ABSTRACT
Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal-epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.
Subject(s)
Autoantigens/metabolism , Epidermolysis Bullosa, Junctional/genetics , Non-Fibrillar Collagens/metabolism , Oligonucleotides, Antisense/genetics , RNA Splicing , Alternative Splicing , Biopsy , Cell Line , Cell Survival , Epidermolysis Bullosa, Junctional/metabolism , Epidermolysis Bullosa, Junctional/therapy , Exons , Genotype , Homozygote , Humans , Keratinocytes/cytology , Liposomes/chemistry , Mutation , Organ Culture Techniques , RNA, Messenger/metabolism , Collagen Type XVIIABSTRACT
RNA interference has emerged as a powerful tool for therapeutic gene silencing, as it offers the possibility to silence virtually any known pathology-causing gene. However, in vivo delivery of RNAi molecules is hampered by their unfavourable physicochemical characteristics and susceptibility to degradation by endogenous enzymes. To overcome these limitations, we recently developed an elastic liposomal formulation, called DDC642, as topical delivery system of therapeutic RNAi molecules for skin disorders. In this study, we validated the therapeutic efficacy of DDC642-encapsulated RNAi molecules in the treatment of psoriasis using 3 different in vitro models: a standardized keratinocyte monolayer culture, psoriasis-induced keratinocytes and a psoriasis-reconstructed skin model. Four genes (IL22RA1, KRT17, DEFB4 and TSLP), known to be upregulated in psoriatic lesions, and thereby key players in psoriasis pathogenesis were selected. Moreover, the possibility of using a combined siRNA therapy in the topical treatment of psoriasis was explored. Results indicate a successful gene silencing of each different target, both at mRNA and protein levels. Additionally, siRNA-DDC642 treatment resulted in a reduced expression of specific psoriasis markers, indicating their potential in future therapeutic approach. The examined siRNA combination (ie simultaneous knockdown of KRT17, DEFB4 and TSLP) showed an enhanced reduction in TSLP expression, whereas the decrease in K17 protein expression was impaired in psoriatic keratinocytes. Although the here examined siRNA combination could still be further improved, our study proved already in vitro the clinical potential of targeting multiple genes at once, each playing a different role in a complex disease such as psoriasis.
Subject(s)
In Vitro Techniques , Models, Biological , Psoriasis/therapy , RNAi Therapeutics , Adult , Biomarkers/metabolism , Humans , Keratinocytes/metabolism , Proof of Concept StudyABSTRACT
BACKGROUND: Antibodies blocking programmed cell death 1 (PD-1) have encouraging responses in patients with metastatic melanoma. Response to anti-PD-1 treatment requires pre-existing CD8+ T cells that are negatively regulated by PD-1-mediated adaptive immune resistance. Unfortunately, less than half of melanoma tumours have these characteristics. Combining anti-PD-1 treatment with other immunomodulating treatments to activate CD8+ T cells is therefore of vital importance to increase response rates and long-term survival benefit in melanoma patients. Both preclinical and retrospective clinical data support the hypothesis that radiotherapy increases the response rates to anti-PD-1 treatment by stimulating the accumulation and activation of CD8+ T cells in the tumour microenvironment. Combining radiotherapy with a PD-1 blocking antibody might therefore increase response rates and even induce long-term survival. The current phase II study will be testing these hypotheses and aims to improve local and distant tumour responses by exploiting the pro-immunogenic effects of radiotherapy in addition to anti-PD-1 treatment. METHODS: The trial will be conducted in patients with metastatic melanoma. Nivolumab or pembrolizumab, both antibodies that target PD-1, will be administrated according to the recommended dosing schedule. Prior to the 2nd cycle, radiotherapy will be delivered in three fractions of 8 Gy to the largest FDG-avid metastatic lesion. The primary endpoint is the proportion of patients with a partial or complete response in non-irradiated metastases according to RECIST v1.1. Secondary endpoints include response rate according to immune related response criteria, metabolic response, local control and survival. To identify peripheral blood biomarkers, peripheral blood mononuclear cells and serum samples will be collected prospectively before, during and after treatment and subjected to flow cytometry and cytokine measurement. DISCUSSION: The current phase II trial aims at exploring the suggested benefits of combining anti-PD-1 treatment and radiotherapy. The translational focus on immunologic markers might be suitable for predicting efficacy and monitoring the effect so to improve patient selection for future clinical applications. ClinicalTrials.gov Identifier NCT02821182.
