Lesional Skin Biopsy Specimens Research Articles

Profibrotic Subsets of SPP1+ Macrophages and POSTN+ Fibroblasts Contribute to Fibrotic Scarring in Acne Keloidalis

Acne keloidalis is a primary scarring alopecia characterized by longstanding inflammation in the scalp causing keloid-like scar formation and hair loss. Histologically, acne keloidalis is characterized by mixed leukocytic infiltrates in the acute stage followed by a granulomatous reaction and extensive fibrosis in the later stages. To further explore its pathogenesis, bulk RNA sequencing, single-cell RNA sequencing, and spatial transcriptomics were applied to occipital scalp biopsy specimens of lesional and adjacent no-lesional skin in patients with clinically active disease. Unbiased clustering revealed 19 distinct cell populations, including 2 notable populations: POSTN+ fibroblasts with enriched extracellular matrix signatures and SPP1+ myeloid cells with an M2 macrophage phenotype. Cell communication analyses indicated that fibroblasts and myeloid cells communicated by SPP1 signaling networks in lesional skin. A reverse transcriptomics in silico approach identified corticosteroids as possessing the capability to reverse the gene expression signatures of SPP1+ myeloid cells and POSTN+ fibroblasts. Intralesional corticosteroid injection greatly reduced SPP1 and POSTN gene expression as well as acne keloidalis disease activity. Spatial transcriptomics and immunofluorescence staining verified microanatomic specificity of SPP1+ myeloid cells and POSTN+ fibroblasts with disease activity. In summary, the communication between POSTN+ fibroblasts and SPP1+ myeloid cells by SPP1 axis may contribute to the pathogenesis of acne keloidalis.

The neglected twin: Nummular eczema is a variant of atopic dermatitis with codominant TH2/TH17 immune response

Nummular eczema (NE) is a common chronic inflammatory skin disease characterized by multiple, pruritic, discoid-shaped lesions. Since the underlying immune mechanisms are not fully understood, it is unclear whether NE should be regarded as variant of atopic dermatitis (AD) or a distinct disease. We compared the clinical, histopathologic, and molecular signatures of NE with that of type 2 and type 3 skin diseases. We performed bulk RNA sequencing as well as histologic and clinical studies in lesional and nonlesional skin biopsy specimens from NE (n= 50), AD (n= 47), and psoriasis (n= 90) patients. NE displayed typical hallmarks of AD, such as an impaired epidermal barrier, microbial colonization, spongiosis, and eosinophil infiltration, but also aspects of psoriasis, including increased epidermal thickness, number of Ki-67+ cells, and neutrophilic infiltration. At the gene expression level, neutrophil-attracting cytokines (IL19, CXCL8, CXCL5) were upregulated, whereas TH2-related cytokines (IL13, CCL17, CCL18, CCL26, CCL27) were similarly expressed in NE compared to AD. Principal component analysis of transcriptome data from lesional skin showed that AD and NE cluster together distinct of psoriasis. In line with this, an established molecular classifier identified NE as AD rather than psoriasis. Finally, we demonstrated clinical and molecular efficacy of dupilumab treatment in NE. NE shows overlapping type 2 and type 3 immune signatures, while type 2 immunity predominates and should be the primary target of specific therapeutic interventions. This supports the view of NE as a variant of AD.

Mycobacterium leprae and host immune transcriptomic signatures for reactional states in leprosy.

Mycobacterium leprae transcriptomic and human host immune gene expression signatures that demonstrate a plausible association with type I (T1R) and type II reactions (T2R) aid in early diagnosis, prevention of nerve damage and consequent demyelinating neuropathy in leprosy. The aim of the study is to identify M. leprae and host-associated gene-expression signatures that are associated with reactional states in leprosy. The differentially expressed genes from the whole transcriptome of M. leprae were determined using genome-wide hybridization arrays with RNA extracted from skin biopsies of 20 T1R, 20 T2R and 20 non reactional controls (NR). Additionally, human immune gene-expressions were profiled using RT2-PCR profiler arrays and real-time qPCRs. The RNA quality was optimal in 16 NR, 18 T1R and 19 T2R samples. Whole transcriptome expression array of these samples revealed significant upregulation of the genes that encode integral and intrinsic membrane proteins, hydrolases and oxidoreductases. In T1R lesional skin biopsy specimens, the top 10 significantly upregulated genes are ML2064, ML1271, ML1960, ML1220, ML2498, ML1996, ML2388, ML0429, ML2030 and ML0224 in comparison to NR. In T2R, genes ML2498, ML1526, ML0394, ML1960, ML2388, ML0429, ML0281, ML1847, ML1618 and ML1271 were significantly upregulated. We noted ML2664 was significantly upregulated in T1R and repressed in T2R. Conversely, we have not noted any genes upregulated in T2R and repressed in T1R. In both T1R and T2R, ML2388 was significantly upregulated. This gene encodes a probable membrane protein and epitope prediction using Bepipred-2.0 revealed a distinct B-cell epitope. Overexpression of ML2388 was noted consistently across the reaction samples. From the host immune gene expression profiles, genes for CXCL9, CXCL10, CXCL2, CD40LG, IL17A and CXCL11 were upregulated in T1R when compared to the NR. In T2R, CXCL10, CXCL11, CXCL9, CXCL2 and CD40LG were upregulated when compared to the NR group. A gene set signature involving bacterial genes ML2388, ML2664, and host immune genes CXCL10 and IL-17A can be transcriptomic markers for reactional states in leprosy.

