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The Sf9 and Sf21 cell lines derived from ovarian tissues of the wide-host-range phytophagous lepidopteran Spodoptera frugiperda are widely used for research and commercial-scale production of recombinant proteins. These cell lines are chronically infected with a rhabdovirus (Sf-RV) that does not cause any overt cytopathic effects. We demonstrate that wild populations of S. frugiperda in the eastern United States and Caribbean are infected with genetically diverse strains of Sf-RV and that this virus is also capable of infecting cells of Spodoptera exigua, Heliothis subflexa, and Bombyx mori Feeding studies demonstrated the ability of S. frugiperda larvae to deposit Sf-RV onto human-consumed vegetables during feeding. Although no evidence for replication in two species of plant cells was detected, subcellular localization studies demonstrated that the Sf-RV nucleocapsid was targeted to plasmodesmata, while two forms of the accessory protein were differentiated on the basis of their ability to localize to nuclei. Collectively, the results from this study suggest that environmental exposure of humans to Sf-RV is likely to be commonplace and frequent, but its inability to replicate in plant or human cells suggests that there is no substantial risk to human health.IMPORTANCE Insect-derived cell lines are widely used commercially for the production of vaccines and protein-based pharmaceuticals. After decades of safe and beneficial use, it was a surprise to the biotechnology industry to discover an endemic rhabdovirus in Sf9 cells. This discovery was made possible only by the substantial advancements in DNA sequencing technologies. Given the public health concerns associated with many rhabdovirus species, several initiatives were undertaken to establish that Spodoptera frugiperda rhabdovirus (Sf-RV) does not pose a threat to humans. Such actions include the generation of cell lines that have been cleared of Sf-RV. Given that Sf9 is derived from a moth whose larvae feed on human-edible foods, we explored the prevalence of Sf-RV in its wild and lab-grown populations, as well as its ability to be deposited on food items during feeding. Collectively, our data suggest that there is no overt risk from exposure to Sf-RV.
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Especificidade de Hospedeiro/fisiologia , Rhabdoviridae/fisiologia , Spodoptera/virologia , Animais , Linhagem Celular , Humanos , Insetos/virologia , Larva/metabolismo , Larva/virologia , Plantas/virologia , Proteínas Recombinantes/metabolismo , Rhabdoviridae/metabolismo , Células Sf9 , Spodoptera/metabolismo , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Previous studies have highlighted the role of the brain reward and cognitive control systems in the etiology of anorexia nervosa (AN). In an attempt to disentangle the relative contribution of these systems to the disorder, we used functional magnetic resonance imaging (fMRI) to investigate hemodynamic responses to reward-related stimuli presented both subliminally and supraliminally in acutely underweight AN patients and age-matched healthy controls (HC). METHODS: fMRI data were collected from a total of 35 AN patients and 35 HC, while they passively viewed subliminally and supraliminally presented streams of food, positive social, and neutral stimuli. Activation patterns of the group × stimulation condition × stimulus type interaction were interrogated to investigate potential group differences in processing different stimulus types under the two stimulation conditions. Moreover, changes in functional connectivity were investigated using generalized psychophysiological interaction analysis. RESULTS: AN patients showed a generally increased response to supraliminally presented stimuli in the inferior frontal junction (IFJ), but no alterations within the reward system. Increased activation during supraliminal stimulation with food stimuli was observed in the AN group in visual regions including superior occipital gyrus and the fusiform gyrus/parahippocampal gyrus. No group difference was found with respect to the subliminal stimulation condition and functional connectivity. CONCLUSION: Increased IFJ activation in AN during supraliminal stimulation may indicate hyperactive cognitive control, which resonates with clinical presentation of excessive self-control in AN patients. Increased activation to food stimuli in visual regions may be interpreted in light of an attentional food bias in AN.
