RESUMEN
Plasmodium coatneyi has been proposed as an animal model for human Plasmodium falciparum malaria as it appears to replicate many aspects of pathogenesis and clinical symptomology. As part of the ongoing evaluation of the rhesus macaque model of severe malaria, a detailed ultrastructural analysis of the interaction between the parasite and both the host erythrocytes and the microvasculature was undertaken. Tissue (brain, heart and kidney) from splenectomized rhesus macaques and blood from spleen-intact animals infected with P. coatneyi were examined by electron microscopy. In all three tissues, similar interactions (sequestration) between infected red blood cells (iRBC) and blood vessels were observed with evidence of rosette and auto-agglutinate formation. The iRBCs possessed caveolae similar to P. vivax and knob-like structures similar to P. falciparum. However, the knobs often appeared incompletely formed in the splenectomized animals in contrast to the intact knobs exhibited by spleen intact animals. Plasmodium coatneyi infection in the monkey replicates many of the ultrastructural features particularly associated with P. falciparum in humans and as such supports its use as a suitable animal model. However, the possible effect on hostparasite interactions and the pathogenesis of disease due to the use of splenectomized animals needs to be taken into consideration.
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Malaria , Plasmodium , Animales , Eritrocitos/parasitología , Interacciones Huésped-Parásitos , Macaca mulatta/parasitología , Malaria/parasitologíaRESUMEN
BACKGROUND: The Ehlers-Danlos syndromes (EDS) consist of 13 subtypes with overlapping features including joint hypermobility, skin and vascular fragility and generalized connective tissue friability. As DNA analysis has become the gold standard for investigation of EDS, transmission electron microscopy (TEM) in clinical practice is decreasing. However, owing to the use of next-generation sequencing, the frequency of variants of uncertain significance (VUS) identified using DNA analysis is increasing. We hypothesized that TEM can provide evidence for or against pathogenicity of VUS. OBJECTIVES: The aim of this study was to evaluate the role of TEM in the diagnosis of EDS subtypes. METHODS: Data were collected from patients who underwent a skin biopsy between October 2012 and March 2017 at the London EDS National Diagnostic Service. TEM biopsies were categorized as 'normal' or 'abnormal' according to the description and conclusion in the TEM reports. Definitive diagnoses were reached via a combination of clinical features, structural and functional studies and DNA investigations. RESULTS: The analysis included 177 patients, comprising 30 abnormal and 147 normal TEM reports. A definitive diagnosis of monogenic EDS subtypes was made in 24 patients. Overall, 17 of these 24 patients (71%) had an abnormal biopsy report and seven (29%) had a normal biopsy report. No TEM findings were specifically associated with any EDS subtype, although collagen flowers were present in most patients with a genetically confirmed diagnosis of classical EDS. CONCLUSIONS: TEM analysis of collagen structure may have the potential to provide evidence for or against the pathogenicity of a VUS, but more work is needed to establish a clear role for TEM in this process. What's already known about this topic? Collagen fibril abnormalities can be seen in several Ehlers-Danlos syndrome (EDS) subtypes. What does this study add? This study provides clinical data, transmission electron microscopy (TEM) data and molecular data of one of the largest groups of patients suspected to have a monogenetic EDS subtype. No TEM findings were specifically associated with an EDS subtype. There was a higher percentage (71%) of abnormal biopsy findings in patients with a definitive diagnosis of a monogenetic EDS subtype and where a class 4/5 genetic variant was present.
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Síndrome de Ehlers-Danlos , Colágeno , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/genética , Humanos , Londres , Microscopía Electrónica , SíndromeRESUMEN
Ehlers-Danlos syndrome (EDS) encompasses a genetically and clinically heterogeneous group of connective tissue disorders, characterized by joint hypermobility, skin hyperextensibility and tissue fragility. It is a rare condition, and inheritance is either autosomal dominant or recessive. Previously grouped into 11 different subtypes, with increasing knowledge of the underlying molecular defects, it was reclassified in 1997 into 6 major groups, with type VIII excluded from this classification. Type VIII EDS is a very rare subtype, characterized by severe, early-onset periodontitis, skin fragility and abnormal scarring. Voice abnormalities have occasionally been described in other forms of the condition, and may be due to defects in the collagen of the vocal ligament. We report two cases of patients with EDS type VIII and hoarseness.
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Síndrome de Ehlers-Danlos/complicaciones , Síndrome de Ehlers-Danlos/patología , Ronquera/etiología , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to â¼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to â¼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of 'standard' 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.
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Exosomas/química , Citometría de Flujo/métodos , Trofoblastos/química , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Biomarcadores/metabolismo , Western Blotting , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrifugación , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Femenino , Filtración , Citometría de Flujo/instrumentación , Fluorescencia , Expresión Génica , Humanos , Microscopía Electrónica de Transmisión , Nanopartículas/química , Nanopartículas/ultraestructura , Perfusión , Embarazo , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Trofoblastos/metabolismoRESUMEN
Stable isotopes are ideal labels for studying biological processes because they have little or no effect on the biochemical properties of target molecules. The NanoSIMS is a tool that can image the distribution of stable isotope labels with up to 50 nm spatial resolution and with good quantitation. This combination of features has enabled several groups to undertake significant experiments on biological problems in the last decade. Combining the NanoSIMS with other imaging techniques also enables us to obtain not only chemical information but also the structural information needed to understand biological processes. This article describes the methodologies that we have developed to correlate atomic force microscopy and backscattered electron imaging with NanoSIMS experiments to illustrate the imaging of stable isotopes at molecular, cellular, and tissue scales. Our studies make it possible to address 3 biological problems: (1) the interaction of antimicrobial peptides with membranes; (2) glutamine metabolism in cancer cells; and (3) lipoprotein interactions in different tissues.
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Glutamina/metabolismo , Microscopía de Fuerza Atómica/métodos , Neoplasias/metabolismo , Espectrometría de Masa de Ion Secundario/métodos , Péptidos Catiónicos Antimicrobianos/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Marcaje Isotópico/métodos , Lipoproteínas/metabolismo , Nanotecnología/métodos , Neoplasias/patología , Distribución TisularRESUMEN
Neisseria meningitidis causes half a million cases of septicemia and meningitis globally each year. The opacity (Opa) integral outer membrane proteins from N. meningitidis are polymorphic and highly immunogenic. Particular combinations of Opa proteins are associated with the hyperinvasive meningococcal lineages that have caused the majority of serogroup B and C meningococcal disease in industrialized countries over the last 60 years. For the first time, this genetic structuring of a diverse outer membrane protein family has been used to select a novel combination of representative antigens for immunogenicity testing. Fourteen recombinant Opa variants were produced and used in murine immunizations inducing an increase in specific antimeningococcal total IgG levels. All 14 Opa proteins elicited bactericidal antibodies against at least one hyperinvasive meningococcal isolate, and most isolates from each hyperinvasive lineage were killed by at least one Opa antiserum at a titer of 1:16 or greater. Cross-reactive bactericidal antibody responses were observed among clonal complexes. A theoretical coverage of 90% can be achieved by using a particular combination of 6 Opa proteins against an isolate collection of 227 recent United Kingdom disease cases. This study indicates the potential of Opa proteins to provide broad coverage against multiple meningococcal hyperinvasive lineages.
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Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Meningococicas/inmunología , Neisseria meningitidis/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Neisseria meningitidis/genética , Neisseria meningitidis/patogenicidad , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7(-/-) erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119(+)/CD71(High) stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.
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Anemia/etiología , Autofagia/fisiología , Mitocondrias/fisiología , Animales , Autofagia/genética , Proteína 7 Relacionada con la Autofagia , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/fisiología , Médula Ósea/crecimiento & desarrollo , Médula Ósea/fisiología , Codón/genética , Células Eritroides/metabolismo , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/fisiología , Integrasas/genética , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas Proto-Oncogénicas c-vav/deficiencia , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/fisiología , Transcripción GenéticaRESUMEN
The first clue to the elucidation of the complete life cycle of Toxoplasma gondii was the identification of an infectious form in cat faeces that could be transmitted orally and could survive in the external environment for extended periods. This personal review describes the scientist (W.M. Hutchison) and the background to the initial discovery and covers the period to the complete elucidation of the life cycle of T. gondii.
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Enfermedades de los Gatos/transmisión , Heces/parasitología , Estadios del Ciclo de Vida , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/transmisión , Animales , Animales Domésticos/parasitología , Enfermedades de los Gatos/historia , Enfermedades de los Gatos/parasitología , Gatos , Historia del Siglo XX , Humanos , Ratones , Oocistos/crecimiento & desarrollo , Conejos , Enfermedades de los Roedores/historia , Enfermedades de los Roedores/parasitología , Toxoplasma/ultraestructura , Toxoplasmosis/historia , Toxoplasmosis Animal/historia , Toxoplasmosis Animal/transmisiónRESUMEN
A young Russian man presented with increasing shortness of breath and signs of worsening aortic regurgitation. A diagnosis of infective endocarditis was made before emergency valve replacement. The infective cause was not discovered by routine culture but was suggested by electron microscopy and confirmed by serology and PCR testing.
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Insuficiencia de la Válvula Aórtica/etiología , Infecciones por Bartonella/diagnóstico , Endocarditis Bacteriana/diagnóstico , Fiebre de las Trincheras/diagnóstico , Antibacterianos/uso terapéutico , Insuficiencia de la Válvula Aórtica/cirugía , Infecciones por Bartonella/tratamiento farmacológico , Bartonella quintana/aislamiento & purificación , Disnea/etiología , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/microbiología , Válvulas Cardíacas/microbiología , Humanos , MasculinoRESUMEN
Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.
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Eimeria tenella/metabolismo , Ácido Graso Desaturasas/metabolismo , Acido Graso Sintasa Tipo II/metabolismo , Orgánulos/metabolismo , Secuencia de Aminoácidos , Animales , Eimeria tenella/genética , Eimeria tenella/ultraestructura , Ácido Graso Desaturasas/genética , Genes Protozoarios/genética , Genoma de Protozoos/genética , Células Germinativas/crecimiento & desarrollo , Inmunohistoquímica/métodos , Estadios del Ciclo de Vida , Merozoítos/ultraestructura , Microscopía Electrónica/métodos , Microscopía Inmunoelectrónica/métodos , Datos de Secuencia Molecular , Orgánulos/ultraestructura , Filogenia , Esporozoítos/ultraestructuraRESUMEN
In order to improve the technique and protocols of cryopreservation of articular cartilage, a study was carried out to assess the effects of rapid cooling on the intact articular cartilage. Cartilage slices with a thickness ranging from 0.2 to 0.5 mm taken from bovine metacarpal-phalangeal joints were subjected to rapid cooling by immersing them in liquid nitrogen with and without treatment of the VS55 cryoprotective agent (CPA). The ultrastructure, chondrocyte viability, swelling property, and glycosaminoglycan (GAG) content were then examined before and after cryopreservation to give qualitative and quantitative evaluation on the functional state of both chondrocytes and extracellular matrix. The transmission electron microscopy study demonstrated that damage to chondrocytes without CPA was far more pronounced than those with VS55 protection while the structure of the extracellular matrix altered little in either group. The cell viability assay showed that although the exposure to VS55 led to about 36% chondrocytes losing membrane integrity, the VS55 could provide protection to chondrocytes during rapid cooling and thawing, with approximately 51% of the cells having survived rapid cooling compared to fewer than 5% in the absence of CPA. There were no significant differences in degrees of swelling or the GAG contents of cartilage slices after cryopreservation indicating rapid freezing caused little damage to the matrix. Future research activities include searching improved CPA formulation, optimising the treatment protocol and investigating the long-term effects of rapid cooling on articular cartilage.
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Cartílago Articular , Condrocitos , Criopreservación , Crioprotectores/farmacología , Dimetilsulfóxido/farmacología , Formamidas/farmacología , HEPES/farmacología , Glicoles de Propileno/farmacología , Animales , Bovinos , Supervivencia Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/patología , Condrocitos/ultraestructura , Congelación , Glicosaminoglicanos/análisis , Microscopía Electrónica de TransmisiónRESUMEN
The quiescent cell population of tumours poses a barrier to the success of many cancer therapies. Most chemotherapeutic drugs target proliferating cells, but the growth fraction of many tumours is low. Based on the multicellular tumour spheroid model, a system was developed using human colon adenocarcinoma (DLD-1) cells to mimic the microenvironment of quiescent microregions of solid tumours. The quiescent tumour spheroids (TS(Q)) showed decreased expression of the proliferation marker Ki-67 and increased expression of the quiescence marker p27(kip1) compared to proliferating spheroids (TS(P)). The quiescent status of the TS(Q) was confirmed by long-term growth assessment. The quiescence was completely reversible demonstrating that the TS(Q) retained the ability to proliferate and morphological assessment by light microscopy confirmed the absence of significant apoptosis. When the efficacy of widely used chemotherapeutic drugs was determined, vinblastine, doxorubicin, cisplatin and 5-fluorouracil (5-FU) all produced significant cell death in the TS(P). However, while still effective, the potencies of doxorubicin and cisplatin were significantly reduced in TS(Q). In contrast, 5-FU and vinblastine did not produce cell death in the TS(Q). In summary, TS(Q) show considerable resistance to a panel of established chemotherapeutic agents and represent a useful model for evaluating the efficacy of drugs and other cancer therapies in quiescent tumours.
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Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/fisiología , Modelos Biológicos , Esferoides Celulares/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , HumanosRESUMEN
A feature of black African hair is an apparent increased fragility of the hair shaft compared to other ethnic groups (as measured by the tensile force needed to break the hair fibre). This has certain similarities to that reported for trichorrhexis nodosa (weathering secondary to physical damage) and trichothiodystrophy [a genetic disorder associated with reduced cystine (sulphur)-rich proteins and increased fragility]. In the present study, the distribution of the cystine-rich proteins in the hair of black Africans was compared to that of Caucasian and Asian volunteers, plus patients with trichorrhexis nodosa and trichothiodystrophy, using transmission electron microscopy and specific silver stains. It was found that the silver staining pattern of the hair shafts of black Africans was similar to that observed for Caucasians, Asians and also patients with trichorrhexis nodosa. The cuticular cells exhibited an electron dense A layer and exocuticle, and in the cortex the microfibrils forming the macrofibres were outlined by electron-dense material. This contrasts with the abnormal distribution of the cystine-rich proteins seen in trichothiodystrophy. This study is the first formal comparison of the cystine-rich proteins in the various racial groups and shows that there is no abnormality in their distribution in black African hair shafts compared to the other ethnic groups. Therefore, the excessive structural damage observed in the African hair shafts is consistent with physical trauma (resulting from grooming) rather than an inherent weakness due to any structural abnormality.
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Cistina/química , Cistina/metabolismo , Cabello/patología , Cabello/ultraestructura , África , Pueblo Asiatico , Población Negra , Enfermedades del Cabello/patología , Humanos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Tinción con Nitrato de Plata , Población BlancaRESUMEN
BACKGROUND: Pili annulati is an inherited hair shaft abnormality with a wide range of clinical expression. OBJECTIVE: We have examined closely three kindreds to reveal levels and character of expression of the phenotype and supplement current literature on the threshold for detection and aspects of hair shaft fragility. PATIENTS AND METHODS: Eleven cases of pili annulati from three families were included in a clinical and morphological study. All cases were assessed clinically and by light and scanning electron microscopy (SEM) of hair shafts. In addition, transmission electron microscopy (TEM) (four patients) and amino acid analysis (three patients) were undertaken on clinically overt cases. Results Examination by light microscopy with a fluid mountant was more sensitive than clinical examination, increasing the detection rate by 120%. Microscopic examination revealed that the characteristic periodic bands become less frequent distally in the hair shaft. Microscopic features of weathering were found in two cases, adding pili annulati to the list of structural hair shaft dystrophies that may weaken hair and dispose to weathering. Amino acid analysis of the hair of three patients with pili annulati showed elevated lysine and decreased cystine content compared to 12 normal controls, consistent with the reduced threshold for weathering. CONCLUSION: Careful light microscopy with fluid-mounted hair is needed to detect subjects mildly affected by pili annulati. Expression of the phenotype varies widely between individuals, between hairs and within hairs of the same individual, where ageing of the hair diminishes detectable features.
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Enfermedades del Cabello/diagnóstico , Enfermedades del Cabello/genética , Aminoácidos/análisis , Femenino , Cabello/anomalías , Cabello/ultraestructura , Enfermedades del Cabello/patología , Humanos , Masculino , Microscopía , Microscopía Electrónica de Rastreo , Linaje , FenotipoRESUMEN
Heterotopic bone formation in soft tissues occurs commonly in Paget's disease patients following a primary total hip arthroplasty (THA). The nature of this heterotopic bone has not been documented. In this report, we show that the heterotopic bone removed 14 years after primary THA in a case of Paget's disease was sclerotic, contained prominent mosaic cement lines and showed increased remodelling activity on the bone surface. In addition to these typically Pagetic histological features, it was noted ultrastructurally that the osteoclasts contained characteristic intranuclear viral-like inclusions. In contrast, the foreign body macrophages found in the joint pseudocapsule and pseudomembrane, which are a population of mononuclear precursor cells from which osteoclasts can be formed, did not contain viral-like inclusions. These findings are of interest regarding the pathogenesis of heterotopic bone formation following hip arthroplasty and the ontogeny of Pagetic osteoclasts.
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Artroplastia de Reemplazo de Cadera/efectos adversos , Osificación Heterotópica/diagnóstico por imagen , Osteítis Deformante/diagnóstico por imagen , Anciano , Humanos , Masculino , Osificación Heterotópica/complicaciones , Osificación Heterotópica/patología , Osteítis Deformante/complicaciones , Osteítis Deformante/patología , RadiografíaRESUMEN
Human serum high-density lipoprotein (HDL) is necessary and sufficient for the short-term maintenance of Plasmodium falciparum in in vitro culture. However, at high concentrations it is toxic to the parasite. A heat-labile component is apparently responsible for the stage-specific toxicity to parasites within infected erythrocytes 12-42 h after invasion, i.e. during trophozoite maturation. The effects of HDL on parasite metabolism (as determined by nucleic acid synthesis) are evident at about 30 h after invasion. Parasites treated with HDL show gross abnormalities by light and electron microscopy.
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Lipoproteínas HDL/farmacología , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Animales , Eritrocitos/parasitología , Eritrocitos/ultraestructura , Citometría de Flujo , Humanos , Hipoxantina/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacología , Microscopía Electrónica , Plasmodium falciparum/metabolismo , Plasmodium falciparum/ultraestructuraRESUMEN
BACKGROUND: Pili annulati is a rare autosomal dominant inherited hair shaft abnormality in which clinical examination reveals alternating light and dark bands leading to a shiny appearance of the hair. The clinically light bands are the abnormal areas due to cavities within the cortex. The pathogenesis remains unknown. OBJECTIVES: To investigate the expression of the basement membrane zone (BMZ) components in pili annulati hair follicles of the scalp. METHODS: Transmission electron microscopy (TEM) was carried out on scalp sections of six individuals with pili annulati and six controls. Longitudinal sections of scalp tissues from four individuals with pili annulati and six normal controls were studied by immunohistochemistry with a panel of monoclonal antibodies to the following BMZ components: alpha(6)beta(4) integrin, laminin 5, LH39 antigen, laminin 1, collagen IV and collagen VII. RESULTS: Using TEM, pili annulati scalp specimens exhibited a reduplicated lamina densa in the region of the root bulb in comparison with the single thin electron-dense band in controls. Using immunohistochemistry, there was a wavy BMZ in pili annulati follicles with antibodies to components of the lamina lucida, lamina densa and anchoring fibrils, whereas the BMZ in control hair follicles was as a smooth linear band. The expression of the hemidesmosome-associated alpha(6)beta(4) integrin was linear in both pili annulati and control hair follicles. CONCLUSIONS: Our results suggest that the genetic defect may be a mutation in proteins involved in signalling and regulation of formation and degradation of the lamina densa and sublamina densa region resulting in abnormal assembly or remodelling of the BMZ.
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Folículo Piloso/anomalías , Adulto , Anticuerpos Monoclonales , Membrana Basal/patología , Femenino , Folículo Piloso/patología , Humanos , Inmunohistoquímica/métodos , Microscopía Electrónica/métodos , Persona de Mediana Edad , Cuero Cabelludo/patologíaRESUMEN
Toxoplasma gondii has a complex life cycle involving definite (cat) and intermediate (all warm blooded animals) hosts. This gives rise to four infectious forms each of which has a distinctive biological role. Two (tachyzoite and merozoite) are involved in propagation within a host and two (bradyzoite and sporozoite) are involved in transmission to new hosts. The various forms can be identified by their structure, host parasite relationship and distinctive developmental processes. In the present in vivo study, the various stages have been evaluated by electron microscopy and immunocytochemistry using a panel of molecular markers relating to surface and cytoplasmic molecules, metabolic iso-enzymes and secreted proteins that can differentiate between tachyzoite, bradyzoite and coccidian development. Tachyzoites were characterised as being positive for surface antigen 1, enolase isoenzyme 2, lactic dehydrogenase isoenzyme 1 and negative for bradyzoite antigen 1. In contrast, bradyzoites were negative for SAG1 but positive for BAG1, ENO1 and LDH2. When stage conversion was followed in brain lesion at 10 and 15 days post-infection, tachyzoites were predominant but a number of single intermediate organisms displaying tachyzoite and certain bradyzoite markers were observed. At later time points, small groups of organisms displaying only bradyzoite markers were also present. A number (9) of dense granule proteins (GRA1-8, NTPase) have also been identified in both tachyzoites and bradyzoites but there were differences in their location during parasite development. All the dense granule proteins extensively label the parasitophorous vacuole during tachyzoite development. In contrast the tissue cyst wall displays variable staining for the dense granule proteins, which also expresses an additional unique cyst wall protein. The molecular differences could be identified at the single cell stage consistent with conversion occurring at the time of entry into a new cell. These molecular differences were reflected in the structural differences in the parasitophorous vacuoles observed by electron microscopy. Stage conversion to enteric (coccidian) development was limited to the enterocytes of the cat small intestine. Although no specific markers were available, this form of development can be identified by the absence of specific tachyzoite (SAG1) and bradyzoite (BAG1) markers although the isoenzymes ENO2 and LHD1 were expressed. There was also a significant difference in the expression of the dense granule proteins. The coccidian stages and merozoites only expressed two (GRA7 and NTPase) of the nine dense granule proteins and this was reflected in significant differences in the structure of the parasitophorous vacuole. The coccidian stages also undergo conversion from asexual to sexual development. The mechanism controlling this process is unknown but does not involve any change in the host cell type or parasitophorous vacuole and may be pre-programmed, since the number of asexual cycles was self-limiting. In conclusion, it was possible using a combination of molecular markers to identify tachyzoite, bradyzoite and coccidian development in tissue sections.
Asunto(s)
Toxoplasma/crecimiento & desarrollo , Toxoplasmosis Animal/parasitología , Animales , Biomarcadores/análisis , Encéfalo/parasitología , Encéfalo/ultraestructura , Gatos , Técnicas para Inmunoenzimas , Intestino Delgado/parasitología , Intestino Delgado/ultraestructura , Estadios del Ciclo de Vida , Pulmón/parasitología , Pulmón/ultraestructura , Ratones , Microscopía Inmunoelectrónica , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Toxoplasma/ultraestructura , Toxoplasmosis Animal/metabolismo , Toxoplasmosis Animal/patologíaRESUMEN
We have identified, and followed the development of three macrogamete organelles involved in the formation of the oocyst wall of Eimeria maxima. The first were small lucent vacuoles that cross-reacted with antibodies to the apple domains of the Toxoplasma gondii microneme protein 4. They appeared early in development and were secreted during macrogamete maturation to form an outer veil and were termed veil forming bodies. The second were the wall forming bodies type 1, large, electron dense vacuoles that stained positively only with antibodies raised to an enriched preparation of the native forms of 56 (gam56), 82 (gam82) and 230 kDa (gam230) gametocyte antigens (termed anti-APGA). The third were the wall forming bodies type 2, which appeared before the wall forming bodies type 1 but remain enclosed within the rough endoplasmic reticulum and stained positively with antibodies raised to recombinant versions of gam56 (anti-gam56), gam82 (anti-gam82) and gam230 (anti-gam230) plus anti-APGA. At the initiation of oocyst wall formation, the anti-T. gondii microneme protein 4 positive outer veil detached from the surface. The outer layer of the oocyst wall was formed by the release of the contents of wall forming bodies type 1 at the surface to form an electron dense, anti-APGA positive layer. The wall forming bodies type 2 appeared, subsequently, to give rise to the electron lucent inner layer. Thus, oocyst wall formation in E. maxima represents a sequential release of the contents of the veil forming bodies, wall forming bodies types 1 and 2 and this may be controlled at the level of the rough endoplasmic reticulum/Golgi body.