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1.
Front Oncol ; 10: 608, 2020.
Article in English | MEDLINE | ID: mdl-32582525

ABSTRACT

Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information. Large tumor-derived EVs (tdEVs, >1 µm) in blood from cancer patients are associated with poor outcome, and changes in their number can be used to monitor therapy effectiveness. Whereas, small tumor-derived EVs (<1 µm) are likely to outnumber their larger counterparts, thereby offering better statistical significance, identification and quantification of small tdEVs are more challenging. In the blood of cancer patients, a subpopulation of EVs originate from tumor cells, but these EVs are outnumbered by non-EV particles and EVs from other origin. In the Dutch NWO Perspectief Cancer-ID program, we developed and evaluated detection and characterization techniques to distinguish EVs from non-EV particles and other EVs. Despite low signal amplitudes, we identified characteristics of these small tdEVs that may enable the enumeration of small tdEVs and extract relevant information. The insights obtained from Cancer-ID can help to explore the full potential of tdEVs in the clinic.

2.
J Extracell Vesicles ; 8(1): 1643671, 2019.
Article in English | MEDLINE | ID: mdl-31489142

ABSTRACT

Extracellular vesicles (EVs) in plasma are commonly identified by staining with antibodies and generic dyes, but the specificity of antibodies and dyes to stain EVs is often unknown. Previously, we showed that platelet-depleted platelet concentrate contains two populations of particles >200 nm, one population with a refractive index (RI) < 1.42 that included the majority of EVs, and a second population with an RI > 1.42, which was thought to include lipoproteins. In this study, we investigated whether EVs can be distinguished from lipoproteins by the RI and whether the RI can be used to determine the specificity of antibodies and generic dyes used to stain plasma EVs. EVs and lipoproteins present in platelet-depleted platelet concentrate were separated by density gradient centrifugation. The density fractions were analyzed by Western blot and transmission electron microscopy, the RI of particles was determined by Flow-SR. The RI was used to evaluate the staining specificity of an antibody against platelet glycoprotein IIIa (CD61) and the commonly used generic dyes calcein AM, calcein violet, di-8-ANEPPS, and lactadherin in plasma. After density gradient centrifugation, EV-enriched fractions (1.12 to 1.07 g/mL) contained the highest concentration of particles with an RI < 1.42, and the lipoprotein-enriched fractions (1.04 to 1.03 g/mL) contained the highest concentration of particles with an RI > 1.42. Application of the RI showed that CD61-APC had the highest staining specificity for EVs, followed by lactadherin and calcein violet. Di-8-ANEPPS stained mainly lipoproteins and calcein AM stained neither lipoproteins nor EVs. Taken together, the RI can be used to distinguish EVs and lipoproteins, and thus allows evaluation of the specificity of antibodies and generic dyes to stain EVs.

3.
Clin Exp Allergy ; 34(3): 488-96, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15005745

ABSTRACT

BACKGROUND: Respiratory viral infections in early childhood may interact with the immune system and modify allergen sensitization and/or allergic manifestations. In mice, respiratory syncytial virus (RSV) infection during allergic provocation aggravates the allergic T helper (Th) 2 immune response, characterized by the production of IL-4, IL-5, and IL-13, and inflammatory infiltrates. However, it is unclear whether the RSV-enhanced respiratory allergic response is a result of non-specific virus-induced damage of the lung, or virus-specific immune responses. OBJECTIVE: In the present study we investigated whether RSV, pneumonia virus of mice (PVM) and influenza A virus similarly affect the allergic response. METHODS: BALB/c mice were sensitized and challenged with ovalbumin (OVA), and inoculated with virus during the challenge period. Pulmonary inflammation, lung cytokine mRNA responses, and IgE production in serum were assessed after the last OVA-challenge. RESULTS: Like RSV, PVM enhanced the OVA-induced pulmonary IL-4, IL-5, and IL-13 mRNA expression, which was associated with enhanced perivascular inflammation. In addition, PVM increased the influx of eosinophils in lung tissue. In contrast, influenza virus decreased the Th2 cytokine mRNA expression in the lungs. However, like PVM, influenza virus enhanced the pulmonary eosinophilic infiltration in OVA-allergic mice. CONCLUSION: The Paramyxoviruses RSV and PVM both are able to enhance the allergic Th2 cytokine response and perivascular inflammation in BALB/c mice, while the Orthomyxovirus influenza A is not.


