RESUMEN
BACKGROUND: The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by effector memory T cells (TEM) and plays an important role in their activation and proliferation. Mycosis fungoides (MF), the most common subtype of cutaneous T-cell lymphoma (CTCL), was recently proposed to be a malignancy of skin-resident TEM. However, the expression of Kv1.3 in CTCL has not been investigated. OBJECTIVES: This study aims to examine the expression of Kv1.3 in situ and in vitro in CTCL. METHODS: The expression of Kv1.3 was examined by immunohistochemistry in skin lesions from 38 patients with MF, 4 patients with Sézary syndrome (SS), and 27 patients with benign dermatosis. In 4 malignant T-cell lines of CTCL (Myla2059, PB2B, SeAx, and Mac2a) and a non-malignant T-cell line (MyLa1850), the expression of Kv1.3 was determined by flow cytometry. The proliferation of those cell lines treated with various concentrations of Kv1.3 inhibitor ShK was measured by 3H-thymdine incorporation. RESULTS: Half of the MF patients (19/38) displayed partial Kv1.3 expression including 1 patient with moderate Kv1.3 positivity, while the other half (19/38) exhibited Kv1.3 negativity. An almost identical distribution was observed in patients with benign conditions, that is, 44.4% (12/27) were partially positive for Kv1.3 including 1 patient with moderate Kv1.3 positivity, while 55.6% (15/27) were Kv1.3 negative. In contrast, 3 in 4 SS patients displayed partial Kv1.3 positivity including 2 patients with weak staining and 1 with moderate staining, while 1 in 4 SS patients was Kv1.3 negative. In addition, all malignant T-cell lines, and a non-malignant T-cell line, displayed low Kv1.3 surface expression with a similar pattern. Whereas 2 cell lines (PB2B and Mac2a) were sensitive to Kv1.3 blockade, the other 2 (Myla2059 and SeAx) were completely resistant. CONCLUSIONS: We provide the first evidence of a heterogeneous Kv1.3 expression in situ in CTCL lesions.
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Dermatitis/metabolismo , Canal de Potasio Kv1.3/biosíntesis , Linfoma Cutáneo de Células T/metabolismo , Neoplasias Cutáneas/metabolismo , Piel/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Línea Celular Tumoral , Niño , Dermatitis/patología , Femenino , Humanos , Inmunohistoquímica , Canal de Potasio Kv1.3/antagonistas & inhibidores , Linfoma Cutáneo de Células T/patología , Masculino , Persona de Mediana Edad , Piel/patología , Neoplasias Cutáneas/patología , Adulto JovenRESUMEN
In osteosarcoma, a primary mesenchymal bone cancer occurring predominantly in younger patients, invasive tumour growth leads to extensive bone destruction. This process is insufficiently understood, cannot be efficiently counteracted and calls for novel means of treatment. The endocytic collagen receptor, uPARAP/Endo180, is expressed on various mesenchymal cell types and is involved in bone matrix turnover during normal bone growth. Human osteosarcoma specimens showed strong expression of this receptor on tumour cells, along with the collagenolytic metalloprotease, MT1-MMP. In advanced tumours with ongoing bone degeneration, sarcoma cells positive for these proteins formed a contiguous layer aligned with the degradation zones. Remarkably, osteoclasts were scarce or absent from these regions and quantitative analysis revealed that this scarcity marked a strong contrast between osteosarcoma and bone metastases of carcinoma origin. This opened the possibility that sarcoma cells might directly mediate bone degeneration. To examine this question, we utilized a syngeneic, osteolytic bone tumour model with transplanted NCTC-2472 sarcoma cells in mice. When analysed in vitro, these cells were capable of degrading the protein component of surface-labelled bone slices in a process dependent on MMP activity and uPARAP/Endo180. Systemic treatment of the sarcoma-inoculated mice with a mouse monoclonal antibody that blocks murine uPARAP/Endo180 led to a strong reduction of bone destruction. Our findings identify sarcoma cell-resident uPARAP/Endo180 as a central player in the bone degeneration of advanced tumours, possibly following an osteoclast-mediated attack on bone in the early tumour stage. This points to uPARAP/Endo180 as a promising therapeutic target in osteosarcoma, with particular prospects for improved neoadjuvant therapy.