Treatment of Discoid Lupus Erythematosus in a Dog with Human Intravenous Immunoglobulin

Background: Discoid lupus erythematosus (DLE) is a common canine autoimmune skin disease, in which systemic manifestations are absent. Skin Lesions are usually present on the nasal planum, and characterised by erythema, depigmentation, erosion, ulceration, and crusting. The diagnosis is based on histopathological results, which should demonstrate lymphoplasmacytic lichenoid-interface dermatitis. Human intravenous immunoglobulin (hIVIg) has been used in veterinary medicine to treat cutaneous diseases including erythema multiforme, PF, and severe adverse cutaneous drug reactions. In human medicine, it has been effective to treat DLE. This report firstly describes the clinical response to hIVIg in a dog with DLE resistant to common immunosuppressive drugs.Case: A 5-year-old, intact female Shih Tzu presented with a 1-month history of slowly progressive black crusting on the nasal planum, chin, and claw. Based on the results of a dermatologic examination, superficial pyoderma was diagnosed. The skin lesions did not improve during and after anti-infective treatment. After removing the crusts, a skin biopsy was obtained from the muzzle. Histopathology of lesional skin biopsy specimens revealed lymphoplasmacytic interface dermatitis at the dermoepidermal junction. Microscopic examination also revealed vacuolar changes and pigmentary incontinence of the basal layer as a lichenoid tissue reaction. No mites or fungi were detected on the skin section. The absence of acantholytic cells excluded pemphigus foliaceus, which is also characterised by the lesions of the nasal planum. Based on the distribution of the lesions, histopathology and exclusion of other dermatoses, the dog was diagnosed with DLE. The skin lesions temporarily improved after treatment with prednisolone (2 mg/kg PO q12h). However, after tapering the dose of prednisolone, new black crusts developed on the nasal planum and claw. Although the dog was successively treated with other immunosuppressive drugs, including azathioprine, cyclosporin with dexamethasone, and mycophenolate mofetil, black crusts still remained. Due to the low efficacy of these immunosuppressive drugs, hIVIg was administered at 0.5 g/kg once daily for 4 days, for a total dose of 2 g/kg. During hIVIg administration, the crusted lesions gradually improved. After the hIVIg administration, the dog was treated with prednisolone (1 mg/kg PO q12h). The lesions were almost in complete remission at 21 days after an additional application of prednisolone. The skin lesions did not recur, and the treatment was eventually discontinued after 6 weeks of additional prednisolone application.Discussion: The standard treatment of canine DLE includes glucocorticoids, and second-line immunosuppressive drugs, such as azathioprine and cyclosporine, are usually added in cases resistant to steroids. This case suggests that hIVIg may be beneficial as an adjunctive treatment option for canine DLE, especially when the application of standard immunosuppressive drugs is limited due to adverse effects or low efficacy. There is evidence from several studies that the steroid-sparing effect of hIVIg is significant in human patients. In the current case, the effective dose of prednisolone was reduced to 2 mg/kg/day after hIVIg administration, and prednisolone therapy was finally discontinued completely. The hIVIg appears to lower the daily steroid dose requirement in this dog. Keywords: autoimmune skin disease, discoid lupus erythematosus, canine dermatology, immunosuppressive drug, human intravenous immunoglobulin.

Identification of a TH 2-high psoriasis cluster based on skin biomarker analysis in a Chinese psoriasis population.

