RESUMEN
Efferocytosis, the clearing of dead or dying cells from living tissues, is a highly programmed, vital process to maintain the healthy functioning of every organism. Disorders of efferocytosis have been linked to several chronic diseases including atherosclerosis and auto-immune diseases. To date several different assays to determine phagocytosis, using microscopy or FACS analysis with labelled targets, have been developed. However, many of these are unable to differentiate between cells that have truly been phagocytosed and those still present on the surface of the macrophages hindering exact assessment of efferocytotic capacity. We herein describe AnxA5-pHrodo and its negative control M1234-pHrodo as new molecular probes to measure in vitro as well as ex-vivo efferocytotic capacity.
Asunto(s)
Anexina A5/metabolismo , Fagocitosis/fisiología , Animales , Apoptosis/fisiología , Aterosclerosis/metabolismo , Línea Celular , Humanos , Células Jurkat , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Sondas Moleculares/metabolismoRESUMEN
PURPOSE: The purpose of this paper is to study molecular imaging of apoptosis and necrosis, two key players in atherosclerosis instability, using a multimodal imaging approach combining single photon emission computed tomography (SPECT), positron emission tomography (PET), and computed tomography (CT). PROCEDURES: Collar-induced carotid atherosclerosis ApoE knockout mice were imaged with (99m)Tc-AnxAF568 SPECT-CT to study apoptosis and sequentially with PET-CT following (124)I-Hypericin ((124)I-Hyp) injection to visualize necrosis. RESULTS: SPECT depicted increased (99m)Tc-AnxAF568 uptake in both atherosclerotic carotid arteries, whereas our data suggest that this uptake is not merely apoptosis related. Although PET of (124)I-Hyp was hampered by the slow blood clearance in atherosclerotic mice, (124)I-Hyp was able to target necrosis in the atherosclerotic plaque. CONCLUSION: Both (99m)Tc-AnxAF568 and (124)I-Hyp uptake are increased in atherosclerotic carotid vasculature compared to control arteries. While apoptosis imaging remains challenging, necrosis imaging can be feasible after improving the biodistribution characteristics of the probe.
Asunto(s)
Apoptosis , Imagen Molecular/métodos , Imagen Multimodal , Placa Aterosclerótica/diagnóstico por imagen , Tomografía de Emisión de Positrones , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada por Rayos X , Animales , Anexina A5/metabolismo , Antracenos , Humanos , Ratones , Necrosis , Perileno/análogos & derivados , Placa Aterosclerótica/patología , Tecnecio , Distribución TisularRESUMEN
Impaired efferocytosis has been shown to be associated with, and even to contribute to progression of, chronic inflammatory diseases such as atherosclerosis. Enhancing efferocytosis has been proposed as strategy to treat diseases involving inflammation. Here we present the strategy to increase 'eat me' signals on the surface of apoptotic cells by targeting cell surface-expressed phosphatidylserine (PS) with a variant of annexin A5 (Arg-Gly-Asp-annexin A5, RGD-anxA5) that has gained the function to interact with α(v)ß(3) receptors of the phagocyte. We describe design and characterization of RGD-anxA5 and show that introduction of RGD transforms anxA5 from an inhibitor into a stimulator of efferocytosis. RGD-anxA5 enhances engulfment of apoptotic cells by phorbol-12-myristate-13-acetate-stimulated THP-1 (human acute monocytic leukemia cell line) cells in vitro and resident peritoneal mouse macrophages in vivo. In addition, RGD-anxA5 augments secretion of interleukin-10 during efferocytosis in vivo, thereby possibly adding to an anti-inflammatory environment. We conclude that targeting cell surface-expressed PS is an attractive strategy for treatment of inflammatory diseases and that the rationally designed RGD-anxA5 is a promising therapeutic agent.