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Neoplasias Óseas/patología , Osteólisis/metabolismo , Osteosarcoma/patología , Receptores Mitogénicos/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Invasividad Neoplásica , Osteoclastos/patología , Osteólisis/etiología , Osteólisis/patologíaRESUMEN
Atopic dermatitis (AD) is a common inflammatory skin disease with underlying defects in epidermal function and immune responses. In this study, we used microarray analysis to investigate differences in gene expression in lesional skin from patients with mild extrinsic or intrinsic AD compared to skin from healthy controls and from lesional psoriasis skin. The primary aim was to identify differentially expressed genes involved in skin barrier formation and inflammation, and to compare our results with those reported for patients with moderate and severe AD. In contrast to severe AD, expression of the majority of genes associated with skin barrier formation was unchanged or upregulated in patients with mild AD compared to normal healthy skin. Among these, no significant differences in the expression of filaggrin (FLG) and loricrin at both mRNA and protein level were found in lesional skin from patients with mild AD, despite the presence of heterozygous FLG mutations in the majority of patients with mild extrinsic AD. Several inflammation-associated genes such as S100A9, MMP12, CXCL10 and CCL18 were highly expressed in lesional skin from patients with mild psoriasis and were also increased in patients with mild extrinsic and intrinsic AD similar to previous reports for severe AD. Interestingly, expression of genes involved in inflammatory responses in intrinsic AD resembled that of psoriasis more than that of extrinsic AD. Overall, differences in expression of inflammation-associated genes found among patients with mild intrinsic and extrinsic AD correlated with previous findings for patients with severe intrinsic and extrinsic AD.
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Dermatitis Atópica/metabolismo , Perfilación de la Expresión Génica , Psoriasis/metabolismo , Adulto , Estudios de Casos y Controles , Dermatitis Atópica/clasificación , Dermatitis Atópica/patología , Proteínas Filagrina , Humanos , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Piel/metabolismo , Piel/patología , Adulto JovenRESUMEN
Rearrangement of the skin during wound healing depends on plasmin and plasminogen, which serve to degrade fibrin depositions in the provisional matrix and thereby facilitate keratinocyte migration. In the current study, we investigated whether plasmin and plasminogen likewise played a role during the development of skin cancer. To test this, we set up a chemically induced skin tumor model in a cohort of mice and found that skin tumor growth in Plg(-/-) male mice was reduced by 52% compared with wild-type controls. Histological analyses suggested that the growth-restricting effect of plasminogen deficiency was due to thrombosis and lost patency of the tumor vasculature, resulting in tumor necrosis. The connection between plasmin-dependent fibrinolysis, vascular patency, and tumor growth was further substantiated as the effect of plasminogen deficiency on tumor growth could be reverted by superimposing heterozygous fibrinogen deficiency on Plg(-/-) mice. Tumors derived from these Fib(-/+);Plg(-/-) mice displayed a significantly decreased level of tumor thrombosis compared with Plg(-/-) mice. In summary, these data indicate that plasmin-driven fibrinolysis facilitates tumor growth by maintaining patency of the tumor vasculature.
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Fibrinolisina/metabolismo , Fibrinólisis/fisiología , Neoplasias Cutáneas/patología , Animales , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Femenino , Fibrina/genética , Fibrina/metabolismo , Fibrinógeno/genética , Fibrinógeno/metabolismo , Fibrinolisina/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Laminina/genética , Laminina/metabolismo , Masculino , Ratones , Ratones Noqueados , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/metabolismo , Ovariectomía , Factores Sexuales , Neoplasias Cutáneas/metabolismo , Trombosis/metabolismoRESUMEN
Fibrosis of the liver and its end-stage, cirrhosis, represent major health problems worldwide. In these fibrotic conditions, activated fibroblasts and hepatic stellate cells display a net deposition of collagen. This collagen deposition is a major factor leading to liver dysfunction, thus making it crucially important to understand both the collagen synthesis and turnover mechanisms in this condition. Here we show that the endocytic collagen receptor, uPARAP/Endo180, is a major determinant in governing the balance between collagen deposition and degradation. Cirrhotic human livers displayed a marked up-regulation of uPARAP/Endo180 in activated fibroblasts and hepatic stellate cells located close to the collagen deposits. In a hepatic stellate cell line, uPARAP/Endo180 was shown to be active in, and required for, the uptake and intracellular degradation of collagen. To evaluate the functional importance of this collagen receptor in vivo, liver fibrosis was induced in uPARAP/Endo180-deficient mice and littermate wild-type mice by chronic CCl(4) administration. A strong up-regulation of uPARAP/Endo180 was observed in wild-type mice, and a quantitative comparison of collagen deposits in the two groups of mice clearly revealed a fibrosis protective role of uPARAP/Endo180. This effect appeared to directly reflect the activity of the collagen receptor, since no compensatory events were noted when comparing the mRNA expression profiles of the two groups of mice in an array system focused on matrix-degrading components. This function of uPARAP/Endo180 defines a novel role of intracellular collagen turnover in fibrosis protection.