Subject(s)
Clinical Trials, Phase II as Topic , Melanoma/secondary , Melanoma/therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Radiosurgery , Endpoint Determination , Follow-Up Studies , Humans , Melanoma/immunology , Outcome Assessment, Health Care , Programmed Cell Death 1 Receptor/metabolism , Sample Size , Treatment OutcomeABSTRACT
BACKGROUND: Current first-line standard of therapy for metastatic urothelial carcinoma is platinum-based combination chemotherapy. Pembrolizumab in phase III has demonstrated a promising overall response rate of 21.1% in patients with progression or recurrence after platinum-based chemotherapy. Preclinical and clinical evidence suggests that radiotherapy has a systemic anti-cancer immune effect and can increase the level of PD-L1 and tumor infiltrating lymphocytes in the tumor microenvironment. These findings gave rise to the hypothesis that the combination of radiotherapy with anti-PD1 treatment could lead to a synergistic effect, hereby enhancing response rates. METHODS: The phase I part will assess the dose limiting toxicity of the combination treatment of stereotactic body radiotherapy (SBRT) with four cycles of pembrolizumab (200 mg intravenously, every 3 weeks) in patients with metastatic urothelial carcinoma. The dose of both pembrolizumab and SBRT will be fixed, yet the patients will be randomized to receive SBRT either before the first cycle of pembrolizumab or before the third cycle of pembrolizumab. SBRT will be delivered (24 Gy in 3 fractions every other day) to the largest metastatic lesion. Secondary objectives include response rate according to RECIST v1.1 and immune related response criteria, progression-free survival and overall survival. The systemic immune effect triggered by the combination therapy will be monitored on various time points during the trial. The PD-L1/TIL status of the tumors will be analyzed via immunohistochemistry and response rates in the subgroups will be analyzed separately. A Simon's two-stage optimum design is used to select the treatment arm associated with the best response rate and with acceptable toxicity to proceed to the phase II trial. In this phase, 13 additional patients will be accrued to receive study treatment. DISCUSSION: The progress made in the field of immunotherapy has lead to promising breakthroughs in various solid malignancies. Unfortunately, the majority of patients do not respond. The current trial will shed light on the toxicity and potential anti-tumor activity of the combination of radiotherapy with anti-PD1 treatment and may identify potential new markers for response and resistance to therapy. Trial registration this trial is registered on clinicaltrials.gov (NCT02826564).
Subject(s)
Antibodies, Monoclonal, Humanized/adverse effects , Antibodies, Monoclonal, Humanized/therapeutic use , Radiosurgery/adverse effects , Urologic Neoplasms/immunology , Urologic Neoplasms/therapy , Urothelium/pathology , Combined Modality Therapy , Dose-Response Relationship, Radiation , Female , Follow-Up Studies , Humans , Male , Neoplasm Metastasis , Sample Size , Statistics as Topic , Urologic Neoplasms/drug therapy , Urologic Neoplasms/radiotherapyABSTRACT
BACKGROUND: Immune markers in the peripheral blood of melanoma patients could provide prognostic information. However, there is currently no consensus on which circulating cell types have more clinical impact. We therefore evaluated myeloid-derived suppressor cells (MDSC), dendritic cells (DC), cytotoxic T-cells and regulatory T-cells (Treg) in a series of blood samples of melanoma patients in different stages of disease. METHODS: Flow cytometry was performed on peripheral blood mononuclear cells of 69 stage I to IV melanoma patients with a median follow-up of 39 months after diagnosis to measure the percentage of monocytic MDSCs (mMDSCs), polymorphonuclear MDSCs (pmnMDSCs), myeloid DCs (mDCs), plasmacytoid DCs (pDCs), cytotoxic T-cells and Tregs. We also assessed the expression of PD-L1 and CTLA-4 in cytotoxic T-cells and Tregs respectively. The impact of cell frequencies on prognosis was tested with multivariate Cox regression modelling. RESULTS: Circulating pDC levels were decreased in patients with advanced (P = 0.001) or active (P = 0.002) disease. Low pDC levels conferred an independent negative impact on overall (P = 0.025) and progression-free survival (P = 0.036). Even before relapse, a decrease in pDC levels was observed (P = 0.002, correlation coefficient 0.898). High levels of circulating MDSCs (>4.13%) have an independent negative prognostic impact on OS (P = 0.012). MDSC levels were associated with decreased CD3+ (P < 0.001) and CD3 + CD8+ (P = 0.017) T-cell levels. Conversely, patients with high MDSC levels had more PD-L1+ T-cells (P = 0.033) and more CTLA-4 expression by Tregs (P = 0.003). pDCs and MDSCs were inversely correlated (P = 0.004). The impact of pDC levels on prognosis and prediction of the presence of systemic disease was stronger than that of MDSC levels. CONCLUSION: We demonstrated that circulating pDC and MDSC levels are inversely correlated but have an independent prognostic value in melanoma patients. These cell types represent a single immunologic system and should be evaluated together. Both are key players in the immunological climate in melanoma patients, as they are correlated with circulating cytotoxic and regulatory T-cells. Circulating pDC and MDSC levels should be considered in future immunoprofiling efforts as they could impact disease management.
