RESUMEN
Inflammatory conditions can lead to debilitating and persistent pain. This hyperalgesia reflects sensitization of peripheral terminals and facilitation of pain signaling at the spinal level. Studies of peripheral systems show that tissue injury triggers not only inflammation but also a well-orchestrated series of events that leads to reversal of the inflammatory state. In this regard, lipoxins represent a unique class of lipid mediators that promote resolution of inflammation. The antiinflammatory role of peripheral lipoxins raises the hypothesis that similar neuraxial systems may also down-regulate injury-induced spinal facilitation of pain processing. We report that the lipoxin A(4) receptor is expressed on spinal astrocytes both in vivo and in vitro and that spinal delivery of lipoxin A(4), as well as stable analogues, attenuates inflammation-induced pain. Furthermore, activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase in astrocytes, which has been indicated to play an important role in spinal pain processing, was attenuated in the presence of lipoxins. This linkage opens the possibility that lipoxins regulate spinal nociceptive processing though their actions upon astrocytic activation. Targeting mechanisms that counterregulate the spinal consequences of persistent peripheral inflammation provide a novel endogenous mechanism by which chronic pain may be controlled.
Asunto(s)
Aspirina/metabolismo , Hiperalgesia/tratamiento farmacológico , Lipoxinas/uso terapéutico , Dolor/tratamiento farmacológico , Dolor/fisiopatología , Animales , Astrocitos/metabolismo , Western Blotting , Carragenina/toxicidad , Hiperalgesia/inducido químicamente , Inmunohistoquímica , Inflamación/complicaciones , Mediadores de Inflamación/metabolismo , Lipoxinas/metabolismo , Masculino , Dolor/etiología , Ratas , Ratas Sprague-Dawley , Receptores de Lipoxina/metabolismo , Médula Espinal/citologíaRESUMEN
Clinical and experimental evidence indicates that inflammatory processes contribute to the pathophysiology of epilepsy, but underlying mechanisms remain mostly unknown. Using immunohistochemistry for CD45 (common leukocyte antigen) and CD3 (T-lymphocytes), we show here microglial activation and infiltration of leukocytes in sclerotic tissue from patients with mesial temporal lobe epilepsy (TLE), as well as in a model of TLE (intrahippocampal kainic acid injection), characterized by spontaneous, nonconvulsive focal seizures. Using specific markers of lymphocytes, microglia, macrophages, and neutrophils in kainate-treated mice, we investigated with pharmacological and genetic approaches the contribution of innate and adaptive immunity to kainate-induced inflammation and neurodegeneration. Furthermore, we used EEG analysis in mutant mice lacking specific subsets of lymphocytes to explore the significance of inflammatory processes for epileptogenesis. Blood-brain barrier disruption and neurodegeneration in the kainate-lesioned hippocampus were accompanied by sustained ICAM-1 upregulation, microglial cell activation, and infiltration of CD3(+) T-cells. Moreover, macrophage infiltration was observed, selectively in the dentate gyrus where prominent granule cell dispersion was evident. Unexpectedly, depletion of peripheral macrophages by systemic clodronate liposome administration affected granule cell survival. Neurodegeneration was aggravated in kainate-lesioned mice lacking T- and B-cells (RAG1-knock-out), because of delayed invasion by Gr-1(+) neutrophils. Most strikingly, these mutant mice exhibited early onset of spontaneous recurrent seizures, suggesting a strong impact of immune-mediated responses on network excitability. Together, the concerted action of adaptive and innate immunity triggered locally by intrahippocampal kainate injection contributes seizure-suppressant and neuroprotective effects, shedding new light on neuroimmune interactions in temporal lobe epilepsy.
Asunto(s)
Encéfalo/patología , Epilepsia del Lóbulo Temporal/etiología , Leucocitos/patología , Microglía/patología , Inmunidad Adaptativa , Análisis de Varianza , Animales , Biomarcadores/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/metabolismo , Epilepsia del Lóbulo Temporal/patología , Humanos , Inmunidad Innata , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/metabolismo , Ácido Kaínico , Antígenos Comunes de Leucocito/metabolismo , Recuento de Leucocitos , Leucocitos/metabolismo , Masculino , Ratones , Ratones Noqueados , Microglía/metabolismo , Neuronas/metabolismo , Neuronas/patología , Esclerosis/metabolismo , Esclerosis/patologíaRESUMEN
Intra-hippocampal injection of kainic acid (KA) in adult mice causes a focal lesion in the CA1 area and hilus of the dentate gyrus, as well as pronounced granule cell hypertrophy and dispersion. The lesion results in chronic focal seizures, with a two-week delay following KA-induced status epilepticus. Furthermore, seizures are preceded by infiltration of T lymphocytes into the lesioned tissue and of macrophage-like cells, strongly immunopositive for the monocyte marker F4/80, into the dentate gyrus, where they regulate granule cell dispersion and survival. Unexpectedly, depletion of CD4(+) and/or CD8(+) T lymphocytes by targeted gene deletion results in a marked shortening of the delay prior to seizure onset, suggesting a role of adaptive immunity in epileptogenesis (Zattoni et al. 2011, J. Neurosci. 31, 4037). Here, we investigated the specific role of adaptive immunity in this TLE model by adoptive i.v. transfer of immunopurified T cells in mutant mice lacking either CD4(+) T cells (MHCII-knockout), CD8(+) T cells (ß2-microglobulin-knockout), or both populations (RAG1-knockout mice). EEG analysis in mutants mice injected with KA two days after the T cell transfer revealed that grafting of the missing T cell population had no influence on seizure onset, but strongly influenced F4/80(+) macrophage-like cell infiltration in the dentate gyrus. Specifically, CD8(+) T cells in ß2-microgloblin-knockout mice enhanced macrophage recruitment, whereas CD4(+) T cells transferred in MHCII-knockout and in RAG1-knockout mice blocked macrophage infiltration, leading to reduced granule cell dispersion and survival, thereby worsening the KA-induced lesion. These results suggest that intact adaptive immunity is required for delayed seizure onset in this mouse model of TLE and unravel complex interactions between T cells and mononuclear phagocytes for the control of neuronal integrity and survival in the lesioned brain.
