Skin in the game: a review of single-cell and spatial transcriptomics in dermatological research.

Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) are two emerging research technologies that uniquely characterize gene expression microenvironments on a cellular or subcellular level. The skin, a clinically accessible tissue composed of diverse, essential cell populations, serves as an ideal target for these high-resolution investigative approaches. Using these tools, researchers are assembling a compendium of data and discoveries in healthy skin as well as a range of dermatologic pathophysiologies, including atopic dermatitis, psoriasis, and cutaneous malignancies. The ongoing advancement of single-cell approaches, coupled with anticipated decreases in cost with increased adoption, will reshape dermatologic research, profoundly influencing disease characterization, prognosis, and ultimately clinical practice.

Short cell cycle duration is a phenotype of human epidermal stem cells.

BACKGROUND: A traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs). METHODS: We examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing. RESULTS: We found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC. CONCLUSIONS: Contrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro.

A single-cell atlas of IL-23 inhibition in cutaneous psoriasis distinguishes clinical response.

Psoriasis vulgaris and other chronic inflammatory diseases improve markedly with therapeutic blockade of interleukin-23 (IL-23) signaling, but the genetic mechanisms underlying clinical responses remain poorly understood. Using single-cell transcriptomics, we profiled immune cells isolated from lesional psoriatic skin before and during IL-23 blockade. In clinically responsive patients, a psoriatic transcriptional signature in skin-resident memory T cells was strongly attenuated. In contrast, poorly responsive patients were distinguished by persistent activation of IL-17-producing T (T17) cells, a mechanism distinct from alternative cytokine signaling or resistance isolated to epidermal keratinocytes. Even in IL-23 blockade-responsive patients, we detected a recurring set of recalcitrant, disease-specific transcriptional abnormalities. This irreversible immunological state may necessitate ongoing IL-23 inhibition. Spatial transcriptomic analyses also suggested that successful IL-23 blockade requires dampening of >90% of IL-17-induced response in lymphocyte-adjacent keratinocytes, an unexpectedly high threshold. Collectively, our data establish a patient-level paradigm for dissecting responses to immunomodulatory treatments.

Tarsal Buckling and Entropion after Levator Aponeurosis Advancement in Patients with Floppy Eyelid Syndrome.

Two patients with floppy eyelid syndrome presented with severe eye pain and foreign body sensation after recent levator aponeurosis advancement. The examination in both patients was notable for entropion of the upper eyelid, and upper lid eversion revealed deformity of the tarsus in both patients. Surgical revision with full-thickness horizontal tarsotomy and limited excision of the abnormal tarsus corrected the deformity. The authors propose a mechanism for this phenomenon and a modification of surgical technique that may prevent this complication.

STmut: a framework for visualizing somatic alterations in spatial transcriptomics data of cancer.

Spatial transcriptomic technologies, such as the Visium platform, measure gene expression in different regions of tissues. Here, we describe new software, STmut, to visualize somatic point mutations, allelic imbalance, and copy number alterations in Visium data. STmut is tested on fresh-frozen Visium data, formalin-fixed paraffin-embedded (FFPE) Visium data, and tumors with and without matching DNA sequencing data. Copy number is inferred on all conditions, but the chemistry of the FFPE platform does not permit analyses of single nucleotide variants. Taken together, we propose solutions to add the genetic dimension to spatial transcriptomic data and describe the limitations of different datatypes.

GLUT3 promotes macrophage signaling and function via RAS-mediated endocytosis in atopic dermatitis and wound healing.

The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow-derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4Rα subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.

Efficacy of teprotumumab therapy in patients with long-duration thyroid eye disease.

