RESUMEN
Immune cells and epithelium form sophisticated barrier systems in symbiotic relationships with microbiota. Evidence suggests that immune cells can sense microbes through intact barriers, but regulation of microbial commensalism remain largely unexplored. Here, we uncovered spatial compartmentalization of skin-resident innate lymphoid cells (ILCs) and modulation of sebaceous glands by a subset of RORγt+ ILCs residing within hair follicles in close proximity to sebaceous glands. Their persistence in skin required IL-7 and thymic stromal lymphopoietin, and localization was dependent on the chemokine receptor CCR6. ILC subsets expressed TNF receptor ligands, which limited sebocyte growth by repressing Notch signaling pathway. Consequently, loss of ILCs resulted in sebaceous hyperplasia with increased production of antimicrobial lipids and restricted commensalism of Gram-positive bacterial communities. Thus, epithelia-derived signals maintain skin-resident ILCs that regulate microbial commensalism through sebaceous gland-mediated tuning of the barrier surface, highlighting an immune-epithelia circuitry that facilitates host-microbe symbiosis.
Asunto(s)
Linfocitos/inmunología , Glándulas Sebáceas/metabolismo , Glándulas Sebáceas/microbiología , Animales , Bacterias/metabolismo , Citocinas/metabolismo , Epitelio/inmunología , Folículo Piloso/metabolismo , Folículo Piloso/microbiología , Inmunidad Innata , Interleucina-7/metabolismo , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microbiota/inmunología , Receptores CCR6/metabolismo , Receptores Notch/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Glándulas Sebáceas/inmunología , Piel/metabolismo , Fenómenos Fisiológicos de la Piel , Simbiosis , Linfopoyetina del Estroma TímicoRESUMEN
Hypodermis is the predominant site of Staphylococcus aureus infections that cause cellulitis. Given the importance of macrophages in tissue remodeling, we examined the hypodermal macrophages (HDMs) and their impact on host susceptibility to infection. Bulk and single-cell transcriptomics uncovered HDM subsets with CCR2-dichotomy. HDM homeostasis required the fibroblast-derived growth factor CSF1, ablation of which abrogated HDMs from the hypodermal adventitia. Loss of CCR2- HDMs resulted in accumulation of the extracellular matrix component, hyaluronic acid (HA). HDM-mediated HA clearance required sensing by the HA receptor, LYVE-1. Cell-autonomous IGF1 was required for accessibility of AP-1 transcription factor motifs that controlled LYVE-1 expression. Remarkably, loss of HDMs or IGF1 limited Staphylococcus aureus expansion via HA and conferred protection against cellulitis. Our findings reveal a function for macrophages in the regulation of HA with an impact on infection outcomes, which may be harnessed to limit the establishment of infection in the hypodermal niche.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/fisiología , Celulitis (Flemón)/metabolismo , Macrófagos/metabolismo , Matriz ExtracelularRESUMEN
Hair follicles (HFs) function as hubs for stem cells, immune cells, and commensal microbes, which must be tightly regulated during homeostasis and transient inflammation. Here we found that transmembrane endopeptidase ADAM10 expression in upper HFs was crucial for regulating the skin microbiota and protecting HFs and their stem cell niche from inflammatory destruction. Ablation of the ADAM10-Notch signaling axis impaired the innate epithelial barrier and enabled Corynebacterium species to predominate the microbiome. Dysbiosis triggered group 2 innate lymphoid cell-mediated inflammation in an interleukin-7 (IL-7) receptor-, S1P receptor 1-, and CCR6-dependent manner, leading to pyroptotic cell death of HFs and irreversible alopecia. Double-stranded RNA-induced ablation models indicated that the ADAM10-Notch signaling axis bolsters epithelial innate immunity by promoting ß-defensin-6 expression downstream of type I interferon responses. Thus, ADAM10-Notch signaling axis-mediated regulation of host-microbial symbiosis crucially protects HFs from inflammatory destruction, which has implications for strategies to sustain tissue integrity during chronic inflammation.