Asunto(s)
Traslado Adoptivo/métodos , Epilepsia del Lóbulo Temporal/inmunología , Epilepsia del Lóbulo Temporal/fisiopatología , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/fisiopatología , Enfermedades Neurodegenerativas/inmunología , Enfermedades Neurodegenerativas/fisiopatología , Subgrupos de Linfocitos T/inmunología , Animales , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/etiología , Síndromes de Inmunodeficiencia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades Neurodegenerativas/etiología , Subgrupos de Linfocitos T/trasplanteRESUMEN
Urethral reconstruction has received much attention in recent years, due to pathologies such as recurrence of urethral strictures after treatments. Various surgical techniques have been developed to obtain the best risk-benefit ratio, such as autologous grafts taken from the oral cavity. Tissue engineering and stem cells, growing in a tissue from a small biopsies, can further improve surgery, reducing invasiveness and morbidity. To determine whether urethra or other epithelia can be equally useful for urethra engineering, a comparison of clonogenic ability, proliferative potential and stem cell markers should be obtained. In this study, 19 biopsies from urethra, and 21 from oral mucosa were obtained from patients, during reconstructive surgery. Urethral and oral tissues were removed from the same donor, to develop primary cultures and cell characterization. The long term regenerative properties of both tissues were investigated in vitro by life span, clonal analysis and markers of different clonal types. Results revealed the same high proliferative potential for urethra and oral mucosa cultures, but maintenance of specific markers. Karyotype and growth factor dependence confirmed the normal phenotype of cultured cells. Clonal analysis of the proliferative compartment highlighted a very different proportion of stem and transient amplifying cells, characterised by dissimilar cell size profile and marker expression. In conclusion, both tissues can be cultured and preserve their stem cells in vitro. Few differences appeared in oral mucosa vs urethra, suggesting that they can be equally useful for tissue engineering of the urethral tract.
Asunto(s)
Mucosa Bucal/citología , Regeneración/fisiología , Células Madre/citología , Uretra/citología , Uretra/fisiología , Células 3T3 , Animales , Biomarcadores/metabolismo , Proliferación Celular , Tamaño de la Célula , Células Cultivadas , Células Clonales , Células Epiteliales/citología , Humanos , Queratinocitos/citología , Ratones , Complejo Represivo Polycomb 1/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
One of the main putative causes of therapy refractory epilepsy in mesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis is the overexpression of multidrug transporters (MDTs) at the blood-brain barrier (BBB). It steps up the removal of antiepileptic drugs (AEDs) out of the brain cells across the BBB resulting in a low concentration of AEDs within the target cells. Some of the mechanisms of AED resistance are most likely to be genetically determined. To obtain more information about the underlying pathophysiology of intractability in epilepsy, we compared the global gene expression profile of human hippocampus and hippocampal-derived microvascular endothelial cells from MTLE with HS patients and controls. At the level of MDT, a significant up-regulation was found for ABCB1 (P-gp), ABCB2, ABCB3, and ABCB4, which was mainly related to endothelial cells. The data on the MDT were validated and extended by quantitative RT-PCR. Surprisingly, inflammatory factors such as interleukins (IL-1α, IL-1ß, IL-6, and IL-18) and cytokines (TNF-α and TGF-ß1) were found to be up-regulated in hippocampal parenchyma. The overexpression of P-gp, IL-1ß, and IL-6 was also confirmed by immunohistochemistry (IHC). Our results suggest that complex expression changes of ABC-transporters may play a decisive role in pharmacoresistance in MTLE. Further studies on the new and unexpected overexpression of inflammatory cytokines may unlock hitherto undiscovered pathways of the underlying pathophysiology of human MTLE.
RESUMEN
Pharmacological studies indicate that spinal p38 mitogen-activated protein kinase plays a role in the development of hyperalgesia. We investigated whether either the spinal isoform p38alpha or p38beta is involved in peripheral inflammation evoked pain state and increased expression of spinal COX-2. Using intrathecal antisense oligonucleotides, we show that hyperalgesia is prevented by downregulation of p38beta but not p38alpha, whereas increases in spinal COX-2 protein expression at 8 hours are mediated by both p38alpha and beta isoforms. These data suggest that early activation of spinal p38beta isoform may affect acute facilitatory processing, and both p38beta and alpha isoforms mediate temporally delayed upregulation of spinal COX-2.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Hiperalgesia/prevención & control , Dolor/metabolismo , Médula Espinal/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Western Blotting , Carragenina/administración & dosificación , Carragenina/farmacología , Ciclooxigenasa 1/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Hiperalgesia/inducido químicamente , Inflamación , Inyecciones Espinales , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Proteínas de la Membrana/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , Dolor/inducido químicamente , Dolor/fisiopatología , Dimensión del Dolor/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Factores de TiempoRESUMEN
Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha, a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation, inhibited proliferation in vitro and in mice, and suppressed leukemogenesis. Moreover, activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells, respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis, as indicated by the effects of p42 C/EBPalpha, which were more potent than those of K298E C/EBPalpha, a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC, including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation, also induced granulocyte differentiation. Thus, these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors, and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation.