High-throughput surface epitope immunoaffinity isolation of extracellular vesicles and downstream analysis.

Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high-throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilizes affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high-throughput isolation of EVs from biofluids and downstream analysis of RNA, protein, and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new SEI isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. microRNA (miRNA) sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high-throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high-throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high-throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.

A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry.

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.

[Rapid differential diagnosis of Orthopoxviruses and Herpesviruses based upon multiplex real-time PCR]. / Un nuovo metodo rapido, basato su real-time PCR multiplex, per la diagnosi differenziale di infezioni da Orthopoxvirus e Herpesvirus.

OBJECTIVE: Variola virus, belonging to Orthopoxviridae family, is one of the most dangerous human pathogens that could be used as biological weapon. We have developed a new rapid assay, based upon Real-time PCR and melting temperatures analysis of amplicons, for the contemporary detection of Orthopoxvirus, VZV and HSV1-2, that are the most important infectious agents to be considered for differential diagnosis. METHODS: The target for detection of orthopoxvirus DNA has been a region of the crmB gene which is common to Variola virus and to other old world orthopoxviruses pathogenic for humans. The targets for VZV and HSV1-2 have been ORF 29 and DNA polymerase, respectively. Suitability of the amplified fragments to RFLP or sequencing analysis, to recognize the involved viral species, has been also tested. RESULT: The selected primers have showed high sensitivity, specificity and compatibility with common amplification conditions. A mean melting temperature difference of 8.7 degree C was observed between the amplicons from the two virus types. Further identification of individual pathogens was made using RFLP analysis. CONCLUSION: The PCR-based protocol set up in this study for presumptive differential diagnosis of variola and herpesviral infections is rapid and specific and it can be used also to detect other orthopoxviral infections, like monkeypox.

Rapid, differential diagnosis of orthopox- and herpesviruses based upon real-time PCR product melting temperature and restriction enzyme analysis of amplicons.

Orthopoxviruses tend to have non-specific early symptoms that cannot be differentiated readily from other infectious exanthemas, such as varicella-zoster virus (VZV) or disseminated herpes simplex virus (HSV) infections. A rapid assay was developed for the differential diagnosis of orthopoxviruses and herpesviruses based upon the melting temperatures of real-time PCR amplicons. A mean melting temperature difference of 8.7 degrees C was observed between the products amplified from the two virus families. Further identification of individual pathogens was made using restriction enzyme analysis. The assay was able to identify correctly viruses from quality control panels of herpes and orthopoxviruses.

CXCR4-dependent HIV-1 infection of differentiated epithelial cells.

Epithelial cells constitute a physical barrier to sexual transmission of HIV, but are also a source of cytokines that could alter infection efficiency. We studied HIV infection of the human colonic epithelial cell line HCT116, which is a model for differentiation of intestinal mucosal epithelium. Differentiated HCT116 cells had increased expression of cell surface C-X-C chemokine receptor type-4 (CXCR4) that mediated HIV entry, despite the apparent absence of cell surface CD4. HIV infection in differentiated HCT116 cells increased the levels of IL-1alpha, and IFN-alpha mRNA even though only 1% of cells had integrated provirus. The inefficient, CXCR4-mediated infection of differentiated HCT116 cells supports the view that epithelial cells are a barrier and not a portal for HIV transmission. However, low level infection of epithelial cells could trigger the release of cytokines that indirectly increase the transmission rate.

Efficacy of three surface disinfectants against spores of Clostridium difficile ribotype 027.

BACKGROUND: The emergence of Clostridium difficile ribotype 027 raised the question of sporicidal surface disinfectants are also effective against spores of C. difficile ribotype 027. MATERIALS AND METHODS: Three surface disinfectants based on magnesium monoperoxyphthalate hexahydrate (Dismozon pur), a combination of (ethylenedioxy)dimethanol, glutaral and benzyl-C12-18-alkyldimethylammonium chlorides (Kohrsolin extra) and a combination of glutaral, benzyl-C12-18-alkyldimethylammonium chlorides and didecyl-dimethylammonium chloride (Kohrsolin FF) were tested in a suspension test in various concentrations and contact times against spores of three C. difficile strains including ribotype 027. RESULTS: All three surface disinfectant reduced the number of spores by ≥4 log(10) steps, e.g. Dismozon pur at 1.5% and 2 h exposure time, Kohrsolin extra at 2% and 4 h exposure time, and Kohrsolin FF at 2% and 6 h exposure time. Spores of ribotype 027 did not show a lower susceptibility to Dismozon pur compared to the other two C. difficile strains. CONCLUSIONS: All three tested surface disinfectants should be effective for surface disinfection in outbreaks caused by C. difficile ribotype 027.