Asunto(s)
Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Disbiosis/inmunología , Folículo Piloso/patología , Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Receptores Notch/inmunología , Piel/microbiología , Alopecia/inmunología , Alopecia/patología , Animales , Corynebacterium , Disbiosis/patología , Femenino , Folículo Piloso/inmunología , Inmunidad Innata , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Ratones , Transducción de Señal/inmunología , Piel/inmunología , Piel/patologíaRESUMEN
Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1f/f:HoxB7-Cre and Runx1f/f:R26-CreERT2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal , Riñón , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Riñón/metabolismo , Riñón/embriología , Riñón/crecimiento & desarrollo , Ratones , Macaca fascicularis , Regulación del Desarrollo de la Expresión Génica , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , División del ARN , Thermus thermophilus/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Secuencia de Bases , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/ultraestructura , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Endorribonucleasas/ultraestructura , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Thermus thermophilus/enzimologíaRESUMEN
Conventional dendritic cells (cDCs) derive from bone marrow (BM) precursors that undergo cascades of developmental programs to terminally differentiate in peripheral tissues. Pre-cDC1s and pre-cDC2s commit in the BM to each differentiate into CD8α+/CD103+ cDC1s and CD11b+ cDC2s, respectively. Although both cDCs rely on the cytokine FLT3L during development, mechanisms that ensure cDC accessibility to FLT3L have yet to be elucidated. Here, we generated mice that lacked a disintegrin and metalloproteinase (ADAM) 10 in DCs (Itgax-cre × Adam10-fl/fl; ADAM10∆DC) and found that ADAM10 deletion markedly impacted splenic cDC2 development. Pre-cDC2s accumulated in the spleen with transcriptomic alterations that reflected their inability to differentiate and exhibited abrupt failure to survive as terminally differentiated cDC2s. Induced ADAM10 ablation also led to the reduction of terminally differentiated cDC2s, and restoration of Notch signaling, a major pathway downstream of ADAM10, only modestly rescued them. ADAM10∆DC BM failed to generate cDC2s in BM chimeric mice with or without cotransferred ADAM10-sufficient BM, indicating that cDC2 development required cell-autonomous ADAM10. We determined cDC2s to be sources of soluble FLT3L, as supported by decreased serum FLT3L concentration and the retention of membrane-bound FLT3L on cDC2 surfaces in ADAM10∆DC mice, and by demonstrating the release of soluble FLT3L by cDC2 in ex vivo culture supernatants. Through in vitro studies utilizing murine embryonic fibroblasts, we determined FLT3L to be a substrate for ADAM10. These data collectively reveal cDC2s as FLT3L sources and highlight a cell-autonomous mechanism that may enhance FLT3L accessibility for cDC2 development and survival.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Diferenciación Celular/inmunología , Células Dendríticas/fisiología , Proteínas de la Membrana/metabolismo , Bazo/citología , Proteína ADAM10/genética , Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Animales , Trasplante de Médula Ósea , Membrana Celular/inmunología , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/inmunología , Micropartículas Derivadas de Células/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Fibroblastos , Células Madre Hematopoyéticas/fisiología , Inmunidad Celular , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Bazo/inmunología , Quimera por TrasplanteRESUMEN
The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , ARN Bacteriano/metabolismo , Ribonucleasas/metabolismo , Thermus thermophilus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Secuenciación de Nucleótidos de Alto Rendimiento , Microscopía Electrónica , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína , ARN Bacteriano/química , ARN Bacteriano/genética , Ribonucleasas/química , Ribonucleasas/genética , Análisis de Secuencia de ARN , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Thermus thermophilus/genéticaRESUMEN
[Purpose] To determine the effects of lower limb ergometer exercise on the spasticity and joint range of motion of the lower extremity and gait function in patients with cerebral palsy and spastic paralysis. [Participants and Methods] This study included 8 participants with cerebral palsy and spastic paralysis (GMFCS levels I to IV) who received care at the outpatient clinic. After a 5-min rest, the lower limb ergometer exercises were performed for 10â min. We measured the participants' arterial blood pressure, pulse rate, passive range of knee joint extension, muscle tone using the Modified Ashworth scale (MAS) and Modified Tardieu scale (MTS), and 10-m walk test (10MWT). Measurements were collected three times (at baseline before exercise, immediately at the end of exercise, and 5â min after exercise during recovery). [Results] The 10-min lower limb ergometer exercise significantly improved the knee joint extension, MAS and MTS scores, and reduced lower extremity spasticity. Furthermore, it significantly increased the range of knee joint extension and decreased the 10MWT score. [Conclusion] The results showed that the 10-minute lower limb ergometer exercise is beneficial in reducing the spasticity of the lower limb muscles and in increasing the range of motion of knee extension in paraplegic patients with cerebral palsy, suggesting that its implementation in young children could prevent spasticity and enhance motor function.
