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INTRODUCTION: Sensory nerve endings transmit mechanical stimuli into afferent neural signals and form the basis of proprioception, giving rise to the self-perception of dynamic stability of joints. We aimed to analyze the three-dimensional structure of periarticular corpuscular sensory nerve endings in a carpal ligament to enhance our understanding of their microstructure. METHODS: Two dorsal parts of the scapholunate ligament were excised from two human cadaveric wrist specimens. Consecutive cryosections were stained with immunofluorescence markers protein S100B, neurotrophin receptor p75, protein gene product 9.5 (PGP 9.5), and 4',6-diamidino-2-phenylindole. Three-dimensional images of sensory nerve endings were obtained using confocal laser scanning microscopy, and subsequent analysis was performed using Imaris software. RESULTS: Ruffini endings were characterized by a PGP 9.5-positive central axon, with a median diameter of 4.63 µm and a median of 25 cells. The p75-positive capsule had a range in thickness of 0.94 µm and 15.5 µm, consisting of single to three layers of lamellar cells. Ruffini endings were significantly smaller in volume than Pacini corpuscles or Golgi-like endings. The latter contained a median of three intracorpuscular structures. Ruffini endings and Golgi-like endings presented a similar structural composition of their capsule and subscapular space. The central axon of Pacini corpuscles was surrounded by S100-positive cells forming the inner core which was significantly smaller than the outer core, which was immunoreactive for p75 and PGP 9.5. CONCLUSION: This study reports new data regarding the intricate outer and intracorpuscular three-dimensional morphology of periarticular sensory nerve endings, including the volume, number of cells, and structural composition. These results may form a basis to differ between normal and pathological morphological changes in periarticular sensory nerve endings in future studies.
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Trichophyton (T.) verrucosum is a highly pathogenic dermatophyte causing zoonotic bovine ringworm that is transmissible to humans. The virulence factors subtilisin (Sub)3 and Sub6 are discussed to contribute to disease manifestation but no protein expression study is available for T. verrucosum. We used customized antibodies (against Trichophyton-species, Sub3 and Sub6) to examine skin biopsies of infected cattle via immunofluorescence stainings. Both virulence factors Sub3 and 6 were solely expressed by conidia and not only found in epidermal but also in dermal and hair structures. The anti-T-antibody reliably detected the fungus and proved more sensitive compared to histological stains. LAY SUMMARY: We examined the zoonotic dermatophyte Trichophyton (T.) verrucosum in bovine skin and studied two important virulence factors called subtilisin (Sub)3 and Sub6 that T. verrucosum produces and secretes using immunolabeling.
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Enfermedades de los Bovinos/microbiología , Piel/microbiología , Subtilisina/genética , Tiña/veterinaria , Trichophyton/genética , Trichophyton/patogenicidad , Animales , Biopsia/veterinaria , Bovinos , Enfermedades de los Bovinos/diagnóstico , Técnica del Anticuerpo Fluorescente , Piel/patología , Esporas Fúngicas/genética , Esporas Fúngicas/patogenicidad , Subtilisina/clasificación , Tiña/microbiología , Factores de Virulencia/genética , Zoonosis/microbiologíaRESUMEN
Age-related macular degeneration (AMD) is a leading cause of blindness in people over 50â years of age in many developed countries. Drusen are yellowish extracellular deposits beneath retinal pigment epithelium (RPE) found in aging eyes and considered as a biomarker of AMD. However, the biogenesis of drusen has not been elucidated. We reported previously that multicellular spheroids of human RPE cells constructed a well-differentiated monolayer of RPE with a Bruch's membrane. We determined that RPE spheroids exhibited drusen formation between the RPE and Bruch's membrane with expression of many drusen-associated proteins, such as amyloid ß and complement components, the expression of which was altered by a challenge with oxidative stress. Artificial lipofuscin-loaded RPE spheroids yielded drusen more frequently. In the current study, we showed that drusen originates from the RPE. This culture system is an attractive tool for use as an in vitro drusen model, which might help elucidate the biogenesis of drusen and the pathogenesis of related diseases, such as AMD.