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Anorexia Nervosa/fisiopatologia , Córtex Cerebral/fisiopatologia , Alimentos , Neuroimagem Funcional/métodos , Reconhecimento Visual de Modelos/fisiologia , Recompensa , Estimulação Subliminar , Adolescente , Adulto , Anorexia Nervosa/diagnóstico por imagem , Córtex Cerebral/diagnóstico por imagem , Feminino , Humanos , Imageamento por Ressonância Magnética , Adulto JovemRESUMO
Recent progress in high-performance computing and data informatics has opened up numerous opportunities to aid the design of advanced materials. Herein, we demonstrate a computational workflow that includes rapid population of high-fidelity materials datasets via petascale computing and subsequent analyses with modern data science techniques. We use a first-principles approach based on density functional theory to derive the segregation energies of 34 microalloying elements at the coherent and semi-coherent interfaces between the aluminium matrix and the θ'-Al2Cu precipitate, which requires several hundred supercell calculations. We also perform extensive correlation analyses to identify materials descriptors that affect the segregation behaviour of solutes at the interfaces. Finally, we show an example of leveraging machine learning techniques to predict segregation energies without performing computationally expensive physics-based simulations. The approach demonstrated in the present work can be applied to any high-temperature alloy system for which key materials data can be obtained using high-performance computing.
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Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were â¼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes.
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Variação Antigênica , Antígenos de Protozoários/imunologia , Epitopos/imunologia , Vacinas Antimaláricas , Proteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Variação Antigênica/genética , Variação Antigênica/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Células Cultivadas , Sequência Conservada/imunologia , Mapeamento de Epitopos , Epitopos/genética , Imunidade Humoral , Vacinas Antimaláricas/química , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Modelos Moleculares , Plasmodium berghei/genética , Plasmodium berghei/imunologia , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologiaRESUMO
The placenta from an embryo transfer-recipient mare and live foal was examined. The placenta was effaced by multifocal masses, which ranged from less than 1 cm to 14 cm in diameter. The foal represented at 52 days for lethargy, ataxia, and urine dribbling; due to a poor prognosis, the foal was euthanized. At necropsy, the liver was effaced by multifocal, pale, irregular nodules. The lumbar vertebrae and other skeletal sites had multifocal lytic lesions. The placenta had 4 populations of neoplastic cells, including a spindle cell population, tall columnar and transitional epithelial cell populations, and an undifferentiated polygonal cell population. The foal's liver had similar populations and patterns of cells as those in the placenta. The lesion in the placenta and the masses in the foal were diagnosed as a mixed germ cell tumor and metastatic mixed germ cell tumor, respectively.
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Biomarcadores Tumorais/metabolismo , Doenças dos Cavalos/patologia , Neoplasias Hepáticas/veterinária , Neoplasias Embrionárias de Células Germinativas/veterinária , Animais , Animais Recém-Nascidos , Feminino , Cavalos , Fígado/patologia , Neoplasias Hepáticas/secundário , Metástase Neoplásica , Neoplasias Embrionárias de Células Germinativas/patologia , Placenta/patologia , GravidezRESUMO
Stimulus visibility is associated with neural signals in multiple brain regions, ranging from visual cortex to prefrontal regions. Here we used functional magnetic resonance imaging (fMRI) to investigate to which extent the perceived visibility of a "low-level" grating stimulus is reflected in the brain activity in high-level brain regions. Oriented grating stimuli were presented under varying visibility conditions created by backward masking. Visibility was manipulated using four different stimulus onset asynchronies (SOAs), which created a continuum from invisible to highly visible target stimuli. Brain activity in early visual areas, high-level visual brain regions (fusiform gyrus), as well as parietal and prefrontal brain regions was significantly correlated with subjects' psychometric visibility functions. In addition, increased stimulus visibility was reflected in the functional coupling between low and high-level visual areas. Specifically, neuroimaging signals in the middle occipital gyrus were significantly more correlated with signals in the inferior temporal gyrus when subjects successfully perceived the target stimulus than when they did not. These results provide evidence that not only low-level visual but also high-level brain regions reflect visibility of low-level grating stimuli and that changes in functional connectivity reflect perceived stimulus visibility.