RESUMEN
Disposal of apoptotic cells is important for tissue homeostasis. Defects in this process in immune tissues may lead to breakdown of self-tolerance against intracellular molecules, including nuclear components. Development of diverse anti-nuclear Abs (ANAs) is a hallmark of lupus, which may arise, in part, due to impaired apoptotic cell clearance. In this work, we demonstrate that spontaneous germinal centers (GCs) in lupus-prone mice contain significantly elevated levels of unengulfed apoptotic cells, which are otherwise swiftly engulfed by tingible body macrophages. We indicate that osteopontin (OPN) secreted by CD153(+) senescence-associated T cells, which selectively accumulate in the GCs of lupus-prone mice, interferes with phagocytosis of apoptotic cells specifically captured via MFG-E8. OPN induced diffuse and prolonged Rac1 activation in phagocytes via integrin αvß3 and inhibited the dissolution of phagocytic actin cup, causing defective apoptotic cell engulfment. In wild-type B6 mice, administration of TLR7 ligand also caused spontaneous GC reactions with increasing unengulfed apoptotic cells and ANA production, whereas B6 mice deficient for Spp1 encoding OPN showed less apoptotic cells and developed significantly reduced ANAs in response to TLR7 ligand. Our results suggest that OPN secreted by follicular CD153(+) senescence-associated T cells in GCs promotes a continuous supply of intracellular autoantigens via apoptotic cells, thus playing a key role in the progression of the autoreactive GC reaction and leading to pathogenic autoantibody production in lupus-prone mice.
Asunto(s)
Anticuerpos Antinucleares/biosíntesis , Apoptosis , Centro Germinal/fisiología , Lupus Eritematoso Sistémico/inmunología , Osteopontina/fisiología , Animales , Ligando CD30/análisis , Células Cultivadas , Integrina alfaVbeta3/fisiología , Lupus Eritematoso Sistémico/patología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/fisiología , Fagocitosis , Receptor Toll-Like 7/fisiología , Proteína de Unión al GTP rac1/fisiologíaRESUMEN
Immune aging results in diminished adaptive immunity and increased risk for autoimmunity. We previously reported a unique PD-1(+) CD44(high)CD4(+) T cell population that increases with age in normal mice. In this study, we indicate that the age-dependent PD-1(+) CD44(high)CD4(+) T cells develop as unique T follicular (TF) cells in a B cell-dependent manner and consist of two subpopulations, as follows: CD153(+) cells preferentially secreting abundant osteopontin on TCR stimulation and CD153(-) cells that are apparently TCR anergic. These unique TF cells with essentially similar features increase much earlier and are accumulated in the spontaneous germinal centers (GCs) in lupus-prone female BWF1 (f-BWF1) mice. These TF cells showed characteristic cell-senescence features and developed in association with extensive CD4(+) T cell proliferation in vivo, suggesting replicative senescence. Although the CD153(+) TF cells were defective in proliferation capacity, they were quite stable and specifically responded to self GC-B cells to secret abundant osteopontin, which inhibited B cell receptor-induced GC-B cell apoptosis in f-BWF1 mice. Transfer of CD153(+) PD-1(+) CD4(+) T cells promoted the growth of spontaneous GCs, whereas administration of anti-osteopontin Ab suppressed GC enlargement and anti-nuclear Ab production and ameliorated clinical lupus nephritis of f-BWF1 mice. Current results suggest that senescent CD153(+) TF cells generated as a consequence of extensive endogenous CD4(+) T cell proliferation play an essential, if not sufficient, role in lupus pathogenesis in lupus-prone genetic background and may also contribute to an increased autoimmunity risk with age.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Senescencia Celular/inmunología , Nefritis Lúpica/inmunología , Nefritis Lúpica/metabolismo , Osteopontina/biosíntesis , Animales , Apoptosis , Linfocitos B/inmunología , Linfocitos B/metabolismo , Ligando CD30/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Femenino , Centro Germinal/inmunología , Centro Germinal/metabolismo , Inmunofenotipificación , Nefritis Lúpica/patología , Activación de Linfocitos/inmunología , Masculino , Ratones , Fenotipo , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
