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1.
Cell Tissue Res ; 393(2): 393-399, 2023 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-37314493

RESUMEN

In the past years, it has become clear that the family of Mas-related G protein-coupled receptors plays a central role in neuro-immune communication at mucosal barrier surfaces, in particular in the skin. Remarkably, MRGPR expression at other mucosal surfaces remains poorly characterized. To fill this gap in our understanding, the present study was undertaken to screen and verify the expression of the human MRGPR family members in the mucosal biopsies of the human gastrointestinal (GI) tract. Our findings revealed that, of all human MRGPRs family members, only MRGPRF mRNA is expressed at detectable levels in human mucosal biopsies of both terminal ileum and sigmoid colon. Furthermore, immunohistochemical stainings revealed that MRGPRF is specifically expressed by mucosal entero-endocrine cells (EECs). Overall, this study showed for the first time that the human ileum and colonic mucosa represent a novel expression site for the orphan MRGPRF, more specifically in EECs.


Asunto(s)
Células Endocrinas , Mucosa Intestinal , Humanos , Mucosa Intestinal/metabolismo , Tracto Gastrointestinal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Colon/metabolismo , Células Endocrinas/metabolismo , Células Enteroendocrinas/metabolismo
2.
Cell Commun Signal ; 20(1): 58, 2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35513867

RESUMEN

BACKGROUND: The contribution of native or modified oligodendroglia-derived extracellular vesicles (OL-EVs) in controlling chronic inflammation is poorly understood. In activated microglia, OL-EVs contribute to the removal of cytotoxic proteins following a proteotoxic stress. Intracellular small heat shock protein B8 (HSPB8) sustain this function by facilitating autophagy and protecting cells against oxidative stress mediated cell death. Therefore, secretion of HSPB8 in OL-EVs could be beneficial for neurons during chronic inflammation. However, how secreted HSPB8 contribute to cellular proteostasis remains to be elucidated. METHODS: We produced oligodendroglia-derived EVs, either native (OL-EVs) or HSPB8 modified (OL-HSPB8-EVs), to investigate their effects in controlling chronic inflammation and cellular homeostasis. We analyzed the impact of both EV subsets on either a resting or activated microglial cell line and on primary mixed neural cell culture cells. Cells were activated by stimulating with either tumor necrosis factor-alpha and interleukin 1-beta or with phorbol-12-myristate-13-acetate. RESULTS: We show that OL-EVs and modified OL-HSPB8-EVs are internalized by C20 microglia and by primary mixed neural cells. The cellular uptake of OL-HSPB8-EVs increases the endogenous HSPB8 mRNA expression. Consistently, our results revealed that both EV subsets maintained cellular homeostasis during chronic inflammation with an increase in the formation of autophagic vesicles. Both EV subsets conveyed LC3B-II and BAG3 autophagy markers with an enhanced effect observed for OL-HSPB8-EVs. Moreover, stimulation with either native or modified OL-HSPB8-EVs showed a significant reduction in ubiquitinated protein, reactive oxygen species and mitochondrial depolarization, with OL-HSPB8-EVs exhibiting a more protective effect. Both EV subsets did not induce cell death in the C20 microglia cell line or the primary mixed neural cultures. CONCLUSION: We demonstrate that the functions of oligodendroglia secreted EVs enriched with HSPB8 have a supportive role, comparable to the native OL-EVs. Further development of engineered oligodendroglia derived EVs could be a novel therapeutic strategy in countering chronic inflammation. Video Abstract.