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Lobo Occipital/fisiologia , Lobo Temporal/fisiologia , Adulto , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Estimulação Luminosa , Córtex Visual/fisiologia , Adulto JovemRESUMO
The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.
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Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Sítios de Ligação , Sequência Conservada/genética , Cisteína/metabolismo , Citocalasina D/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Merozoítos/efeitos dos fármacos , Merozoítos/ultraestrutura , Modelos Biológicos , Dados de Sequência Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/ultraestrutura , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/química , Relação Estrutura-AtividadeRESUMO
White alder (Alnus rhombifolia) is a fast-growing tree native to the western United States and is planted frequently in landscapes. In September 2010, mature leaves of white alder with small, orange-yellow pustules were collected in a commercial nursery in Santa Cruz County, CA. Approximately 25 white alder trees were affected. Collected leaves were sent to the California Department of Food and Agriculture Plant Pest Diagnostics Laboratory. Young uredinial pustules were bullate, with urediniospores emerging from a single pore in the pustule. Spiny cells lined the ostiole. With age, pustules broke open to release more spores. Urediniospores were obovate to oval and measured from 14 to 20 × 27 to 41 µm (17.1 × 32.2 µm average, n = 62). Spores were uniformly echinulate and contained a nearly hyaline cell wall measuring from 1 to 2 µm (1.5 µm average) in thickness. A portion of the 28S ribosomal subunit (GenBank Accession No. KC313888) and the internal transcribed spacer regions (KC313889) were amplified and sequenced from DNA extracted from urediniospores using primers LR6 and rust2inv (1) and ITS1-F and ITS4-B (2), respectively. Our ITS sequence had 99% identity to GenBank accession EF564164, Melampsoridium hiratsukanum. In September 2011, white alder leaves with similar symptoms were collected from a commercial nursery in Santa Barbara County, CA. The spore morphology matched the white alder sample previously collected in Santa Cruz County, CA, in 2010. At that time, pathogenicity assays were conducted on three 1-year-old, 61-cm white alder trees planted in 3.8-liter pots. Six detached leaves with visible rust pustules were rubbed gently onto both the apical and distal side of moistened leaves of the healthy alders. Each infected leaf was used to inoculate a total of 6 to 10 healthy leaves by rubbing two leaves per tree before moving to the next tree. Leaves on three additional white alder trees were rubbed with healthy leaves as controls. Trees were incubated in a dew chamber for 3 days in darkness at 24°C, then placed in a growth chamber at 22°C with a 12-h photoperiod. Twelve days after inoculation, small lesions were visible on a few of the leaf undersides of each inoculated tree. Not all inoculated leaves developed pustules. No lesions developed on the control trees. M. hiratsukanum has been reported in Canada, Europe, and eastern Asia (3). There are no published reports of this rust in the United States, but there is an unpublished specimen from white alder in the USDA Systematic Mycology Herbarium (BPI 028048) collected from California in 1931, which was identified as M. hiratsukanum by G. B. Cummins using morphological criteria. We are unaware if older specimens of this rust exist because we were unable to search other herbaria in the United States. To the best of our knowledge, this rust has been present in California since 1931, but has only recently been found causing disease in nursery plants. There have been no reports of the serious foliar disease symptoms on trees in California wild lands as have been reported in Europe, presumably due to dry summer and fall seasons in white alder's natural habitat. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (3) J. Hatula et al. Mycologia 101:622, 2009.
