RESUMO
Identification of synaptic partners is a fundamental task for systems neuroscience. To date, few reliable techniques exist for whole brain labeling of downstream synaptic partners in a cell-type-dependent and monosynaptic manner. Herein, we describe a novel monosynaptic anterograde tracing system based on the deletion of the gene UL6 from the genome of a cre-dependent version of the anterograde Herpes Simplex Virus 1 strain H129. Given that this knockout blocks viral genome packaging and thus viral spread, we reasoned that co-infection of a HSV H129 ΔUL6 virus with a recombinant adeno-associated virus expressing UL6 in a cre-dependent manner would result in monosynaptic spread from target cre-expressing neuronal populations. Application of this system to five nonreciprocal neural circuits resulted in labeling of neurons in expected projection areas. While some caveats may preclude certain applications, this system provides a reliable method to label postsynaptic partners in a brain-wide fashion.
Assuntos
Herpesvirus Humano 1 , Herpesvirus Humano 1/genética , Neurônios , EncéfaloRESUMO
Immunoregulatory effects of IL-4 and IL-13 and alterations of keratinocyte (KC) differentiation are important factors in the pathogenesis of atopic dermatitis. This study investigated the role of IL-4 and IL-13 in KC responses to changes in extracellular calcium (Ca2+) and analyzed differentiation signals elicited via a Ca2+ sensor, SMOC1. Real-time dynamics of transmembrane Ca2+ influx were assessed in live KCs by flow cytometry and microscopy. Exposure of KCs to a high Ca2+ environment (1.3 mM) triggered a rapid intracellular Ca2+ influx, whereas IL-4- and IL-13-treated cells exhibited a significant decrease in the peak amplitude of Ca2+ influx (P < 0.01). IL-17A and IL-22 did not elicit such responses. Evaluation of intracellular Ca2+ dynamics by microscopy confirmed these observations and revealed heterogeneity of individual KC responses. IL-4 and IL-13 significantly inhibited the expression of Ca2+-binding protein SMOC1 (P < 0.001). Inhibition of epidermal differentiation markers were also observed in SMOC1 small interfering RNA-transfected KCs. Concurrently, the deletion of SMOC1 increased the amplitude of Ca2+ peak response (P < 0.05). In conclusion, our results provide innovative data that IL-4 and IL-13 regulate KC sensitivity to microenvironmental Ca2+ changes and inhibit Ca2+-induced KC differentiation signals. SMOC1 inhibition by IL-4 and IL-13 alters Ca2+ transport in KCs and inhibits differentiation, suggesting a new target for treatment of atopic dermatitis.
Assuntos
Cálcio/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Queratinócitos/metabolismo , Osteonectina/metabolismo , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Perfilação da Expressão Gênica , Humanos , Microscopia Intravital , Queratinócitos/imunologia , Osteonectina/genética , Cultura Primária de Células , Análise de Célula ÚnicaRESUMO
BACKGROUND: Monitoring the effects of biologic therapies in skin diseases will benefit from alternative noninvasive skin sampling techniques to evaluate immune pathways in diseased tissue early and longitudinally. OBJECTIVE: To establish a minimally invasive profiling of skin cytokines for diagnosis, therapeutic response monitoring, and clinical research in atopic dermatitis (AD) and other skin diseases, particularly in pediatric cohorts. METHODS: We developed a novel method for cytokine profiling in the epidermis using skin tape strips (STSs) in a setting designed to maximize the efficiency of protein extraction from STSs. This method was applied to analyze STS protein extracts from the lesional skin of children having AD (n = 41) and normal, healthy controls (n = 22). A total of 20 cytokines were probed with the ultrasensitive Mesoscale multiplex cytokine assay. RESULTS: A significant increase in interleukin (IL)-1b (P < .01), IL-18 (P < .001), and IL-8 (P < .001) with a decrease in IL-1a (P < .001) in the stratum corneum of AD lesional skin was found. Concurrently, an increase in markers associated with type 2 inflammatory response was readily detectable in AD lesional skin, including C-C motif chemokine ligand (CCL) 22, CCL 17, and thymic stromal lymphopoietin (TSLP). The levels of IL-1b, IL-18, and TSLP exhibited positive correlations with the AD severity index (Scoring AD index) and skin transepidermal water loss (TEWL), whereas an inverse correlation between IL-1a and Scoring AD index and IL-1a and TEWL was found. The levels of CCL17, CCL22, TSLP, IL-22, and IL-17a correlated with skin TEWL measurements. CONCLUSION: Using minimally invasive STS analysis, we identified cytokine profiles easily sampled in AD lesional skin. The expression of these markers correlated with disease severity and reflected changes in TEWL in lesional skin. These markers suggest new response assessment targets for AD skin. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03168113.
Assuntos
Biomarcadores , Citocinas/metabolismo , Dermatite Atópica/etiologia , Dermatite Atópica/metabolismo , Mediadores da Inflamação/metabolismo , Fitas Reagentes , Dermatite Atópica/diagnóstico , Humanos , Índice de Gravidade de Doença , Avaliação de SintomasRESUMO
In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or "leak" expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinase-specific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant "cross-over insensitive" recognition sites combined with an "ATG-out" design. We show that these CIAO (cross-over insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.