Asunto(s)
Vesículas Extracelulares , Proteínas de Choque Térmico , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Vesículas Extracelulares/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Inflamación/metabolismo , Chaperonas Moleculares/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/patología , Estrés Oxidativo
3.
Adv Exp Med Biol ; 1383: 259-269, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36587165

RESUMEN

Over the past 15 years, the research field on Mas-related G protein-coupled receptors (Mrgprs), a relatively new family of rhodopsin A-like G protein-coupled receptors, has expanded enormously, and a plethora of recent studies have provided evidence that several of these Mrgpr family members play an important role in the underlying mechanisms of itch and pain, as well as in the initiation and modulation of inflammatory/allergic responses. Initial studies mainly focused on the skin, but more recently also visceral organs such as the respiratory and gastrointestinal (GI) tracts emerged as sites for Mrgpr involvement. It has become clear that the gastrointestinal tract and its innervation in close association with the immune system represent a novel expression site for Mrgprs where they contribute to the interoceptive mechanisms maintaining homeostasis and might constitute promising targets in chronic abdominal pain disorders. In this short review, we provide an update of our current knowledge on the expression, distribution, and function of members of this Mrgpr family in intrinsic and extrinsic neuro-immune pathways related to the gastrointestinal tract, their mediatory role(s) in gut neuro-immune signaling, and their involvement in visceral afferent (nociceptive) pathways.


Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Prurito , Dolor , Piel/metabolismo
4.
Small ; 17(5): e2006786, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33448084

RESUMEN

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.


Asunto(s)
Células Endoteliales , Vesículas Extracelulares , Análisis por Conglomerados , Humanos , Plasma , Aprendizaje Automático no Supervisado
5.
Int J Mol Sci ; 22(24)2021 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-34948051

RESUMEN

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist ß-alanine, we observed a release of IL-6. ß-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the Gαq/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-κB) and stimulation of IL-6 release. This IL-6 release could be blocked by a Gαq inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for ß-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay.


Asunto(s)
Interleucina-6/metabolismo , FN-kappa B/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta-Alanina/farmacología , Animales , Estrenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Péptidos Cíclicos/farmacología , Pirrolidinonas/farmacología , Receptores Acoplados a Proteínas G/agonistas , Transducción de Señal/efectos de los fármacos
6.
Neurobiol Dis ; 143: 105011, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32653674

RESUMEN

Progressive accumulation of hyperphosphorylated tau is a hallmark of various neurodegenerative disorders including Alzheimer's disease. However, to date, the functional effects of tau pathology on brain network connectivity remain poorly understood. To directly interrogate the impact of tau pathology on functional brain connectivity, we conducted a longitudinal experiment in which we monitored a fibril-seeded hTau.P301L mouse model using correlative whole-brain microscopy and resting-state functional MRI. Despite a progressive aggravation of tau pathology across the brain, the major resting-state networks appeared unaffected up to 15 weeks after seeding. Targeted analyses also showed that the connectivity of regions with high levels of hyperphosphorylated tau was comparable to that observed in controls. In line with the ostensible retention of connectivity, no behavioural changes were detected between seeded and control hTau.P301L mice as determined by three different paradigms. Our data indicate that seeded tau pathology, with accumulation of tau aggregates throughout different regions of the brain, does not alter functional connectivity or behaviour in this mouse model. Additional correlative functional studies on different mouse models should help determine whether this is a generalizable trait of tauopathies.


Asunto(s)
Encéfalo/fisiopatología , Red Nerviosa/fisiopatología , Vías Nerviosas/fisiopatología , Agregación Patológica de Proteínas/fisiopatología , Proteínas tau/metabolismo , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Humanos , Imagen por Resonancia Magnética , Ratones , Red Nerviosa/patología , Vías Nerviosas/patología , Agregación Patológica de Proteínas/patología
7.
J Vasc Res ; 57(6): 348-354, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32610324

RESUMEN

Multiple lines of evidence suggest that intraplaque (IP) neovascularization promotes atherosclerotic plaque growth, destabilization, and rupture. However, pharmacological inhibition of IP neovascularization remains largely unexplored due to the limited number of animal models that develop IP neovessels and the lack of reliable methods for visualizing IP angiogenesis. Here, we applied 3D confocal microscopy with an optimized tissue-clearing process, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, to visualize IP neovessels in apolipoprotein E-deficient (ApoE-/-) mice carrying a heterozygous mutation (C1039+/-) in the fibrillin-1 gene. Unlike regular ApoE-/- mice, this mouse model is characterized by the presence of advanced plaques with evident IP neovascularization. Plaques were stained with antibodies against endothelial marker CD31 for 3 days, followed by incubation with fluorescently labeled secondary antibodies. Subsequent tissue clearing with dichloromethane (DCM)/methanol, DCM, and dibenzyl ether allowed easy visualization and 3D reconstruction of the IP vascular network while plaque morphology remained intact.


