Therapeutic base editing and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). For fibroblasts from two patients (Pat1 and Pat2), we applied adenine base editors (ABEs) to correct the pathogenic mutation of COL7A1 or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the COL7A1 mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that transfer of edited patient-derived skin equivalents (i.e., RDEB keratinocytes and PE-corrected RDEB fibroblasts from the RDEB patient) into the skin of immunodeficient mice led to C7 deposition and anchoring fibril formation within the dermal-epidermal junction, suggesting that base editing and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.

Mohs Micrographic Surgery With Digital Pathology Improves Surgical Quality and Efficiency: A Retrospective Cohort Study.

BACKGROUND: Mohs micrographic surgery, involving pathology of the surgical margin, has the lowest recurrence rate for skin cancer. Moreover, because of technological advances, digital pathology systems are gradually being adopted in hospitals. Yongin Severance Hospital was the first hospital to construct a fully digitalized pathology system in Korea. OBJECTIVE: To evaluate the efficiency and characteristics of the digital pathology system for Mohs micrographic surgery. METHODS: The medical records of 80 patients with skin cancer who underwent Mohs micrographic surgery from March 2020 to August 2022 were analyzed for the number of frozen margins, number of stages, operation time, and recurrence rate to compare cases based on the pathology system. RESULTS: Overall, 23 and 57 patients were examined using the conventional and digital pathology systems, respectively. The mean number of final stages was 0.494 lower ( p -value = .008), the time from the previous to the next stage was 0.687-fold shorter ( p = .002), and the rate of switching from positive to negative margins was 1.990 times higher ( p = .044) in the digital than the conventional group. LIMITATIONS: Retrospective single-center experience; short follow-up time. CONCLUSION: Digital pathology reduces operative time and increases accuracy in Mohs micrographic surgery.

Two cases of KRT1 mutation-associated epidermolytic ichthyosis without typical epidermolytic hyperkeratosis in the neonatal skin lesions.

Epidermolytic ichthyosis (EI) is a rare genetic disorder of keratinization caused by mutations in either KRT1 or KRT10. Histopathologically, epidermolytic hyperkeratosis (EHK) is a hallmark of EI. Here, we report two EI cases in which KRT1 mutation was confirmed by molecular study, but without typical EHK present on skin biopsies performed within 1 week of age. Our cases demonstrate that EHK may not be evident in EI if skin biopsy is performed during the neonatal period.

Behavioral evidence for geomagnetic imprinting and transgenerational inheritance in fruit flies.

Certain long-distance migratory animals, such as salmon and sea turtles, are thought to imprint on the magnetic field of their natal area and to use this information to help them return as adults. Despite a growing body of indirect support for such imprinting, direct experimental evidence thereof remains elusive. Here, using the fruit fly as a magnetoreceptive model organism, we demonstrate that exposure to a specific geographic magnetic field during a critical period of early development affected responses to a matching magnetic field gradient later in life. Specifically, hungry flies that had imprinted on a specific magnetic field from 1 of 3 widely separated geographic locations responded to the imprinted field, but not other magnetic fields, by moving downward, a geotactic behavior associated with foraging. This same behavior occurred spontaneously in the progeny of the next generation: female progeny moved downward in response to the field on which their parents had imprinted, whereas male progeny did so only in the presence of these females. These results represent experimental evidence that organisms can learn and remember a magnetic field to which they were exposed during a critical period of development. Although the function of the behavior is not known, one possibility is that imprinting on the magnetic field of a natal area assists flies and their offspring in recognizing locations likely to be favorable for foraging and reproduction.

Targeting inducible costimulator expressed on CXCR5+PD-1+ TH cells suppresses the progression of pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune bullous disease mediated by autoantibodies against desmoglein 3 (DSG3). Inducible costimulator (ICOS) is a costimulatory receptor expressed on T cells and influences the activity of T follicular helper (TFH) cells in various autoimmune diseases, but the roles of ICOS and TFH cells in PV remain unclear. OBJECTIVE: We examined the immunological characteristics, antigen specificity, and pathogenicity of CD4+ T-cell subpopulations, as well as the therapeutic effect of anti-ICOS blocking antibodies in PV. METHODS: A mouse model of PV was established by adoptive transfer of immune cells from the skin-draining lymph nodes or spleens of DSG3-expressing skin-grafted Dsg3-/- mice into Rag1-/- mice. The TFH cells and CD4+ T cells in PBMCs from PV patients were examined by flow cytometry. RESULTS: Among CD4+ T cells from the mouse model, ICOS-positive TFH cells were associated with B-cell differentiation and were required for disease induction. Using an MHC class II tetramer, DSG3-specific ICOS+ TFH cells were found to be associated with anti-DSG3 antibody production and expanded in the absence of B cells. In human PV, the frequency of ICOS+CXCR5+PD-1+ memory CD4+ T cells correlated with the autoantibody level. Treatment with anti-ICOS blocking antibodies targeting ICOS+ TFH cells decreased the anti-DSG3 antibody level and delayed disease progression in vivo. CONCLUSIONS: Mouse Dsg3-specific ICOS+ TFH cells and human ICOS+CXCR5+PD-1+ TH cells are associated with the anti-DSG3 antibody response in PV. ICOS expressed on CXCR5+PD-1+ TH cells may be a therapeutic target for PV.

