Indirect immunofluorescence for bullous pemphigoid using ex vivo confocal laser scanning microscopy.
The Journal of dermatology
View details for DOI 10.1111/1346-8138.16773
View details for PubMedID 36914975
In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial.
Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1, the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain-severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.
View details for DOI 10.1038/s41591-022-01737-y
View details for PubMedID 35347281
Simultaneous immunofluorescence and histology in pemphigus vulgaris using ex vivo confocal laser scanning microscopy.
Journal of biophotonics
BACKGROUND: Ex vivo confocal laser scanning microscopy (ex vivo CLSM) provides rapid, high-resolution imaging and immunofluorescence examinations of the excised tissues.OBJECTIVES: Evaluating the applicability of ex vivo CLSM in histomorphological and direct immunofluorescence (DIF) examination of pemphigus vulgaris (PV).METHODS: 20 PV sections were stained with fluorescent-labelled anti- IgG and anti-C3 using various dilutions and incubation periods. Subsequently, the determined ideal staining protocol was applied on 20 additional PV and 20 control sections.RESULTS: Ex vivo CLSM identified intraepidermal blisters and acantholytic cells in 80% and 60% of PV patients, respectively. The sensitivity of ex vivo CLSM in detecting intraepidermal fluorescence was 90% both with IgG and C3. The specificity of staining for IgG and C3 was 70% and 90%, respectively.CONCLUSION: Histomorphological and immunofluorescence features of PV could be detected within the same ex vivo CSLM session showing a comparable performance to conventional histopathology and DIF microscopy. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/jbio.202000509
View details for PubMedID 33491324
In vivo examination of healthy human skin after short-time treatment with moisturizers using confocal Raman spectroscopy and optical coherence tomography: Preliminary observations.
Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI)
Skin is our barrier against environmental damage. Moisturizers are widely used to increase hydration and barrier integrity of the skin; however, there are contrasting observations on their in vivo effects in real-life settings. In cosmetic studies, corneometers and tewameters are traditionally used to assess skin hydration. In this study, two novel noninvasive diagnostic techniques, optical coherence tomography (OCT) and confocal Raman spectroscopy, were used to analyze stratum corneum and epidermal thickness (ET), water content, blood flow in function of depth, skin roughness, attenuation coefficient, natural moisturizing factor, ceramides and free fatty acids, cholesterol, urea, and lactates in 20 female subjects aged between 30 and 45 before and after 2 weeks application of a commercially available moisturizing lotion on one forearm. The untreated forearm served as control. A third measurement was conducted 1 week after cessation of moisturizing to verify whether the changes in the analyzed parameters persisted. We noticed a reduction in skin roughness, an increase in ceramides and free fatty acids and a not statistically significant increase in ET. As a conclusion, short time moisturizing appears insufficient to provide significant changes in skin morphology and composition, as assessed by OCT and RS. Novel noninvasive imaging methods are suitable for the evaluation of skin response to topical moisturizers. Further studies on larger sample size and longer treatment schedules are needed to analyze changes under treatment with moisturizers and to standardize the use of novel noninvasive diagnostic techniques.
View details for DOI 10.1111/srt.13101
View details for PubMedID 34555219
New generation diagnostics in inflammatory skin diseases: immunofluorescence and histopathological assessment using ex vivo confocal laser scanning microscopy in cutaneous lupus erythematosuss.
BACKGROUND: Ex vivo confocal laser scanning microscopy (CLSM) offers real-time examination of excised tissue in reflectance, fluorescence and digital hematoxylin-eosin (H&E)-like staining modes enabling application of fluorescent-labelled antibodies.OBJECTIVES: To assess the diagnostic performance of ex vivo CLSM in identifying histopathological features and lupus band test in cutaneous lupus erythematosus (CLE) with comparison to conventional histopathology and direct immunofluorescence (DIF).MATERIAL AND METHODS: 72 sections of 18 CLE patients were stained with acridine orange (AO), anti-IgG, -IgM and -IgA; 21 control samples were stained with AO. Subsequently, ex vivo CLSM examination of all samples was performed in reflectance, fluorescence and digital H&E-like staining modes.RESULTS: Superficial and deep perivascular inflammatory infiltration (94.4%), interface dermatitis (88.9%), spongiosis (83.3%) and vacuolar degeneration (77.7%) were the most common features detected with ex vivo CLSM. Kappa test revealed a level of agreement ranging within "perfect" to "good" between ex vivo CLSM and conventional histopathology. ROC analysis showed that the combination of perivascular infiltration, interface dermatitis and spongiosis detected by ex vivo CLSM has the potential to distinguish between CLE and controls. Basement membrane immunoreactivity with IgG, IgM and IgA was identified in 88.8% (n=15), 55.5% (n=10) and 55.5% (n=10) of the CLE samples using ex vivo CLSM, respectively. Whereas, DIF showed IgG, IgM and IgA positivity in 94.4% (n=17), 100% (n=18) and 88.9% (n=16) of patients, respectively.CONCLUSION: Ex vivo CLSM enables simultaneous histopathological and immunofluorescence examination in CLE showing a high agreement with conventional histopathology, albeit with a lower performance than conventional DIF.
View details for DOI 10.1111/exd.14265
View details for PubMedID 33345402
Immunofluorescence and histopathological assessment using ex-vivo confocal laser scanning microscopy in lichen planus.
Journal of biophotonics
Ex-vivo confocal laser scanning microscopy (CLSM) provides rapid, high-resolution imaging, fluorescence detection and digital hematoxylin-eosin (H&E)-like staining.Assessing the performance of ex-vivo CLSM in identifying histomorphology and immunoreactivity in lichen planus (LP) and comparing its accuracy with conventional histopathology and direct immunofluorescence (DIF).33 sections of 17 LP patients stained with acridine orange (AO) and FITC-labelled anti-fibrinogen antibody and 21 control samples stained with AO were examined using ex-vivo CLSM.Ex-vivo CLSM was in perfect agreement with conventional histopathology in identifying interface dermatitis, vacuolar degeneration and band-like infiltration. ROC analysis showed that the presence of vacuolar degeneration, interface dermatitis and band-like infiltration was useful to distinguish LP sections from controls (p<0.0001). The detection rates of fibrinogen deposition using DIF and ex-vivo CLSM were 93.8% and 62.5%, respectively.Ex-vivo CLSM enables histopathological and immunofluorescence examination in LP with the advantage of digital H&E-like staining. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/jbio.202000328
View details for PubMedID 33025741