BACKGROUND: Spasticity is a major disabling symptom in stroke patients. Clinically, one of the goals of management of stroke patients should be to reduce spasticity. Recent evidence suggests that motor recovery after stroke comprises a hierarchical, dynamic framework of interacting mechanisms in brain cortex. We hypothesized that unaffected arm exercise can stimulate the ipsilateral motor cortex and change the affected upper limb function and spasticity in stroke patients. To test the hypothesis, we evaluated the effects of unaffected arm exercise on spasticity of the affected upper limb and motor function in stroke patients. METHODS: The study was performed in 41 chronic stroke patients with upper limb hemiparesis. Affected upper limb spasticity and function were assessed at baseline and after each intervention by the modified Ashworth Scale and Fugl-Meyer Assessment, respectively. Patients were also evaluated clinically by the modified Rankin Scale, Functional Independence Measurement and National Institutes of Health Stroke Scale. Subjects stood for 10 min during the control period, and then cycled an arm crank ergometer at 50% of maximum work load for 10 min by the unaffected arm in standing position. RESULTS: The mean age at study entry was 64.6 ± 1.7 years. The latency between onset of stroke and the study was 109.0 ± 17.0 months (range, 6-495). The cause of hemiparesis was cerebral infarction (n = 21), intracerebral hemorrhage (n = 17) or subarachnoid hemorrhage (n = 3). Exercise significantly improved the modified Ashworth Scale compared with baseline (p < 0.0001). No such change was noted after the control intervention. The Fugl-Meyer Assessment score did not change after exercise compared with baseline (p = 0.95). CONCLUSIONS: We conclude that 10 min of unaffected arm exercise improves the affected upper limb spasticity in stroke patients. Further studies are needed to determine the exact mechanism of such improvement and the long-term effects of unaffected arm exercise on motor performance.
Asunto(s)
Brazo/fisiopatología , Terapia por Ejercicio , Espasticidad Muscular/fisiopatología , Espasticidad Muscular/rehabilitación , Paresia/rehabilitación , Rehabilitación de Accidente Cerebrovascular , Anciano , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Espasticidad Muscular/etiología , Recuperación de la Función/fisiología , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/fisiopatología , Resultado del TratamientoRESUMEN
OVOL1 is an important transcription factor for epidermal keratinization, which suppresses proliferation and switches on the differentiation of keratinocytes. A recent genome-wide association study has revealed that OVOL1 is one of the genes associated with susceptibility to atopic dermatitis. Although it is known to be expressed in murine skin and hair follicles, no investigations have focused on its localization in human skin. In the present study, we thus immunolocalized the expression of OVOL1 in normal and diseased human skin. In normal human skin, OVOL1 was preferentially expressed in the suprabasal layer of the epidermis, inner root sheath of hair, mature sebocytes and the ductal portion of the eccrine glands. Compared to this, no remarkable change in the expression of OVOL1 was observed among inflammatory skin diseases. The expression of OVOL1 was evident in eccrine poroma and hidradenoma. Moreover, it was overexpressed in Bowen's disease and sebaceous adenoma, in sharp contrast to its downregulation in their more malignant counterparts, squamous cell carcinoma and sebaceous carcinoma. OVOL1 may play an important role in human skin morphogenesis and tumorigenesis.