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Lámina Basal de la Coroides/metabolismo , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pigmentos Retinianos/metabolismo , Péptidos beta-Amiloides/metabolismo , Biomarcadores/metabolismo , Células Epiteliales/metabolismo , Humanos , Imagenología Tridimensional/métodos , Degeneración Macular/metabolismo , Degeneración Macular/patología , Retina/patología , Epitelio Pigmentado de la Retina/patología , Esferoides Celulares/metabolismoRESUMEN
Chronic inflammatory enteropathy (CIE) in dogs, a spontaneous model of human inflammatory bowel disease (IBD), is associated with a high rate of cobalamin deficiency. The etiology of hypocobalaminemia in human IBD and canine CIE remains unknown, and compromised intestinal uptake of cobalamin resulting from ileal cobalamin receptor deficiency has been proposed as a possible cause. Here, we evaluated the intestinal expression of the cobalamin receptor subunits, amnionless (AMN) and cubilin (CUBN), and the basolateral efflux transporter multi-drug resistance protein 1 (MRP1) in 22 dogs with CIE in comparison to healthy dogs. Epithelial CUBN and AMN levels were quantified by confocal laser scanning microscopy using immunohistochemistry in endoscopic ileal biopsies from dogs with (i) CIE and normocobalaminemia, (ii) CIE and suboptimal serum cobalamin status, (iii) CIE and severe hypocobalaminemia, and (iv) healthy controls. CUBN and MRP1 expression was quantified by RT-qPCR. Receptor expression was evaluated for correlation with clinical patient data. Ileal mucosal protein levels of AMN and CUBN as well as mRNA levels of CUBN and MRP1 were significantly increased in dogs with CIE compared to healthy controls. Ileal cobalamin receptor expression was positively correlated with age, clinical disease activity index (CCECAI) score, and lacteal dilation in the ileum, inversely correlated with serum folate concentrations, but was not associated with serum cobalamin concentrations. Cobalamin receptor downregulation does not appear to be the primary cause of hypocobalaminemia in canine CIE. In dogs of older age with severe clinical signs and/or microscopic intestinal lesions, intestinal cobalamin receptor upregulation is proposed as a mechanism to compensate for CIE-associated hypocobalaminemia. These results support oral supplementation strategies in hypocobalaminemic CIE patients.
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Enfermedades de los Perros , Enfermedades Inflamatorias del Intestino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Deficiencia de Vitamina B 12 , Humanos , Perros , Animales , Vitamina B 12 , Regulación hacia Arriba , Deficiencia de Vitamina B 12/genética , Deficiencia de Vitamina B 12/veterinaria , Enfermedades Inflamatorias del Intestino/patología , Íleon/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Enfermedades de los Perros/genéticaRESUMEN
Jamaican fruit bats (Artibeus jamaicensis) naturally harbor a wide range of viruses of human relevance. These infections are typically mild in bats, suggesting unique features of their immune system. To better understand the immune response to viral infections in bats, we infected male Jamaican fruit bats with the bat-derived influenza A virus (IAV) H18N11. Using comparative single-cell RNA sequencing, we generated single-cell atlases of the Jamaican fruit bat intestine and mesentery. Gene expression profiling showed that H18N11 infection resulted in a moderate induction of interferon-stimulated genes and transcriptional activation of immune cells. H18N11 infection was predominant in various leukocytes, including macrophages, B cells, and NK/T cells. Confirming these findings, human leukocytes, particularly macrophages, were also susceptible to H18N11, highlighting the zoonotic potential of this bat-derived IAV. Our study provides insight into a natural virus-host relationship and thus serves as a fundamental resource for future in-depth characterization of bat immunology.