Asunto(s)
Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/patología , Imagenología Tridimensional , Microscopía Confocal , Neovascularización Patológica , Placa Aterosclerótica , Animales , Biomarcadores/metabolismo , Complejo CD3/metabolismo , Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibrilina-1/genética , Fibrilina-1/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Ratones Noqueados para ApoE , Mutación
8.
Neurobiol Dis ; 127: 398-409, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30878534

RESUMEN

We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.


Asunto(s)
Encéfalo/metabolismo , Microglía/metabolismo , Tauopatías/metabolismo , Proteínas tau/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ratones , Ratones Transgénicos , Microglía/patología , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Tauopatías/patología
9.
Cell Tissue Res ; 378(3): 555-558, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31473818

RESUMEN

Due to their pivotal role in nociception and mast cell biology, the family of Mas-related G protein-coupled receptors (Mrgprs) has recently gained attention for their possible expression and role(s) in the gastrointestinal tract. In this context, based on immunocytochemical stainings using a commercial antibody, a recent study by Zhou et al. reported that the murine Mrgprd member is expressed in mouse gut lamina propria immune cells and in the outer smooth muscle layers pointing to a potential role for MrgprD in inflammatory responses and intestinal immunity. Immunohistochemical staining for G protein-coupled receptors (GPCRs), however, remains challenging and should be cautiously interpreted using appropriate specificity controls. Using the same antibody with an identical dilution, we did observe a similar staining in the same wild-type mouse strain, but an identical staining pattern was also found in mice lacking the MrgprD receptor, indicating that this antibody recognizes epitopes other than those of MrgprD. Moreover, in situ hybridization for MrgprD further indicated the absence of receptor mRNA expression in lamina propria immune cells and in the outer smooth muscle layers. Therefore, the results and conclusions regarding the presence of MrgprD at protein level within the GI wall as described in the study of Zhou and collaborators should be interpreted with strong caution and should be reconsidered in the light of the emerging possible roles of MrgprD and therapeutic perspectives in gastrointestinal pathophysiology.


Asunto(s)
Íleon/metabolismo , Membrana Mucosa/metabolismo , Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL
10.
Respir Res ; 19(1): 207, 2018 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-30367659

RESUMEN

BACKGROUND: The microenvironment (ME) of neuroepithelial bodies (NEBs) harbors densely innervated groups of pulmonary neuroendocrine cells that are covered by Clara-like cells (CLCs) and is believed to be important during development and for adult airway epithelial repair after severe injury. Yet, little is known about its potential stem cell characteristics in healthy postnatal lungs. METHODS: Transient mild lung inflammation was induced in mice via a single low-dose intratracheal instillation of lipopolysaccharide (LPS). Bronchoalveolar lavage fluid (BALF), collected 16 h after LPS instillation, was used to challenge the NEB ME in ex vivo lung slices of control mice. Proliferating cells in the NEB ME were identified and quantified following simultaneous LPS instillation and BrdU injection. RESULTS: The applied LPS protocol induced very mild and transient lung injury. Challenge of lung slices with BALF of LPS-treated mice resulted in selective Ca2+-mediated activation of CLCs in the NEB ME of control mice. Forty-eight hours after LPS challenge, a remarkably selective and significant increase in the number of divided (BrdU-labeled) cells surrounding NEBs was observed in lung sections of LPS-challenged mice. Proliferating cells were identified as CLCs. CONCLUSIONS: A highly reproducible and minimally invasive lung inflammation model was validated for inducing selective activation of a quiescent stem cell population in the NEB ME. The model creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.


