RESUMEN
Recessive dystrophic epidermolysis bullosa (RDEB) patients develop poorly healing skin wounds that are frequently colonized with microbiota. Because T cells play an important role in clearing such pathogens, we aimed to define the status of adaptive T cell-mediated immunity in RDEB wounds. Using a non-invasive approach for sampling of wound-associated constituents, we evaluated microbial contaminants in cellular fraction and exudates obtained from RDED wounds. Infectivity and intracellular trafficking of inactivated Staphylococcus aureus was accessed in RDEB keratinocytes. S. aureus and microbial antigen-specific activation of RDEB wound-derived T cells were investigated by fluorescence-activated cell sorting-based immune-phenotyping and T-cell functional assays. We found that RDEB wounds and epithelial cells are most frequently infected with Staphylococcus sp. and Pseudomonas sp. and that S. aureus essentially infects more RDEB keratinocytes and RDEB-derived squamous cell carcinoma cells than keratinocytes from healthy donors. The RDEB wound-associated T cells contain populations of CD4+ and CD8+ peripheral memory T cells that respond to soluble microbial antigens by proliferating and secreting interferon gamma (IFNγ). Moreover, CD8+ cytotoxic T lymphocytes recognize S. aureus-infected RDEB keratinocytes and respond by producing interleukin-2 (IL-2) and IFNγ and degranulating and cytotoxically killing infected cells. Prolonged exposure of RDEB-derived T cells to microbial antigens in vitro does not trigger PD-1-mediated T-cell exhaustion but induces differentiation of the CD4high population into CD4high CD25+ FoxP3+ regulatory T cells. Our data demonstrated that adaptive T cell-mediated immunity could clear infected cells from wound sites, but these effects might be inhibited by PD-1/Treg-mediated immuno-suppression in RDEB.
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Infecciones Bacterianas , Epidermólisis Ampollosa Distrófica , Linfocitos T , Antígenos , Colágeno Tipo VII , Epidermólisis Ampollosa Distrófica/patología , Humanos , Queratinocitos/patología , Activación de Linfocitos , Receptor de Muerte Celular Programada 1 , Staphylococcus aureus , Linfocitos T/inmunologíaRESUMEN
Vitiligo is an acquired pigmentary skin disorder that currently lacks standardized treatment and validated biomarkers to objectively evaluate disease state or therapeutic response. Although prior studies have linked vitiligo autoimmunity with CXCL10/CXCL9-mediated recruitment of leukocytes to the skin, only limited clinical data are available regarding CXCL10 as vitiligo biomarker. To evaluate the utility of systemic CXCL10 as a predictor of disease progression and treatment response on a large cohort of vitiligo patients. CXCL10 levels in lesional, perilesional, and unaffected skin of vitiligo patient (n = 30) and in the serum (n = 51) were measured by quantitative ELISA. CXCL10 expression, recruitment of leukocytes, and inflammatory infiltrates were evaluated by histochemical (n = 32) and immunofluorescence (n = 10) staining. Rigorous cross-sectional and longitudinal biostatistical analysis were employed to correlate CXCL10 levels with disease variables, treatment response, and outcome. We demonstrated that elevated CXCL10 level (2 pg/mm2 and higher) in lesional skin correlates with increased leukocytic infiltrate, disease duration (< 2 year), and its higher level in the serum (50 pg/ml and higher). Changes in CXCL10 serum levels in patients treated with psoralen plus UVA (PUVA) phototherapy, narrowband UVB (NB-UVB) phototherapy, and systemic steroids (SS) correlated with changes in the intralesional CXCL10 levels in repigmented skin. NB-UVB and SS regimens provided most consistent CXCL10 mean change, suggesting that these regimens are most effective in harnessing CXCR3-mediated inflammatory response. Serum CXCL10 is a useful vitiligo biomarker, which predicts lesional skin leukocytic infiltration, and vitiligo treatment response and outcome.