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Quirópteros , Infecciones por Orthomyxoviridae , Análisis de la Célula Individual , Animales , Quirópteros/virología , Quirópteros/inmunología , Quirópteros/genética , Masculino , Humanos , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/veterinaria , Macrófagos/inmunología , Macrófagos/virología , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Perfilación de la Expresión GénicaRESUMEN
Inhibitory (GABAergic) interneurons entrain assemblies of excitatory principal neurons to orchestrate information processing in the hippocampus. Disrupting the dynamic recruitment as well as the temporally precise activity of interneurons in hippocampal circuitries can manifest in epileptiform seizures, and impact specific behavioral traits. Despite the importance of GABAergic interneurons during information encoding in the brain, experimental tools to selectively manipulate GABAergic neurotransmission are limited. Here, we report the selective elimination of GABAergic interneurons by a ribosome inactivation approach through delivery of saporin-conjugated anti-vesicular GABA transporter antibodies (SAVAs) in vitro as well as in the mouse and rat hippocampus in vivo. We demonstrate the selective loss of GABAergic--but not glutamatergic--synapses, reduced GABA release, and a shift in excitation/inhibition balance in mixed cultures of hippocampal neurons exposed to SAVAs. We also show the focal and indiscriminate loss of calbindin(+), calretinin(+), parvalbumin/system A transporter 1(+), somatostatin(+), vesicular glutamate transporter 3 (VGLUT3)/cholecystokinin/CB(1) cannabinoid receptor(+) and neuropeptide Y(+) local-circuit interneurons upon SAVA microlesions to the CA1 subfield of the rodent hippocampus, with interneuron debris phagocytosed by infiltrating microglia. SAVA microlesions did not affect VGLUT1(+) excitatory afferents. Yet SAVA-induced rearrangement of the hippocampal circuitry triggered network hyperexcitability associated with the progressive loss of CA1 pyramidal cells and the dispersion of dentate granule cells. Overall, our data identify SAVAs as an effective tool to eliminate GABAergic neurons from neuronal circuits underpinning high-order behaviors and cognition, and whose manipulation can recapitulate pathogenic cascades of epilepsy and other neuropsychiatric illnesses.
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Neuronas GABAérgicas/fisiología , Hipocampo , Interneuronas , Red Nerviosa/fisiología , Inhibición Neural , Fagocitosis , Animales , Calbindina 2 , Calbindinas , Muerte Celular/genética , Células Cultivadas , Femenino , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/patología , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiología , Interneuronas/metabolismo , Interneuronas/patología , Interneuronas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Red Nerviosa/metabolismo , Red Nerviosa/patología , Inhibición Neural/genética , Fagocitosis/genética , Conejos , Ratas , Ratas Sprague-Dawley , Proteína G de Unión al Calcio S100/genéticaRESUMEN
Toxoplasma (T.) gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection can lead to severe pathological alterations in the brain. To examine the effects of toxoplasmosis in the fetal brain, pregnant guinea pigs are infected with T. gondii oocysts on gestation day 23 and dissected 10, 17 and 25 days afterwards. We show the neocortex to represent a target region of T. gondii and the parasite to infect neural progenitor cells (NPCs), neurons and astrocytes in the fetal brain. Importantly, we observe a significant reduction in neuron number at end-neurogenesis and find a marked reduction in NPC count, indicating that impaired neurogenesis underlies the neuronal decrease in infected fetuses. Moreover, we observe focal microglioses to be associated with T. gondii in the fetal brain. Our findings expand the understanding of the pathophysiology of congenital toxoplasmosis, especially contributing to the development of cortical malformations.
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Neocórtex , Células-Madre Neurales , Toxoplasma , Toxoplasmosis , Embarazo , Femenino , Animales , Cobayas , NeurogénesisRESUMEN
Hepatosteatosis is a common metabolic disorder of dairy cows, especially during early lactation. Currently, there are a few models of bovine hepatic steatosis available, including primary hepatocytes, liver slices, and animal models. Studies that elucidate the influence of single fatty acids on lipid classes, fatty acid pattern, gene expression, and phenotypic changes are still limited. Hence, we investigated the suitability of the fetal bovine hepatocyte-derived cell line BFH12 as a model for hepatosteatosis. To create a steatotic environment, we treated BFH12 with stearic acid, palmitic acid, or oleic acid in non-toxic doses. Thin-layer chromatography and gas chromatography were used to analyze lipid classes and fatty acid pattern, and qPCR was used to quantify gene expression of relevant target genes. Lipid droplets were visualized with confocal laser scanning microscopy and evaluated for number and size. Treatment with oleic acid increased triglycerides, as well as lipid droplet count per cell and upregulated carnitine palmitoyl transferase 1, which correlates with findings of in vivo models. Oleic acid was largely incorporated into triglycerides, phospholipids, and non-esterified fatty acids. Stearic acid was found mainly in non-esterified fatty acids and triglycerides, whereas palmitic acid was mainly desaturated to palmitoleic acid. All three fatty acids downregulated stearyl-CoA-desaturase 1. In conclusion, BFH12 can acquire a steatotic phenotype by incorporating and accumulating fatty acids. Oleic acid is particularly suitable to produce hepatosteatosis. Therefore, BFH12 may be a useful in vitro model to study bovine hepatosteatosis and its underlying molecular mechanisms.
