Biallelic variants in CRIPT cause a Rothmund-Thomson-like syndrome with increased cellular senescence.

PURPOSE: Rothmund-Thomson syndrome (RTS) is characterized by poikiloderma, sparse hair, small stature, skeletal defects, cancer, and cataracts, resembling features of premature aging. RECQL4 and ANAPC1 are the 2 known disease genes associated with RTS in >70% of cases. We describe RTS-like features in 5 individuals with biallelic variants in CRIPT (OMIM 615789). METHODS: Two newly identified and 4 published individuals with CRIPT variants were systematically compared with those with RTS using clinical data, computational analysis of photographs, histologic analysis of skin, and cellular studies on fibroblasts. RESULTS: All CRIPT individuals fulfilled the diagnostic criteria for RTS and additionally had neurodevelopmental delay and seizures. Using computational gestalt analysis, CRIPT individuals showed greatest facial similarity with individuals with RTS. Skin biopsies revealed a high expression of senescence markers (p53/p16/p21) and the senescence-associated ß-galactosidase activity was elevated in CRIPT-deficient fibroblasts. RECQL4- and CRIPT-deficient fibroblasts showed an unremarkable mitotic progression and unremarkable number of mitotic errors and no or only mild sensitivity to genotoxic stress by ionizing radiation, mitomycin C, hydroxyurea, etoposide, and potassium bromate. CONCLUSION: CRIPT causes an RTS-like syndrome associated with neurodevelopmental delay and epilepsy. At the cellular level, RECQL4- and CRIPT-deficient cells display increased senescence, suggesting shared molecular mechanisms leading to the clinical phenotypes.

Postoperative complications in dermatological patients undergoing microscopically controlled surgery in inpatient setting (next-day surgery): A single-center epidemiological study.

BACKGROUND: Surgical site infections (SSI), bleeding, and necrosis are possible complications of dermatological surgery, and their rates are well described for Mohs surgery (same-day surgery). However, there are only limited data on their occurrence in microscopically controlled surgery of the form in which it is practiced in German hospitals (next-day surgery). MATERIALS AND METHODS: We performed a retrospective analysis of patient records of patients hospitalized for microscopically controlled surgery during the year 2017 (12 months) in the Department of Dermatology and Allergology at the University Hospital of the RWTH Aachen (Aachen, Germany). The investigation addressed postoperative outcomes. RESULTS: 319 patients underwent 528 dermatosurgical procedures in the defined period. Bleeding and necrosis occurred in 3.8 % (20/528) and 1.7 % (9/528) of the procedures, respectively. SSI occurred in 5.1 % (27/528) of the cases. The occurrence of bleeding was a statistically significant risk factor for SSI (p  =  0.01). Furthermore, bleeding, SSI, and wound closure with a full-thickness graft were statistically significant risk factors for the development of necrosis (p < 0.05). Diabetes or immunosuppression were not found to be statistically significant risk factors for the development of SSI or necrosis after dermatologic surgery (p > 0.05). CONCLUSIONS: Complication rates in microscopically controlled surgery (next-day surgery) are generally low and similar to those reported for Mohs surgery (same-day surgery). Therefore, it appears that some evidence-based perioperative recommendations that have been developed for Mohs surgery could be applied to German inpatient dermatosurgery. However, prospective studies with larger patient numbers are required to offer concrete recommendations specifically for microscopically controlled surgery (next-day surgery).

Novel Human Full-Thickness Three-Dimensional Nonkeratinized Mucous Membrane Model for Pharmacological Studies in Wound Healing.

INTRODUCTION: Efforts are increasingly aiming to develop in vitro models that can provide effective alternatives to in vivo experiments. The main aim of this study was the establishment of an in vitro model of the nonkeratinized mucous membrane that can be used as a standardized tool to evaluate biological and therapeutic effects of pharmaceuticals for mucosal wound healing. METHODS: We established a full-thickness in vitro model of the nonkeratinized mucous membrane. While histological examination was performed to assess morphological characteristics, we utilized gene expression profiling using microarray and qRT-PCR analyses to identify molecular effects of treatment with a dexpanthenol-containing ointment after laser wounding. RESULTS: Performing histological and immunofluorescence analyses we proved that our model mimics the two distinctive layers of the mucous membrane - the stratified squamous epithelium and the lamina propria. We used this model to investigate molecular effects of a dexpanthenol-containing ointment that is commonly used for the wound treatment of mucous membranes. For that purpose, our model exhibits a unique feature in that dexpanthenol and proliferation-enhancing additives that may interfere with our studies are not required for the maintenance of the model culture. After setting standardized lesions with a nonsequential fractional ultrapulsed CO2 laser, topical treatment with the dexpanthenol-containing ointment enhanced wound closure in the model compared to placebo and untreated controls. Furthermore, microarray analysis revealed that the treatment of the laser-wounded model with the dexpanthenol-containing ointment evoked an upregulated expression of various genes related to accelerated wound healing. CONCLUSION: Overall, we verified that this novel mucous membrane model can be utilized in future to monitor ex vivo effects of various topical therapies on mucosa morphology, physiology, and gene expression. Our findings confirm the potential of the model as an in vitro tool for the replacement of pharmacological in vivo studies regarding mucosal wound healing.