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Proteínas de Unión al ADN/análisis , Cabello/química , Glándulas Sebáceas/química , Neoplasias Cutáneas/química , Factores de Transcripción/análisis , Glándulas Ecrinas/química , Inmunohistoquímica , Piel/química , Enfermedades de la Piel/metabolismoRESUMEN
SbtR is one of the four TetR family transcriptional regulators present in the extremely thermophilic bacterium, Thermus thermophilus HB8. We identified 10 genes controlled by four promoters with negative regulation by SbtR in vitro. The SbtR-regulated gene products include probable transporters, probable enzymes for sugar or amino acid metabolism, and nucleic acid-related enzymes. SbtR binds pseudopalindromic sequences, with the consensus sequence of 5'-TGACCCNNKGGTCA-3' surrounding the promoters, and has a proposed 1:1 dimer binding stoichiometry. The X-ray crystal structure analysis revealed that SbtR comprises either nine or 10 α-helices and forms a dimer, as in the typical TetR family proteins. Similar to many characterized TetR family regulators, SbtR has a predicted ligand-binding pocket at the center of each monomer. Interestingly, the SbtR dimer contains an intermolecular disulfide bridge, formed between the Cys164 residues at the entrance of the pocket. The Cys164Ser and Cys164Ala mutant SbtR proteins formed homodimers similar to that of the wild type, but their thermal stabilities were lower by about 8°C, indicating that the disulfide bridge contributes to the thermal stability of the protein. However, altered repression activity of the mutants was not observed in vitro. From these results, we propose that ligand-binding is essential for SbtR to disengage from DNA, in a similar manner to the other characterized TetR family regulators. The formation and reduction of the disulfide bond might function in controlling the ligand-binding affinity of this transcriptional regulator.
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Proteínas Bacterianas/química , Resistencia a la Tetraciclina/genética , Thermus thermophilus/genética , Factores de Transcripción/química , Transcripción Genética , Secuencia de Bases , Secuencia de Consenso , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , Thermus thermophilus/químicaRESUMEN
Atopic dermatitis (AD) is a multifactorial disease with underlying barrier disruption and altered microbial flora, resulting in dry skin and eczematous inflammation with persistent pruritis. Mouse models have been heavily used to investigate AD pathophysiology. Among various AD mouse models, AD-like inflammation induced by topical calcipotriol, a vitamin D3 analog referred to as MC903 in experimental settings, is a versatile model that can be applied to any strain of mice, which can be used for immunologic and morphologic studies. Herein, we provide basic protocols for the topical application of MC903 and approaches to assess phenotypes. After inducing AD-like inflammation, the skin is harvested for flow cytometry analysis, as well as for histologic and immunofluorescence microscopy analyses. The combination of these approaches enables accurate characterization of the degree of inflammation, type of inflammatory infiltrate, and localization of immune infiltrates. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Application of MC903 and gross phenotype assessment Basic Protocol 2: Processing skin for flow cytometry analysis Support Protocol: Skin immune cell surface staining and flow cytometry analysis Basic Protocol 3: Harvesting skin for histologic analysis Basic Protocol 4: Immunofluorescence staining to identify immune cell infiltrates.
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Dermatitis Atópica , Ratones , Animales , Dermatitis Atópica/genética , Dermatitis Atópica/patología , Piel/metabolismo , Piel/patología , Colecalciferol/efectos adversos , Colecalciferol/metabolismo , Inflamación/metabolismo , Inflamación/patología , FenotipoRESUMEN
The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.