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The deposition of amyloid-ß (Aß) peptides in the walls of leptomeningeal and cortical blood vessels as cerebral amyloid angiopathy (CAA) is present in normal ageing and the majority of Alzheimer's disease (AD) brains. The failure of clearance mechanisms to eliminate Aß from the brain contributes to the development of sporadic CAA and AD. Here, we investigated the effects of CAA and ageing on the pattern of perivascular drainage of solutes in the brains of naïve mice and in the Tg2576 mouse model of AD. We report that drainage of small molecular weight dextran along cerebrovascular basement membranes is impaired in the hippocampal capillaries and arteries of 22-month-old wild-type mice compared to 3- and 7-month-old animals, which was associated with age-dependent changes in capillary density. Age-related alterations in the levels of laminin, fibronectin and perlecan in vascular basement membranes were also noted in wild-type mice. Furthermore, dextran was observed in the walls of veins of Tg2576 mice in the presence of CAA, suggesting that deposition of Aß in vessel walls disrupts the normal route of elimination of solutes from the brain parenchyma. These data support the hypothesis that perivascular solute drainage from the brain is altered both in the ageing brain and as a consequence of CAA. These findings have implications for the success of therapeutic strategies for the treatment of AD that rely upon the health of the ageing cerebral vasculature.
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Envejecimiento/patología , Encéfalo/irrigación sanguínea , Encéfalo/patología , Angiopatía Amiloide Cerebral/complicaciones , Angiopatía Amiloide Cerebral/patología , Factores de Edad , Precursor de Proteína beta-Amiloide/genética , Análisis de Varianza , Animales , Membrana Basal/metabolismo , Membrana Basal/patología , Membrana Basal/fisiopatología , Colágeno Tipo IV/metabolismo , Dextranos , Modelos Animales de Enfermedad , Femenino , Fibronectinas/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Laminina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genéticaRESUMEN
BACKGROUND: Primary cultures of epithelial cells are suitable models for studying epithelial function and, in particular, the regulation of epithelial tightness in vitro. The aim of our study was to develop a protocol for the isolation and culture of porcine colonic epithelial cells and to establish transepithelial electrical resistance (TEER) as a functional parameter for epithelial tightness. METHODS: Epithelial cells were obtained from the proximal colon of piglets by enzymatic dispase digestion. Cells were cultured on collagen-coated membrane supports for 21 days. The epithelial origin of the cells was shown by immunohistochemical detection of cytokeratin and zonula occludens protein 1 (ZO-1). Scanning electron microscopy, transmission electron microscopy and confocal microscopy were used for further morphological characterization. The integrity and tightness of the artificial epithelium were determined by measuring TEER. RESULTS: The cultured epithelial cells were immunoreactive for cytokeratin and ZO-1. They showed dense microvilli on their apical membranes and expression of Na(+)/K(+)-ATPase on their basolateral membranes. Adjacent cells were connected by tight junctions. We observed TEER to continuously increase up to 870 ± 38 Ω·cm(2) during the culture period. TEER correlated with the amount of epithelial cells expressing ZO-1. CONCLUSIONS: The properties of primary cultured epithelial cells resemble the structural properties of polarized colonic epithelium in vivo. Measurement of TEER seems to be suitable for studying epithelial tightness in vitro. We suggest that these primary epithelial cultures be used to investigate the regulation of the epithelial barrier function.