Psoriasis is an immune-mediated, chronic inflammatory disease with diverse phenotypes. However, its biological diversity has not been well-characterized in Chinese psoriasis population. To characterize psoriasis biological heterogenicity using gene expression profiles of lesional skin biopsy specimens in a Chinese psoriasis population. Lesional tissues and blood samples from Chinese psoriasis patients (n=40), atopic dermatitis (AD) patients (n=25) and age-matched healthy controls (n=19) were investigated by using real-time PCR array, histological evaluation and flow cytometry. Unsupervised hierarchical clustering was performed using gene expression profiles of patients with psoriasis. Two distinct psoriasis clusters were identified. Both clusters indicated high TH 17 activation. One cluster (n=6 of 40 consecutive psoriasis patients) indicated a strong TH 2 component in skin lesions, with early onset and low peripheral blood eosinophil level. Significantly higher IL-4, IL-13, IL-25, IL-31 and TSLP gene induction typified this cluster of psoriasis patients, even compared with AD patients. Both psoriasis clusters were characterized by neutrophilic microabscess formation. Histologically, the TH 2 high psoriasis cluster indicated a low percentage of perivascular eosinophils. Two distinct psoriasis clusters were identified. One presented early onset and a low eosinophil level, indicating TH 17 polarization and a strong TH 2 component. These results laid the foundation for further demonstrating the pathogenesis of psoriasis in Chinese population.

Elevated level of B-cell-activating factor in tissues and serum of patients with vitiligo: a proof of autoimmune theory

Background Several studies strongly supported the involvement of autoimmunity in vitiligo pathogenesis, which proposed the alteration of both humoral and cellular immunity leading to the destruction of melanocytes. B-lymphocyte-activating factor (BAFF) is a member of tumor necrosis factor family that has an important role in peripheral B-cell survival and homeostasis. The increased production of BAFF may lead to autoimmunity. Objective To assess BAFF levels in both serum and tissue in patients with vitiligo to detect its possible role in the disease. Patients and methods The present case–control study included 30 patients with vitiligo and 30 age-matched and sex-matched healthy controls. Overall, 5 ml of blood sample was retrieved, and lesional skin biopsy specimen was obtained from the margin of lesional skin from each patient and site-matched control for estimation of BAFF levels by ELISA. Results A higher level of BAFF was detected in tissues and sera of patients with vitiligo (8.3±5.0 and 1.9±1.0, respectively) compared with healthy controls (1.0±0.3 and 1.1±0.5 with P<0.001 and <0.005, respectively). Significant correlation was found between BAFF levels in either tissue or serum with duration of the disease (r=0.9, P<0.001 and r=0.7, P<0.001, respectively). Moreover, significant correlation was found between BAFF in tissue and BAFF in serum (r=0.7 and P<0.001). Conclusion The present study provides further evidence on the role of BAFF in the pathogenesis of vitiligo.

Minimally invasive skin tape strip RNA sequencing identifies novel characteristics of the type 2–high atopic dermatitis disease endotype

Expression profiling of skin biopsy specimens has established molecular features of the skin in patients with atopic dermatitis (AD). The invasiveness of biopsies has prevented their use in defining individual-level AD pathobiological mechanisms (endotypes) in large research studies. We sought to determine whether minimally invasive skin tape strip transcriptome analysis identifies gene expression dysregulation in AD and molecular disease endotypes. We sampled nonlesional and lesional skin tape strips and biopsy specimens from white adult patients with AD (18 male and 12 female patients; age [mean±SE], 36.3±2.2years) and healthy control subjects (9 male and 16 female subjects; age [mean±SE], 34.8±2.2years). AmpliSeq whole-transcriptome sequencing was performed on extracted RNA. Differential expression, clustering/pathway analyses, immunostaining of skin biopsy specimens, and clinical trait correlations were performed. Skin tape expression profiles were distinct from skin biopsy profiles and better sampled epidermal differentiation complex genes. Skin tape expression of 29 immune and epidermis-related genes (false discovery rate<5%) separated patients with AD from healthy subjects. Agnostic gene set analyses and clustering revealed 50% of patients with AD exhibited a type 2 inflammatory signature (type 2-high endotype) characterized by differential expression of 656 genes, including overexpression of IL13, IL4R, CCL22, CCR4 (log2 fold change=5.5, 2.0, 4.0, and 4.1, respectively) and at a pathway level by TH2/dendritic cell activation. Both expression and immunostaining of skin biopsy specimens indicated this type 2-high group was enriched for inflammatory, type 2-skewed dendritic cells expressing FcεRI. The type 2-high endotype group exhibited more severe disease by using both the Eczema Area and Severity Index score and body surface area covered by lesions. Minimally invasive expression profiling of nonlesional skin reveals stratification in AD molecular pathology by type 2 inflammation that correlates with disease severity.