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OBJECTIVE: This article assesses the use of BeneHold Thin Absorbent Skin Adhesive (TASA) wound dressings in a large UK primary care organisation. These wound dressings are thin (0.12 mm), breathable, transparent, and are able to absorb and retain wound exudate. This non-comparative evaluation was undertaken to explore the clinical advantages this differentiated combination of physical properties offered. METHOD: The dressings are CE-marked medical devices, and were used on patients with acute and chronic wounds that were assessed and classified as light to moderately exuding. Clinical performance was evaluated with respect to the dressing's ease of use (application and removal, conformability, mould-ability, rolling and edge-lift), debridement, protection of the peri-wound, wear time, fluid handling, wound bed residue, visibility of the wound, and clinical acceptability. The evaluating clinicians used an agreed audit tool to collect data from case reports to document the progression of wounds of various aetiologies, including chronic and acute, for a maximum period of four weeks. Qualitative feedback on dressing performance was also collected at the evaluation's end, both from the clinicians' and patients' perspectives Results: Some 15 patients were assessed. The wear time was up to seven days in many cases, and on average was 3.9 days longer than their previous dressings. Clinicians perceived that wounds progressed toward healing in all but two cases, where the wounds remained unchanged. Out of five cases where wounds presented with necrosis, all underwent significant autolytic debridement underneath the new dressings. Transparency was a noted benefit from both the clinicians' and patients' perspectives because it enabled continuous monitoring of the full wound bed and peri-wound skin without the need to disrupt the dressing. CONCLUSION: The dressing was well-received by both clinicians and patients in all fifteen cases. The thin absorbent skin adhesive dressing was found to be a promising new technology that could offer significant advantages to improve the quality, cost, and convenience of wound care. Further work is underway to validate these findings in larger and more homogeneous patient groups.
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Curativos Hidrocoloides , Cicatrização , Ferimentos e Lesões/patologia , Ferimentos e Lesões/terapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Exsudatos e Transudatos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Necrose/prevenção & controle , Satisfação Pessoal , Resultado do Tratamento , Reino Unido , Adulto JovemRESUMO
We describe the formation and properties of atomically bonded, optical quality, nanostructured thin glass film coatings on glass plates, utilizing phase separation by spinodal decomposition in a sodium borosilicate glass system. Following deposition via magnetron sputtering, thermal processing and differential etching, these coatings are structurally superhydrophilic (i.e., display anti-fogging functionality) and demonstrate robust mechanical properties and superior abrasion resistance. After appropriate chemical surface modification, the surfaces display a stable, non-wetting Cassie-Baxter state and exhibit exceptional superhydrophobic performance, with water droplet contact angles as large as 172°. As an added benefit, in both superhydrophobic and superhydrophilic states these nanostructured surfaces can block ultraviolet radiation and can be engineered to be anti-reflective with broadband and omnidirectional transparency. Thus, the present approach could be tailored toward distinct coatings for numerous markets, such as residential windows, windshields, specialty optics, goggles, electronic and photovoltaic cover glasses, and optical components used throughout the US military.
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Vidro/química , Nanopartículas/química , Interações Hidrofóbicas e Hidrofílicas , Nanopartículas/ultraestrutura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Propriedades de Superfície , MolhabilidadeRESUMO
The wild and captive koala population of the Mt Lofty Ranges in South Australia has a high level of renal dysfunction in which crystals consistent with calcium oxalate have been observed in the kidneys. This study aimed to describe the pathological features of the renal disease in this population, confirm the composition of renal crystals as calcium oxalate, and determine whether any age or sex predispositions exist for this disease. A total of 51 koalas (28 wild rescues, 23 captive) were examined at necropsy, of which 28 (55%) were found to have gross and/or histological evidence of oxalate nephrosis. Histopathological features included intratubular and interstitial inflammation, tubule dilation, glomerular atrophy, tubule loss, and cortical fibrosis. Calcium oxalate crystals were demonstrated using a combination of polarization microscopy, alizarin red S staining, infrared spectroscopy, and energy-dispersive X-ray analysis with scanning electron microscopy. Uric acid and phosphate deposits were also shown to be present but were associated with minimal histopathological changes. No significant differences were found between the numbers of affected captive and wild rescued koalas; also, there were no sex or age predispositions identified, but it was found that oxalate nephrosis may affect koalas <2 years of age. The findings of this study suggest that oxalate nephrosis is a leading disease in this koala population. Possible causes of this disease are currently under investigation.