Subject(s)
Dendritic Cells/immunology , Melanoma/immunology , Myeloid Cells/immunology , Adult , Cell Movement , Disease-Free Survival , Humans , Logistic Models , Melanoma/pathology , Middle Aged , Neoplasm Staging , Proportional Hazards Models , Skin Neoplasms , Melanoma, Cutaneous MalignantABSTRACT
Crosstalk between the human host and its microbiota is reported to influence various diseases such as mucositis. Fundamental research in this area is however complicated by the time frame restrictions during which host-microbe interactions can be studied in vitro. The model proposed in this paper, consisting of an oral epithelium and biofilm, can be used to study microbe-host crosstalk in vitro in non-infectious conditions up to 72 h. Microbiota derived from oral swabs were cultured on an agar/mucin layer and challenged with monolayers of keratinocytes grown on plastic or collagen type I layers embedded with fibroblasts. The overall microbial biofilm composition in terms of diversity remained representative for the oral microbiome, whilst the epithelial cell morphology and viability were unaffected. Applying the model to investigate wound healing revealed a reduced healing of 30 % in the presence of microbiota, which was not caused by a reduction of the proliferation index (52.1-61.5) or a significantly increased number of apoptotic (1-1.13) or necrotic (32-30.5 %) cells. Since the model allows the separate study of the microbial and cellular exometabolome, the biofilm and epithelial characteristics after co-culturing, it is applicable for investigations within fundamental research and for the discovery and development of agents that promote wound healing.
Subject(s)
Microbiota , Mouth Diseases/physiopathology , Mouth Mucosa/microbiology , Wound Healing , Animals , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Bacterial Physiological Phenomena , Biofilms , Cell Line , Cell Proliferation , Cells, Cultured , Host-Pathogen Interactions , Humans , Keratinocytes/microbiology , Mice , Models, Biological , Mouth Diseases/microbiology , Mouth Mucosa/physiologyABSTRACT
Because current skin whitening agents often have insufficient efficacy and side effects, we aim to develop effective and safe therapeutics using RNA interference (RNAi). We established a pigmented human-reconstructed skin model as a first step in the development of novel siRNA-based depigmenting agents. Histological characterization revealed that our model had a similar morphology as normal human skin, expressed keratinocyte differentiation as well as basement membrane markers, and showed a high degree of pigmentation. The utility of the model to study RNAi-induced depigmentation was validated by incorporation of melanocytes transfected with siRNA against tyrosinase, a key enzyme in skin pigmentation. This resulted in a strong reduction in pigmentation and inhibition of melanin transfer proving that siRNA-mediated gene silencing in melanocytes worked successfully in our model. Therefore, this self-made 3D skin model will be a useful and easy tool to validate the whitening potential of candidate genes with a presumed function in melanin synthesis or transfer.
