after the diagnosis of PNP was made, although the liver mass has slowly enlarged. Our case is clinically and histologically similar to erosive mucosal lichen planus. Immunological studies confirmed the diagnosis of PNP. The results of negative Dsg1 and positive Dsg3 were consistent with clinical features showing severe mucosal involvement without cutaneous erosion. In PNP, the association with non-hematological solid tumor is extremely rare.

Desmocollin 1 and desmoglein 1 expression in human epidermis and keratinizing oral mucosa: a comparative immunohistochemical and molecular study.

Epidermis and keratinizing oral mucosa (KOM) are effective barriers against a wide spectrum of insults. The optimal form of protection provided by each epithelium is determined also by the molecular composition of desmosomes. Up to now, the expression of the "skin type" desmosomal cadherins, i.e. desmocollin 1 (Dsc1) and desmoglein 1 (Dsg1), was correlated with the morphological features of keratinocyte terminal differentiation in epidermis, but not in KOM. The aim of the present study was to investigate Dsc1 and Dsg1 expression in adult human KOM compared to epidermis. Biopsies of epidermis and KOM were obtained from young healthy adults (n=6) and simultaneously processed for immunofluorescence analysis, post-embedding immunogold electron microscopy (immunogold EM), and RT-PCR analysis. For molecular biology analysis, as a negative control, we considered human fibroblasts. By immunofluorescence and immunogold EM, Dsc1 labeling was not detected in any suprabasal layer of KOM, but it was present in the upper spinous/granular layers of epidermis. Immunofluorescence and transmission electron microscopy analysis showed that (i) Dsg1 expression was evident in the spinous, granular, and horny layer of the oral epithelium and (ii) Dsg1 immunoreactivity was always lower in desmosomes between oral keratinocytes than in all epidermal junctions. RT-PCR analysis confirmed that in KOM Dsc1 gene expression was undetectable. On the whole, these observations suggest a weakened adhesion in KOM, allowing oral keratinocytes to undergo a faster transition throughout the living layers of the epithelium. The intrinsic and specific regulation of the molecular composition of desmosomes can contribute in defining a specific keratinocyte phenotype in KOM and in epidermis.