Diagnosis and management of pemphigus: Recommendations of an international panel of experts.

BACKGROUND: Several European countries recently developed international diagnostic and management guidelines for pemphigus, which have been instrumental in the standardization of pemphigus management. OBJECTIVE: We now present results from a subsequent Delphi consensus to broaden the generalizability of the recommendations. METHODS: A preliminary survey, based on the European Dermatology Forum and the European Academy of Dermatology and Venereology guidelines, was sent to a panel of international experts to determine the level of consensus. The results were discussed at the International Bullous Diseases Consensus Group in March 2016 during the annual American Academy of Dermatology conference. Following the meeting, a second survey was sent to more experts to achieve greater international consensus. RESULTS: The 39 experts participated in the first round of the Delphi survey, and 54 experts from 21 countries completed the second round. The number of statements in the survey was reduced from 175 topics in Delphi I to 24 topics in Delphi II on the basis of Delphi results and meeting discussion. LIMITATIONS: Each recommendation represents the majority opinion and therefore may not reflect all possible treatment options available. CONCLUSIONS: We present here the recommendations resulting from this Delphi process. This international consensus includes intravenous CD20 inhibitors as a first-line therapy option for moderate-to-severe pemphigus.

Peptide nucleic acid (PNA) probe-based analysis to detect filaggrin mutations in atopic dermatitis patients.

Atopic dermatitis (AD) is a chronic inflammatory skin disease whose prevalence is increasing worldwide. Filaggrin (FLG) is essential for the development of the skin barrier, and its genetic mutations are major predisposing factors for AD. In this study, we developed a convenient and practical method to detect FLG mutations in AD patients using peptide nucleic acid (PNA) probes labelled with fluorescent markers for rapid analysis. Fluorescence melting curve analysis (FMCA) precisely identified FLG mutations based on the distinct difference in the melting temperatures of the wild-type and mutant allele. Moreover, PNA probe-based FMCA easily and accurately verified patient samples with both heterozygote and homozygote FLG mutations, providing a high-throughput method to reliable screen AD patients. Our method provides a convenient, rapid and accurate diagnostic tool to identify potential AD patients allowing for early preventive treatment, leading to lower incidence rates of AD, and reducing total healthcare expenses.

Programmed cell death ligand 1 alleviates psoriatic inflammation by suppressing IL-17A production from programmed cell death 1-high T cells.

BACKGROUND: Psoriasis is one of the most common chronic inflammatory diseases of the skin. Recently, IL-17-producing T cells have been shown to play a critical role in psoriatic inflammation. Programmed cell death 1 (PD-1) is a coinhibitory receptor expressed on T cells in various chronic inflammatory diseases; however, the expression and function of PD-1 during psoriatic inflammation have not previously been characterized. OBJECTIVE: We examined PD-1 expression on IL-17A-producing T cells from imiquimod-treated mice and patients with psoriasis. Additionally, we investigated the therapeutic effect of recombinant programmed cell death ligand 1 (PD-L1) protein on imiquimod-induced psoriatic inflammation. METHODS: PD-1 expression on IL-17A-producing γδ T cells from imiquimod-treated mice was examined by means of multicolor flow cytometric analysis. In the psoriatic skin of patients, PD-1 and IL-17A expression was analyzed by using immunofluorescence. The therapeutic effect of PD-L1-Fc fusion protein (PD-L1-Fc) was assessed in imiquimod-treated mice ex vivo and in vivo. RESULTS: During imiquimod-induced psoriatic inflammation, PD-1 is overexpressed on CD27(-)Vγ1(-) γδ T cells. Furthermore, PD-1 expression on IL-17A(+) T cells was confirmed in psoriatic skin tissues from patients and imiquimod-treated mice. In the CD27(-)Vγ1(-) γδ T-cell population, Vγ4(-) γδ T cells with Vγ6 mRNA expression showed a high level of PD-1 expression. Furthermore, these PD-1(hi)Vγ4(-) (Vγ6(+)) γδ T cells were specialized for anti-CD3-induced IL-17A production, which was inhibited by PD-L1-Fc treatment. In imiquimod-treated mice PD-L1-Fc reduced psoriatic inflammation when given alone and enhanced the therapeutic effect of anti-p40 when given in combination. CONCLUSION: PD-1 is overexpressed in IL-17A-producing T cells in both imiquimod-treated mice and patients with psoriasis. Moreover, recombinant PD-L1-Fc alleviates psoriatic inflammation in imiquimod-treated mice.