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Interleucina-2 , Factor de Transcripción STAT5 , Animales , Ratones , Elementos de Facilitación Genéticos/genética , Interleucina-2/genética , Interleucina-2/farmacología , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Receptores de Interleucina-2 , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismoRESUMEN
PfmR is one of four TetR family transcriptional regulators found in the extremely thermophilic bacterium, Thermus thermophilus HB8. We identified three promoters with strong negative regulation by PfmR, both in vivo and in vitro. PfmR binds pseudopalindromic sequences, with the consensus sequence of 5'-TACCGACCGNTNGGTN-3' surrounding the promoters. According to the amino acid sequence and three-dimensional structure analyses of the PfmR-regulated gene products, they are predicted to be involved in phenylacetic acid and fatty acid metabolism. In vitro analyses revealed that PfmR weakly cross-regulated with the TetR family repressor T. thermophilus PaaR, which controls the expression of the paa gene cluster putatively involved in phenylacetic acid degradation but not with another functionally identified TetR family repressor, T. thermophilus FadR, which is involved in fatty acid degradation. The X-ray crystal structure of the N-terminal DNA-binding domain of PfmR and the nucleotide sequence of the predicted PfmR-binding site are quite similar to those of the TetR family repressor QacR from Staphylococcus aureus. Similar to QacR, two PfmR dimers bound per target DNA. The bases recognized by QacR within the QacR-binding site are conserved in the predicted PfmR-binding site, and they were important for PfmR to recognize the binding site and properly assemble on it. The center of the PfmR molecule contains a tunnel-like pocket, which may be the ligand-binding site of this regulator.
Asunto(s)
Thermus thermophilus/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: The purpose of this study was to elucidate the clinicoradiologic characteristics of pseudolesions of the liver in patients with hepatocellular carcinoma (HCC) treated with transarterial chemoembolization (TACE) as observed on gadoxetate disodium-enhanced MR images. A particular interest was correlation between the pseudolesion characteristics and TACE-MRI interval, during which sequential changes in pseudolesions may be revealed after TACE. MATERIALS AND METHODS: Forty-eight patients with HCC who underwent gadoxetate disodium-enhanced MRI after TACE were retrospectively recruited. Pseudolesions were defined as areas of decreased signal intensity in treated areas on hepatocellular phase images that were confirmed to be nontumorous areas at follow-up. The prevalence and MRI features of pseudolesions were correlated with various clinical parameters, including TACE-MRI interval. RESULTS: Pseudolesions were found in 14 patients (29%). Within 1 month of TACE, the prevalence of pseudolesions was 83%. All of the pseudolesions had arterial enhancement, mimicking residual HCC. After 1 month, the prevalence of pseudolesions decreased, and these pseudolesions tended to exhibit no abnormality in any sequence other than the hepatocellular phase. Results of multivariate analysis suggested that size of HCC (p < 0.0001), duration of postembolization syndrome (p = 0.012), and TACE-MRI interval (p = 0.038) are independent indicators of the presence of pseudolesions. CONCLUSION: The prevalence and appearance of pseudolesions differ at different intervals from TACE. Radiologists need to recognize the clinicoradiologic characteristics to differentiate pseudolesions from true residual or recurrent HCC.
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Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Quimioembolización Terapéutica/métodos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Imagen por Resonancia Magnética/métodos , Anciano , Anciano de 80 o más Años , Antineoplásicos/administración & dosificación , Medios de Contraste , Femenino , Gadolinio DTPA , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Estadísticas no Paramétricas , Resultado del TratamientoRESUMEN
OBJECTIVES: To compare plasma concentrations of oxidized low-density lipoprotein (oxLDL) and adrenaline during exercise between persons with spinal cord injury (SCI) and able-bodied (AB) individuals. DESIGN: Randomized controlled study. SETTING: Human laboratory at a medical university. PARTICIPANTS: Persons with SCI (n=7) and AB individuals (n=9). INTERVENTION: Two-hour arm crank ergometer exercise at 60% maximum oxygen consumption. MAIN OUTCOME MEASURES: Plasma oxLDL and adrenaline levels. RESULTS: Exercise significantly increased plasma adrenaline levels in AB persons (mean ± SD: rest, 45.4±32.2 pg/mL; exercise, 200.9±113.7 pg/mL; P<.05) and persons with SCI; however; the magnitude of the increase in those with SCI was attenuated (mean ± SD: rest, 45.4±14.0 pg/mL; exercise, 83.0±55.8 pg/mL; P<.05). Exercise also significantly increased plasma oxLDL levels in AB persons (mean ± SD: rest, 102.2±30.2 U/L; exercise, 179.7±60.0 U/L; P<.05), but not in persons with SCI (mean ± SD: rest, 124.3±66.0 U/L; exercise, 138.9±59.5 U/L). CONCLUSIONS: The results suggest that increases in plasma adrenaline levels during exercise contribute to the increase in plasma oxLDL levels.