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Quimiocina CXCL10/metabolismo , Vitíligo/terapia , Adolescente , Corticoesteroides/uso terapéutico , Adulto , Biomarcadores/sangre , Biomarcadores/metabolismo , Quimiocina CXCL10/sangre , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Terapia PUVA , Valor Predictivo de las Pruebas , Terapia Ultravioleta , Vitíligo/metabolismo , Vitíligo/patología , Adulto JovenRESUMEN
Hereditary epidermolysis bullosa (EB) is a mechanobullous skin fragility disorder characterized by defective epithelial adhesion, leading to mechanical stress-induced skin blistering. Based on the level of tissue separation within the dermal-epidermal junction, EB is categorized into simplex (EBS), junctional (JEB), dystrophic (DEB) and Kindler syndrome. There is no cure for EB, and painful chronic cutaneous wounds are one of the major complications in recessive (RDEB) patients. Although RDEB is considered a cutaneous disease, recent data support the underlying systemic immunological defects. Furthermore, chronic wounds are often colonized with pathogenic microbiota, leading to excessive inflammation and altered wound healing. Consequently, patients with RDEB suffer from a painful sensation of chronic, cutaneous itching/burning and an endless battle with bacterial infections. To improve their quality of life and life expectancy, it is important to prevent cutaneous infections, dampen chronic inflammation and stimulate wound healing. A clear scientific understanding of the immunological events underlying the maintenance of chronic poorly healing wounds in RDEB patients is necessary to improve disease management and better understand other wound healing disorders. In this review, we summarize current knowledge of the role of professional phagocytes, such as neutrophils, macrophages and dendritic cells, the role of T-cell-mediated immunity in lymphoid organs, and the association of microbiota with poor wound healing in RDEB. We conclude that RDEB patients have an underlying immunity defect that seems to affect antibacterial immunity.
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Epidermólisis Ampollosa Distrófica/fisiopatología , Piel/patología , Cicatrización de Heridas , Epidermólisis Ampollosa Distrófica/inmunología , HumanosRESUMEN
The propensity of uveal melanoma to metastasize to the liver hinders the accrual of micro-metastatic and end-stage disease tissue samples and restricts the investigation of metastatic uveal melanoma (MUM). Pre-clinical experimental animal models of MUM can help elucidate the pathophysiology of metastatic lesions and provide a tool for designing new therapeutic approaches for MUM. Here, we present an advanced model of hepatic metastases that enables quantitatively visualizing the development of individual hepatic tumor clones and estimating their growth kinetics and colonization efficiency. Similar to clinically observed liver metastases, these models enable the assessment of growth kinetics of the liver micro-metastases and the testing of therapeutic approaches for the treatment of MUM. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Experimental patient-derived xenograft mouse model of metastatic uveal melanoma Basic Protocol 2: Experimental liver micro-metastatic mouse model using splenic injection of metastatic uveal melanoma cells.
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Neoplasias Hepáticas , Melanoma , Neoplasias de la Úvea , Animales , Xenoinjertos , Humanos , Melanoma/terapia , Ratones , Neoplasias de la Úvea/terapiaRESUMEN
Pathogenic invasion of Staphylococcus aureus is a major concern in patients with chronic skin diseases like atopic dermatitis (AD), epidermolysis bullosa (EB), or chronic diabetic foot and venous leg ulcers, and can result in persistent and life-threatening chronic non-healing wounds. Staphylococcus aureus is generally recognized as extracellular pathogens. However, S. aureus can also invade, hide and persist in skin cells to contribute to wound chronicity. The intracellular life cycle of S. aureus is currently incompletely understood, although published studies indicate that its intracellular escape strategies play an important role in persistent cutaneous infections. This review provides current scientific knowledge about the intracellular life cycle of S. aureus in skin cells, which can be classified into professional and non-professional antigen-presenting cells, and its strategies to escape adaptive defense mechanisms. First, we discuss phenotypic switch of S. aureus, which affects intracellular routing and degradation. This review also evaluates potential intracellular escape mechanism of S. aureus to avoid intracellular degradation and antigen presentation, preventing an immune response. Furthermore, we discuss potential drug targets that can interfere with the intracellular life cycle of S. aureus. Taken together, this review aimed to increase scientific understanding about the intracellular life cycle of S. aureus into skin cells and its strategies to evade the host immune response, information that is crucial to reduce pathogenic invasion and life-threatening persistence of S. aureus in chronic cutaneous infections.