Dual addressing of thymidine synthesis pathways for effective targeting of proliferating melanoma.

Here, we examined the potential of blocking the thymidine de novo synthesis pathways for sensitizing melanoma cells to the nucleoside salvage pathway targeting endogenous DNA irradiation. Expression of key nucleotide synthesis and proliferation enzymes thymidylate synthase (TS) and thymidine kinase 1 (TK1) was evaluated in differentiated (MITFhigh [microphthalmia-associated transcription factor] IGR1) and invasive (MITFmedium IGR37) melanoma cells. For inhibition of de novo pathways cells were incubated either with an irreversible TS inhibitor 5-fluoro-2'-deoxyuridine (FdUrd) or with a competitive dihydrofolate-reductase (DHFR) inhibitor methotrexate (MTX). Salvage pathway was addressed by irradiation-emitting thymidine analog [123/125 I]-5-iodo-4'-thio-2'-deoxyuridine (123/125 I-ITdU). The in vivo targeting efficiency was visualized by single-photon emission computed tomography. Pretreatment with FdUrd strongly increased the cellular uptake and the DNA incorporation of 125 I-ITdU into the mitotically active IGR37 cells. This effect was less pronounced in the differentiated IGR1 cells. In vivo, inhibition of TS led to a high and preferential accumulation of 123 I-ITdU in tumor tissue. This preclinical study presents profound rationale for development of therapeutic approach by highly efficient and selective radioactive targeting one of the crucial salvage pathways in melanomas.

Prospective, Randomized Study on the Efficacy and Safety of Local UV-Free Blue Light Treatment of Eczema.

BACKGROUND: Blue light was shown to reduce the activation of T cells and modulate cytokine release in vitro. Therefore, we investigated the efficacy of blue light in the treatment of eczema. METHODS: A sample of 21 patients with mild to moderate eczema were locally treated with blue LED light (light-emitting diode, emission maximum: 453 nm). They received light treatment 3 times per week for 4 weeks. A contralateral control lesion remained untreated. RESULTS: A total of 20 patients completed the trial with a compliance rate of 100%. The blue light treatment was safe with no adverse events and no side effects. The primary end point change from baseline in the mean sum score of the local Eczema Severity Index (local ESI) was more pronounced for the treated area than for the control area (-1.9 ± 2.02 vs. -1.3 ± 2.24). The treatment difference was statistically significant (p = 0.0152, paired t test, two-sided). CONCLUSION: In this study UV-free blue light was safe and effective in the reduction of eczema lesions.

Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand.

Triple-negative breast cancer (TNBC) is a heterogeneous disease in which the tumors do not express estrogen receptor (ER), progesterone receptor (PgR) or human epidermal growth factor receptor 2 (HER2). Classical receptor-targeted therapies such as tamoxifen or trastuzumab are therefore unsuitable and combinations of surgery, chemotherapy and/or radiotherapy are required. Photoimmunotheranostics is a minimally invasive approach in which antibodies deliver nontoxic photosensitizers that emit light to facilitate diagnosis and produce cytotoxic reactive oxygen species to induce apoptosis and/or necrosis in cancer cells. We developed a panel of photoimmunotheranostic agents against three TNBC-associated cell surface antigens. Antibodies against epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) and chondroitin sulfate proteoglycan 4 (CSPG4) were conjugated to the highly potent near-infrared imaging agent/photosensitizer IRDye®700DX phthalocyanine using SNAP-tag technology achieving clear imaging in both breast cancer cell lines and human biopsies and highly potent phototherapeutic activity with IC50values of 62-165 nM against five different cell lines expressing different levels of EGFR, EpCAM and CSPG4. A combination of all three reagents increased the therapeutic activity against TNBC cells by up to 40%.