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Colon/metabolismo , Células Epiteliales/citología , Animales , Proliferación Celular , Células Cultivadas , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Inmunohistoquímica , Queratinas/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Rastreo , Fosfoproteínas/metabolismo , Porcinos , Proteína de la Zonula Occludens-1RESUMEN
BACKGROUND: Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions. RESULTS: Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures. CONCLUSIONS: This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.
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Bronquios/citología , Mucosa Respiratoria/citología , Animales , Diferenciación Celular , Proliferación Celular , Células Clonales , Medios de Cultivo , Medio de Cultivo Libre de Suero , CaballosRESUMEN
The drastic loss of cholinergic projection neurons in the basal forebrain is a hallmark of Alzheimer's disease (AD), and drugs most frequently applied for the treatment of dementia include inhibitors of the acetylcholine-degrading enzyme acetylcholinesterase (AChE). This protein is known to act as a ligand of beta-amyloid (Abeta) in senile plaques, a further neuropathological sign of AD. Recently, we have shown that the fluorescent, heterodimeric AChE inhibitor PE154 allows for the histochemical staining of cortical Abeta plaques in triple-transgenic (TTG) mice with age-dependent beta-amyloidosis and tau hyperphosphorylation, an established animal model for aspects of AD. In the present study, we have primarily demonstrated the targeting of Abeta-immunopositive plaques with PE154 in vivo for 4 h up to 1 week after injection into the hippocampi of 13-20-month-old TTG mice. Numerous plaques, double-stained for PE154 and Abeta-immunoreactivity, were revealed by confocal laser-scanning microscopy. Additionally, PE154 targeted hippocampal Abeta deposits in aged TTG mice after injection of carboxylated polyglycidylmethacrylate nanoparticles delivering the fluorescent marker in vivo. Furthermore, biodegradable core-shell polystyrene/polybutylcyanoacrylate nanoparticles were found to be suitable, alternative vehicles for PE154 as a useful in vivo label of Abeta. Moreover, we were able to demonstrate that PE154 targeted Abeta, but neither phospho-tau nor reactive astrocytes surrounding the plaques. In conclusion, nanoparticles appear as versatile carriers of AChE inhibitors and other promising drugs for the treatment of AD.
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Acridinas/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Inhibidores de la Colinesterasa/metabolismo , Cromonas/metabolismo , Colorantes Fluorescentes/metabolismo , Hipocampo/metabolismo , Nanopartículas , Acridinas/administración & dosificación , Envejecimiento , Péptidos beta-Amiloides/genética , Animales , Astrocitos/metabolismo , Inhibidores de la Colinesterasa/administración & dosificación , Cromonas/administración & dosificación , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos/métodos , Enbucrilato , Colorantes Fluorescentes/administración & dosificación , Humanos , Ratones , Ratones Transgénicos , Placa Amiloide/metabolismo , Ácidos Polimetacrílicos , Poliestirenos , Presenilina-1/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Borrelia burgdorferi, the agent of Lyme borreliosis, has the ability to undergo morphological transformation from a motile spirochetal to non-motile spherical shape when it encounters unfavorable conditions. However, little information is available on the mechanism that enables the bacterium to change its shape and whether major components of the cells--nucleic acids, proteins, lipids--are possibly modified during the process. Deducing from investigations utilizing electron microscopy, it seems that shape alteration begins with membrane budding followed by folding of the protoplasmatic cylinder inside the outer surface membrane. Scanning electron microscopy confirmed that a deficiency in producing functioning periplasmic flagella did not hinder sphere formation. Further, it was shown that the spirochetes' and spheres' lipid compositions were indistinguishable. Neither phosphatidylcholine nor phosphatidylglycerol were altered by the structural transformation. In addition, no changes in differential protein expression were detected during this process. However, minimal degradation of RNA and a reduced antigen-antibody binding activity were observed with advanced age of the spheres. The results of our comparisons and the failure to generate mutants lacking the ability to convert to spheres suggest that the metamorphosis of B. burgdorferi results in a conditional reconstruction of the outer membrane. The spheres, which appear to be more resistant to unfavorable conditions and exhibit reduced immune reactivity when compared to spirochetes, might allow the B. burgdorferi to escape complete clearance and possibly ensure long-term survival in the host.
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Proteínas Bacterianas/análisis , Borrelia burgdorferi/ultraestructura , Fosfatidilcolinas/análisis , Fosfatidilgliceroles/análisis , ARN Bacteriano/metabolismo , Borrelia burgdorferi/química , Borrelia burgdorferi/genética , Borrelia burgdorferi/fisiología , Electroforesis en Gel Bidimensional , Flagelos/metabolismo , Mutación , Análisis por Matrices de Proteínas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
It is commonly assumed that Dermacentor reticulatus immature life stages are nidicolous and therefore cannot be collected from vegetation. However, in June and July of 2018 and 2019, a total of 47 questing D. reticulatus larvae and two nymphs were collected by the flagging method in two different sites close to the city of Leipzig, Germany. To confirm their role in the transmission of tick-borne pathogens, 45 larvae (pooled by 2 in 21 pools and 1 pool with three individuals) and one nymph were tested either by conventional or real-time PCR for the presence of Bartonella spp., Neoehrlichia mikurensis, Rickettsia spp., and Babesia spp. All samples tested negative for Bartonella spp., N. mikurensis, and Babesia spp.; while the minimal infection rate of larvae for Rickettsia spp. was 42%, and the one tested nymph was also positive. Sequencing partial ompB genes revealed the presence of Rickettsia raoultii in larvae and nymph. Further research needs to be done to determine under which circumstances immature D. reticulatus ticks are found outside the burrows of their hosts and can be collected from vegetation.
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Distribución Animal , Dermacentor/fisiología , Rickettsia/aislamiento & purificación , Animales , Dermacentor/crecimiento & desarrollo , Dermacentor/microbiología , Ecosistema , Alemania , Larva/crecimiento & desarrollo , Larva/microbiología , Larva/fisiología , Ninfa/crecimiento & desarrollo , Ninfa/microbiología , Ninfa/fisiologíaRESUMEN
Surface disinfectants are regularly used in prophylactic and infection control measures. Concern has been raised whether residues of sub-inhibitory disinfectant concentrations may constitute a selective pressure and could contribute to the development of strains which are tolerant and/or resistant to biocides including antibiotics. The current study investigated whether Staphylococcus (S.) aureus ATCC® 29213™ and ATCC® 6538™ would change their growth characteristics and antimicrobial susceptibility profiles after prolonged treatment with sub-inhibitory concentrations of sodium hypochlorite (NaOCl). NaOCl is a fast-acting disinfectant with a broad-spectrum activity, inexpensive and widely used in healthcare and the food production industry. Minimum inhibitory concentration (MIC) for NaOCl was determined by broth macrodilution according to the guidelines for disinfectant efficacy testing provided by the German Veterinary Medical Society. Serial passages after 24 h and 72 h, respectively, in defined sub-inhibitory concentrations of NaOCl resulted in a number of phenotypic variants. Two of these variants, derived from S. aureus ATCC® 29213™, showed elevated MICs of oxacillin and were considered as in vitro-generated borderline oxacillin-resistant S. aureus (BORSA). Transmission electron microscopy revealed a significantly thickened cell wall in these isolates, a phenomenon that has also been described for Listeria monocytogenes after low-level exposure to NaOCl. Whole genome sequencing revealed an early stop codon in the gene coding for the GdpP protein and thereby abolishing the function of this gene. GdpP represents a phosphodiesterase that regulates gene expression, and loss of function of the GdpP protein has been described in association with borderline oxacillin resistance. Our findings suggest that a mutation in the GdpP protein gene and morphological changes of the cell wall were induced by repeated exposure to sub-lethal NaOCl concentrations, and most likely accounted for a BORSA phenotype in two variants derived from S. aureus ATCC® 29213™.
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Neurotransmitter uptake into synaptic vesicles is mediated by vesicular neurotransmitter transporters. Although these transporters belong to different families, they all are thought to share a common overall topology with an even number of transmembrane domains. Using epitope-specific antibodies and mass spectrometry we show that the vesicular GABA transporter (VGAT) possesses an uneven number of transmembrane domains, with the N terminus facing the cytoplasm and the C terminus residing in the synaptic vesicle lumen. Antibodies recognizing the C terminus of VGAT (anti-VGAT-C) selectively label GABAergic nerve terminals of live cultured hippocampal and striatal neurons as confirmed by immunocytochemistry and patch-clamp electrophysiology. Injection of fluorochromated anti-VGAT-C into the hippocampus of mice results in specific labeling of GABAergic synapses in vivo. Overall, our data open the possibility of studying novel GABA release sites, characterizing inhibitory vesicle trafficking, and establishing their contribution to inhibitory neurotransmission at identified GABAergic synapses.
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Inmunohistoquímica/métodos , Prosencéfalo/metabolismo , Coloración y Etiquetado/métodos , Sinapsis/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/química , Ácido gamma-Aminobutírico/metabolismo , Animales , Especificidad de Anticuerpos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/ultraestructura , Endocitosis/fisiología , Exocitosis/fisiología , Hipocampo/metabolismo , Hipocampo/ultraestructura , Espectrometría de Masas , Ratones , Inhibición Neural/fisiología , Técnicas de Placa-Clamp , Prosencéfalo/ultraestructura , Estructura Terciaria de Proteína/fisiología , Sinapsis/ultraestructura , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestructura , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/inmunología , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
Interaction between epithelial cells and fibroblasts play a key role in wound repair and remodelling in the asthmatic airway epithelium. We present the establishment of a co-culture model using primary equine bronchial epithelial cells (EBECs) and equine bronchial fibroblasts (EBFs). EBFs at passage between 4 and 8 were seeded on the bottom of 24-well plates and treated with mitomycin C at 80% confluency. Then, freshly isolated (P0) or passaged (P1) EBECs were seeded on the upper surface of membrane inserts that had been placed inside the EBF-containing well plates and grown first under liquid-liquid interface (LLI) then under air-liquid interface (ALI) conditions to induce epithelial differentiation. Morphological, structural and functional markers were monitored in co-cultured P0 and P1 EBEC monolayers by phase-contrast microscopy, scanning and transmission electron microscopy, hematoxylin-eosin, immunocytochemistry as well as by measuring the transepithelial electrical resistance (TEER) and transepithelial transport of selected drugs. After about 15-20 days of co-culture at ALI, P0 and P1 EBEC monolayers showed pseudo-stratified architecture, presence of ciliated cells, typically honeycomb-like pattern of tight junction protein 1 (TJP1) expression, and intact selective barrier functions. Interestingly, some notable differences were observed in the behaviour of co-cultured EBECs (adhesion to culture support, growth rate, differentiation rate) as compared to our previously described EBEC mono-culture system, suggesting that cross-talk between epithelial cells and fibroblasts actually takes place in our current co-culture setup through paracrine signalling. The EBEC-EBF co-culture model described herein will offer the opportunity to investigate epithelial-mesenchymal cell interactions and underlying disease mechanisms in the equine airways, thereby leading to a better understanding of their relevance to pathophysiology and treatment of equine and human asthma.