PURPOSE OF REVIEW: Teprotumumab, an inhibitor of the insulin-like growth factor 1 receptor (IGF-1R), was approved by the US Food and Drug Administration in January 2020 for the treatment of thyroid eye disease (TED). The clinical trials leading to its approval enrolled patients with recent disease onset and significant inflammatory symptoms and signs. Subsequent real-world teprotumumab use in patients with longer duration of disease also may be effective, and there have been several publications reporting on experience in these patient groups. RECENT FINDINGS: TED results in disfiguring changes such as ocular proptosis and affects visual function by altering extraocular muscle function, leading to diplopia. Compressive optic neuropathy also may occur, and disease manifestations may persist for years. Teprotumumab treatment in cases of TED in which prior interventions (medical or surgical) had failed, or in treatment-naïve patients whose disease had been stable for years, has been reported to improve both clinical signs and symptoms (proptosis, diplopia) and to reduce the pathologic orbital changes as assessed by orbital imaging. SUMMARY: Teprotumumab may be an appropriate treatment for TED regardless of disease duration and irrespective of the presence or absence of markers of active inflammation within the orbit.

Computational modeling and minimization of unintended neuronal excitation in a LIFU stimulation.

The neuromodulation effect of low-intensity focused ultrasound (LIFU) is highly target-specific. Unintended off-target neuronal excitation can be elicited when the beam focusing accuracy and resolution are limited, whereas the resulted side effect has not been evaluated quantitatively. There is also a lack of methods addressing the minimization of such side effects. Therefore, this work introduces a computational model of unintended neuronal excitation during LIFU neuromodulation, which evaluates the off-target activation area (OTAA) by integrating an ultrasound field model with the neuronal spiking model. In addition, a phased array beam focusing scheme called constrained optimal resolution beamforming (CORB) is proposed to minimize the off-target neuronal excitation area while ensuring effective stimulation in the target brain region. A lower bound of the OTAA is analytically approximated in a simplified homogeneous medium, which could guide the selection of transducer parameters such as aperture size and operating frequency. Simulations in a human head model using three transducer setups show that CORB markedly reduces the OTAA compared with two benchmark beam focusing methods. The high neuromodulation resolution demonstrates the capability of LIFU to effectively limit the side effects during neuromodulation, allowing future clinical applications such as treatment of neuropsychiatric disorders.

Noninvasive Brain Stimulation Techniques for Treatment-Resistant Depression: Transcranial Magnetic Stimulation and Transcranial Direct Current Stimulation.

Transcranial magnetic stimulation is a safe, effective, and well-tolerated intervention for depression; it is currently approved for treatment-resistant depression. This article summarizes the mechanism of action, evidence of clinical efficacy, and the clinical aspects of this intervention, including patient evaluation, stimulation parameters selection, and safety considerations. Transcranial direct current stimulation is another neuromodulation treatment for depression; although promising, the technique is not currently approved for clinical use in the United States. The final section outlines the open challenges and future directions of the field.

Isolation of human cutaneous immune cells for single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-seq) allows for high-resolution analysis of transcriptionally dysregulated cell subpopulations in inflammatory diseases. However, it can be challenging to properly isolate viable immune cells from human skin for scRNA-seq due to its barrier properties. Here, we present a protocol to isolate high-viability human cutaneous immune cells. We describe steps for obtaining and enzymatically dissociating a skin biopsy specimen and isolating immune cells using flow cytometry. We then provide an overview of downstream computational techniques to analyze sequencing data. For complete details on the use and execution of this protocol, please refer to Cook et al. (2022)1 and Liu et al. (2022).2.

Digital Medicine System in Veterans With Severe Mental Illness: Feasibility and Acceptability Study.