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Enfermedades de la Piel/inmunología , Enfermedades de la Piel/microbiología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Autofagia , Humanos , Staphylococcus aureusRESUMEN
BACKGROUND: Poorly healing wounds are one of the major complications in patients suffering from recessive dystrophic epidermolysis bullosa (RDEB). At present, there are no effective means to analyze changes in cellular and molecular networks occurring during RDEB wound progression to predict wound outcome and design betted wound management approaches. OBJECTIVES: To better define mechanisms influencing RDEB wound progression by evaluating changes in molecular and cellular networks. METHODS: We developed a non-invasive approach for sampling and analysis of wound-associated constituents using wound-covering bandages. Cellular and molecular components from seventy-six samples collected from early, established and chronic RDEB wounds were evaluated by FACS-based immuno-phenotyping and ELISA. RESULTS: Our cross-sectional analysis determined that progression of RDEB wounds to chronic state is associated with the accumulation (up to 90 %) of CD16+CD66b+ mature neutrophils, loss of CD11b+CD68+ macrophages, and a significant increase (up to 50 %) in a number of CD11c+CD80+CD86+ activated professional antigen presenting cells (APC). It was also marked by changes in activated T cells populations including a reduction of CD45RO+ peripheral memory T cells from 80 % to 30 % and an increase (up to 70 %) in CD45RA+ effector T cells. Significantly higher levels of MMP9, VEGF-A and cathepsin G were also associated with advancing of wounds to poorly healing state. CONCLUSIONS: Our data demonstrated that wound-covering bandages are useful for a non-invasive sampling and analysis of wound-associated constituents and that transition to poorly healing wounds in RDEB patients as associated with distinct changes in leukocytic infiltrates, matrix-remodeling enzymes and pro-angiogenic factors at wound sites.
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Epidermólisis Ampollosa Distrófica/complicaciones , Leucocitos/inmunología , Piel/patología , Cicatrización de Heridas/inmunología , Adolescente , Adulto , Anciano , Niño , Preescolar , Estudios Transversales , Epidermólisis Ampollosa Distrófica/inmunología , Epidermólisis Ampollosa Distrófica/patología , Femenino , Humanos , Lactante , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Receptores CCR2/metabolismo , Receptores de Interleucina-8B/metabolismo , Piel/citología , Piel/inmunología , Adulto JovenRESUMEN
BACKGROUND: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness. The two most prevalent forms of CMD, collagen VI-related myopathies (COL6RM) and laminin α2 deficient CMD type 1A (MDC1A), are both caused by deficiency or dysfunction of extracellular matrix proteins. Previously, we showed that an intramuscular transplantation of human adipose-derived stem cells (ADSC) into the muscle of the Col6a1-/- mice results in efficient stem cell engraftment, migration, long-term survival, and continuous production of the collagen VI protein, suggesting the feasibility of the systemic cellular therapy for COL6RM. In order for this therapeutic approach to work however, stem cells must be efficiently targeted to the entire body musculature. Thus, the main goal of this study is to test whether muscle homing of systemically transplanted ADSC can be enhanced by employing muscle-specific chemotactic signals originating from CMD-affected muscle tissue. METHODS: Proteomic screens of chemotactic molecules were conducted in the skeletal muscles of COL6RM- and MDC1A-affected patients and CMD mouse models to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also using a pre-clinical animal model, recapitulating mild Ullrich congenital muscular dystrophy (UCMD), the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations. RESULTS: Comprehensive proteomic screens evaluating relevant human and mouse skeletal muscle biopsies offered chemotactic axes to enhance directional migration of systemically transplanted cells into CMD-affected muscles, including CCL5-CCR1/3/5, CCL2-CCR2, CXCL1/2-CXCR1,2, and CXCL7-CXCR2. Also, the specific populations of ADSC selected with an affinity for the chemokines being released by damaged muscle showed efficient migration to injured site and presented their therapeutic effect. CONCLUSIONS: Collectively, identified molecules provided insight into the mechanisms governing directional migration and intramuscular trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for CMD treatment.
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Células Madre Adultas , Distrofias Musculares , Animales , Quimiocinas , Humanos , Laminina , Ratones , Músculo Esquelético , Distrofias Musculares/genética , Distrofias Musculares/terapia , ProteómicaRESUMEN
Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin ß3) mutations account for 80% of patients with H-JEB, and â¼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin ß3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin ß3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin ß3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6ß4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.
Asunto(s)
Moléculas de Adhesión Celular , Codón sin Sentido , Epidermólisis Ampollosa de la Unión , Gentamicinas/farmacología , Queratinocitos/metabolismo , Mutagénesis/efectos de los fármacos , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Epidermólisis Ampollosa de la Unión/genética , Epidermólisis Ampollosa de la Unión/metabolismo , Epidermólisis Ampollosa de la Unión/patología , Células HEK293 , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Queratinocitos/patología , KalininaRESUMEN
Purpose: The standard treatment for organ-confined prostate cancer is surgery or radiation, and locally advanced prostate cancer is typically treated with radiotherapy alone or in combination with androgen deprivation therapy. Here, we investigated whether Stat5a/b participates in regulation of double-strand DNA break repair in prostate cancer, and whether Stat5 inhibition may provide a novel strategy to sensitize prostate cancer to radiotherapy.Experimental Design: Stat5a/b regulation of DNA repair in prostate cancer was evaluated by comet and clonogenic survival assays, followed by assays specific to homologous recombination (HR) DNA repair and nonhomologous end joining (NHEJ) DNA repair. For HR DNA repair, Stat5a/b regulation of Rad51 and the mechanisms underlying the regulation were investigated in prostate cancer cells, xenograft tumors, and patient-derived prostate cancers ex vivo in 3D explant cultures. Stat5a/b induction of Rad51 and HR DNA repair and responsiveness to radiation were evaluated in vivo in mice bearing prostate cancer xenograft tumors.Results: Stat5a/b is critical for Rad51 expression in prostate cancer via Jak2-dependent mechanisms by inducing Rad51 mRNA levels. Consistent with this, genetic knockdown of Stat5a/b suppressed HR DNA repair while not affecting NHEJ DNA repair. Pharmacologic Stat5a/b inhibition potently sensitized prostate cancer cell lines and prostate cancer tumors to radiation, while not inducing radiation sensitivity in the neighboring tissues.Conclusions: This work introduces a novel concept of a pivotal role of Jak2-Stat5a/b signaling for Rad51 expression and HR DNA repair in prostate cancer. Inhibition of Jak2-Stat5a/b signaling sensitizes prostate cancer to radiation and, therefore, may provide an adjuvant therapy for radiation to reduce radiation-induced damage to the neighboring tissues. Clin Cancer Res; 24(8); 1917-31. ©2018 AACR.
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Reparación del ADN , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Recombinasa Rad51/metabolismo , Tolerancia a Radiación/genética , Factor de Transcripción STAT5/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Modelos Animales de Enfermedad , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/radioterapia , ARN Interferente Pequeño/genética , Recombinasa Rad51/genética , Tolerancia a Radiación/efectos de los fármacos , Radiación Ionizante , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Colágeno Tipo VII/inmunología , Epidermólisis Ampollosa/diagnóstico , Epidermólisis Ampollosa/inmunología , Queratina-14/inmunología , Queratina-5/inmunología , Anticuerpos/inmunología , Estudios Transversales , Epidermólisis Ampollosa Distrófica/diagnóstico , Epidermólisis Ampollosa Distrófica/inmunología , Epidermólisis Ampollosa Simple/diagnóstico , Epidermólisis Ampollosa Simple/inmunología , Epidermólisis Ampollosa de la Unión/diagnóstico , Epidermólisis Ampollosa de la Unión/inmunología , Técnica del Anticuerpo Fluorescente , HumanosRESUMEN
Hereditary epidermolysis bullosa (EB) is associated with skin blistering and the development of chronic nonhealing wounds. Although clinical studies have shown that cell-based therapies improve wound healing, the recruitment of therapeutic cells to blistering skin and to more advanced skin lesions remains a challenge. Here, we analyzed cytokines and chemokines in blister fluids of patients affected by dystrophic, junctional, and simplex EB. Our analysis revealed high levels of CXCR1, CXCR2, CCR2, and CCR4 ligands, particularly dominant in dystrophic and junctional EB. In vitro migration assays demonstrated the preferential recruitment of CCR4+ lymphocytes and CXCR1+, CXCR2+, and CCR2+ myeloid cells toward EB-derived blister fluids. Immunophenotyping of skin-infiltrating leukocytes confirmed substantial infiltration of EB-affected skin with resting (CD45RA+) and activated (CD45RO+) T cells and CXCR2+ CD11b+ cells, many of which were identified as CD16b+ neutrophils. Our studies also showed that abundance of CXCR2 ligand in blister fluids also creates a favorable milieu for the recruitment of the CXCR2+ stem cells, as validated by in vitro and in-matrix migration assays. Collectively, this study identified several chemotactic pathways that control the recruitment of leukocytes to the EB-associated skin lesions. These chemotactic axes could be explored for the refinement of the cutaneous homing of the therapeutic stem cells.