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Animais Selvagens/metabolismo , Animais de Zoológico/metabolismo , Oxalato de Cálcio/metabolismo , Nefrose/epidemiologia , Nefrose/veterinária , Phascolarctidae , Fatores Etários , Animais , Antraquinonas , Rim/metabolismo , Rim/ultraestrutura , Microscopia Eletrônica de Varredura/veterinária , Nefrose/metabolismo , Nefrose/patologia , Austrália do Sul/epidemiologia , Espectrometria por Raios X/veterinária , Espectrofotometria Infravermelho/veterináriaRESUMO
This article outlines a strategic process for the evaluation of wound management products and the development of an algorithm as an implementation model for wound management. Wound management is an increasingly complex process given the variety of interactive dressings and other devices available. This article discusses the procurement process, access to wound management dressings and the use of wound management formularies within the UK. We conclude that the current commissioners of tissue viability within healthcare organisations need to adopt a proactive approach to ensure appropriate formulary evaluation and product selection, in order to achieve the most beneficial clinical and financial outcomes.
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Algoritmos , Benchmarking/métodos , Análise Custo-Benefício/métodos , Modelos Estatísticos , Medicina Estatal/economia , Ferimentos e Lesões/economia , Ferimentos e Lesões/terapia , Humanos , Reino Unido , CicatrizaçãoRESUMO
The cervical spinal cords of 2 horses with equine degenerative myeloencephalopathy (EDM) were evaluated for evidence of oxidative damage to the central nervous system (CNS) using immunohistochemical staining for 3-nitrotyrosine (3-NT) and 4-hydroxynonenol (4-HNE). Neurons of the CNS from horses with EDM had positive immunohistochemical staining, whereas control samples did not, thus supporting the theory that oxidative damage is a potential underlying factor in horses with EDM. In addition, serum vitamin E concentration was low in both EDM-affected horses, and vitamin E concentration was also deficient in the cerebrospinal fluid in 1 EDM horse, further supporting the association between low vitamin E concentrations and oxidative damage to the CNS. Continued research is necessary to further define the pathophysiologic mechanisms of EDM.
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Encefalopatias/veterinária , Doenças dos Cavalos/diagnóstico , Doenças Neurodegenerativas/veterinária , Doenças da Medula Espinal/veterinária , Aldeídos/análise , Animais , Ataxia/veterinária , Encefalopatias/diagnóstico , Encefalopatias/patologia , Sistema Nervoso Central/patologia , Feminino , Doenças dos Cavalos/patologia , Cavalos , Imuno-Histoquímica/veterinária , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/patologia , Estresse Oxidativo , Doenças da Medula Espinal/diagnóstico , Doenças da Medula Espinal/patologia , Tirosina/análogos & derivados , Tirosina/análise , Vitamina E/líquido cefalorraquidiano , Deficiência de Vitamina E/veterináriaRESUMO
Cosmos (Cosmos bipinnatus Cav.) is an annual that is grown for cut flowers or as a landscape bedding plant. In late July 2009, cosmos plants were collected from a 0.4-ha field in Santa Barbara County, CA and submitted to the California Department of Food and Agriculture's Plant Pest Diagnostics Laboratory. Plants showed symptoms of chlorosis, wilting, necrosis, and death. Symptomatic plants comprised approximately 50% of the crop. Roots and stems appeared entirely discolored. Pieces (4 mm3) were taken from the edges of the discolored tissue of roots and stems, surface sterilized in 0.6% NaOCl for 2 min, and placed onto one-half-strength acidified potato dextrose agar (APDA). Fungal colonies consisted of fine, hyaline hyphae with verticillate conidiophores producing hyaline conidia, measuring 4.2 to 7.0 × 1.8 to 3.0 µm (5.13 × 2.44 µm average), in slimy masses. Microsclerotia (30.0 to 137.5 × 15.0 to 60.0 µm, 57.6 × 33.7 µm average) formed after 1 week in culture, causing the center of the colony to darken. Morphological characteristics were consistent with