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Colágeno/metabolismo , Endocitosis/fisiología , Cirrosis Hepática Experimental/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Línea Celular , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Humanos , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/prevención & control , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos , Ratones Noqueados , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Regulación hacia ArribaRESUMEN
Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9, gelatinase B) have separately been recognized to play important roles in various tissue remodeling processes. In this study, we demonstrate that deficiency for MMP9 in combination with ablation of either uPA- or tissue-type plasminogen activator (tPA)-catalyzed plasminogen activation is critical to accomplish normal gestation in mice. Gestation was also affected by simultaneous lack of MMP9 and the uPA receptor (uPAR). Interestingly, uPA-deficiency additionally exacerbated the effect of MMP9-deficiency on bone growth and an additive effect caused by combined lack in MMP9 and uPA was observed during healing of cutaneous wounds. By comparison, MMP9-deficiency combined with absence of either tPA or uPAR resulted in no significant effect on wound healing, indicating that the role of uPA during wound healing is independent of uPAR, when MMP9 is absent. Notably, compensatory upregulation of uPA activity was seen in wounds from MMP9-deficient mice. Taken together, these studies reveal essential functional dependency between MMP9 and uPA during gestation and tissue repair.
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Metaloproteinasa 9 de la Matriz/deficiencia , Embarazo/fisiología , Fenómenos Fisiológicos de la Piel , Activador de Plasminógeno de Tipo Uroquinasa/deficiencia , Cicatrización de Heridas/fisiología , Animales , Western Blotting , Pesos y Medidas Corporales , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Femenino , Técnicas Histológicas , Hibridación in Situ , Ratones , Cicatrización de Heridas/genéticaRESUMEN
Besides their evident importance in host defense, macrophages have been shown to play a detrimental role in different pathological conditions, including chronic inflammation, atherosclerosis, and cancer. Regardless of the exact situation, macrophage activation and migration are intimately connected to extracellular matrix degradation. This process is accomplished by multiple proteolytic enzymes, including serine proteases and members of the matrix metalloproteinase family. In this study, we have utilized qPCR arrays to simultaneously analyze the temporal expression pattern of a range of genes involved in extracellular matrix metabolism in the mouse derived-macrophage cell line RAW 264.7 following stimulation with LPS. Our results revealed that LPS induces the expression of matrix metalloproteinases while at the same time decreased the expression of matrix metalloproteinase inhibitors. The opposite scenario was found for the genes encoding serine proteases, which were downregulated while their inhibitors were upregulated. In addition, intergenic comparison of the expression levels of related proteases revealed large differences in their basal expression level. These data highlight the complexity of the gene expression regulation implicated in macrophage-dependent matrix degradation and furthermore emphasize the value of qPCR array techniques for the investigation of the complex regulation of the matrix degradome.
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Regulación Enzimológica de la Expresión Génica , Lipopolisacáridos/metabolismo , Macrófagos/enzimología , Péptido Hidrolasas/biosíntesis , Inhibidores de Proteasas/metabolismo , ARN/biosíntesis , Algoritmos , Animales , Línea Celular , Dosificación de Gen , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reconocimiento de Normas Patrones Automatizadas , Péptido Hidrolasas/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Transducción de Señal , Factores de Tiempo , Transcripción GenéticaRESUMEN
INTRODUCTION: Increasing demand for interprofessional collaboration in health care settings has led to a greater focus on how conditions influence the success of interprofessional collaboration, but little is known about the magnitude of the interactions between different conditions. This paper aims to examine the relationships of intervention conditions and context conditions at the professional and organisational level and examine how they influence the staff's perceived success of the interprofessional collaboration. METHODS: The study was conducted as a multilevel cross-sectional survey in March of 2019 in the second largest municipality in Denmark, Aarhus. The study population was all frontline-staff members and managers in nursing homes, home care units and health care units. The final sample consisted of 498 staff members and 27 managers. Confirmatory path analysis was used to analyse the data. RESULTS: The results indicate that context conditions greatly influence intervention conditions at the professional and organisational level and that the professional and organisational levels moderately co-variate. Professional level context conditions have the biggest influence on staff's perceived success, partly because its influence is confounded by intervention conditions. CONCLUSION: Practice and research in health care settings should re-focus their attention from a broad understanding of context as unchangeable and inconsequential, to understanding context as an important condition type for interprofessional collaboration that needs to be further understood and researched.