Subject(s)
Hypersensitivity/immunology , Hypersensitivity/virology , Influenza A virus , Lung/immunology , Murine pneumonia virus , Respiratory Syncytial Virus, Human , Virus Diseases/immunology , Animals , Female , Immunoglobulin E/blood , Interleukin-13/genetics , Interleukin-4/genetics , Interleukin-5/genetics , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/immunology , Ovalbumin , Pneumovirus Infections/immunology , Pulmonary Eosinophilia , RNA, Messenger/analysis , Respiratory Syncytial Virus Infections/immunology
4.
Clin Exp Allergy ; 32(3): 463-71, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11940079

ABSTRACT

BACKGROUND: Th2 lymphocyte responses are associated with inflammation and disease during allergic responses. Exposure to particular environmental factors during the expression of allergy could result in more pronounced Th2-like immune responses and more severe disease. One factor might be a respiratory virus infection. OBJECTIVE: The aim of our study was to investigate the influence of respiratory syncytial virus (RSV) infection on the expression of ovalbumin (OVA)-induced allergy in BALB/c mice. METHODS: We determined OVA-specific IgE in serum, cytokine profiles and histopathological lesions in lungs of OVA-allergic mice after RSV infection. RESULTS: OVA sensitization and challenge induced OVA-specific IgE in serum, Th2 cytokine mRNA expression, and mononuclear and eosinophilic inflammation in the lungs. RSV inoculation during the challenge period enhanced OVA-induced IL-4 and IL-5 mRNA expression in lung tissue. RSV further enhanced the OVA-induced hypertrophy of mucous cells and eosinophilic infiltration in lung tissue. Surprisingly, RSV infection decreased Th2 cytokine secretion and eosinophilic influx in bronchoalveolar lavage of OVA-allergic mice. Because inactivated RSV did not influence these responses, replication of RSV appeared essential for the modification of OVA-induced Th2 cytokine expression. RSV did not change OVA-specific IgE levels in serum. Furthermore, the RSV-induced IL-12 mRNA expression in lung tissue of OVA-allergic mice was diminished, but IFN-gamma mRNA expression was not affected. CONCLUSION: RSV infection enhanced particular OVA-induced Th2 cytokine mRNA responses and pulmonary lesions in allergic mice and thus aggravated allergic respiratory disease.


Subject(s)
Cytokines/biosynthesis , Cytokines/immunology , Pneumonia/immunology , Pneumonia/physiopathology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/physiopathology , Respiratory Syncytial Virus, Human , Animals , Antibody Specificity/immunology , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Disease Models, Animal , Female , Hypersensitivity/immunology , Hypersensitivity/physiopathology , Immunoglobulin E/blood , Immunoglobulin E/immunology , Lung/blood supply , Lung/cytology , Lung/metabolism , Mice , Mice, Inbred BALB C , Ovalbumin/adverse effects , Ovalbumin/immunology , RNA, Messenger/biosynthesis , RNA, Messenger/immunology , Severity of Illness Index , Time Factors , Ultraviolet Rays
5.
Arch Virol ; 145(3): 541-51, 2000.
Article in English | MEDLINE | ID: mdl-10795521

ABSTRACT

In Europe measles incidence remains high and in some parts the disease is likely to be still endemic due to insufficient vaccination. Luxembourg experienced an outbreak with at least 110 cases in 1996, and cases continued to be reported throughout 1997. We used molecular epidemiology to investigate this apparent endemicity. On the basis of their N gene sequences, the isolates were assigned to the typical European C2 and D6 genotypes. Sequence diversity within the outbreak was 0.2%. The nucleotide distance between the C2-viruses of the outbreak and the other C2 isolates was at least three or four times higher, suggesting an independent origin of the latter viruses. Similarly, the four D6 viruses found in Luxembourg were thought to be of at least two or three origins. Thus, we propose here to use intra-outbreak sequence diversity to differentiate between sporadic endemic cases and a "pseudo-outbreak" of multiple unrelated imported cases.


Subject(s)
Genetic Variation , Measles virus/genetics , Measles/epidemiology , Molecular Epidemiology , Belgium/epidemiology , Humans , Luxembourg/epidemiology , Measles/virology , Measles virus/classification , Molecular Sequence Data , Netherlands/epidemiology , Nucleocapsid Proteins , Nucleoproteins/genetics , Phylogeny , Viral Proteins/genetics
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