those of Verticillium dahliae (2). The internal transcribed spacer region (ITS) of rDNA was amplified for one isolate from cosmos using ITS1 and ITS4 primers as described by White et al. (3), and the amplicon was sequenced (GenBank Accession No. GU99602). BLAST analysis of the 455-bp amplicon showed 100% identity with the ITS sequence of V. dahliae from cosmos in Italy (GenBank Accession No. GQ130129). Pathogenicity of the California cosmos isolate of V. dahliae was determined by inoculating 10 1-month-old seedlings (each approximately 20 cm high) of C. bipinnatus 'Sensation Mix' with this isolate. Plants were inoculated with spores harvested by flooding 2-week-old cultures of V. dahliae grown on APDA medium with sterile distilled water. Plant root tips were trimmed and submerged in a spore suspension (1.3 × 106 spores/ml) for 5 min. Ten plants were dipped in water as the negative control treatment. Plants were then planted in a commercial potting mix in 10-cm-diameter pots and kept in a growth chamber at 25°C with a 12-h photoperiod. Inoculated plants were chlorotic and wilted when root and stem isolations were performed 1 month after inoculation. V. dahliae grew from stems and roots of 7 and 2 of the 10 inoculated plants, respectively. The inoculation experiment was repeated on six 5-month-old plants with similar results, except V. dahliae was isolated from the roots and stems of six and five plants, respectively. No symptoms developed on plants dipped in water, and Verticillium spp. did not grow from isolated root or stem pieces from the noninoculated plants in either experiment. On the basis of morphological and ITS sequence information, the fungus was identified as V. dahliae. V. dahliae is an economically important pathogen with a wide host range worldwide including cosmos in Italy (1). The affected field in California had a history of vegetable and flower seed crops, as well as vegetables for consumption. Infection of cosmos may have been from soilborne microsclerotia from previous susceptible crops. To our knowledge, this is the first report of Verticillium wilt on cosmos in California. References: (1) A. Carlucci et al. Plant Dis. 93:846, 2009. (2) D. L. Hawksworth and P. W. Talboys. No. 256 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1970. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.
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Antibodies against apical membrane antigen 1 (AMA1) inhibit invasion of Plasmodium merozoites into red cells, and a large number of single nucleotide polymorphisms on AMA1 allow the parasite to escape inhibitory antibodies. The availability of a crystal structure makes it possible to test protein engineering strategies to develop a monovalent broadly reactive vaccine. Previously, we showed that a linear stretch of polymorphic residues (amino acids 187 to 207), localized within the C1 cluster on domain 1, conferred the highest level of escape from inhibitory antibodies, and these were termed antigenic escape residues (AER). Here we test the hypothesis that immunodampening the C1 AER will divert the immune system toward more conserved regions. We substituted seven C1 AER of the FVO strain Plasmodium falciparum AMA1 with alanine residues (ALA). The resulting ALA protein was less immunogenic than the native protein in rabbits. Anti-ALA antibodies contained a higher proportion of cross-reactive domain 2 and domain 3 antibodies and had higher avidity than anti-FVO. No overall enhancement of cross-reactive inhibitory activity was observed when anti-FVO and anti-ALA sera were compared for their ability to inhibit invasion. Alanine mutations at the C1 AER had shifted the immune response toward cross-strain-reactive epitopes that were noninhibitory, refuting the hypothesis but confirming the importance of the C1 cluster as an inhibitory epitope. We further demonstrate that naturally occurring polymorphisms that fall within the C1 cluster can predict escape from cross-strain invasion inhibition, reinforcing the importance of the C1 cluster genotype for antigenic categorization and allelic shift analyses in future phase 2b trials.