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Prestación Integrada de Atención de Salud , Relaciones Interprofesionales , Estudios Transversales , Dinamarca , Humanos , Atención Primaria de SaludRESUMEN
During healing of incisional skin wounds, migrating keratinocytes dissect their way under the crust to re-epithelialize the wounded area. The efficiency of this tissue remodelling process depends on the concomitant activity of several extracellular proteases, including members of the plasminogen activation (PA) system and the matrix metalloproteinase (MMP) family. Treatment with the broad spectrum MMP inhibitor, galardin, delays wound healing in wildtype mice and completely arrest wound healing in plasminogen (Plg)-deficient mice, indicating a functional overlap between plasmin- and galardin-sensitive MMPs during wound healing. To address whether MMP2 is accountable for the galardin-induced healing deficiency in wildtype and Plg-deficient mice, incisional skin wounds were generated in MMP2 single-deficient mice and in MMP2/Plg double-deficient mice and followed until healed. Alternatively, tissue was isolated 7 days post wounding for histological and biochemical analyses. No difference was found in the time from wounding to overt gross restoration of the epidermal surface between MMP2-deficient and wildtype control littermate mice. MMP2/Plg double-deficient mice were viable and fertile, and displayed an unchallenged general phenotype resembling that of Plg-deficient mice, including development of rectal prolapses. MMP2/Plg double-deficient mice displayed a slight increase in the wound length throughout the healing period compared with Plg-deficient mice. However, the overall time to complete healing was not significantly different between Plg-deficient and MMP2/Plg double-deficient mice. These results show that MMP2 activity is not essential for wound healing and indicate that lack of MMP2 only marginally potentiates the effect of Plg deficiency.
Asunto(s)
Metaloproteinasa 2 de la Matriz/deficiencia , Plasminógeno/deficiencia , Piel/lesiones , Cicatrización de Heridas/fisiología , Animales , Dipéptidos/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Animales , Plasminógeno/genética , Plasminógeno/metabolismo , Inhibidores de Proteasas/farmacología , ARN Mensajero/metabolismo , Piel/metabolismoRESUMEN
Prostate, breast and lung cancers readily develop bone metastases which lead to fractures, hypercalcemia and pain. Malignant growth in the bones depends on osteoclast-mediated bone resorption and in this regard bisphosphonate compounds, which have high-bone affinity and inhibit osteoclast activity, have been found to alleviate bone cancer symptoms. In this study, the bisphosphonate risedronate and its phosphonocarboxylate derivative NE-10790 was tested in a murine bone cancer pain model. Risedronate decreased bone cancer-related bone destruction and pain-related behavior and decreased the spinal expression of glial fibrillary acidic protein, whereas NE-10790 had no effect on these parameters. Furthermore, risedronate but not NE-10790 induced dose-dependent toxicity in NCTC-2472 cells in vitro. Furthermore, the direct toxic effect of risedronate on tumor cells observed in vitro opens the possibility that a direct toxic effect on tumor cells may also be present in vivo and be related to the efficacy of bisphosphonate compounds. In conclusion, these results suggest that risedronate treatment may lead to an increased life quality, in patient suffering from bone cancer, in terms of decreased osteolysis and pain, and merits further study.
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Conservadores de la Densidad Ósea/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Resorción Ósea/prevención & control , Difosfonatos/uso terapéutico , Ácido Etidrónico/análogos & derivados , Dolor/tratamiento farmacológico , Piridinas/uso terapéutico , Fosfatasa Ácida/sangre , Animales , Conducta Animal , Neoplasias Óseas/secundario , Proliferación Celular , Células Cultivadas , Ácido Etidrónico/uso terapéutico , Fibroblastos/citología , Fibroblastos/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Técnicas para Inmunoenzimas , Isoenzimas/sangre , Masculino , Ratones , Ratones Desnudos , Ácido Risedrónico , Fosfatasa Ácida Tartratorresistente , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pathological pain has been subjected to intense research to shed light on the underlying mechanisms of key symptoms, such as allodynia and hyperalgesia. The main focus has by and large concerned plasticity of spinal cord neurons and the primary afferent nerves relaying peripheral information to the spinal cord. Animal pain models display an increased presence of reactive astrocytes in the spinal cord, but in contrast to neurons, little is known about how they contribute to abnormal pain sensation. However, astrocytes are now beginning to receive greater attention, and as new information is emerging, it appears that astrocytes undertake critical roles in manifesting pathological pain. Through the secretion of diffusible transmitters, such as interleukins, ATP, and NO, astrocytes may augment primary afferent neuronal signaling or sensitize second order neurons in the spinal cord. In addition, astrocytes might lead to altered pain perception by a direct modulation of synaptic transmission between neurons in the nociceptive pathway or through the creation of astrocytic networks capable of transducing signals for extended distances across and along the spinal cord. Future research in astrocyte activation and signaling may therefore reveal novel drug targets for managing pathological pain.