Subject(s)
Models, Biological , RNA Interference , Skin Pigmentation/genetics , Bleaching Agents , Gene Knockdown Techniques , Humans , Imaging, Three-Dimensional , Keratinocytes/cytology , Keratinocytes/metabolism , Melanocytes/cytology , Melanocytes/metabolism , Monophenol Monooxygenase/antagonists & inhibitors , Monophenol Monooxygenase/genetics , RNA, Small Interfering/genetics , Skin/cytology , Skin/metabolism , Skin Pigmentation/physiologyABSTRACT
BACKGROUND: Malignant transformation of melanocytes is frequently attended by a switch in cadherin expression profile as shown for E- and N-cadherin. For P-cadherin, downregulation in metastasizing melanoma has been demonstrated, and over-expression of P-cadherin in melanoma cell lines has been shown to inhibit invasion. The strong invasive and metastatic nature of cutaneous melanoma implies a deregulated interplay between intercellular adhesion and migration-related molecules RESULTS: In this study we performed a microarray analysis to compare the mRNA expression profile of an invasive BLM melanoma cell line (BLM LIE) and the non-invasive P-cadherin over-expression variant (BLM P-cad). Results indicate that nonmuscle myosin II-B is downregulated in BLM P-cad. Moreover, myosin II-B plays a major role in melanoma migration and invasiveness by retracting the tail during the migratory cycle, as shown by the localization of myosin II-B stress fibers relative to Golgi and the higher levels of phosphorylated myosin light chain. Analysis of P-cadherin and myosin II-B in nodular melanoma sections and in a panel of melanoma cell lines further confirmed that there is an inverse relationship between both molecules. CONCLUSIONS: Therefore, we conclude that P-cadherin counteracts the expression and function of myosin II-B, resulting in the suppression of the invasive and migratory behaviour of BLM melanoma cells.
Subject(s)
Cadherins/metabolism , Melanoma/metabolism , Nonmuscle Myosin Type IIB/metabolism , Actins/genetics , Actins/metabolism , Blotting, Western , Cadherins/genetics , Cell Movement/genetics , Cell Movement/physiology , Electrophoresis, Polyacrylamide Gel , Humans , Immunohistochemistry , Immunoprecipitation , Melanoma/genetics , Microscopy, Confocal , Nonmuscle Myosin Type IIB/genetics , Polymerase Chain Reaction , RNA, Small Interfering , Tumor Cells, CulturedABSTRACT
BACKGROUND: RhoC overexpression in tumor cells promotes invasive and metastatic behavior. RhoC expression levels have been correlated with tumor progression and metastasis in multiple human cancers. In melanoma, RhoC is upregulated in highly metastatic tumors. Induced expression in melanoma cell lines resulted in invasion and metastasis, whereas inhibition of RhoC reversed the metastatic phenotype both in vitro and in vivo. METHODS: RhoC mRNA and protein expression in two human melanoma cell lines (DX3aza and MeWo) and pooled primary melanocytes were investigated by means of real-time quantitative polymerase chain reaction and western blotting. RhoC protein expression was evaluated in 123 primary cutaneous melanoma samples by the use of immunohistochemistry and correlated with known prognostic features. RESULTS: RhoC upregulation was observed in the highly metastatic DX3aza cell line, whereas in MeWo, only low expression levels could be detected. RhoC expression in primary cutaneous melanoma was strongly associated with thicker and ulcerated tumors. RhoC expression was associated with the presence of lymphatic metastases at the time of diagnosis and shorter disease-free and overall survival rates, without being an independent predictor. CONCLUSION: These results further support a role for RhoC in growth and metastasis of melanoma.