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Quimiocinas/genética , Citocinas/genética , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/patología , Receptores CXCR/genética , Vesícula/patología , Movimiento Celular/genética , Células Cultivadas , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Biología Molecular , Pronóstico , Muestreo , Sensibilidad y Especificidad , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
Heterologous prime-boost immunization with plasmid DNA and viral vector vaccines is an emerging approach to elicit CD8+ T cell-mediated immunity targeting pathogens and tumor Ags that is superior to either monotherapy. Yet, the mechanisms underlying the synergy of prime-boost strategies remain incompletely defined. In this study, we examine a DNA and adenovirus (Ad5) combination regimen targeting guanylyl cyclase C (GUCY2C), a receptor expressed by intestinal mucosa and universally expressed by metastatic colorectal cancer. DNA immunization efficacy was optimized by i.m. delivery via electroporation, yet it remained modest compared with Ad5. Sequential immunization with DNA and Ad5 produced superior antitumor efficacy associated with increased TCR avidity, whereas targeted disruption of TCR avidity enhancement eliminated GUCY2C-specific antitumor efficacy, without affecting responding T cell number or cytokine profile. Indeed, functional TCR avidity of responding GUCY2C-specific CD8+ T cells induced by various prime or prime-boost regimens correlated with antitumor efficacy, whereas T cell number and cytokine profile were not. Importantly, although sequential immunization with DNA and Ad5 maximized antitumor efficacy through TCR avidity enhancement, it produced no autoimmunity, reflecting sequestration of GUCY2C to intestinal apical membranes and segregation of mucosal and systemic immunity. Together, TCR avidity enhancement may be leveraged by prime-boost immunization to improve GUCY2C-targeted colorectal cancer immunotherapeutic efficacy and patient outcomes without concomitant autoimmune toxicity.
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Biomarcadores de Tumor/metabolismo , Linfocitos T CD8-positivos/fisiología , Neoplasias Colorrectales/terapia , Inmunoterapia Adoptiva/métodos , Mucosa Intestinal/fisiología , Receptores Acoplados a la Guanilato-Ciclasa/metabolismo , Receptores de Péptidos/metabolismo , Vacunas de ADN/inmunología , Adenoviridae/genética , Animales , Biomarcadores de Tumor/genética , Linfocitos T CD8-positivos/trasplante , Células Cultivadas , Neoplasias Colorrectales/inmunología , Citotoxicidad Inmunológica , Inmunidad Mucosa , Inmunización Secundaria , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa/genética , Receptores de Péptidos/genética , Carga TumoralRESUMEN
Generalized nonsegmental vitiligo is often associated with the activation of melanocyte-specific autoimmunity. Because chemokines play an important role in the maintenance of immune responses, we examined chemotactic signatures in cultured vitiligo melanocytes and skin samples of early (≤2 months) and advanced (≥6 months) vitiligo. Analysis showed that melanocytes in early lesions have altered expression of several chemotaxis-associated molecules, including elevated secretion of CXCL12 and CCL5. Higher levels of these chemokines coincided with prominent infiltration of the skin with antigen presenting cells (APCs) and T cells. Most of the intralesional APCs expressed the CD86 maturation marker and co-localized with T cells, particularly in early vitiligo lesions. These observations were confirmed by in vivo animal studies showing preferential recruitment of APCs and T cells to CXCL12- and CCL5-expressing transplanted melanocytes, immunotargeting of the chemokine-positive cells, continuous loss of the pigment-producing cells from the epidermis, and development of vitiligo-like lesions. Taken together, our studies show that melanocyte-derived CXCL12 and CCL5 support APC and T-cell recruitment, antigen acquisition, and T-cell activation in early vitiligo and reinforce the role of melanocyte-derived CXCL12 and CCL5 in activation of melanocyte-specific immunity and suggest inhibition of these chemotactic axes as a strategy for vitiligo stabilization.