Asunto(s)
Anexina A5/farmacología , Oligopéptidos/farmacología , Fosfatidilserinas/metabolismo , Animales , Anexina A5/farmacocinética , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Adhesión Celular/fisiología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Células Jurkat , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Terapia Molecular Dirigida , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Oligopéptidos/farmacocinética , Fagocitosis/efectos de los fármacos , Fagocitosis/fisiología , Fosfatidilserinas/biosíntesisRESUMEN
In an effort to understand better the transmission risk as well for the animal African trypanosomosis (AAT) as for the human trypanosomosis (HAT) in the peri-urban zone of Kinshasa, a serologic study was carried out in local pig farms from 2003 to 2005. An indirect ELISA was used to detect the presence of trypanosome antibodies in 1,240 pigs originating from 404 farms. Seropositivity was recorded in 155 farms (38%), but varied considerably according to the district. In 6% of the farms TAA could be confirmed by parasitological examination. Trapping sites (n = 367) established in the neighbourhood of pig farms made it possible to capture 1,935 tsetse flies (Glossina fuscipes quanzensis). Among 562 dissected flies 23 were found to harbour trypanosomes resulting in an infection rate of 4.1%. In the majority of the districts the transmission risk for animal trypanosomosis anticipated from the apparent vector densities was corroborated by the serology. Zones with strong indications of local AAT transmission were identified in several quarters of three peri-urban districts of Kinshasa: Mount-Ngafula, Ngaliema and N'Sele. An intensification of tsetse control activities in those sites of increased transmission risk is essential.
Asunto(s)
Enfermedades de los Porcinos/epidemiología , Tripanosomiasis Africana/veterinaria , Animales , Anticuerpos Antiprotozoarios/sangre , República Democrática del Congo/epidemiología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Humanos , Insectos Vectores/parasitología , Masculino , Factores de Riesgo , Salud Suburbana , Porcinos , Enfermedades de los Porcinos/parasitología , Enfermedades de los Porcinos/transmisión , Trypanosoma/inmunología , Tripanosomiasis Africana/epidemiología , Tripanosomiasis Africana/transmisión , Moscas Tse-Tse/parasitologíaRESUMEN
Parasitic food-borne diseases are generally underrecognised, however they are becoming more common. Globalization of the food supply, increased international travel, increase of the population of highly susceptible persons, change in culinary habits, but also improved diagnostic tools and communication are some factors associated with the increased diagnosis of food-borne parasitic diseases worldwide. This paper reviews the most important emerging food-borne parasites, with emphasis on transmission routes. In a first part, waterborne parasites transmitted by contaminated food such as Cyclospora cayetanensis, Cryptosporidium and Giardia are discussed. Also human fasciolosis, of which the importance has only been recognised in the last decades, with total numbers of reported cases increasing from less than 3000 to 17 million, is looked at. Furthermore, fasciolopsiosis, an intestinal trematode of humans and pigs belongs to the waterborne parasites as well. A few parasites that may be transmitted through faecal contamination of foods and that have received renewed attention, such as Toxoplasma gondii, or that are (re-)emerging, such as Trypanosoma cruzi and Echinococcus spp., are briefly reviewed. In a second part, meat-borne parasite infections are reviewed. Humans get infected by eating raw or undercooked meat infected with cyst stages of these parasites. Meat inspection is the principal method applied in the control of Taenia spp. and Trichinella spp. However, it is often not very sensitive, frequently not practised, and not done for T. gondii and Sarcocystis spp. Meat of reptiles, amphibians and fish can be infected with a variety of parasites, including trematodes (Opisthorchis spp., Clonorchis sinensis, minute intestinal flukes), cestodes (Diphyllobothrium spp., Spirometra), nematodes (Gnathostoma, spp., anisakine parasites), and pentastomids that can cause zoonotic infections in humans when consumed raw or not properly cooked. Another important zoonotic food-borne trematode is the lungfluke (Paragonimus spp.). Traditionally, these parasitic zoonoses are most common in Asia because of the particular food practices and the importance of aquaculture. However, some of these parasites may emerge in other continents through aquaculture and improved transportation and distribution systems. Because of inadequate systems for routine diagnosis and monitoring or reporting for many of the zoonotic parasites, the incidence of human disease and parasite occurrence in food is underestimated. Of particular concern in industrialised countries are the highly resistant waterborne protozoal infections as well as the increased travel and immigration, which increase the exposure to exotic diseases. The increased demand for animal proteins in developing countries will lead to an intensification of the production systems in which the risk of zoonotic infections needs to be assessed. Overall, there is an urgent need for better monitoring and control of food-borne parasites using new technologies.