Asunto(s)
Proliferación Celular/fisiología , Células Neuroepiteliales/metabolismo , Mucosa Respiratoria/metabolismo , Nicho de Células Madre/fisiología , Células Madre/metabolismo , Animales , Femenino , Pulmón/citología , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Mucosa Respiratoria/citología
11.
Heart Vessels ; 33(1): 80-88, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29098407

RESUMEN

Neuroglobin (NGB) is an oxygen-binding protein that is mainly expressed in nervous tissues where it is considered to be neuroprotective during ischemic brain injury. Interestingly, transgenic mice overexpressing NGB reveal cytoprotective effects on tissues lacking endogenous NGB, which might indicate a therapeutic role for NGB in a broad range of ischemic conditions. In the present study, we investigated the effect of NGB overexpression on survival as well as on the size and occurrence of myocardial infarctions (MI) in a mouse model of acute MI (AMI) and a model of advanced atherosclerosis (ApoE -/- Fbn1 C1039G+/- mice), in which coronary plaques and MI develop in mice being fed a Western-type diet. Overexpression of NGB significantly enhanced post-AMI survival and reduced MI size by 14% 1 week after AMI. Gene expression analysis of the infarction border showed reduction of tissue hypoxia and attenuation of hypoxia-induced inflammatory pathways, which might be responsible for these beneficial effects. In contrast, NGB overexpression did not affect survival or occurrence of MI in the atherosclerotic mice although the incidence of coronary plaques was significantly reduced. In conclusion, NGB proved to act cytoprotectively during MI in the acute setting while this effect was less pronounced in the atherosclerosis model.


Asunto(s)
Citoprotección/genética , Regulación de la Expresión Génica , Globinas/genética , Isquemia Miocárdica/genética , Miocardio/patología , Proteínas del Tejido Nervioso/genética , ARN/genética , Enfermedad Aguda , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Globinas/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Miocardio/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Neuroglobina , Estrés Oxidativo , Reacción en Cadena en Tiempo Real de la Polimerasa
12.
Int J Mol Sci ; 19(1)2018 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-29351192

RESUMEN

Metal contaminated soils are increasing worldwide. Metal-tolerant plants growing on metalliferous soils are fascinating genetic and microbial resources. Seeds can vertically transmit endophytic microorganisms that can assist next generations to cope with environmental stresses, through yet poorly understood mechanisms. The aims of this study were to identify the core seed endophyte microbiome of the pioneer metallophyte Crotalaria pumila throughout three generations, and to better understand the plant colonisation of the seed endophyte Methylobacterium sp. Cp3. Strain Cp3 was detected in C. pumila seeds across three successive generations and showed the most dominant community member. When inoculated in the soil at the time of flowering, strain Cp3 migrated from soil to seeds. Using confocal microscopy, Cp3-mCherry was demonstrated to colonise the root cortex cells and xylem vessels of the stem under metal stress. Moreover, strain Cp3 showed genetic and in planta potential to promote seed germination and seedling development. We revealed, for the first time, that the seed microbiome of a pioneer plant growing in its natural environment, and the colonisation behaviour of an important plant growth promoting systemic seed endophyte. Future characterization of seed microbiota will lead to a better understanding of their functional contribution and the potential use for seed-fortification applications.