BACKGROUND: Suboptimal medication adherence is a significant problem for patients with serious mental illness. Measuring medication adherence through subjective and objective measures can be challenging, time-consuming, and inaccurate. OBJECTIVE: The primary purpose of this feasibility and acceptability study was to evaluate the impact of a digital medicine system (DMS) among Veterans (patients) with serious mental illness as compared with treatment as usual (TAU) on medication adherence. METHODS: This open-label, 2-site, provider-randomized trial assessed aripiprazole refill adherence in Veterans with schizophrenia, schizoaffective disorder, bipolar disorder, or major depressive disorder. We randomized 26 providers such that their patients either received TAU or DMS for a period of 90 days. Semistructured interviews with patients and providers were used to examine the feasibility and acceptability of using the DMS. RESULTS: We enrolled 46 patients across 2 Veterans Health Administration sites: 21 (46%) in DMS and 25 (54%) in TAU. There was no difference in the proportion of days covered by medication refill over 3 and 6 months (0.82, SD 0.24 and 0.75, SD 0.26 in DMS vs 0.86, SD 0.19 and 0.82, SD 0.21 in TAU, respectively). The DMS arm had 0.85 (SD 0.20) proportion of days covered during the period they were engaged with the DMS (mean 144, SD 100 days). Interviews with patients (n=14) and providers (n=5) elicited themes salient to using the DMS. Patient findings described the positive impact of the DMS on medication adherence, challenges with the DMS patch connectivity and skin irritation, and challenges with the DMS app that affected overall use. Providers described an overall interest in using a DMS as an objective measure to support medication adherence in their patients. However, providers described challenges with the DMS dashboard and integrating DMS data into their workflow, which decreased the usability of the DMS for providers. CONCLUSIONS: There was no observed difference in refill rates. Among those who engaged in the DMS arm, the proportion of days covered by refills were relatively high (mean 0.85, SD 0.20). The qualitative analyses highlighted areas for further refinement of the DMS. TRIAL REGISTRATION: ClinicalTrials.gov NCT03881449; https://clinicaltrials.gov/ct2/show/NCT03881449.

Defining Patient-Level Molecular Heterogeneity in Psoriasis Vulgaris Based on Single-Cell Transcriptomics.

Identifying genetic variation underlying human diseases establishes targets for therapeutic development and helps tailor treatments to individual patients. Large-scale transcriptomic profiling has extended the study of such molecular heterogeneity between patients to somatic tissues. However, the lower resolution of bulk RNA profiling, especially in a complex, composite tissue such as the skin, has limited its success. Here we demonstrate approaches to interrogate patient-level molecular variance in a chronic skin inflammatory disease, psoriasis vulgaris, leveraging single-cell RNA-sequencing of CD45+ cells isolated from active lesions. Highly psoriasis-specific transcriptional abnormalities display greater than average inter-individual variance, nominating them as potential sources of clinical heterogeneity. We find that one of these chemokines, CXCL13, demonstrates significant correlation with severity of lesions within our patient series. Our analyses also establish that genes elevated in psoriatic skin-resident memory T cells are enriched for programs orchestrating chromatin and CDC42-dependent cytoskeleton remodeling, specific components of which are distinctly correlated with and against Th17 identity on a single-cell level. Collectively, these analyses describe systematic means to dissect cell type- and patient-level differences in cutaneous psoriasis using high-resolution transcriptional profiles of human inflammatory disease.

A single-cell transcriptional gradient in human cutaneous memory T cells restricts Th17/Tc17 identity.

The homeostatic mechanisms that fail to restrain chronic tissue inflammation in diseases, such as psoriasis vulgaris, remain incompletely understood. We profiled transcriptomes and epitopes of single psoriatic and normal skin-resident T cells, revealing a gradated transcriptional program of coordinately regulated inflammation-suppressive genes. This program, which is sharply suppressed in lesional skin, strikingly restricts Th17/Tc17 cytokine and other inflammatory mediators on the single-cell level. CRISPR-based deactivation of two core components of this inflammation-suppressive program, ZFP36L2 and ZFP36, replicates the interleukin-17A (IL-17A), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon gamma (IFNγ) elevation in psoriatic memory T cells deficient in these transcripts, functionally validating their influence. Combinatoric expression analysis indicates the suppression of specific inflammatory mediators by individual program members. Finally, we find that therapeutic IL-23 blockade reduces Th17/Tc17 cell frequency in lesional skin but fails to normalize this inflammatory-suppressive program, suggesting how treated lesions may be primed for recurrence after withdrawal of treatment.

Personalizing repetitive transcranial magnetic stimulation for precision depression treatment based on functional brain network controllability and optimal control analysis.

Brain neuromodulation effectively treats neurological diseases and psychiatric disorders such as Depression. However, due to patient heterogeneity, neuromodulation treatment outcomes are often highly variable, requiring patient-specific stimulation protocols throughout the recovery stages to optimize treatment outcomes. Therefore, it is critical to personalize neuromodulation protocol to optimize the patient-specific stimulation targets and parameters by accommodating inherent interpatient variability and intersession alteration during treatments. The study aims to develop a personalized repetitive transcranial magnetic stimulation (rTMS) protocol and evaluate its feasibility in optimizing the treatment efficiency using an existing dataset from an antidepressant experimental imaging study in depression. The personalization of the rTMS treatment protocol was achieved by personalizing both stimulation targets and parameters via a novel approach integrating the functional brain network controllability analysis and optimal control analysis. First, the functional brain network controllability analysis was performed to identify the optimal rTMS stimulation target from the effective connectivity network constructed from patient-specific resting-state functional magnetic resonance imaging data. The optimal control algorithm was then applied to optimize the rTMS stimulation parameters based on the optimized target. The performance of the proposed personalized rTMS technique was evaluated using datasets collected from a longitudinal antidepressant experimental imaging study in depression (n = 20). Simulation models demonstrated that the proposed personalized rTMS protocol outperformed the standard rTMS treatment by efficiently steering a depressive resting brain state to a healthy resting brain state, indicated by the significantly less control energy needed and higher model fitting accuracy achieved. The node with the maximum average controllability of each patient was designated as the optimal target region for the personalized rTMS protocol. Our results also demonstrated the theoretical feasibility of achieving comparable neuromodulation efficacy by stimulating a single node compared to stimulating multiple driver nodes. The findings support the feasibility of developing personalized neuromodulation protocols to more efficiently treat depression and other neurological diseases.

EMvelop stimulation: minimally invasive deep brain stimulation using temporally interfering electromagnetic waves.

Objective.Recently, the temporal interference stimulation (TIS) technique for focal noninvasive deep brain stimulation (DBS) was reported. However, subsequent computational modeling studies on the human brain have shown that while TIS achieves higher focality of electric fields than state-of-the-art methods, further work is needed to improve the stimulation strength. Here, we investigate the idea of EMvelop stimulation, a minimally invasive DBS setup using temporally interfering gigahertz (GHz) electromagnetic (EM) waves. At GHz frequencies, we can create antenna arrays at the scale of a few centimeters or less that can be endocranially implanted to enable longitudinal stimulation and circumvent signal attenuation due to the scalp and skull. Furthermore, owing to the small wavelength of GHz EM waves, we can optimize both amplitudes and phases of the EM waves to achieve high intensity and focal stimulation at targeted regions within the safety limit for exposure to EM waves.Approach.We develop a simulation framework investigating the propagation of GHz EM waves generated by line current antenna elements and the corresponding heat generated in the brain tissue. We propose two optimization flows to identify antenna current amplitudes and phases for either maximal intensity or maximal focality transmission of the interfering electric fields with EM waves safety constraint.Main results.A representative result of our study is that with two endocranially implanted arrays of size4.2 cm×4.7 cmeach, we can achieve an intensity of 12 V m-1with a focality of3.6 cmat a target deep in the brain tissue.Significance.In this proof-of-principle study, we show that the idea of EMvelop stimulation merits further investigation as it can be a minimally invasive way of stimulating deep brain targets and offers benefits not shared by prior methodologies of electrical or magnetic stimulation.

Out-of-hospital management of unresponsive, apneic, witnessed opioid overdoses: a case series from a supervised consumption site.