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Epinefrina/sangre , Ejercicio Físico/fisiología , Lipoproteínas LDL/sangre , Paraplejía/sangre , Traumatismos de la Médula Espinal/sangre , Adulto , Análisis de Varianza , Prueba de Esfuerzo , Humanos , Masculino , Consumo de Oxígeno/fisiologíaRESUMEN
Isometric muscle contraction (static exercise) induces circulatory response. Static exercise in individuals with thoracic spinal cord injury (TSCI) induces cardiovascular response and blood redistribution to the non-exercising muscles. The aim of our study was to determine the circulatory response during arm static exercise in individuals with TSCI and able-bodied (AB) controls. Mean blood pressure (MBP), heart rate (HR), cardiac output (CO), leg skin blood flow (SBF), and leg muscle blood flow (MBF) were recorded noninvasively, total peripheral resistance (TPR) was estimated by dividing MBP by CO, and hormonal changes were measured before, during and after static 35% maximal voluntary contraction (MVC) of the arm flexor muscles in seven male individuals with TSCI (T7-T11) and seven age-comparable AB control (32.2 ± 7.6 and 31.0 ± 4.7 years, respectively). The 35% MVC was similar in TSCI and AB individuals (107.3 ± 28.2 and 101.0 ± 22.5 N, respectively). HR, CO, MBP, TPR, SBF and MBF increased in both groups during arm static exercise. Plasma epinephrine concentration increased during arm static exercise in AB controls only (P < 0.05). Circulation to leg muscles was similar in TSCI and AB individuals and the lack of sympathetic vasoconstriction in the paralyzed leg area did not alter the cardiovascular responses during 35% MVC of arm static exercise. We conclude that sympathetic vasoconstriction in the resting leg area did not contribute to the pressor reflex during 35% MVC of arm static exercise.
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Brazo/fisiopatología , Arterias/fisiopatología , Contracción Isométrica , Músculo Esquelético/fisiopatología , Esfuerzo Físico , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/rehabilitación , Adulto , Velocidad del Flujo Sanguíneo , Presión Sanguínea , Terapia por Ejercicio , Frecuencia Cardíaca , Humanos , Masculino , Vértebras Torácicas/lesiones , Vértebras Torácicas/fisiopatologíaRESUMEN
This study investigated the renal and endocrine responses to arm exercise in persons with spinal cord injury (SCI) under euhydration conditions (ad libitum drinking of water) and determined the physiological effects of exercise on renal function in these subjects. Eleven SCI (spinal lesions between T6 and L1, American Spinal Injury Association impairment scale A) and 14 able-bodied (AB) persons first rested for 1 h in a sitting position, before undergoing 2-h arm-crank ergometer exercise at 60% of maximum oxygen consumption followed by a 2-h recovery period. On another day, all subjects participated in a time control study (5 h of rest condition in sitting position). Urine and blood samples were collected hourly. There were no differences in mean blood pressure between the two groups. SCI patients showed attenuated increase in plasma adrenaline and increase in plasma aldosterone compared with AB controls, but similar changes in human atrial natriuretic polypeptide, plasma renin activity and plasma antidiuretic hormone following the exercise. Creatinine clearance, osmolal clearance, free water clearance and fractional excretion of Na(+) did not change during exercise in any of the subjects. These findings suggested that activated aldosterone and attenuated adrenaline responses during exercise in SCI could be due to adaptation to disordered sympathetic nervous system triggered to maintain renal function. The results showed no exercise-related adverse effects on renal function in hydrated subjects with SCI.