Asunto(s)
Crotalaria/microbiología , Methylobacterium/metabolismo , Microbiota/genética , Semillas/microbiología , Crotalaria/crecimiento & desarrollo , Crotalaria/metabolismo , Endófitos/crecimiento & desarrollo , Endófitos/metabolismo , Contaminación Ambiental , Metales/metabolismo , Metales/toxicidad , Desarrollo de la Planta , Raíces de Plantas/química , Semillas/metabolismo , Microbiología del Suelo , Contaminantes del Suelo/metabolismo , Contaminantes del Suelo/toxicidad , Simbiosis
13.
J Cell Mol Med ; 21(6): 1206-1216, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-27997763

RESUMEN

The mast/stem cell growth factor receptor KIT has long been assumed to be a specific marker for interstitial cells of Cajal (ICC) in the bladder, with possible druggable perspectives. However, several authors have challenged the presence of KIT+ ICC in recent years. The aim of this study was therefore to attempt to clarify the conflicting reports on KIT expression in the bladder of human beings, rat, mouse and guinea pig and to elucidate the possible role of antibody-related issues and interspecies differences in this matter. Fresh samples were obtained from human, rat, mouse and guinea pig cystectomies and processed for single/double immunohistochemistry/immunofluorescence. Specific antibodies against KIT, mast cell tryptase (MCT), anoctamin-1 (ANO1) and vimentin were used to characterize the cell types expressing KIT. Gut (jejunum) tissue was used as an external antibody control. Our results revealed KIT expression on mast cells but not on ICC in human, rat, mouse and guinea pig bladder. Parallel immunohistochemistry showed KIT expression on ICC in human, rat, mouse and guinea pig gut, which confirmed the selectivity of the KIT antibody clones. In conclusion, we have shown that KIT+ cells in human, rat, mouse and guinea pig bladder are mast cells and not ICC. The present report is important as it opposes the idea that KIT+ ICC are present in bladder. In this perspective, functional concepts of KIT+ ICC being involved in sensory and/or motor aspects of bladder physiology should be revised.


Asunto(s)
Proteínas Proto-Oncogénicas c-kit/genética , Células Madre/metabolismo , Vejiga Urinaria/metabolismo , Animales , Regulación de la Expresión Génica/genética , Cobayas , Humanos , Células Intersticiales de Cajal/citología , Células Intersticiales de Cajal/metabolismo , Mastocitos/metabolismo , Ratones , Músculo Liso/crecimiento & desarrollo , Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas , Vejiga Urinaria/citología
14.
Bioinformatics ; 32(23): 3691-3693, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27503222

RESUMEN

Deep tissue imaging is increasingly used for non-destructive interrogation of intact organs and small model organisms. An intuitive approach to increase the imaging depth by almost a factor of 2 is to record a sample from two sides and fuse both image stacks. However, imperfect three-dimensional alignment of both stacks presents a computational challenge. We have developed a FIJI plugin, called BiDiFuse, which merges bi-directionally recorded image stacks via 3D rigid transformations. The method is broadly applicable, considering it is compatible with all optical sectioning microscopes and it does not rely on fiducial markers for image registration. AVAILABILITY AND IMPLEMENTATION: The method is freely available as a plugin for FIJI from https://github.com/JanDetrez/BiDiFuse/ CONTACT: winnok.devos@uantwerpen.be.


Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional , Microscopía , Programas Informáticos , Humanos
15.
Cell Tissue Res ; 368(3): 459-467, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28190088

RESUMEN

Being continuously exposed to a plethora of antigens ranging from food antigens to potential pathogenic organisms, the gastrointestinal (GI) tract harbors the largest collection of immune cells in the mammalian body. This immune system has to maintain a delicate balance between mounting an active immune response and maintaining tolerance. The GI tract is also home to an elaborate intrinsic nervous system, the enteric nervous system (ENS). Various in vitro studies of neuro-immune communication have suggested that vasoactive intestinal peptide (VIP), an important GI neurotransmitter, modulates mononuclear phagocytes (MNPs), i.e., dendritic cells and macrophages. Using a combined approach of reverse transcription plus the polymerase chain reaction, immunofluorescence, three-dimensional maximum intensity projections and immunoelectron microscopy, we investigate the interaction between the enteric innervation and MNPs in the ileal lamina propria (LP). We demonstrate that VIP-ergic fibers of the ENS lie adjacent to CX3CR1+ MNPs and that VPAC1 is constitutively expressed on ileal CX3CR1+ cells in the LP of the mouse. We also identify, for the first time, CX3CR1+ immune cells in the LP at the ultrastructural level. Our data thus reveal the in situ presence of the molecular components that are necessary for a VIP-mediated neuro-immune interaction between the ENS and CX3CR1-expressing immune cells in the LP of the ileum.