A specific photoimmunotheranostics agent to detect and eliminate skin cancer cells expressing EGFR.

PURPOSE: The term "theranostics" represents a new paradigm in medicine especially for cancer treatment. This term was coined by Funkhouser in 2002 and defines a reagent that combines therapeutic and diagnostic properties. It is widely believed that theranostics agents will have considerable impact on healthcare before, during, and after disease by improving cancer prognosis and management simultaneously. Current theranostics approaches still rely on passive tumor targeting strategies, which have scattergun effects and tend to damage both neoplastic and non-neoplastic cells. METHODS: Here we describe a simple, controlled, and efficient method to generate homogeneous photoimmunotheranostics reagents. This method combines molecular optical imaging, photodynamic therapy, and immunotherapy using SNAP-tag technology. SNAP-tag is a derivative of the O(6)-alkylguanine-DNA alkyltransferase (AGT) which has the ability to efficiently conjugate to O(6)-benzylguanine (BG) molecules under physiological conditions depending on its folding pattern. RESULTS: The theranostics agent was able to specifically recognize various epidermal growth factor receptor (EGFR)-expressing skin cancer cell lines using flow cytometry analysis and confocal microscopy and eliminate them at EC50's of 32-55 nM. CONCLUSIONS: These experiments provide a framework for using SNAP-tag technology to generate homogeneous photoimmunotheranostics reagents with unified pharmacokinetic and therapeutic profiles. Furthermore, the reagent generated in this work could be used to simultaneously monitor and suppress the growth of skin squamous carcinoma and melanoma cells expressing EGFR.

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies.

Macrophages are key players in controlling the immune response that can adapt to microenvironmental signals. This results in distinct polarization states (classical M1 or alternative M2), that play a differential role in immune regulation. In general, the M1 contribute to onset of inflammation, whereas the M2 orchestrate resolution and repair, whereby failure to switch from predominantly M1 to M2 reinforces a pro-inflammatory environment and chronic inflammation. Here, we show selective elimination of M1 macrophages in vitro by a range of CD64-targeted immunotoxins, including H22(scFv)-ETA'. After re-polarization of already polarized macrophages, still only M1 polarization showed sensitivity toward CD64-directed immunotoxins. The selectivity for M1 was found linked to reduced endosomal protease activity in M1 macrophages as demonstrated by inhibition of endosomal proteases. Using the H22(scFv)-ETA' in a transgenic mouse model for chronic cutaneous inflammation, the M1 specificity was confirmed in vivo and a beneficial effect on inflammation demonstrated. Also ex vivo on skin biopsies from atopic dermatitis and diabetes type II patients with chronically-inflamed skin, a clear M1 specific effect was found. This indicates the potential relevance for human application. Our data show that targeting M1 macrophages through CD64 can be instrumental in developing novel intervention strategies for chronic inflammatory conditions.

Prospective Randomized Long-Term Study on the Efficacy and Safety of UV-Free Blue Light for Treating Mild Psoriasis Vulgaris.

BACKGROUND: Blue light irradiation reduces the proliferation of keratinocytes and modulates T-cell immune response in vitro and has been shown to reduce the severity of psoriasis vulgaris (Pv) in two clinical trials. OBJECTIVE: Evaluation of safety and efficacy of long-term UV-free blue light treatment at home for mild Pv. METHODS: Forty-seven patients with mild Pv were randomized for receiving high-intensity blue light treatment (HI: 453 nm LED, 200 mW/cm(2), n = 24) and low-intensity treatment (LI: 453 nm LED, 100 mW/cm(2), n = 23) of one Pv plaque for 12 weeks. A contralateral control plaque remained untreated. RESULTS: Patient compliance and satisfaction were high. The primary endpoint, change from baseline (CfB) of the Local Psoriasis Severity Index, revealed a significant improvement of the target compared to the control plaques (ΔCfB for the HI group: -0.92 ± 1.10, p = 0.0005; for the LI group: -0.74 ± 1.18, p = 0.0064). CONCLUSION: UV-free blue light home treatment is safe and improves Pv plaques.

Tumor volume as a prognostic factor in resectable malignant melanoma.