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Quimiocina CCL5/metabolismo , Quimiocina CXCL12/metabolismo , Melanocitos/metabolismo , Vitíligo/patología , Animales , Células Presentadoras de Antígenos/inmunología , Autoinmunidad , Línea Celular , Quimiocina CCL5/inmunología , Quimiocina CXCL12/inmunología , Quimiotaxis/inmunología , Progresión de la Enfermedad , Humanos , Melanocitos/inmunología , Ratones , Linfocitos T/inmunología , Vitíligo/inmunologíaRESUMEN
CXCR1 and CXCR2 chemokine receptors and their ligands (CXCL1/2/3/7/8) play an important role in tumor progression. Tested to date CXCR1/2 antagonists and chemokine-targeted antibodies were reported to affect malignant cells in vitro and in animal models. Yet, redundancy of chemotactic signals and toxicity hinder further clinical development of these approaches. In this pre-clinical study we investigated the capacity of a novel small molecule dual CXCR1/2 inhibitor, Ladarixin (LDX), to attenuate progression of experimental human melanomas. Our data showed that LDX-mediated inhibition of CXCR1/2 abrogated motility and induced apoptosis in cultured cutaneous and uveal melanoma cells and xenografts independently of the molecular defects associated with the malignant phenotype. These effects were mediated by the inhibition of AKT and NF-kB signaling pathways. Moreover, systemic treatment of melanoma-bearing mice with LDX also polarized intratumoral macrophages to M1 phenotype, abrogated intratumoral de novo angiogenesis and inhibited melanoma self-renewal. Collectively, these studies outlined the pre-requisites of the successful CXCR1/2 inhibition on malignant cells and demonstrated multifactorial effects of Ladarixin on cutaneous and uveal melanomas, suggesting therapeutic utility of LDX in treatment of various melanoma types.
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Antineoplásicos/farmacología , Melanoma Experimental/tratamiento farmacológico , Receptores de Interleucina-8A/antagonistas & inhibidores , Receptores de Interleucina-8B/antagonistas & inhibidores , Sulfonamidas/farmacología , Microambiente Tumoral/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiotaxis , Humanos , Interleucina-8/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Desnudos , FN-kappa B , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Dystrophic epidermolysis bullosa (DEB), a rare genodermatosis, is characterized by the formation of intra-epidermal blistering and the development of chronic nonhealing skin wounds. Recently, attempts have been made to develop cell-based therapies for this currently intractable disorder. The molecular mechanisms that govern directional migration of the adult stem cells, allowing their efficient and controlled homing to the skin affected with DEB, are poorly understood. The key mechanism that regulates recruitment of leukocytes and progenitor stem cells to distal anatomical tissues affected with disease is chemotaxis, which depends on the signaling molecules, chemokines, and acts primarily as part of the host defense and repair mechanism. METHODS: Comprehensive proteomic screening of chemokines in the blister fluids of DEB-affected mice was conducted to define the inflammatory and immune activities, thus providing potential to examine local biological mechanisms and define the protein signature within lesional skin as a potential marker of disease activity. Also, the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations. RESULTS: Assessment of blister fluid-derived chemokines showed a persistent presence of several chemotactic molecules, including CXCL1 + 2 and CXCL5. The majority of blister-originated chemotactic signals were associated with preferential recruitment of CD45(+)CXCR2(+) and CD11b(+)CXCR2(+) leukocytes. Systemic transplantation of an enriched CXCR2 population of mouse adipose-derived stem cells (mADSC) into DEB-affected mice demonstrated effective recruitment of cells to the blistering skin under the influence of blister-derived ligands and deposition of therapeutic type VII collagen. CONCLUSIONS: Collectively, these studies demonstrate that recruitment of mADSC into DEB skin is tightly controlled by disease-site chemotactic activities and suggest a potential mechanism for effective application of therapeutic stem cells for DEB.