Condylomata lata on the ankle: an unusual location.

A 43-year-old Japanese man presented with reddish nodules on the ankle. The nodules had a yellowish crust and eroded surface. Dermoscopy revealed red to milky-red globules at the periphery and some glomerular vessels in the center and a whitish-pink network, which corresponded to capillary dilatation in the papillary dermis and prominent acanthosis, respectively. These structures were surrounded by a yellowish peripheral structureless area and multiple white, small, round structures in the center, corresponding to the macerated horny layer and keratin plugs. Blood samples were positive for rapid plasma reagin (1:64), Treponema pallidum hemagglutination assay (1:20480), and fluorescent treponemal antibody-absorption (1:1280). A lesional skin biopsy specimen showed irregular acanthosis and papillomatosis. The Warthin-Starry and anti-Treponema pallidum antibody stains on the biopsy specimen revealed many spirochetes in the lower epidermis and the papillary dermis. A diagnosis of secondary syphilis with condylomata lata was made. After one week of treatment with oral benzylpenicillin benzathine hydrate (Bicillin® G granules 400,000 units; Banyu Pharmaceutical Co., Ltd, Tokyo, Japan), 1.6 million units (U) daily, the ankle lesions had resolved with a small ulcer and pigmentation. Although syphilis is a relatively common disease, this case study reports an unusual presentation as well as dermoscopy findings.

Guselkumab (an IL-23–specific mAb) demonstrates clinical and molecular response in patients with moderate-to-severe psoriasis

IL-23 expression is increased in psoriatic lesions and might regulate TH17 T-cell counts in patients with psoriasis. We sought to test a novel IL-23-specific therapeutic agent for the treatment of psoriasis. In this randomized, double-blind, placebo-controlled study the safety, tolerability, and clinical response of guselkumab, an anti-IL-23-specific mAb, were evaluated in patients with moderate-to-severe plaque psoriasis. A total of 24 patients were randomized to receive a single dose of placebo or 10, 30, 100, or 300 mg of guselkumab. Clinical response was assessed by using the Psoriasis Area and Severity Index (PASI). Additionally, histologic analysis and gene expression in skin biopsy specimens from guselkumab-treated patients were compared with those from placebo-treated patients. At week 12, 50% (10 mg), 60% (30 and 100 mg), and 100% (300 mg) of guselkumab-treated patients, respectively, achieved a 75% improvement in PASI scores from baseline compared with 0% of placebo-treated patients. Improvements in PASI scores were generally maintained through week 24 in all guselkumab-treated patients. The proportion of patients experiencing an adverse event was comparable between the combined guselkumab (13/20 [65.0%]) and placebo (2/4 [50.0%]) groups through week 24. Analysis of lesional and nonlesional skin biopsy specimens demonstrated decreases in epidermal thickness and T-cell and dendritic cell expression in guselkumab-treated patients compared with values seen in placebo-treated patients. At week 12, significant reductions in psoriasis gene expression and serum IL-17A levels were observed in guselkumab-treated patients. IL-23 inhibition with a single dose of guselkumab results in clinical responses in patients with moderate-to-severe psoriasis, suggesting that neutralization of IL-23 alone is a promising therapy for psoriasis.

Expression of IFN‐γ, IL‐4, and IL‐17 in cutaneous schistosomal granuloma

Cutaneous schistosomal granuloma (CSG) is a rare dermatological disease, the clinical and histopathological features of which are well defined. Although a panoramic picture of its immunopathogenesis in humans is not yet available, it is believed to be induced by T helper1 (Th1), Th2, or Th17 cytokines in animals. This study evaluated the expression of different types of Th cytokines, including Th1 cytokine interferon-γ (IFN-γ), Th2 cytokine interleukin-4 (IL-4), and Th17 cytokine IL-17, in human CSG. This study included nine patients with CSG. Dermatological examinations were conducted in all subjects. Skin biopsy specimens were stained with hematoxylin and eosin (H&E). Immunohistochemical examination was performed using three monoclonal anti-human antibodies against IFN-γ, IL-4, and IL-17 to evaluate Th1, Th2, and Th17 cytokines, respectively. The most common site of CSG manifestation was the paraumbilical area, which was affected in 66.7% of patients. All lesional skin biopsy specimens revealed multiple dermal granulomas surrounding schistosomal eggs. Positive immunoreactivity for IFN-γ, IL-4, and IL-17 was present in dermal inflammatory infiltrate in 88.9, 11.1, and 88.9% of subjects, respectively. There were statistically significant negative correlations between the duration of disease and both IFN-γ and IL-17 (P≤0.05), and a statistically significant positive correlation between IFN-γ and IL-17 (P≤0.05). This study suggests that CSG is formed by the action of both Th1 (IFN-γ) and Th17 (IL-17) cytokines, which have been shown to be directed against the schistosomal egg to induce a cell-mediated immune response.