Subject(s)
Melanoma/metabolism , Melanoma/pathology , Neoplasm Invasiveness/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , rho GTP-Binding Proteins/metabolism , Blotting, Western , Cell Line, Tumor , Female , Gene Expression , Humans , Immunohistochemistry , Male , Melanoma/genetics , Middle Aged , Neoplasm Invasiveness/pathology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Skin Neoplasms/genetics , rho GTP-Binding Proteins/genetics , rhoC GTP-Binding ProteinABSTRACT
PURPOSE: To report the results of a phase 1 trial evaluating the safety of the ipilimumab/radiation therapy combination in patients with metastatic melanoma. PATIENTS AND METHODS: Thirteen patients with metastatic melanoma were enrolled. Trial treatment consisted of 4 cycles of ipilimumab in combination with concurrent dose-escalated high-dose radiation therapy to 1 lesion administered before the third cycle of ipilimumab. RESULTS: Grade 3 or 4 ipilimumab-related adverse events occurred in 25% of patients. The maximum tolerated radiation therapy dose was not reached. Local control of the irradiated lesions was achieved in 11 of 12 irradiated patients (1 patient had progressive disease before irradiation and dropped out of the trial). Evaluation of the nonirradiated lesions demonstrated that 3 of 13 patients experienced clinical benefit, with 1 patient developing a partial response and 2 patients having confirmed stable disease. Immunomonitoring data showed that in patients without clinical benefit, factors linked to immunotolerance increased early after the initiation of ipilimumab, suggesting that early initiation of radiation therapy might be more effective if combined with ipilimumab. CONCLUSIONS: Our findings suggest that the combination of ipilimumab and high-dose radiation therapy is feasible and safe.
Subject(s)
Antineoplastic Agents, Immunological/administration & dosage , Ipilimumab/administration & dosage , Melanoma/secondary , Melanoma/therapy , Radiosurgery/methods , Adult , Aged , Aged, 80 and over , Antineoplastic Agents, Immunological/adverse effects , Combined Modality Therapy/adverse effects , Combined Modality Therapy/methods , Female , Humans , Immune Tolerance , Ipilimumab/adverse effects , Male , Maximum Tolerated Dose , Melanoma/immunology , Middle Aged , Radiotherapy Dosage , Response Evaluation Criteria in Solid Tumors , Young AdultABSTRACT
INTRODUCTION: In the treatment of dermatological disorders, topical drug administration is a mainstay. However, nanoparticle-based carrier systems could improve and expand the current therapeutic range via localized delivery of active ingredients. Areas covered: This review gives a detailed description of lipid- and surfactant-based drug delivery systems which have been explored for topical drug administration. To guide researchers in their choice of delivery system, an informative decision tree is included. Moreover, this review provides a complete overview of the topical or transdermal drug products, currently on the market or under clinical investigation, delivered via the discussed carriers, in the treatment of skin disorders. Expert opinion: Conventional liposomes are still popular in the domain of topical or transdermal drug delivery and dominate the market landscape. However, several other carriers, such as exosomes and niosomes, are being explored which offer distinct advantages over liposomes and should therefore not be disregarded when selecting a proper drug delivery system.
Subject(s)
Drug Delivery Systems , Nanoparticles , Skin Diseases/drug therapy , Administration, Cutaneous , Humans , Lipids/chemistry , Liposomes/metabolism , Skin/metabolism , Skin Absorption , Surface-Active Agents/chemistryABSTRACT
Psoriasis is a complex chronic immune-mediated inflammatory cutaneous disease associated with the development of inflammatory plaques on the skin. Studies proved that the disease results from a deregulated interplay between skin keratinocytes, immune cells and the environment leading to a persisting inflammatory process modulated by pro-inflammatory cytokines and activation of T cells. However, a major hindrance to study the pathogenesis of psoriasis more in depth and subsequent development of novel therapies is the lack of suitable pre-clinical models mimicking the complex phenotype of this skin disorder. Recent advances in and optimization of three-dimensional skin equivalent models have made them attractive and promising alternatives to the simplistic monolayer cultures, immunological different in vivo models and scarce ex vivo skin explants. Moreover, human skin equivalents are increasing in complexity level to match human biology as closely as possible. Here, we critically review the different types of three-dimensional skin models of psoriasis with relevance to their application potential and advantages over other models. This will guide researchers in choosing the most suitable psoriasis skin model for therapeutic drug testing (including gene therapy via siRNA molecules), or to examine biological features contributing to the pathology of psoriasis. However, the addition of T cells (as recently applied to a de-epidermized dermis-based psoriatic skin model) or other immune cells would make them even more attractive models and broaden their application potential. Eventually, the ultimate goal would be to substitute animal models by three-dimensional psoriatic skin models in the pre-clinical phases of anti-psoriasis candidate drugs. Impact statement The continuous development of novel in vitro models mimicking the psoriasis phenotype is important in the field of psoriasis research, as currently no model exists that completely matches the in vivo psoriasis skin or the disease pathology. This work provides a complete overview of the different available in vitro psoriasis models and suggests improvements for future models. Moreover, a focus was given to psoriatic skin equivalent models, as they offer several advantages over the other models, including commercial availability and validity. The potential and reported applicability of these models in psoriasis pre-clinical research is extensively discussed. As such, this work offers a guide to researchers in their choice of pre-clinical psoriasis model depending on their type of research question.