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Células Madre Adultas/fisiología , Quimiotaxis/fisiología , Epidermólisis Ampollosa Distrófica/terapia , Células Madre Adultas/metabolismo , Animales , Vesícula/metabolismo , Vesícula/terapia , Movimiento Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Quimiocinas/metabolismo , Colágeno Tipo VII/metabolismo , Modelos Animales de Enfermedad , Epidermólisis Ampollosa Distrófica/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Piel/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR's role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/tratamiento farmacológico , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Citoplasma/metabolismo , Doxiciclina/química , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Femenino , Silenciador del Gen , Humanos , Ratones , Nanopartículas/química , Invasividad Neoplásica , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas/tratamiento farmacológico , Fenotipo , Análisis de Componente Principal , ARN/metabolismo , Procesamiento Postranscripcional del ARN , Transducción de SeñalRESUMEN
Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.
Asunto(s)
Vasos Sanguíneos/anomalías , Huesos/anomalías , Colágeno Tipo I/metabolismo , Cutis Laxo/patología , Tejido Elástico/anomalías , Proteínas de la Matriz Extracelular/genética , Técnicas de Sustitución del Gen , Piel/patología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Vasos Sanguíneos/patología , Huesos/patología , Colágeno Tipo I/ultraestructura , Reactivos de Enlaces Cruzados/metabolismo , Cutis Laxo/metabolismo , Modelos Animales de Enfermedad , Tejido Elástico/patología , Tejido Elástico/ultraestructura , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/enzimología , Fibroblastos/patología , Miembro Anterior/anomalías , Miembro Anterior/diagnóstico por imagen , Miembro Anterior/patología , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Biosíntesis de Proteínas , Multimerización de Proteína , Proteína-Lisina 6-Oxidasa/metabolismo , Radiografía , Tendones/anomalías , Tendones/patología , Tendones/ultraestructuraRESUMEN
Bone marrow suppression due to exposure to ionizing radiation is a significant clinical problem associated with radiation therapy as well as with nonmedical radiation exposure. Currently, there are no small molecule agents available that can enhance hematopoietic regeneration after radiation exposure. Here, we report on the effective mitigation of acute hematopoietic radiation syndrome in mice by the synthetic triterpenoid, RTA 408. The administration of a brief course of RTA 408 treatment, beginning 24 h after lethal doses of radiation to bone marrow, significantly increased overall survival. Importantly, treatment with RTA 408 led to the full recovery of steady state hematopoiesis with normalization of the frequency of hematopoietic stem and progenitor cells. Moreover, hematopoietic stem cells from RTA 408-mitigated mice showed lineage-balanced, long-term, multilineage potential in serial transplantation assays, indicative of their normal self-renewal activity. The potency of RTA 408 in mitigating radiation-induced bone marrow suppression makes it an attractive candidate for potential clinical use in treating both therapy-related and unanticipated radiation exposure.
Asunto(s)
Síndrome de Radiación Aguda/tratamiento farmacológico , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Triterpenos/administración & dosificación , Síndrome de Radiación Aguda/patología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Hematopoyesis/efectos de la radiación , Células Madre Hematopoyéticas/efectos de la radiación , Ratones , Radiación Ionizante , Radioterapia/efectos adversos , Irradiación Corporal TotalRESUMEN
Melanoma is one of the most aggressive metastatic cancers with resistance to radiation and most chemotherapy agents. This study highlights an alternative treatment for melanoma based on nanosecond pulsed dielectric barrier discharge (nsP DBD). We show that a single nsP DBD treatment, directly applied to a 5 mm orthotopic mouse melanoma tumor, completely eradicates it 66% (n = 6; p ≤ 0.05) of the time. It was determined that reactive oxygen and nitrogen species produced by nsP DBD are the main cause of tumor eradication, while nsP electric field and heat generated by the discharge are not sufficient to kill the tumor. However, we do not discount that potential synergy between each plasma generated component (temperature, electric field and reactive species) can enhance the killing efficacy.