Effects of Extremely Low Frequency Electromagnetic Fields on Melanogenesis through p-ERK and p-SAPK/JNK Pathways in Human Melanocytes.

This study evaluated frequency-dependent effects of extremely low frequency electromagnetic fields (ELF-EMFs) on melanogenesis by melanocytes in vitro. Melanocytes were exposed to 2 mT EMFs at 30-75 Hz for 3 days before melanogenesis was examined. Exposure to ELF-EMFs at 50 and 60 Hz induced melanogenic maturation without cell damage, without changing cell proliferation and mitochondrial activity. Melanin content and tyrosinase activity of cells exposed to 50 Hz were higher than in controls, and mRNA expression of tyrosinase-related protein-2 was elevated relative to controls at 50 Hz. Phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) levels were higher than controls in cells exposed to ELF-EMFs at 50-75 Hz. Immunohistochemical staining showed that melanocyte-specific markers (HMB45, Melan-A) were strongly expressed in cells exposed to EMFs at 50 and 60 Hz compared to controls. Thus, exposure to ELF-EMFs at 50 Hz could stimulate melanogenesis in melanocytes, through activation of p-CREB and p-p38 and inhibition of phosphorylated extracellular signal-regulated protein kinase and phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase. The results may form the basis of an appropriate anti-gray hair treatment or be applied in a therapeutic device for inducing repigmentation in the skin of vitiligo patients.

Cutaneous Lymphoma in Korea: A Nationwide Retrospective Study.

The epidemiological and clinicopathological features of cutaneous lymphoma may vary by geographical area. However, only a few large-scale epidemiological studies of cutaneous lymphoma have been performed, mainly in the USA and Europe. This aim of this study was to determine the recent characteristics of cutaneous lymphoma in Korea according to the WHO/EORTC classification. A total of 422 patients with newly diagnosed cutaneous lymphoma from January 2009 to December 2013 comprising 293 cases of mature T-cell and natural killer (NK)-cell lymphoma and 39 cases of mature B-cell lymphoma were retrospectively reviewed. The incidence of mature B-cell lymphoma was lower in Korea than in Europe and the USA. Diffuse large B-cell lymphoma was more prevalent in Korea than in Western countries. The incidence of extranodal NK/T-cell lymphoma, nasal-type was higher in Korea than in Western countries and Japan.

Protective Effect of 10-Hz, 1-mT Electromagnetic Field Exposure Against Hypoxia/Reoxygenation Injury in HK-2 Cells.

We investigated the protective effects of electromagnetic field (EMF) on the survival of the human renal proximal tubular cell line, HK-2, using an in vitro hypoxia/reoxygenation (H/R) injury model. The survival rate of cells cultured under H/R condition declined significantly, while the intracellular reactive oxygen species (ROS) levels markedly increased. The 10 Hz/1 mT EMF exposure reversed the H/R induced reduction in cell survival and induction of intracellular ROS. Our results suggest that 10 Hz/1 mT EMF exposure could inhibit H/R-induced cell death of HK-2 via suppression of intracellular ROS production and that this treatment might be clinically useful for the amelioration of renal ischemia/reperfusion injury.

Mortality of patients with bullous pemphigoid in Korea.

BACKGROUND: The reported mortality rates and prognoses of bullous pemphigoid (BP) vary among different countries. However, the mortality rate of BP has not been investigated in Korea. OBJECTIVE: We sought to evaluate the mortality rate of Korean patients with BP in comparison to that of an age-matched general population in Korea, and to identify prognostic factors affecting overall survival. METHODS: We conducted a retrospective analysis of 168 patients diagnosed with BP between 1993 and 2013 at Gangnam Severance Hospital in Seoul, Korea. RESULTS: The 1-, 2-, and 5-year mortality rates of BP patients were 19.46% (95% confidence interval [CI], 13.83-27.00), 29.13% (95% CI, 21.91-38.08), and 58.03% (95% CI, 47.04-69.45), respectively. The median age was 73 years (range, 6-99 years). The standardized mortality ratio ranged from 2.43 (95% CI, 0.12-11.96) to 9.56 (95% CI, 2.43-26.02), depending on the age group. In multivariate analysis, old age at the time of diagnosis, stroke, diabetes, and delayed diagnosis were associated with increased mortality. LIMITATIONS: This study was conducted as a retrospective analysis and was based at a single institution. CONCLUSION: The mortality rate of patients with BP is significantly higher than that for the general population.

Effects of extremely low frequency magnetic fields on NGF induced neuronal differentiation of PC12 cells.

Extremely low-frequency magnetic fields (ELF-MFs) affect various cellular processes and systems, such as cell proliferation, differentiation and metabolic pathways. The present study investigated ELF-MFs effect on nerve growth factor (NGF) induced neuronal differentiation of PC12 cells using proteomic applications to understand its role in the enhancement of neuronal differentiation. After 50 Hz, 1 mT ELF-MFs 5-day exposure on NGF induced PC12 cells, it was observed to increase neurite length as well as an increase in the number of neurite bearing cells. It was also discovered that there was a decrease in proliferation activity, which is associated with an increase in differentiated cells. Neuronal differentiation related mRNA levels and protein levels were increased in NGF induced PC12 cells. Compared with NGF induced group, ELF-MFs stimulated PC12 cells had different protein expression as measured with two-dimensional electrophoresis (2-DE) gels. Consequently six differentially expressed spots were detected between the 2-DE maps, which were identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF LC/MS/MS) as: peripherin, neurosecretory protein nerve growth factor inducible (VGF8a) precursor, dnaK-type molecular chaperone sp72-ps1 (HSP72-psI), low molecular weight (Mr) phosphotyrosine protein phosphatase isoenzyme AcP1 (LMW-PTP/ACP1), Tubulin alpha-1A (TUBA1A) chain, outcome predictor in acute leukemia 1 homolog (OPA1L). The identification of these proteins provides clues to the mechanism of ELF-MFs stimulation on NGF induced PC12 cells that occur during neuronal differentiation and may contribute to the development novel treatments for neurodegenerative diseases.

Isolated benign primary cutaneous plasmacytosis in a child.

Isolated benign primary cutaneous plasmacytosis in a child is a very rare and benign disease. Herein we present a case of this condition occurring in a child who showed good response to topical corticosteroid.

A quantitative analysis of gait patterns in vestibular neuritis patients using gyroscope sensor and a continuous walking protocol.

BACKGROUND: Locomotion involves an integration of vision, proprioception, and vestibular information. The parieto-insular vestibular cortex is known to affect the supra-spinal rhythm generators, and the vestibular system regulates anti-gravity muscle tone of the lower leg in the same side to maintain an upright posture through the extra-pyramidal track. To demonstrate the relationship between locomotion and vestibular function, we evaluated the differences in gait patterns between vestibular neuritis (VN) patients and normal subjects using a gyroscope sensor and long-way walking protocol. METHODS: Gyroscope sensors were attached to both shanks of healthy controls (n=10) and age-matched VN patients (n = 10). We then asked the participants to walk 88.8 m along a corridor. Through the summation of gait cycle data, we measured gait frequency (Hz), normalized angular velocity (NAV) of each axis for legs, maximum and minimum NAV, up-slope and down-slope of NAV in swing phase, stride-swing-stance time (s), and stance to stride ratio (%). RESULTS: The most dominant walking frequency in the VN group was not different compared to normal control. The NAVs of z-axis (pitch motion) were significantly larger than the others (x-, y-axis) and the values in VN patients tended to decrease in both legs and the difference of NAV between both group was significant in the ipsi-lesion side in the VN group only (p=0.03). Additionally, the gait velocity of these individuals was decreased relatively to controls (1.11 ± 0.120 and 0.84 ± 0.061 m/s in control and VN group respectively, p<0.01), which seems to be related to the significantly increased stance and stride time of the ipsi-lesion side. Moreover, in the VN group, the maximum NAV of the lesion side was less, and the minimum one was higher than control group. Furthermore, the down-slope and up-slope of NAV decreased on the impaired side. CONCLUSION: The walking pattern of VN patients was highly phase-dependent, and NAV of pitch motion was significantly decreased in the ipsi-lesion side. The change of gait rhythm, stance and stride time, and maximum/minimum NAV of the ipsi-lesion side were characteristics of individuals with VN.