OBJECTIVES: There are conflicting recommendations for lay rescuer management of patients who are unresponsive and apneic due to opioid overdose. We evaluated the management of such patients at an urban supervised consumption site. METHODS: At a single urban supervised consumption site in Vancouver, BC, we conducted a retrospective chart review and administrative database linkage of consecutive patients who were unresponsive and apneic following witnessed opioid overdose between January 1, 2012 and December 31, 2017. We linked these visits with regional hospital records to define the entire care episode, which concluded when the patient was discharged from the supervised consumption site, ED, or hospital, or died. The primary outcome was successful resuscitation, defined as alive and neurologically intact (ambulatory and speaking coherently, or alert and oriented, or Glasgow Coma Scale 15) at the conclusion of the care episode. Secondary outcomes included mortality and predefined complications of resuscitation. RESULTS: We collected 767 patients, with a median age of 43 and 81.6% male, with complete follow-up on 763 patients (99.5%). All patients were managed with oxygen and ventilation (100%, 95% CI 0.995-1.0); 715 (93.2%, 95% CI 0.911-0.949) received naloxone; no patients underwent chest compressions (0%, 95% CI 0-0.005). All patients with complete follow-up were alive and neurologically intact at the end of their care episode (100%, 95% CI 0.994-1.0). Overall, 191 (24.9%) patients were transported to hospital, and 15 (2.0%) patients required additional naloxone after leaving the supervised consumption site; 16 (2.1%) developed complications, and 1 patient was admitted to hospital. CONCLUSIONS: At an urban supervised consumption site, all unresponsive, apneic patients with witnessed opioid overdose were successfully resuscitated with oxygen and/or naloxone. No patients required chest compressions.

RéSUMé: OBJECTIFS: Il existe des recommandations contradictoires concernant la prise en charge par des secouristes non professionnels des patients qui ne réagissent pas et sont apnéiques en raison d'une surdose d'opioïdes. Nous avons évalué la prise en charge de ces patients dans un site urbain de consommation supervisée. MéTHODES: Dans un seul site de consommation supervisée urbain à Vancouver, en Colombie-Britannique, nous avons effectué un examen rétrospectif des dossiers et un couplage de bases de données administratives de patients consécutifs qui étaient insensibles et apnéiques après avoir été témoins d'une surdose d'opioïdes entre le 1er janvier 2012 et le 31 décembre 2017. Le résultat primaire était la réussite de la réanimation, définie comme étant vivante et neurologiquement intacte (ambulatoire et parlant de manière cohérente, ou alerte et orientée, ou échelle de coma de Glasgow 15) à la fin de l'épisode de soins. Les résultats secondaires comprenaient la mortalité et les complications prédéfinies de la réanimation. RéSULTATS: Nous avons recueilli 767 patients, avec un âge médian de 43 ans et 81,6 % d'hommes, avec un suivi complet de 763 patients (99,5 %). Tous les patients ont été pris en charge avec de l'oxygène et la ventilation (100 %, IC à 95 % : 0,995-1,0) ; 715 (93,2 %, IC à 95 % : 0,911-0,949) ont reçu de la naloxone ; aucun patient n'a subi de compressions thoraciques (0 %, IC à 95 % : 0-0,005). Tous les patients ayant fait l'objet d'un suivi complet étaient vivants et intacts sur le plan neurologique à la fin de leur épisode de soins (100 %, IC à 95 % : 0,994-1,0). Dans l'ensemble, 191 (24,9 %) patients ont été transportés à l'hôpital, et 15 (2,0 %) patients ont eu besoin de naloxone supplémentaire après avoir quitté le site de consommation supervisée ; 16 (2,1 %) ont développé des complications, et 1 patient a été admis à l'hôpital. CONCLUSIONS: Dans un centre de consommation supervisée urbain, tous les patients apnéiques non réceptifs ayant été témoins d'une surdose d'opioïdes ont été réanimés avec succès avec de l'oxygène et/ou de la naloxone. Aucun patient n'a eu besoin de compressions thoraciques.