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Astrocitos/patología , Modelos Neurológicos , Dolor/patología , Médula Espinal/patología , Animales , Astrocitos/enzimología , Humanos , Hipersensibilidad/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Dolor/enzimología , Médula Espinal/enzimologíaRESUMEN
Pain due to bone malignancies is one of the most difficult types of cancer pain to fully control and may further decrease the patients' quality of life. Animal models of chronic pain conditions resulting from peripheral inflammatory reactions or nerve injuries are responsive to treatment with cannabinoid agonists. However, the use of cannabinoid agonists in humans may be hampered by CNS related side effects and development of tolerance. In the present study, we investigated the effect of repeated low dose administration of the synthetic cannabinoid agonist WIN 55,212-2 on bone cancer pain and neuropathic pain in mice. In addition, we investigated the development of CNS related side effects and tolerance. We found that 0.5 mg/kg/day for 18 days reduced pain related behavior and expression of spinal glial fibrillary acidic protein in the bone cancer pain model but not in the neuropathic pain model. Furthermore, this treatment strategy was not found to induce measurable CNS related side effects or tolerance. Cancer cell viability assays and bone volume fraction assessed by micro computed tomography (microCT) demonstrated that these effects were not due to changes in cancer progression. The difference in WIN 55,212-2 efficacy between the bone cancer and neuropathic pain models may reflect the different pain generating mechanisms, which may be utilized in designing new therapeutic drugs.
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Analgésicos/uso terapéutico , Benzoxazinas/uso terapéutico , Neoplasias Óseas/complicaciones , Agonistas de Receptores de Cannabinoides , Morfolinas/uso terapéutico , Naftalenos/uso terapéutico , Dolor/tratamiento farmacológico , Dolor/etiología , Enfermedades del Sistema Nervioso Periférico/complicaciones , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Ganglios Espinales/fisiología , Proteína Ácida Fibrilar de la Glía/biosíntesis , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C3H , Actividad Motora/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Estimulación Física , Equilibrio Postural/efectos de los fármacos , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tomografía Computarizada por Rayos X , Soporte de Peso/fisiologíaRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by a dramatic loss of dopaminergic neurons in the substantia nigra (SN). Several pathogenic mechanisms have been implicated in the demise of these cells, including dopamine-dependent oxidative stress, mitochondrial dysfunction, excitotoxicity, and proteasomal impairment. In recent years, the involvement of neuroinflammatory processes in nigral degeneration has gained increasing attention. Not only have activated microglia and increased levels of inflammatory mediators been detected in the striatum of PD patients, but a large body of animal studies points to a contributory role of inflammation in dopaminergic cell loss. For example, post-mortem examination of human subjects exposed to the parkinsonism-inducing toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, revealed the presence of activated microglia decades after drug exposure, suggesting that even a brief pathogenic insult can induce an ongoing inflammatory response. Perhaps not surprisingly, nonsteroidal anti-inflammatory drugs have been shown to reduce the risk of developing PD. In the past few years, various pathways have come to light that could link neurodegeneration and microglial activation, finally ascribing a pathogenic trigger to the chronic inflammatory response characteristic of PD.