BACKGROUND: Vertical tumor thickness according to Breslow and histological ulceration are still the most powerful predictors for the clinical outcome of resectable cutaneous malignant melanoma (MM) without lymph node infiltration. It has been proposed that tumor volume in MM may also be of prognostic relevance. METHODS: We retrospectively analyzed the prognostic impact of tumor volume and other established risk factors in 122 MM patients with a median follow-up period of 39.7 months. RESULTS: We found the logarithmic tumor volume to be a better prognostic factor compared to Breslow tumor thickness in multivariate analysis. MM with a tumor volume below a threshold of 140 mm(3) had a significantly higher relapse-free survival after 5 years of 98% compared to 47% in larger MMs (p < 0.0001). CONCLUSION: In some melanomas with a low tumor thickness, a higher tumor volume appeared to be linked to a higher risk of disease recurrence. Inclusion of tumor volume into the risk assessment of resectable MM may be of benefit in the future.

Prospective randomized study on the efficacy of blue light in the treatment of psoriasis vulgaris.

BACKGROUND: Blue light has no known toxic effects on human skin, but reduces the proliferative capacity of keratinocytes in vitro. We therefore investigated the efficacy of blue light in the treatment of psoriasis vulgaris (PV). METHODS: Forty patients with mild to moderate PV and bilateral plaques were assigned to two groups. Group 1 (n = 20) received irradiation at home with blue light (light-emitting diode, LED, emission maximum: 420 nm) once daily for 4 weeks. In parallel, group 2 (n = 20) performed irradiations with another blue light device (LED emission maximum: 453 nm). The contralateral control plaques remained untreated in both groups. RESULTS: Thirty-seven patients completed the trial. The main study parameter, the difference of Local Psoriasis Severity Index (LPSI) scores of the irradiated plaques compared to the control plaques, showed statistically significant improvement after 4 weeks of treatment in both groups [group 1 (420 nm): n = 17, p = 0.04; group 2 (453 nm): n = 20, p = 0.04]. Accordingly, plaque status as assessed by both the physicians and the patients improved continuously during the 4 weeks of treatment and steadily declined thereafter. CONCLUSION: Blue light appears to be a promising treatment modality in PV that warrants further evaluation in larger studies.

SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells.

Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR(+) cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.

Squamous cell carcinoma arising from a localized vulval lesion of Hailey-Hailey disease after tacrolimus therapy.

Hailey-Hailey disease (HHD) is a rare, autosomal dominant intraepidermal blistering disorder characterized by recurrent vesicles and erosions affecting mostly the intertriginous areas. We report a case of HHD affecting exclusively the vulva from which an invasive squamous cell carcinoma developed after tacrolimus therapy.

Resistance to antiangiogenic therapy is directed by vascular phenotype, vessel stabilization, and maturation in malignant melanoma.

Angiogenesis is not only dependent on endothelial cell invasion and proliferation, it also requires pericyte coverage of vascular sprouts for stabilization of vascular walls. Clinical efficacy of angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathway is still limited to date. We hypothesized that the level of vessel maturation is critically involved in the response to antiangiogenic therapies. To test this hypothesis, we evaluated the vascular network in spontaneously developing melanomas of MT/ret transgenic mice after using PTK787/ZK222584 for anti-VEGF therapy but also analyzed human melanoma metastases taken at clinical relapse in patients undergoing adjuvant treatment using bevacizumab. Both experimental settings showed that tumor vessels, which are resistant to anti-VEGF therapy, are characterized by enhanced vessel diameter and normalization of the vascular bed by coverage of mature pericytes and immunoreactivity for desmin, NG-2, platelet-derived growth factor receptor beta, and the late-stage maturity marker alpha smooth muscle actin. Our findings emphasize that the level of mural cell differentiation and stabilization of the vascular wall significantly contribute to the response toward antiangiogenic therapy in melanoma. This study may be useful in paving the way toward a more rational development of second generation antiangiogenic combination therapies and in providing, for the first time, a murine model to study this.

Photodynamic therapy in the treatment of actinic keratosis.

The efficacy of photodynamic therapy (PDT) with 5-aminolevulinate and methyl aminolevulinate in the treatment of actinic keratosis has been demonstrated in a large number of clinical studies over the last several years. Here, we recapitulate the major findings, comparing the various photosensitizers, light sources and therapeutic regimens, and present a retrospective analysis of 142 own cases treated with 259 PDTs. In addition, we also discuss the value of PDT in comparison with cryotherapy or 5-fluorouracil. The efficacy and the low risk of side effects of PDT have resulted in a high patient preference in clinical trials.