Asunto(s)
Quimiocina CX3CL1/metabolismo , Íleon/inmunología , Íleon/inervación , Fibras Nerviosas/metabolismo , Neuroinmunomodulación , Péptido Intestinal Vasoactivo/metabolismo , Animales , Íleon/metabolismo , Íleon/ultraestructura , Ratones , Ratones Endogámicos C57BL , Sistema Mononuclear Fagocítico/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Transducción de Señal
16.
Cell Tissue Res ; 370(3): 403-416, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28963588

RESUMEN

With most research on interstitial cells (IC) in the bladder being conducted on animal models, it remains unclear whether all structural and functional data on IC from animal models can be translated to the human context. This prompted us to compare the structural and immunohistochemical properties of IC in bladders from mouse, rat and human. Tissue samples were obtained from the bladder dome and subsequently processed for immunohistochemistry and electron microscopy. The ultrastructural properties of IC were compared by means of electron microscopy and IC were additionally characterized with single/double immunohistochemistry/immunofluorescence. Our results reveal a similar organization of the IC network in the upper lamina propria (ULP), the deep lamina propria (DLP) and the detrusor muscle in human, rat and mouse bladders. Furthermore, despite several similarities in IC phenotypes, we also found several obvious inter-species differences in IC, especially in the ULP. Most remarkably in this respect, ULP IC in human bladder predominantly displayed a myoid phenotype with abundant presence of contractile micro-filaments, while those in rat and mouse bladders showed a fibroblast phenotype. In conclusion, the organization of ULP IC, DLP IC and detrusor IC is comparable in human, rat and mouse bladders, although several obvious inter-species differences in IC phenotypes were found. The present data show that translating research data on IC in laboratory animals to the human setting should be carried out with caution.


Asunto(s)
Células Intersticiales de Cajal/ultraestructura , Membrana Mucosa/ultraestructura , Miocitos del Músculo Liso/ultraestructura , Vejiga Urinaria/anatomía & histología , Animales , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Microscopía Electrónica , Ratas , Ratas Sprague-Dawley
17.
Respir Res ; 18(1): 87, 2017 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-28482837

RESUMEN

BACKGROUND: The pulmonary neuroepithelial body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. METHODS: A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. RESULTS: Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. CONCLUSIONS: This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/metabolismo , Proteínas de la Membrana/metabolismo , Cuerpos Neuroepiteliales/citología , Cuerpos Neuroepiteliales/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Pulmón/citología , Ratones , Ratones Transgénicos
18.
Cell Mol Neurobiol ; 37(7): 1257-1267, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28008568

RESUMEN

Calbindin D28 K (CB) and calretinin (CR) are the members of the EF-hand family of calcium-binding proteins that are expressed in neurons and nerve fibers of the enteric nervous system. CB and CR are expressed differentially in neuronal subpopulations throughout the central and peripheral nervous systems and their expression has been used to selectively target specific cell types and isolate neuronal networks. The present study presents an immunohistochemical analysis of CB and CR in the enteric ganglia of small intestine in rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 60-day-old, 1-year-old, and 2-year-old). The data obtained suggest a number of age-dependent changes in CB and CR expression in the myenteric and submucous plexuses. In the myenteric plexus, the lowest percentage of CB-immunoreactive (IR) and CR-IR neurons was observed at birth, after which the number of IR cells increased in the first 10 days of life. In the submucous plexus, CB-IR and CR-IR neurons were observed from 10-day-old onwards. The percentage of CR-IR and CB-IR neurons increased in the first 2 months and in the first 20 days, respectively. In all animals, the majority of the IR neurons colocalized CR and CB. From the moment of birth, the mean of the cross-sectional area of the CB-IR and CR-IR neuronal profiles was larger than that of CB- and CR-negative cells.