Subject(s)
Models, Biological , Organ Culture Techniques/methods , Psoriasis/physiopathology , Skin/pathology , Tissue Engineering/methods , HumansABSTRACT
The recent interest and elucidation of the JAK/STAT signaling pathway created new targets for the treatment of inflammatory skin diseases (ISDs). JAK inhibitors in oral and topical formulations have shown beneficial results in psoriasis and alopecia areata. Patients suffering from other ISDs might also benefit from JAK inhibition. Given the development of specific JAK inhibitors, the expression patterns of JAKs in different ISDs needs to be clarified. We aimed to analyze the expression of JAK/STAT family members in a set of prevalent ISDs: psoriasis, lichen planus (LP), cutaneous lupus erythematosus (CLE), atopic dermatitis (AD), pyoderma gangrenosum (PG) and alopecia areata (AA) versus healthy controls for (p)JAK1, (p)JAK2, (p)JAK3, (p)TYK2, pSTAT1, pSTAT2 and pSTAT3. The epidermis carried in all ISDs, except for CLE, a strong JAK3 signature. The dermal infiltrate showed a more diverse expression pattern. JAK1, JAK2 and JAK3 were significantly overexpressed in PG and AD suggesting the need for pan-JAK inhibitors. In contrast, psoriasis and LP showed only JAK1 and JAK3 upregulation, while AA and CLE were characterized by a single dermal JAK signal (pJAK3 and pJAK1, respectively). This indicates that the latter diseases may benefit from more targeted JAK inhibitors. Our in vitro keratinocyte psoriasis model displayed reversal of the psoriatic JAK profile following tofacitinib treatment. This direct interaction with keratinocytes may decrease the need for deep skin penetration of topical JAK inhibitors in order to exert its effects on dermal immune cells. In conclusion, these results point to the important contribution of the JAK/STAT pathway in several ISDs. Considering the epidermal JAK3 expression levels, great interest should go to the investigation of topical JAK3 inhibitors as therapeutic option of ISDs.
Subject(s)
Janus Kinase 3/metabolism , Molecular Targeted Therapy , Skin Diseases/drug therapy , Administration, Topical , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Gene Expression Regulation, Enzymologic/drug effects , Humans , Infant , Inflammation/complications , Janus Kinase 3/antagonists & inhibitors , Janus Kinase 3/genetics , Male , Middle Aged , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , STAT Transcription Factors/genetics , STAT Transcription Factors/metabolism , Skin/drug effects , Skin/metabolism , Skin/pathology , Skin Diseases/complications , Skin Diseases/enzymology , Young AdultABSTRACT
This article contains original data, figures and methods used in the characterization of the liposomal carrier 'DDC642' for topical applications, described in "An elastic liposomal formulation for RNAi-based topical treatment of skin disorders: proof-of-concept in the treatment of psoriasis" (Desmet et al., 2016) [1]. Several elastic liposomal formulations have been evaluated for their ability to encapsulate and deliver RNA interference (RNAi) molecules to cultured primary skin cells. The efficiency and effectiveness of these liposomes were compared to that of our previously characterized liposomes, the 'SECosomes' (SEC) (Geusens et al., 2010) [2]. After selection of a potential superior carrier, based on encapsulation and transfection efficiency data (Desmet et al., 2016) [1], the selected DDC642 liposomes were characterized more in-depth. Herein, a detailed characterization of the DDC642 liposome and RNAi-loaded lipoplexes is given, including the matching protocols.
ABSTRACT
RNA interference (RNAi) is a rapidly emerging approach for targeted gene silencing to alleviate disease pathology. However, lack of efficient carriers for targeted delivery delays the clinical translation of RNAi. An interesting target for local RNAi therapeutics is the skin as it allows direct access to target cells. Still, applications are limited due to the effective skin barrier which hinders penetration. Herein, a description is given of a liposomal carrier, called 'DDC642', capable of delivering RNAi molecules to the epidermis of impaired and intact human skin, without targeting the dermis or circulatory system. In a psoriasis tissue model, down-regulation of the psoriasis marker human beta-defensin 2 by DDC642-delivered siRNA was confirmed, providing proof-of-concept. These liposomes thus hold great potential as topical delivery system for RNAi therapeutics in the treatment of numerous skin diseases.
Subject(s)
Gene Transfer Techniques , Psoriasis/therapy , RNA Interference , Administration, Topical , Cholesterol/chemistry , Elasticity , Ethanol/chemistry , Fatty Acids, Monounsaturated/chemistry , Humans , Keratinocytes/metabolism , Liposomes , Melanocytes/metabolism , MicroRNAs/administration & dosage , Phosphatidylethanolamines/chemistry , Psoriasis/genetics , Quaternary Ammonium Compounds/chemistry , RNA, Small Interfering/administration & dosage , beta-Defensins/geneticsABSTRACT
Merkel cell carcinoma (MCC) is a rare aggressive skin tumor which shares histopathological and genetic features with small-cell lung carcinoma (SCLC), both are of neuroendocrine origin. Comparable to SCLC, MCC cell lines are classified into two different biochemical subgroups designated as 'Classic' and 'Variant'. With the aim to identify typical gene-expression signatures associated with these phenotypically different MCC cell lines subgroups and to search for differentially expressed genes between MCC and SCLC, we used cDNA arrays to profile 10 MCC cell lines and four SCLC cell lines. Using significance analysis of microarrays, we defined a set of 76 differentially expressed genes that allowed unequivocal identification of Classic and Variant MCC subgroups. We assume that the differential expression levels of some of these genes reflect, analogous to SCLC, the different biological and clinical properties of Classic and Variant MCC phenotypes. Therefore, they may serve as useful prognostic markers and potential targets for the development of new therapeutic interventions specific for each subgroup. Moreover, our analysis identified 17 powerful classifier genes capable of discriminating MCC from SCLC. Real-time quantitative RT-PCR analysis of these genes on 26 additional MCC and SCLC samples confirmed their diagnostic classification potential, opening opportunities for new investigations into these aggressive cancers.
Subject(s)
Carcinoma, Merkel Cell/genetics , Carcinoma, Small Cell/genetics , Gene Expression Profiling/methods , Lung Neoplasms/genetics , Oligonucleotide Array Sequence Analysis , Skin Neoplasms/genetics , Biomarkers, Tumor , Cell Line, Tumor , Cluster Analysis , DNA, Complementary/metabolism , Diagnosis, Differential , Gene Expression Regulation, Neoplastic , Humans , Phenotype , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Interferon-α (IFN-α) is the only approved adjuvant treatment for high-risk melanoma patients in Europe, but the impact on overall survival is low. Although it is believed that IFN-α exerts its effects through immunomodulation, data on its impact on circulating immune cells are scarce. Flow cytometry was performed on peripheral blood mononuclear cells of eight IFN-α2b-treated stage III melanoma patients and 26 untreated stage III melanoma patients as controls to enumerate myeloid and plasmacytoid dendritic cells (mDC and pDC), monocytic and polymorphonuclear myeloid-derived suppressor cells (mMDSC and pmnMDSC) and cytotoxic and regulatory T-cells (Tregs). The expression of several immunosuppressive markers [indoleamine 2,3-dioxygenase (IDO), programmed-death ligand-1 (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA4)] was explored. IDO activity in the blood was confirmed by ultra-performance liquid chromatography. Compared with controls, IFN-α2b treatment was associated with increased IDO expression by pDCs (P=0.021) and an increased kynurenine/tryptophan ratio in the serum (P=0.004), compatible with IDO enzyme activity. Furthermore, IFN-α2b-treated patients had a decreased mDC/DC ratio (P=0.002), decreased CD3+ lymphocytes (P=0.034) and increased circulating Treg (P<0.001) and PD-L1+cytotoxic T-cell (P=0.001) frequencies. IDO expression is upregulated in circulating pDCs of high-risk melanoma patients treated with adjuvant IFN-α2b. This is associated with tryptophan consumption in the patients' serum and higher Treg and PD-L1+cytotoxic T-cell frequencies. We hypothesize that in IFN-α2b-treated patients, IDO activity acts as a negative feedback mechanism and might limit the clinical efficacy of IFN-α2b therapy. The underlying mechanism should be explored as this could lead to more efficient immunotherapies.
Subject(s)
Dendritic Cells/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/immunology , Interferon-alpha/therapeutic use , Melanoma/drug therapy , Myeloid Cells/immunology , Adult , Aged , B7-H1 Antigen/biosynthesis , CTLA-4 Antigen/biosynthesis , Europe , Female , Flow Cytometry , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/biosynthesis , Indoleamine-Pyrrole 2,3,-Dioxygenase/blood , Leukocytes, Mononuclear/immunology , Male , Melanoma/immunology , Middle Aged , Neoplasm Staging , Skin Neoplasms/drug therapy , Skin Neoplasms/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Regulatory/immunology , Melanoma, Cutaneous MalignantABSTRACT
Recently developed quantitative and high-throughput technologies that allow automated and rapid screening of the whole genome, transcriptome and proteome have revolutionized the field of cancer genetics. At the same time, new challenges are met, e.g. the need for improved data analysis and standardization of tumor sample handling. Even if these issues are resolved, an 'old' problem in genetic tumor analysis remains, i.e. contamination of tumor samples by stromal and surrounding normal cells. To overcome this obstacle, laser capture microdissection (LCM) has been developed in order to procure the cells of interest from stained tissue sections with retention of morphology. In this review we describe the possible down-stream applications of LCM in the genetic analysis of neuroblastoma (NB). Special focus is given to MYCN copy number determination using real-time quantitative polymerase chain reaction (Q-PCR), analysis of 1p-, 3p- and 11q-deletions using loss of heterozygosity analysis and Q-PCR expression analysis of microdissected normal neuroblast cells and NB cells.
Subject(s)
Nervous System Neoplasms/genetics , Neuroblastoma/genetics , Precancerous Conditions/genetics , DNA, Neoplasm/analysis , Humans , Lasers , Loss of Heterozygosity , Nervous System Neoplasms/pathology , Neuroblastoma/pathology , Polymerase Chain Reaction/methods , Precancerous Conditions/pathologyABSTRACT
Malignant peripheral nerve sheath tumors (MPNST) are rare soft-tissue malignancies. The genetic basis of these tumors is still poorly understood. Cytogenetic analyses predominantly revealed complex karyotypes, precluding the identification of recurrent chromosomal changes. We report loss of 1p material in a near-diploid karyotype with few or no additional structural chromosome changes in two sporadic cases of MPNST, indicating an important role of 1p loss in MPNST development. In one of these two tumors, a distal 1p deletion (1p31.2 approximately pter) was detected suggesting involvement of a tumor suppressor gene located within this distal region of 1p. Further evidence for recurrent 1p loss in MPNST was obtained by interphase fluorescence in situ hybridization, which showed loss of 1p material in 3 out of 13 tumors. These findings together with data from the literature suggest that loss of a tumor suppressor gene located within distal 1p is implicated in the pathogenesis of MPNST.
Subject(s)
Chromosomes, Human, Pair 1/genetics , Genes, Tumor Suppressor , Nerve Sheath Neoplasms/genetics , Child , Female , Humans , In Situ Hybridization, Fluorescence , Loss of Heterozygosity/genetics , Male , Middle AgedABSTRACT
Hyperpigmentation is a key feature in a variety of inherited and acquired syndromes. Nonetheless, determining the exact diagnosis only on the clinical phenotype can be challenging, and a detailed search for associated symptoms is often of crucial importance. As pigmentation pathways are regulated by complex signaling transduction cascades (e.g. MSH/cAMP, KIT signaling pathways), the underlying defects leading to elevated melanin production are numerous. With regard to treatment, limited therapeutic options exist, each with specific side effects. In acquired hyperpigmentation, the melanin deposition may, however, be reversible after adequate therapy of the underlying disorder or even disappear spontaneously. In this review, we provide an overview of the biology of hyperpigmentation syndromes classified according to the main underlying defect that deregulates physiological melanogenesis. The identification of novel genes or key players involved in hyperpigmentary disorders is becoming increasingly important in view of the development of safer and more efficient treatments.