Asunto(s)
Enfermedades Transmisibles Emergentes/parasitología , Parasitología de Alimentos , Helmintiasis , Infecciones por Protozoos , Zoonosis/parasitología , Animales , HumanosRESUMEN
Taenia solium cysticercosis is a major helminth zoonosis in developing countries. Pigs are the intermediate hosts mediating transmission of infection. Specific assays to diagnose living cysts in pigs are lacking. The monoclonal-based antigen detection ELISA is genus-specific and cross-reactions with Taenia hydatigena hamper the use of this test to screen pigs. We, therefore, aimed to introduce nanobodies, camelid-derived single-domain antibodies specific for T. solium cysticercosis, to develop unambiguous tests. Nanobodies were cloned following immunization of two dromedaries with T. solium antigen and eight T. solium-specific nanobodies were selected after phage display. Their binding characteristics and potential for the diagnosis of porcine cysticercosis were investigated. The nanobodies do not cross-react with T. hydatigena, Taenia saginata, Taenia crassiceps or Trichinella spiralis and were categorized into four epitope-binding groups. The target protein was identified as 14kDa diagnostic glycoprotein (Ts14), but the nanobodies also reacted with other proteins of the same family. Nanobodies were tested in a sandwich ELISA with cyst fluid, and one particular nanobody detected its cognate serum antigens in a species-specific inhibition ELISA. Considering their beneficial production and stability properties, these highly specific nanobodies constitute a promising tool to diagnose cysticercosis after further improvement of the sensitivity and future assay validation.
Asunto(s)
Anticuerpos Antihelmínticos/inmunología , Cisticercosis/veterinaria , Nanopartículas , Enfermedades de los Porcinos/diagnóstico , Taenia solium/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/biosíntesis , Anticuerpos Antihelmínticos/genética , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antígenos Helmínticos/sangre , Antígenos Helmínticos/inmunología , Western Blotting/métodos , Camelus/parasitología , Reacciones Cruzadas , Cisticercosis/diagnóstico , Cisticercosis/parasitología , Ensayo de Inmunoadsorción Enzimática/métodos , Mapeo Epitopo/métodos , Estudios de Factibilidad , Datos de Secuencia Molecular , Pruebas Serológicas/métodos , Especificidad de la Especie , Porcinos , Enfermedades de los Porcinos/parasitologíaRESUMEN
Taenia solium cysticercosis is a significant public health problem in endemic countries. The current serodiagnostic techniques are not able to differentiate between infections with viable cysts and infections with degenerated cysts. The objectives of this study were to identify specific novel biomarkers of these different disease stages in the serum of experimentally infected pigs using ProteinChip technology (Bio-Rad) and to validate these biomarkers by analyzing serum samples from naturally infected pigs. In the experimental sample set 30 discriminating biomarkers (p<0.05) were found, 13 specific for the viable phenotype, 9 specific for the degenerated phenotype and 8 specific for the infected phenotype (either viable or degenerated cysts). Only 3 of these biomarkers were also significant in the field samples; however, the peak profiles were not consistent among the two sample sets. Five biomarkers discovered in the sera from experimentally infected pigs were identified as clusterin, lecithin-cholesterol acyltransferase, vitronectin, haptoglobin and apolipoprotein A-I.
Asunto(s)
Biomarcadores/sangre , Cisticercosis/veterinaria , Análisis por Matrices de Proteínas , Enfermedades de los Porcinos/diagnóstico , Animales , Apolipoproteína A-I/sangre , Cromatografía Liquida , Clusterina/sangre , Cisticercosis/sangre , Cisticercosis/diagnóstico , Femenino , Haptoglobinas/análisis , Masculino , Perú , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos , Enfermedades de los Porcinos/sangre , Enfermedades de los Porcinos/parasitología , Taenia solium , Espectrometría de Masas en Tándem , Vitronectina/sangre , ZambiaRESUMEN
An oral infection model with Taenia solium whole proglottids was used to study host-parasite relationships and the mechanisms underlying resistance to infection in pigs. In addition, an attempt was made to link the parasitological findings to serological data. Groups of six piglets aged 1, 3 and 5 months were infected and slaughtered 3 months p.i. Circulating antibody and antigen levels were monitored weekly. At autopsy total cyst counts were performed. Although the detailed carcass dissection at necropsy revealed a high variation in the number of cysts, the trend was that the number of viable cysts decreased with the age at which the animals were infected. The kinetics of the antigen levels throughout the course of the infection differed markedly between the three age groups of the experimental infection model. In the younger animals, a fast increase in titres of circulating antigen was observed in most animals, reaching a plateau as early as 2 weeks p.i. Besides its faster increase, antigen levels in pigs infected at younger ages also reached higher levels than in older animals and were associated with weaker antibody responses. Results also demonstrated that a relationship exists between the number of cysts and the titre of circulating antigen. This is promising in view of the development of an assay to quantify the progress of an active T. solium infection and would be a useful tool in epidemiological studies to assess the infection burden and the risk of transmission of the disease. The use of specific antibody-detection assays combined with circulating antigen detection could improve our understanding of this relationship.