Overexpression of interleukin-23 and interleukin-17 in the lesion of pemphigus vulgaris: a preliminary study.

IL-23/IL-17 axis has been identified as major factor involved in the pathogenesis of several autoimmune diseases; yet its pathogenetic role in pemphigus vulgaris (PV) remains controversial. The aim of this research was to investigate the potential role of IL-23/IL-17 axis in the immunopathogenesis of PV, and correlation between IL-23+ cells and IL-17+ cells was also evaluated. For this purpose, ten patients with PV, three patients with pemphigus foliaceus (PF), and six healthy individuals were allocated to this research. The lesional skin biopsy specimens were obtained before treatment. Then immunofluorescence staining was performed to analyze the expression of IL-23 and IL-17 in the PV/PF patients and the healthy individuals. The results showed that the numbers of IL-23+ and IL-17+ cells were significantly higher in PV lesions, compared to PF lesions and normal control skins, respectively (all P < 0.05). Moreover, the correlation between IL-23+ cells and IL-17+ cells was significant (r = 0.7546; P < 0.05). Taken together, our results provided evidence that the IL-23/IL-17 axis may play a crucial role in the immunopathogenesis of PV and may serve as novel therapeutic target for PV.

Altered expression of antimicrobial peptide genes in the skin of dogs with atopic dermatitis and other inflammatory skin conditions

Reports indicate that human and canine patients with atopic dermatitis (AD) have reduced production of several skin antimicrobial peptides, but more recent data have called those results into question. To compare the mRNA expression of seven antimicrobial peptide genes in lesional and adjacent nonlesional skin biopsy specimens from dogs with AD with those from normal dogs and from dogs experiencing other inflammatory skin conditions. Normal dogs and patients with AD or other inflammatory skin conditions were enrolled with owner permission and approval of the Institutional Animal Care and Use Committee. Transcripts were measured by quantitative RT-PCR using a standard curve assessment. Normal transcript levels for all seven antimicrobial peptides varied depending on the body site assessed. Transcripts for secretory leukocyte proteinase inhibitor (SLPI) and skin-derived antileucoproteinase (SKALP; also known as elafin) were typically ~10-fold greater in number than transcripts for the canine β-defensins (CBD)-1, -102, -103, -122 and -124. Transcripts for SKALP, SLPI, CBD-1, CBD-103 and CBD-122 were lower in both lesional and adjacent nonlesional skin from dogs with AD in comparison to normal skin. Transcripts were reduced to a similar extent versus normal dogs in skin of dogs with inflammatory skin conditions from both lesional and nonlesional biopsies, except for CBD-122, which was reduced only in lesional skin. Compared with normal dog skin, transcripts for CBD-102 and CBD-124 were unaffected in dogs with AD. Both SKALP and SLPI may be important contributors to skin innate immunity, but their decreased expression in AD patients does not account for increased skin infections compared with other skin conditions.

Evaluation of apoptosis regulatory proteins in response to PUVA therapy for psoriasis

The histopathologic changes characteristic of psoriasis might be related to suppressed apoptosis. One of the actions of psoralen ultraviolet A (PUVA) in psoriasis could be exerted through induction of apoptosis of keratinocytes and lymphocytes; however, its exact molecular mechanism is still confusing. In this study, we evaluated the expression of pro-apoptotic (P53, Fas and Bax) and anti-apoptotic (Bcl-2) proteins correlating it with apoptotic index (AI) and epidermal thickness in psoriatic skin before and after PUVA therapy. Lesional and non-lesional skin biopsy specimens were obtained from 10 patients with generalized plaque psoriasis before and after 8 weeks of PUVA therapy. Histometric measurements of epidermal thickness as well as P53, Fas, Bax and Bcl-2 expressions were evaluated using immunoperoxidase technique and apoptotic cells were detected by terminal deoxynucleotide transferase (TdT) mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. After PUVA therapy, the epidermal thickness of psoriatic skin was significantly decreased (P < 0.001) and keratinocytes of psoriatic skin showed significant increased expression of P53 (P < 0.001), Fas (P < 0.001) and Bcl-2 (P < 0.001) with no significant change in Bax expression (P > 0.05). Apart from significant decrease of Bcl-2 expression (P = 0.01), no significant difference in all previous markers were encountered in lymphocytes (P53, Fas and Bax; P > 0.05) after PUVA therapy. The AI was significantly increased (P = 0.008) after PUVA therapy especially in lymphocytes (P = 0.002). The present study suggests that one of the actions of PUVA therapy in psoriasis might be exerted through induction of apoptosis especially of lymphocytes by suppression of Bcl-2 expression and of keratinocytes through P53 and Fas pathways leading to healing of psoriasis.