Asunto(s)
Calbindina 2/biosíntesis , Calbindinas/biosíntesis , Sistema Nervioso Entérico/metabolismo , Ganglios/metabolismo , Neuronas/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Calbindina 2/análisis , Calbindinas/análisis , Sistema Nervioso Entérico/química , Sistema Nervioso Entérico/crecimiento & desarrollo , Ganglios/química , Ganglios/crecimiento & desarrollo , Neuronas/química , Ratas
19.
Reprod Fertil Dev ; 29(8): 1567-1575, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27677211

RESUMEN

Electrically silent voltage-gated potassium (KvS) channel subunits (i.e. Kv5-Kv6 and Kv8-Kv9) do not form functional homotetrameric Kv channels, but co-assemble with Kv2 subunits, generating functional heterotetrameric Kv2--KvS channel complexes in which the KvS subunits modulate the Kv2 channel properties. Several KvS subunits are expressed in testis tissue but knowledge about their contribution to testis physiology is lacking. Here, we report that the targeted deletion of Kv6.4 in a transgenic mouse model (Kcng4-/-) causes male sterility as offspring from homozygous females were only obtained after mating with wild-type (WT) or heterozygous males. Semen quality analysis revealed that the sterility of the homozygous males was caused by a severe reduction in total sperm-cell count and the absence of motile spermatozoa in the semen. Furthermore, spermatozoa of homozygous mice showed an abnormal morphology characterised by a smaller head and a shorter tail compared with WT spermatozoa. Comparison of WT and Kcng4-/- testicular tissue indicated that this inability to produce (normal) spermatozoa was due to disturbed spermiogenesis. These results suggest that Kv6.4 subunits are involved in the regulation of the late stages of spermatogenesis, which makes them a potentially interesting pharmacological target for the development of non-hormonal male contraceptives.


Asunto(s)
Infertilidad Masculina/genética , Canales de Potasio con Entrada de Voltaje/genética , Espermatogénesis/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Animales , Forma de la Célula/genética , Infertilidad Masculina/metabolismo , Masculino , Ratones , Ratones Noqueados , Canales de Potasio con Entrada de Voltaje/metabolismo , Análisis de Semen , Motilidad Espermática/genética , Espermatozoides/citología
20.
Int J Med Sci ; 14(4): 310-318, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28553162

RESUMEN

Background: Human amniotic membranes (HAMs) are assumed to have a number of unique characteristics including durability, hypoallergenic and anti-inflammatory properties. Materials and Methods: Multilayer HAMs from caesarian sections were applied to repair defined bladder defects in male Sprague-Dawley rats. The animals were sacrificed at 7, 21 and 42 days after implantation. Bladder volume capacity after grafting was measured. Histological analyses were performed to asses a number of parameters including HAM degradation, inflammatory reaction, graft rejection and smooth muscle ingrowth. Results: One rat died from sepsis in the treated group. No severe complications or signs of leakage were observed. Bladder capacity did not change over time. The initially increased inflammation in the HAM group diminished significantly over time (p<0.05). No signs of HAM degradation were observed and smooth muscle staining increased over time. Conclusions: HAMs appear to be durable and hypoallergenic grafts. The assumed suitability for the reconstruction of urinary tract justifies further research on detailed immunological process in larger grafts.


Asunto(s)
Amnios/trasplante , Procedimientos de Cirugía Plástica/métodos , Regeneración , Vejiga Urinaria/trasplante , Animales , Modelos Animales de Enfermedad , Rechazo de Injerto/fisiopatología , Xenoinjertos , Humanos , Inflamación/fisiopatología , Músculo Liso/crecimiento & desarrollo , Ratas , Vejiga Urinaria/fisiopatología
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