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1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Dopamina/metabolismo , Intoxicación por MPTP/metabolismo , Microglía/metabolismo , Estrés Oxidativo/efectos de los fármacos , Sustancia Negra/metabolismo , Antiinflamatorios no Esteroideos/uso terapéutico , Enfermedad Crónica , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Intoxicación por MPTP/tratamiento farmacológico , Intoxicación por MPTP/patología , Microglía/patología , Factores de Riesgo , Sustancia Negra/patologíaRESUMEN
In the present study, we first report an in vivo characterization of flexor responses induced by three distinct sine-wave stimuli in the electrical stimulation-induced paw flexion (EPF) test in mice. The fixed sine-wave electric stimulations of 5 Hz (C-fiber), 250 Hz (Adelta-fiber) and 2000 Hz (Abeta-fiber) to the hind paw of mice induced a paw-flexion response and vocalization. The average threshold for paw flexor responses by sine-wave stimulations was much lower than that for vocalization. Neonatally (P3) pretreatment with capsaicin to degenerate polymodal substance P-ergic C-fiber neurons increased the threshold to 5 Hz (C-fiber) stimuli, but not to 250 Hz (Adelta-fiber) and 2000 Hz (Abeta-fiber). The flexor responses to 5 Hz stimuli were significantly blocked by intrathecal (i.t.) pretreatment with both CP-99994 and MK-801, an NK1 and NMDA receptor antagonist, respectively, but not by CNQX, an AMPA/kainate receptor antagonist. On the other hand, the flexor responses induced by 250 Hz stimuli were blocked by MK-801 (i.t.) but not by CP-99994 or CNQX. In contrast, flexor responses induced by 2000 Hz stimuli were only blocked by CNQX treatment. These data suggest that we have identified three pharmacologically different categories of responses mediated through different primary afferent fibers. Furthermore, we also carried out characterization of the in vivo functional sensitivity of each of the sensory fiber types in nerve-injured mice using the EPF test, and found that the threshold to both 250 Hz and 2000 Hz stimulations were markedly decreased, whereas the threshold to 5 Hz stimulations was significantly increased. Thus we found opposing effects on specific sensory fiber-mediated responses as a result of nerve injury in mice. These results also suggest that the EPF analysis is useful for the evaluation of plasticity in sensory functions in animal disease models.
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Neuronas Aferentes/fisiología , Nociceptores/fisiopatología , Umbral del Dolor/fisiología , Dolor/fisiopatología , Nervios Periféricos/fisiopatología , Raíces Nerviosas Espinales/fisiopatología , Animales , Animales Recién Nacidos , Capsaicina/efectos adversos , Modelos Animales de Enfermedad , Estimulación Eléctrica/efectos adversos , Estimulación Eléctrica/métodos , Pie/inervación , Pie/fisiopatología , Miembro Posterior/inervación , Miembro Posterior/fisiopatología , Masculino , Ratones , Fibras Nerviosas Amielínicas/efectos de los fármacos , Fibras Nerviosas Amielínicas/metabolismo , Antagonistas del Receptor de Neuroquinina-1 , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Neuronas Aferentes/clasificación , Neuronas Aferentes/efectos de los fármacos , Nociceptores/efectos de los fármacos , Nociceptores/lesiones , Dolor/inducido químicamente , Dimensión del Dolor/métodos , Umbral del Dolor/efectos de los fármacos , Traumatismos de los Nervios Periféricos , Nervios Periféricos/efectos de los fármacos , Valor Predictivo de las Pruebas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Neuroquinina-1/metabolismo , Reflejo/efectos de los fármacos , Reflejo/fisiología , Raíces Nerviosas Espinales/efectos de los fármacos , Raíces Nerviosas Espinales/lesiones , Sustancia P/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
BACKGROUND AND AIM: Ingenol mebutate (IngMeb) is an effective treatment for actinic keratosis. In this study, we hypothesized that repeated treatments with IngMeb may prevent progression of UV-induced photodamage, and that concurrent application of a corticosteroid may reduce IngMeb-induced local skin responses (LSR). METHODS: Hairless mice (n = 60; 3 groups of 20 mice) were irradiated with solar simulated ultraviolet radiation (UVR) throughout the study. Five single treatments with IngMeb were given at 4-week intervals (Days 21, 49, 77, 105, and 133). Clobetasol propionate (CP) was applied once daily for 5 days prior to each IngMeb application, as well as 6 h and 1 day post treatment. One week after IngMeb treatment No. 1, 3, and 5 (Days 28, 84, and 140), biopsies from four mice in each group were collected for histological evaluation of UV-damage on a standardized UV-damage scale (0-12). LSR (0-24) were assessed once daily (Days 1-7) after each IngMeb treatment. RESULTS: IngMeb prevented progression of photodamage in terms of keratosis grade, epidermal hypertrophy, dysplasia, and dermal actinic damage with a lower composite UV-damage score on day 140 (UVR 10.25 vs. UVR+IngMeb 6.00, p = 0.002) compared to UVR alone. IngMeb induced LSR, including erythema, flaking, crusting, bleeding, vesiculation, and ulceration. Concurrent CP increased LSR (max LSR Tx 1-5: UVR+IngMeb+CP 3.6-5.5 vs. UVR+IngMeb 2.6-4.3) and provided better prevention of photodamage compared to IngMeb alone (Day 140: UVR+IngMeb 6.00 vs. UVR+IngMeb+CP 3.00 p < 0.001). CONCLUSION: Repeated field-directed treatments with IngMeb prevent progression of cutaneous photodamage in hairless mice, while CP cannot be used to alleviate IngMeb-induced LSR. The findings suggest that IngMeb may potentially serve as a prophylactic treatment for UV-induced tumors.
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Diterpenos/administración & dosificación , Queratosis Actínica/tratamiento farmacológico , Rayos Ultravioleta , Animales , Clobetasol/administración & dosificación , Progresión de la Enfermedad , Femenino , Queratosis Actínica/patología , Ratones PeladosRESUMEN
UNLABELLED: The rapid and strong clinical efficacy of the first-in-class, ingenol mebutate, against actinic keratosis (AK) has resulted in its recent approval. We conducted the first comprehensive analysis of the cellular and molecular mode of action of topical ingenol mebutate 0.05% gel in both AK and uninvolved skin of 26 patients in a phase I, single-center, open-label, within-patient comparison. As early as 1 day after application, ingenol mebutate induced profound epidermal cell death, along with a strong infiltrate of CD4+ and CD8+ T-cells, neutrophils, and macrophages. Endothelial ICAM-1 activation became evident after 2 days. The reaction pattern was significantly more pronounced in AK compared with uninvolved skin, suggesting a tumor-preferential mode of action. Extensive molecular analyses and transcriptomic profiling of mRNAs and microRNAs demonstrated alterations in gene clusters functionally associated with epidermal development, inflammation, innate immunity, and response to wounding. Ingenol mebutate reveals a unique mode of action linking directly to anti-tumoral effects. TRIAL REGISTRATION: ClinicalTrials.gov NCT01387711.
Asunto(s)
Diterpenos/farmacología , Epidermis/inmunología , Epidermis/metabolismo , Queratosis Actínica/etiología , Queratosis Actínica/metabolismo , Administración Tópica , Adulto , Biomarcadores , Muerte Celular/efectos de los fármacos , Análisis por Conglomerados , Diterpenos/uso terapéutico , Epidermis/efectos de los fármacos , Epidermis/patología , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata , Queratosis Actínica/tratamiento farmacológico , Queratosis Actínica/patología , Leucocitos/inmunología , Leucocitos/metabolismo , Leucocitos/patología , MicroARNs/genética , ARN Mensajero/genéticaRESUMEN
The fibrinolytic activity of plasmin plays a fundamental role in resolution of blood clots and clearance of extravascular deposited fibrin in damaged tissues. These vital functions of plasmin are exploited by malignant cells to accelerate tumor growth and facilitate metastases. Mice lacking functional plasmin thus display decreased tumor growth in a variety of cancer models. Interestingly, this role of plasmin has, in regard to skin cancer, been shown to be restricted to male mice. It remains to be clarified whether gender also affects other phenotypic characteristics of plasmin deficiency or if this gender effect is restricted to skin cancer. To investigate this, we tested the effect of gender on plasmin dependent immune cell migration, accumulation of hepatic fibrin depositions, skin composition, and skin wound healing. Gender did not affect immune cell migration or hepatic fibrin accumulation in neither wildtype nor plasmin deficient mice, and the existing differences in skin composition between males and females were unaffected by plasmin deficiency. In contrast, gender had a marked effect on the ability of plasmin deficient mice to heal skin wounds, which was seen as an accelerated wound closure in female versus male plasmin deficient mice. Further studies showed that this gender effect could not be reversed by ovariectomy, suggesting that female sex-hormones did not mediate the accelerated skin wound healing in plasmin deficient female mice. Histological examination of healed wounds revealed larger amounts of fibrotic scars in the provisional matrix of plasmin deficient male mice compared to female mice. These fibrotic scars correlated to an obstruction of cell infiltration of the granulation tissue, which is a prerequisite for wound healing. In conclusion, the presented data show that the gender dependent effect of plasmin deficiency is tissue specific and may be secondary to already established differences between genders, such as skin thickness and composition.
Asunto(s)
Plasminógeno/deficiencia , Caracteres Sexuales , Cicatrización de Heridas/fisiología , Análisis de Varianza , Animales , Movimiento Celular/fisiología , Femenino , Fibrina/metabolismo , Tejido de Granulación/citología , Tejido de Granulación/metabolismo , Técnicas Histológicas , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , OvariectomíaRESUMEN
A well-coordinated remodeling of uncalcified collagen matrices is a pre-requisite for bone development and homeostasis. Collagen turnover proceeds through different pathways, either involving extracellular reactions exclusively, or being dependent on endocytic processes. Extracellular collagen degradation requires the action of secreted or membrane attached collagenolytic proteases, whereas the alternative collagen degradation pathway proceeds intracellularly after receptor-mediated uptake and delivery to the lysosomes. In this study we have examined the functional interplay between the extracellular collagenase, MMP-2, and the endocytic collagen receptor, uPARAP, by generating mice with combined deficiency of both components. In both uPARAP-deficient and MMP-2-deficient adult mice the length of the tibia and femur was decreased, along with a reduced bone mineral density and trabecular bone quality. An additional decrease in bone length was observed when combining the two deficiencies, pointing to both components being important for the remodeling processes in long bone growth. In agreement with results found by others, a different effect of MMP-2 deficiency was observed in the distinct bone structures of the calvaria. These membranous bones were found to be thickened in MMP-2-deficient mice, an effect likely to be related to an accompanying defect in the canalicular system. Surprisingly, both of the latter defects in MMP-2-deficient mice were counteracted by concurrent uPARAP deficiency, demonstrating that the collagen receptor does not support the same matrix remodeling processes as the MMP in the growth of the skull. We conclude that both uPARAP and MMP-2 take part in matrix turnover processes important for bone growth. However, in some physiological situations, these two components do not support the same step in the growth process.
Asunto(s)
Huesos/fisiología , Homeostasis/genética , Metaloproteinasa 2 de la Matriz/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Animales , Densidad Ósea/genética , Femenino , Crecimiento y Desarrollo/genética , Masculino , Ratones , Ratones Noqueados , Tamaño de los Órganos/genética , Osteogénesis/genética , Cráneo/crecimiento & desarrolloRESUMEN
The glycosylphosphatidylinositol (GPI)-anchored C4.4A was originally identified as a metastasis-associated protein by differential screening of rat pancreatic carcinoma cell lines. C4.4A is accordingly expressed in various human carcinoma lesions. Although C4.4A is a structural homolog of the urokinase receptor (uPAR), which is implicated in cancer invasion and metastasis, no function has so far been assigned to C4.4A. To assist future studies on its function in both physiological and pathophysiological conditions, the present study provide a global survey on C4.4A expression in the normal mouse by a comprehensive immunohistochemical mapping. This task was accomplished by staining paraffin-embedded tissues with a specific rabbit polyclonal anti-C4.4A antibody. In the adult mouse, C4.4A was predominantly expressed in the suprabasal layers of the squamous epithelia of the oral cavity, esophagus, non-glandular portion of the rodent stomach, anus, vagina, cornea, and skin. This epithelial confinement was particularly evident from the abrupt termination of C4.4A expression at the squamo-columnar transition zones found at the ano-rectal and utero-vaginal junctions, for example. During mouse embryogenesis, C4.4A expression first appears in the developing squamous epithelium at embryonic day 13.5. This anatomical location of C4.4A is thus concordant with a possible functional role in early differentiation of stratified squamous epithelia.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Desarrollo Embrionario , Células Epiteliales/citología , Proteínas Ligadas a GPI/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptores del Activador de Plasminógeno Tipo Uroquinasa/química , Homología de Secuencia de Aminoácido , Animales , Moléculas de Adhesión Celular/química , Células Epiteliales/metabolismo , Femenino , Proteínas Ligadas a GPI/química , Humanos , Inmunohistoquímica , Masculino , Ratones , Fenotipo , Transporte de Proteínas , Ratas , ReproducciónRESUMEN
The family of matrix metalloproteinases (MMPs) is responsible for extracellular matrix degradation during physiological and pathophysiological tissue remodeling processes such as embryogenesis, tissue repair and cancer progression. Despite these important roles of MMPs, inhibition or ablation of individual members of the MMP family in animal models have been shown to have little effect. It has been speculated that this results from a functional overlap between individual MMPs and (as-yet-unclassified) functional overlaps between MMPs and other protease systems. We here present genetic data showing that concomitant ablation of MMP9 (gelatinase B) and the serine protease plasmin results in lethal inflammatory mass lesions in the colon. These lesions possessed several histological attributes that are characteristic of mucosal prolapse seen in humans, and they were found to be associated with splenomegaly, enlarged mesenteric lymph nodes, decreased thymus size and altered populations of circulating immune cells. A time-course study provided evidence that the massive lymphoid hyperplasia and reactive changes were secondary to discrete fibrinous lesions also observed in mice only deficient for plasminogen (Plg), the zymogen for plasmin. These data demonstrate a non-appreciated vital protective role for MMP9 in the absence of Plg.