Phase I Clinical Trial of O6-Benzylguanine and Topical Carmustine in the Treatment of Cutaneous T-Cell Lymphoma, Mycosis Fungoides Type

To evaluate the toxic effects and maximum tolerated dose of topical carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea] following intravenous O6-benzylguanine in the treatment of cutaneous T-cell lymphoma (CTCL), and to determine pharmacodynamics of O6-alkylguanine DNA alkyltransferase activity in treated CTCL lesions. Open-label, dose-escalation, phase I trial. Dermatology outpatient clinic and clinical research unit at a university teaching hospital. A total of 21 adult patients (11 male, 10 female)with early-stage (IA-IIA) refractory CTCL, mycosis fungoides type, treated with topical carmustine following intravenous O6-benzylguanine. Treatment once every 2 weeks with 120 mg/m(2) intravenous O6-benzylguanine followed 1 hour later by whole-body, low-dose topical carmustine starting at 10 mg, with 10-mg incremental dose-escalation in 3 patient cohorts. Cutaneous T-cell lymphoma lesional skin biopsy specimens were taken at baseline and 6 hours, 24 hours, and 1 week after the first O6-benzylguanine infusion for analysis of O6-alkylguanine-DNA alkyltransferase activity. Clinical response measured by physical examination and severity-weighted assessment tool measurements, safety data acquired by review of adverse events at study visits, and O6-alkylguanine-DNA alkyltransferase activity in treated lesion skin biopsy specimens. A minimal toxic effect was observed through the 40-mg carmustine dose level with 76% of adverse events being grade 1 based on the National Cancer Institute Common Terminology Criteria for Adverse Events. Mean baseline O6-alkylguanine-DNA alkyltransferase activity in CTCL lesions was 3 times greater than in normal controls and was diminished by a median of 100% at 6 and 24 hours following O6-benzylguanine with recovery at 1 week. Clinical disease reduction correlated positively with O6-alkylguanine-DNA alkyltransferase activity at 168 hours (P=.02) and inversely with area under the curve of O6-alkylguanine-DNA alkyltransferase over 1 week (P=.01). Twelve partial responses and 4 complete responses were observed (overall response, 76% [95% CI, 0.55-0.89]). Five patients discontinued therapy owing to adverse events with a possible, probable, or definite relationship to the study drug. O6-benzylguanine significantly depletes O6-alkylguanine-DNA alkyltransferase in CTCL lesions and in combination with topical carmustine is well tolerated and shows meaningful clinical responses in CTCL at markedly reduced total carmustine treatment doses.

Mechanisms of IFN-γ–induced apoptosis of human skin keratinocytes in patients with atopic dermatitis

Enhanced apoptosis of keratinocytes is the main cause of eczema and spongiosis in patients with the common inflammatory skin disease atopic dermatitis (AD). The aim of the study was to investigate molecular mechanisms of AD-related apoptosis of keratinocytes. Primary keratinocytes isolated from patients with AD and healthy donors were used to study apoptosis by using annexin V/7-aminoactinomycin D staining. Illumina mRNA Expression BeadChips, quantitative RT-PCR, and immunofluorescence were used to study gene expression. In silico analysis of candidate genes was performed on genome-wide single nucleotide polymorphism data. We demonstrate that keratinocytes of patients with AD exhibit increased IFN-γ-induced apoptosis compared with keratinocytes from healthy subjects. Further mRNA expression analyses revealed differential expression of apoptosis-related genes in AD keratinocytes and skin and the upregulation of immune system-related genes in skin biopsy specimens of chronic AD lesions. Three apoptosis-related genes (NOD2, DUSP1, and ADM) and 8 genes overexpressed in AD skin lesions (CCDC109B, CCL5, CCL8, IFI35, LYN, RAB31, IFITM1, and IFITM2) were induced by IFN-γ in primary keratinocytes. The protein expression of IFITM1, CCL5, and CCL8 was verified in AD skin. In line with the functional studies and AD-related mRNA expression changes, in silico analysis of genome-wide single nucleotide polymorphism data revealed evidence of an association between AD and genetic markers close to or within the IFITM cluster or RAB31, DUSP1, and ADM genes. Our results demonstrate increased IFN-γ responses in skin of patients with AD and suggest involvement of multiple new apoptosis- and inflammation-related factors in the development of AD.