Asunto(s)
Bronquios/citología , Diferenciación Celular , Células Epiteliales/citología , Fibroblastos/citología , Animales , Atenolol/metabolismo , Transporte Biológico/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Electricidad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Fibroblastos/efectos de los fármacos , Caballos , Mitomicina/farmacología , Fenotipo , Propranolol/metabolismo , Ratas , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Factores de TiempoRESUMEN
A fovea is a pitted invagination in the inner retinal tissue (fovea interna) that overlies an area of photoreceptors specialized for high acuity vision (fovea externa). Although the shape of the vertebrate fovea varies considerably among the species, there are two basic types. The retina of many predatory fish, reptilians, and birds possess one (or two) convexiclivate fovea(s), while the retina of higher primates contains a concaviclivate fovea. By refraction of the incoming light, the convexiclivate fovea may function as image enlarger, focus indicator, and movement detector. By centrifugal displacement of the inner retinal layers, which increases the transparency of the central foveal tissue (the foveola), the primate fovea interna improves the quality of the image received by the central photoreceptors. In this review, we summarize â with the focus on Müller cells of the human and macaque fovea â data regarding the structure of the primate fovea, discuss various aspects of the optical function of the fovea, and propose a model of foveal development. The "Müller cell cone" of the foveola comprises specialized Müller cells which do not support neuronal activity but may serve optical and structural functions. In addition to the "Müller cell cone", structural stabilization of the foveal morphology may be provided by the 'z-shaped' Müller cells of the fovea walls, via exerting tractional forces onto Henle fibers. The spatial distribution of glial fibrillary acidic protein may suggest that the foveola and the Henle fiber layer are subjects to mechanical stress. During development, the foveal pit is proposed to be formed by a vertical contraction of the centralmost Müller cells. After widening of the foveal pit likely mediated by retracting astrocytes, Henle fibers are formed by horizontal contraction of Müller cell processes in the outer plexiform layer and the centripetal displacement of photoreceptors. A better understanding of the molecular, cellular, and mechanical factors involved in the developmental morphogenesis and the structural stabilization of the fovea may help to explain the (patho-) genesis of foveal hypoplasia and macular holes.
Asunto(s)
Fóvea Central , Animales , Astrocitos/citología , Astrocitos/fisiología , Células Ependimogliales/citología , Células Ependimogliales/fisiología , Fóvea Central/anatomía & histología , Fóvea Central/embriología , Fóvea Central/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Macaca , Enfermedades de la Retina/patologíaRESUMEN
The results obtained from the outer ear skin of female pigs (German Landrace) by light microscopy (LM), transmission electron microscopy (TEM), and cryo scanning electron microscopy (cryo SEM) methods, in particular relying on careful and artefact-free tissue processing, exhibited that the stratum superficiale dermidis of the auricle had a very homogeneous and compact construction, especially in one area (central dorsum auriculae). Based upon the important measurements made [average thickness of stratum superficiale dermidis: 94 (+/-16) microm, region A: 81 (+/-10); average thickness of collagen fibre bundles: 12 (+/-2) microm, region A: 13 (+/-0.5); average density of subepidermal capillaries: 3134 (+/-459) loops/cm2, region A: 3497 (+/-247)], this impression was confirmed by low standard deviations for all parameters, in comparison to marginal locations studied. The capillary system present was analysed by LM and TEM for specific structural features, whereby it generally compared to the microvasculature in human skin. Moreover, a regular pattern of diffusion-relevant punctiform contacts of the capillary loop apex with the epidermal basement membrane became obvious. Cryo SEM, particularly offering the advantages of dispensation of chemical fixation, dehydration and solvents during processing, highlighted delicate structures without shrinkage and without loss of soluble sample components. Thus rather real spatial conditions in the region of the epidermo-dermal junction and the upper dermis were visualized, whereby very regular arrangements of the structures present became obvious. This pertained also to a correct demonstration of all components of the epidermal basement membrane, in particular the lamina lucida. In addition, the water-based stable character of the entire stratum superficiale dermidis could be emphasized as a basic feature for controlled diffusion processes.
Asunto(s)
Células Epidérmicas , Animales , Capilares/citología , Capilares/ultraestructura , Microscopía por Crioelectrón , Epidermis/ultraestructura , Humanos , Modelos Animales , Piel/irrigación sanguínea , PorcinosRESUMEN
Pseudoalteromonas phage vB_PspS-H40/1 is a lytic phage that infects Pseudoalteromonas sp. strain H40. Both, the phage and its host were isolated in the 1970s from seawater samples collected from the North Sea near the island of Helgoland, Germany. The phage particle has an icosahedral capsid with a diameter of ~43 to 45 nm and a long non-contractile tail of ~68 nm in length, a typical morphology for members of the Siphoviridae family. The linear dsDNA genome of Pseudoalteromonas phage vB_PspS-H40/1 has a sequence length of 45,306 bp and a GC content of 40.6%. The genome has a modular structure and contains a high proportion of sequence information for hypothetical proteins, typically seen in phage genome sequences. This is the first report of the complete genome sequence of this lytic phage, which has been frequently used since the 1990s as biological tracer in hydrogeological transport studies.