Risk factors of rituximab-induced thrombocytopenia in patients with autoimmune bullous diseases.

Rituximab has been the mainstay treatment for autoimmune bullous diseases (AIBDs). Among the side effects of rituximab, rituximab-induced thrombocytopenia (RIT) is a rare but critical complication. However, there have been no reports or identification of risk factors for RIT in patients with AIBD. In our retrospective study, we compared rituximab-treated AIBD in patients with and without thrombocytopenia to explore the risk factors. In addition, we compared two different rituximab protocols (rheumatoid arthritis [RA] and lymphoma) in terms of the incidence and severity of thrombocytopenia. A total of 222 patients were enrolled, and 46 patients (20.7%) developed RIT. Multivariate logistic regression analysis identified age and chronic kidney disease (CKD) as significant factors for RIT. We also found that patients treated with the lymphoma protocol demonstrated a significantly higher mean post-rituximab platelet count compared with those on the RA protocol. This was the first analysis, to our knowledge, of risk factors for RIT in patients with AIBD. Individuals aged 70 or older and those with multiple comorbidities, particularly CKD, should be closely monitored for thrombocytopenia. For patients with CKD, it may be safer to use the lymphoma protocol for rituximab administration as it results in a lesser reduction in post-rituximab platelet count.

Efficacy of oral JAK1 or JAK1/2 inhibitor for treating refractory pruritus in dystrophic epidermolysis bullosa: A retrospective case series.

Refractory pruritus is the most distressing, disease-related symptom in patients with dystrophic epidermolysis bullosa (DEB), inducing an itch-scratch-blister cycle. Chronic inflammation is a hallmark of DEB, thus upregulation of inflammatory cytokines and Janus kinase (JAK) signaling may play a role in DEB-related pruritus. We retrospectively reviewed the medical records of DEB patients with refractory pruritus who were treated with either baricitinib, a JAK1/2 inhibitor, or upadacitinib, a selective JAK1 inhibitor. Patients received baricitinib (4 mg) or upadacitinib (15 mg) once a day for 2-32 weeks. A total of 12 DEB patients (six recessive DEB and six dominant DEB) were included in this study. The mean±SD baseline pruritus visual analog scale (VAS) score was 7.5 ± 1.7. Upadacitinib or baricitinib treatment resulted in a rapid and sustained decrease in itch. Four out of 12 patients (33.3%) and seven out of 10 patients (70%) showed a decrease of at least 3 points in the pruritus VAS score from baseline at weeks 2 and 4, respectively. The mean percentage changes from baseline in pruritus VAS scores at weeks 2 and 4 were -42.9% and -52.7%, respectively. Subgroup analysis showed greater reductions in the pruritus VAS score in the baricitinib group (n = 5) compared to the upadacitinib group (n = 7), and in patients with epidermolysis bullosa pruriginosa (n = 3) compared to other subtypes of DEB (n = 9); however, these differences did not reach statistical significance. Three out of 10 (33.3%) patients showed at least a 2-point reduction in pain intensity from baseline at week 4. Eight out of 12 patients (66.7%) also showed a reduction in the number of new blisters, which correlated with a reduction in the pruritus score. No patient discontinued treatment because of serious adverse events. Our results suggest that JAK1 or JAK1/2 inhibitors could be a promising treatment option for DEB-related pruritus. Long-term safety should be assessed in future studies.

Comparative study of efficacy and safety: Biosimilar rituximab versus originator rituximab in the treatment of pemphigus.

Rituximab is a monoclonal antibody that targets CD20 antigen in B cells. For pemphigus, rituximab has been highly effective in steroid-sparing therapy for moderate to severe cases. Originator rituximab has demonstrated favorable treatment effects in patients with pemphigus, but its high cost remains a challenge. Biosimilar rituximab is expected to offer a potential solution. However, it is required for the comparative study of efficacy and safety between biosimilar and originator because all biosimilars may not be identical to the originator. In this study, we compared the treatment effects and safety of biosimilar (Truxima) and originator (MabThera) rituximab in patients with pemphigus. A final cohort of 52 patients in the MabThera group and 72 patients in the Truxima group was enrolled. Except for the intravenous immunoglobulin administration rate, there were no differences in baseline characteristics between the two groups, and for the purpose of comparing efficacy, investigations into time to complete remission, total steroid intake to complete remission, and total steroid intake for 6 months following rituximab treatment revealed no significant differences between the two groups. Truxima can be considered a relatively affordable alternative treatment option for pemphigus, offering cost-effectiveness to patients who are indicated for the treatment with MabThera.