Reversal of atopic dermatitis with narrow-band UVB phototherapy and biomarkers for therapeutic response

Atopic dermatitis (AD) is a common inflammatory skin disease exhibiting a predominantly T(H)2/"T22" immune activation and a defective epidermal barrier. Narrow-band UVB (NB-UVB) is considered an efficient treatment for moderate-to-severe AD. In patients with psoriasis, NB-UVB has been found to suppress T(H)1/T(H)17 polarization, with subsequent reversal of epidermal hyperplasia. The immunomodulatory effects of this treatment are largely unknown in patients with AD. We sought to evaluate the effects of NB-UVB on immune and barrier abnormalities in patients with AD, aiming to establish reversibility of disease and biomarkers of therapeutic response. Twelve patients with moderate-to-severe chronic AD received NB-UVB phototherapy 3 times weekly for up to 12 weeks. Lesional and nonlesional skin biopsy specimens were obtained before and after treatment and evaluated by using gene expression and immunohistochemistry studies. All patients had at least a 50% reduction in SCORAD index scores with NB-UVB phototherapy. The T(H)2, T22, and T(H)1 immune pathways were suppressed, and measures of epidermal hyperplasia and differentiation normalized. The reversal of disease activity was associated with elimination of inflammatory leukocytes and T(H)2/T22- associated cytokines and chemokines and normalized expression of barrier proteins. Our study shows that resolution of clinical disease in patients with chronic AD is accompanied by reversal of both the epidermal defects and the underlying immune activation. We have defined a set of biomarkers of disease response that associate resolved T(H)2 and T22 inflammation in patients with chronic AD with reversal of barrier pathology. By showing reversal of the AD epidermal phenotype with a broad immune-targeted therapy, our data argue against a fixed genetic phenotype.

Canonical Wnt signaling induces skin fibrosis and subcutaneous lipoatrophy: A novel mouse model for scleroderma?

Because aberrant Wnt signaling has been linked with systemic sclerosis (SSc) and pulmonary fibrosis, we sought to investigate the effect of Wnt-10b on skin homeostasis and differentiation in transgenic mice and in explanted mesenchymal cells. The expression of Wnt-10b in patients with SSc and in a mouse model of fibrosis was investigated. The skin phenotype and biochemical characteristics of Wnt-10b-transgenic mice were evaluated. The in vitro effects of ectopic Wnt-10b were examined in explanted skin fibroblasts and preadipocytes. The expression of Wnt-10b was increased in lesional skin biopsy specimens from patients with SSc and in those obtained from mice with bleomycin-induced fibrosis. Transgenic mice expressing Wnt-10b showed progressive loss of subcutaneous adipose tissue accompanied by dermal fibrosis, increased collagen deposition, fibroblast activation, and myofibroblast accumulation. Wnt activity correlated with collagen gene expression in these biopsy specimens. Explanted skin fibroblasts from transgenic mice demonstrated persistent Wnt/β-catenin signaling and elevated collagen and α-smooth muscle actin gene expression. Wnt-10b infection of normal fibroblasts and preadipocytes resulted in blockade of adipogenesis and transforming growth factor β (TGFβ)-independent up-regulation of fibrotic gene expression. SSc is associated with increased Wnt-10b expression in the skin. Ectopic Wnt-10b causes loss of subcutaneous adipose tissue and TGFβ-independent dermal fibrosis in transgenic mice. These findings suggest that Wnt-10b switches differentiation of mesenchymal cells toward myofibroblasts by inducing a fibrogenic transcriptional program while suppressing adipogenesis. Wnt-10b-transgenic mice represent a novel animal model for investigating Wnt signaling in the setting of fibrosis.