Therapy of chronic wounds with water-filtered infrared-A (wIRA).

The central portion of chronic wounds is often hypoxic and relatively hypothermic, representing a deficient energy supply of the tissue, which impedes wound healing or even makes it impossible. Water-filtered infrared-A (wIRA) is a special form of heat radiation with a high tissue penetration and a low thermal load to the skin surface. wIRA produces a therapeutically usable field of heat and increases temperature, oxygen partial pressure and perfusion of the tissue. These three factors are decisive for a sufficient tissue supply with energy and oxygen and consequently as well for wound healing, especially in chronic wounds, and infection defense. wIRA acts both by thermal and thermic as well as by non-thermal and non-thermic effects. wIRA can advance wound healing or improve an impaired wound healing process and can especially enable wound healing in non-healing chronic wounds. wIRA can considerably alleviate the pain and diminish wound exudation and inflammation and can show positive immunomodulatory effects.In a prospective, randomized, controlled study of 40 patients with chronic venous stasis ulcers of the lower legs irradiation with wIRA and visible light (VIS) accelerated the wound healing process (on average 18 vs. 42 days until complete wound closure, residual ulcer area after 42 days 0.4 cm(2) vs. 2.8 cm(2)) and led to a reduction of the required dose of pain medication in comparison to the control group of patients treated with the same standard care (wound cleansing, wound dressing with antibacterial gauze, and compression garment therapy) without the concomitant irradiation. Another prospective study of 10 patients with non-healing chronic venous stasis ulcers of the lower legs included extensive thermographic investigation. Therapy with wIRA(+VIS) resulted in a complete or almost complete wound healing in 7 patients and a marked reduction of the ulcer size in another 2 of the 10 patients, a clear reduction of pain and required dose of pain medication, and a normalization of the thermographic image. In a current prospective, randomized, controlled, blinded study patients with non-healing chronic venous stasis ulcers of the lower legs are treated with compression garment therapy, wound cleansing, wound dressings and 30 minutes irradiation five times per week over 9 weeks. A preliminary analysis of the first 23 patients of this study has shown in the group with wIRA(+VIS) compared to a control group with VIS an advanced wound healing, an improved granulation and in the later phase of treatment a decrease of the bacterial burden. Some case reports have demonstrated that wIRA can also be used for mixed arterial-venous ulcers or arterial ulcers, if irradiation intensity is chosen appropriately low and if irradiation is monitored carefully. wIRA can be used concerning decubital ulcers both in a preventive and in a therapeutic indication. wIRA can improve the resorption of topically applied substances also on wounds. An irradiation with VIS and wIRA presumably acts with endogenous protoporphyrin IX (or protoporphyrin IX of bacteria) virtually similar as a mild photodynamic therapy (endogenous PDT-like effect). This could lead to improved cell regeneration and wound healing and to antibacterial effects. In conclusion, these results indicate that wIRA generally should be considered for the treatment of chronic wounds.

Interleukin-6 gene ablation in a transgenic mouse model of malignant skin melanoma.

Interleukin (IL)-6 is a pleiotropic cytokine that has been shown to inhibit the growth of early stage and to promote the proliferation of advanced stage melanoma cells in vitro. In patients with metastasizing melanomas, highly increased IL-6 blood levels correlate with a poor response to chemotherapy and a worse overall prognosis, suggesting that IL-6 promotes melanoma progression in vivo. Here, we analyzed the role of IL-6 in melanoma development and progression in a transgenic mouse model. We bred IL-6-deficient mice with MT-ret transgenic animals predisposed for melanomas. While MT-ret transgenic animals develop severe melanosis of the skin and subcutis and subsequent melanomas at an incidence of 80% during their first year of life, MT-ret mice devoid of IL-6 developed preneoplastic melanosis and consecutive melanomas significantly less frequently (47%; P < 0.05). Moreover, the tumors were significantly smaller in the groups of MT-ret mice lacking one (P < 0.05) or both (P < 0.01) copies of the IL-6 gene. Immunoblot analysis revealed that ret transgene expression was not reduced in the skin of mice lacking IL-6, indicating that the observed decrease of melanoma incidence and of tumor sizes was not because of a down-regulation of transgene expression. Taken together, these results indicate that IL-6 enhances both the development of melanoma precursor lesions and the subsequent growth of the resulting tumors in the MT-ret model of melanoma development.