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Antígenos de Protozoários/imunologia , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Alanina , Animais , Anticorpos Antiprotozoários/imunologia , Especificidade de Anticorpos , Antígenos de Protozoários/química , Western Blotting , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/química , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Estrutura Quaternária de Proteína , Proteínas de Protozoários/química , CoelhosRESUMO
The multiple antigen peptide (MAP) approach is an effective method to chemically synthesize and deliver multiple T-cell and B-cell epitopes as the constituents of a single immunogen. Here we report on the design, chemical synthesis, and immunogenicity of three Plasmodium falciparum MAP vaccines that incorporated antigenic epitopes from the sporozoite, liver, and blood stages of the life cycle. Antibody and cellular responses were determined in three inbred (C57BL/6, BALB/c, and A/J) strains, one congenic (HLA-A2 on the C57BL/6 background) strain, and one outbred strain (CD1) of mice. All three MAPs were immunogenic and induced both antibody and cellular responses, albeit in a somewhat genetically restricted manner. Antibodies against MAP-1, MAP-2, and MAP-3 had an antiparasite effect that was also dependent on the mouse major histocompatibility complex background. Anti-MAP-1 (CSP-based) antibodies blocked the invasion of HepG2 liver cells by P. falciparum sporozoites (highest, 95.16% in HLA-A2 C57BL/6; lowest, 11.21% in BALB/c). Furthermore, antibodies generated following immunizations with the MAP-2 (PfCSP, PfLSA-1, PfMSP-1(42), and PfMSP-3b) and MAP-3 (PfRAP-1, PfRAP-2, PfSERA, and PfMSP-1(42)) vaccines were able to reduce the growth of blood stage parasites in erythrocyte cultures to various degrees. Thus, MAP-based vaccines remain a viable option to induce effective antibody and cellular responses. These results warrant further development and preclinical and clinical testing of the next generation of candidate MAP vaccines that are based on the conserved protective epitopes from Plasmodium antigens that are widely recognized by populations of divergent HLA types from around the world.
Assuntos
Antígenos de Protozoários , Vacinas Antimaláricas , Malária Falciparum/prevenção & controle , Vacinas de Subunidades Antigênicas , Vacinas Sintéticas , Sequência de Aminoácidos , Animais , Animais não Endogâmicos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Desenho de Fármacos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Eritrócitos/parasitologia , Feminino , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Hepatócitos/parasitologia , Humanos , Imunização , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/síntese química , Vacinas Antimaláricas/química , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos , Dados de Sequência Molecular , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/síntese química , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologiaRESUMO
Plasmodium falciparum malaria merozoites invade human erythrocytes bearing sialic acid in a multistage process involving the sialic acid-dependent binding of a malaria molecule, the 175-kD erythrocyte binding antigen (EBA-175). We show here that after the initial interaction of EBA-175 with its sialic acid-containing erythrocyte determinant, endogenous proteases can cleave EBA-175 to 65-kD fragment(s), whose binding to erythrocytes is sialic acid independent. A 65-kD fragment was immunoprecipitated by antibodies against peptides between residues 354 and 1061 but not beyond residue 1062. Binding experiments utilizing combinations of native protein, expression-PCR-synthesized EBA-175 polypeptides, peptide synthesis, and antibodies, demonstrated that sialic acid-independent binding could be further mapped to a small (about 40-amino acid) homologous part of the dimorphic allelic region of EBA-175, residues 898-938 (Camp strain numbering). These data support a two-step binding hypothesis and are discussed in relation to the formation of a junction between the merozoite and the erythrocyte, and the finding that after the interaction of some viruses with specific cellular receptors, they undergo conformational changes or cleavage permitting membrane fusion with the host cell.