The role of human herpesvirus‐6, Epstein–Barr virus and cytomegalovirus infections in the etiopathogenesis of different types of cutaneous drug reactions

Recently, it has been recognized that drug-induced hypersensitivity syndrome (DIHS) is associated with reactivation of human herpesvirus-6 (HHV-6), Epstein–Barr virus (EBV) and cytomegalovirus (CMV). However, whether those viruses have a role in the development of cutaneous drug reactions (CDRs) other than DIHS is not known. To investigate the role of HHV-6, EBV and CMV infections in the etiopathogenesis of different types of CDRs. Eighteen patients with diagnosis of CDR according to the clinical and histopathological findings were evaluated. Real-time polymerase chain reaction (PCR) was used for the detection of EBV, CMV, and HHV-6 DNA in lesional skin biopsy specimens; EBV and CMV DNA in serum samples; and HHV-6 DNA in peripheral blood mononuclear cells. The genome of HHV-6 was detected only in the lesional skin of two patients with DIHS. Epstein–Barr virus and CMV DNA in the skin lesions, EBV and CMV genomes in the serum samples, and HHV-6 DNA in the peripheral blood mononuclear cells were negative in all patients. The patient population was small and did not include all types of CDRs. Also, we had only two patients with DIHS. We had not been able to measure the increase in anti-viral IgG titers in serial serum samples. Epstein–Barr virus and CMV infections do not seem to have a role in the etiopathogenesis of CDRs including DIHS. The association between HHV-6 infection and CDRs is likely to be limited to DIHS.

Identification of TNF-related apoptosis-inducing ligand and other molecules that distinguish inflammatory from resident dendritic cells in patients with psoriasis

Previous work has identified CD11c(+)CD1c(-) dendritic cells (DCs) as the major "inflammatory" dermal DC population in patients with psoriasis vulgaris and CD1c(+) DCs as the "resident" cutaneous DC population. We sought to further define molecular differences between these 2 myeloid dermal DC populations. Inflammatory and resident DCs were single-cell sorted from lesional skin biopsy specimens of patients with psoriasis, and the transcriptome of CD11c(+)CD1c(-) versus CD1c(+) DCs was determined. Results were confirmed with RT-PCR, flow cytometry, immunohistochemistry, and double-labeled immunofluorescence. Human keratinocytes were cultured for functional studies. TNF-related apoptosis-inducing ligand (TRAIL), Toll-like receptors 1 and 2, S100A12/ENRAGE, CD32, and many other inflammatory products were differentially expressed in inflammatory DCs compared with resident DCs. Flow cytometry and immunofluorescence confirmed higher protein expression on CD1c(-) versus CD1c(+) DCs. TRAIL receptors, death receptor 4, and decoy receptor 2 were expressed in keratinocytes and dermal cells. In vitro culture of keratinocytes with TRAIL induced CCL20 chemokine. CD11c(+)CD1c(-) inflammatory DCs in psoriatic lesional skin express a wide range of inflammatory molecules compared with skin-resident CD1c(+) DCs. Some molecules made by inflammatory DCs, including TRAIL, could have direct effects on keratinocytes or other skin cell types to promote disease pathogenesis.

Comparison of the radiometric BACTEC 460 TB culture system and Löwenstein–Jensen medium for the isolation of mycobacteria in cutaneous tuberculosis and their drug susceptibility pattern

Mycobacterial isolation from cutaneous tuberculosis on Löwenstein-Jensen (L-J) medium has been reported to be low. The radiometric BACTEC 460 TB culture system (BACTEC system) has shown better isolation rates in pulmonary tuberculosis. There has been a progressive increase in the prevalence of multidrug resistance in pulmonary tuberculosis, but similar studies are lacking for cutaneous tuberculosis. Therefore, this study was undertaken to compare mycobacterial isolation on conventional L-J medium vs. the BACTEC system, and to determine the prevalence of multidrug resistance in cutaneous tuberculosis. Thirty-five untreated, clinically diagnosed, and histopathologically documented patients with cutaneous tuberculosis constituted the study material. Lesional skin biopsy specimens were cultured on both L-J medium and the BACTEC system. The isolates obtained were identified and subjected to sensitivity to rifampicin, isoniazid, ethambutol, and streptomycin using the BACTEC system. Twenty-six mycobacterial isolates were recovered from 35 patients. Nine isolates (25.7%) grew on L-J medium after a mean period of 31.5 days, and 22 (62.8%) on the BACTEC system in 17.3 days. All of the isolates were identified as Mycobacterium tuberculosis. Drug susceptibility testing demonstrated 12 isolates to be resistant, seven multidrug resistant. The BACTEC system demonstrated an improved mycobacterial isolation rate and substantially reduced detection time when compared with L-J medium. The combined isolation rate on both media was 74.3% (26/35), greater than that of either used separately. Drug resistance was observed in 46.2% of isolates. Radiometric liquid culture medium together with conventional L-J medium may be recommended in practice to enable the institution of appropriate antituberculous therapy modifications in drug-resistant cases of cutaneous tuberculosis.