Anti-severe acute respiratory syndrome coronavirus immune responses: the role played by V gamma 9V delta 2 T cells.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV) strain. Analyses of T cell repertoires in health care workers who survived SARS-CoV infection during the 2003 outbreak revealed that their effector memory V gamma 9V delta 2 T cell populations were selectively expanded ~3 months after the onset of disease. No such expansion of their alpha beta T cell pools was detected. The expansion of the V gamma 9V delta 2 T cell population was associated with higher anti-SARS-CoV immunoglobulin G titers. In addition, in vitro experiments demonstrated that stimulated V gamma 9V delta 2 T cells display an interferon- gamma -dependent anti-SARS-CoV activity and are able to directly kill SARS-CoV-infected target cells. These findings are compatible with the possibility that V gamma 9V delta 2 T cells play a protective role during SARS.

BeadCons: detection of nucleic acid sequences by flow cytometry.

Molecular beacons are single-stranded nucleic acid structures with a terminal fluorophore and a distal, terminal quencher. These molecules are typically used in real-time PCR assays, but have also been conjugated with solid matrices. This unit describes protocols related to molecular beacon-conjugated beads (BeadCons), whose specific hybridization with complementary target sequences can be resolved by cytometry. Assay sensitivity is achieved through the concentration of fluorescence signal on discrete particles. By using molecular beacons with different fluorophores and microspheres of different sizes, it is possible to construct a fluid array system with each bead corresponding to a specific target nucleic acid. Methods are presented for the design, construction, and use of BeadCons for the specific, multiplexed detection of unlabeled nucleic acids in solution. The use of bead-based detection methods will likely lead to the design of new multiplex molecular diagnostic tools.

Long-lasting CD3+ T-cell deficiency after cord blood stem cell transplantation in a human herpesvirus 6-infected child.

We report a long-lasting (8-month) reactivation of human herpesvirus 6 (HHV-6) infection in child who had undergone cord blood stem cell transplantation. The reactivation was characterized by high viral loads and by immediate-early mRNA positivity. HHV-6 infection was associated with a deep depletion of CD3, while the CD4/CD8 ratio remained substantially unchanged.

Human herpesvirus-6 modulates RANTES production in primary human endothelial cell cultures.

Human herpesvirus 6 (HHV6) is a beta-herpesvirus capable of infecting several cell types from different origins. HHV6 is the etiological agent of exantem subitum and has been associated with several diseases, all characterized by an inflammatory response triggered by chemokines. We show that strain U1102 of HHV6 is able to infect persistently human endothelial cells obtained from umbilical veins, adult aorta and adult heart microvessels, without apparent cytopathic effect. Analysis by in situ PCR showed that HHV6 sequences were present in 20% of HUVEC, 10% of aortic, and 1% of heart microvascular endothelial cells. Regardless of endothelial cell origin, HHV6 infection induced de novo synthesis of the RANTES CC-chemokine. It was found, however, that microvascular endothelial cells, despite their lower susceptibility to HHV6 infection, showed the highest RANTES expression. Chemokine production occurred also in the absence of viral DNA synthesis. Furthermore, RANTES synthesis required an active viral genome, as UV-inactivated HHV6 infection of endothelial cells did not lead to chemokine production. We investigated the expression of HHV6 U51 gene, which encodes a chemokine receptor that is already known to sequester and down modulate RANTES in epithelial cells. HHV6-infected endothelial cells co-expressed RANTES and U51 mRNAs starting from 12 hr up to 48 hr post-infection. Then, RANTES transcripts disappeared whereas U51 messages continued to be expressed. In conclusion, this study highlights the major role of HHV6 in endothelial cell biology and the development of inflammatory processes.

Flow cytometry and T-cell response monitoring after smallpox vaccination.

Orthopoxvirus zoonosis or smallpox as result of bioterrorism or biological warfare represents a risk for epidemic spread. By monitoring T-cell responses by flow cytometry, we observed a recall response after recent vaccination against smallpox. When the high similarity between the orthopoxviruses is considered, this rapid assay that uses vaccinia antigens could identify recently exposures.