Assuntos
Antígenos de Protozoários/metabolismo , Proteínas de Transporte/metabolismo , Eritrócitos/metabolismo , Expressão Gênica , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/isolamento & purificação , Sequência de Bases , Proteínas de Transporte/biossíntese , Proteínas de Transporte/isolamento & purificação , Primers do DNA , DNA de Protozoário/metabolismo , Eletroforese em Gel de Poliacrilamida , Eritrócitos/parasitologia , Humanos , Soros Imunes , Immunoblotting , Macaca mulatta , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da Polimerase , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/isolamento & purificação , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
In this report and (R. Schmidt-Ullrich, L. H. Miller, and D. F. H. Wallach. Manuscript in preparation.), we have demonstrated that malaria proteins on the surface of merozoites and infected erythrocytes cross-react between at least two primate malarias, Plasmodium knowlesi and P. falciparum. Sera from five Gambian adults who were highly immune to P. falciparum were used as a reagent to study the cross-reactivity between P. falciparum schizonts and surface proteins on P. knowlesi merozoites. Although the sera bound to the surface of viable, intact P. knowlesi merozoites, the sera did not block invasion of rhesus erythrocytes. 125I-lactoperoxidase-labeled surface proteins on merozoites formed complexes with the antibody. All major protein bands seen in the electrophoresis of the original Triton extract were bound by the immune sera. Because Gambians have never been exposed to P. knowlesi malaria, the antibodies that reacted with P. knowlesi merozoites must be directed against antigens of another parasite such as P. falciparum. We tested this hypothesis by competition for antibody in a Gambian serum between Triton-extracted antigens from P. falciparum schizont-infected erythrocytes and from surface-labeled P. knowlesi merozoites. P. falciparum inhibited the reaction, thus indicating cross-reaction between antigens in P. falciparum schizonts and P. knowlesi merozoites.
Assuntos
Antígenos de Superfície/análise , Plasmodium/imunologia , Animais , Especificidade de Anticorpos , Reações Cruzadas , Epitopos , Humanos , Imunidade , Larva/imunologia , Malária/imunologia , Plasmodium falciparum/imunologiaRESUMO
A 135-kD parasite protein, a minor component of the Plasmodium knowlesi malaria radiolabeled proteins released into culture supernatant at the time of merozoite release and reinvasion, specifically bound to human erythrocytes that are invaded and carry a Duffy blood group determinant (Fya or Fyb), but did not bind to human erythrocytes that are not invaded and do not carry a Duffy determinant (FyFy). Specific anti-Duffy antibodies blocked the binding of the 135-kD protein to erythrocytes carrying that specific Duffy determinant. Purified 135-kD protein bound specifically to the 35-45-kD Duffy glycoprotein on a blot of electrophoretically separated membrane proteins from Fya and Fyb erythrocytes but not from FyFy erythrocytes. Binding of the 135-kD protein was consistently greater to Fyb than to Fya both on the blot and on intact erythrocytes. The 135-kD protein also bound to rhesus erythrocytes that are Fyb and are invaded, but not to rabbit or guinea pig erythrocytes that are Duffy-negative and are not invaded. Cleavage of the Duffy determinant by pretreating Fyb human erythrocytes with chymotrypsin greatly reduced both invasion and binding of the 135-kD protein, whereas pretreating Fyb erythrocytes with trypsin had little effect on the Duffy antigen, the 135-kD protein binding, or on invasion. However, instances of invasion of other enzyme-treated erythrocytes that are Duffy-negative and do not bind the 135-kD protein suggest that alternative pathways for invasion do exist.
Assuntos
Antígenos de Protozoários/fisiologia , Antígenos de Grupos Sanguíneos/imunologia , Sistema do Grupo Sanguíneo Duffy/imunologia , Eritrócitos/parasitologia , Malária/sangue , Plasmodium/patogenicidade , Animais , Humanos , Técnicas In Vitro , Malária/parasitologia , Peso Molecular , Ligação Proteica , Receptores de Superfície Celular/fisiologiaRESUMO
Human erythrocytes lacking various blood group determinants were susceptible to invasion by Plasmodium falciparum including Duffy-negative erythrocytes that are refractory to invasion by Plasmodium knowlesi. Erythrocytes treated with trypsin or neuraminidase had reduced susceptibility of P. falciparum and normal susceptibility to P. knowlesi. Chymotrypsin treatment (0.1 mg/ml) blocked invasion only by P. knowlesi. The differential effect of enzymatic cleavage of determinats from the erythrocyte surface on invasion by these parasites suggests that P. falciparum and P. knowlesi interact with different determinants on the erythrocyte surface.