Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 9 de 9
Filter
Add more filters










Database
Language
Publication year range
1.
Virology ; 504: 63-72, 2017 04.
Article in English | MEDLINE | ID: mdl-28157546

ABSTRACT

Whether respiratory syncytial virus (RSV) induces severe infantile pulmonary disease may depend on viral strain and expression of types I and III interferons (IFNs). These IFNs impact disease severity by inducing expression of many anti-viral IFN-stimulated genes (ISGs). To investigate the impact of RSV strain on IFN and ISG expression, we stimulated human monocyte-derived DCs (MDDCs) with either RSV A2 or Line 19 and measured expression of types I and III IFNs and ISGs. At 24h, A2 elicited higher ISG expression than Line 19. Both strains induced MDDCs to express genes for IFN-ß, IFN-α1, IFN-α8, and IFN-λ1-3, but only A2 induced IFN-α2, -α14 and -α21. We then show that IFN-α8 and IFN-α14 most potently induced MDDCs and bronchial epithelial cells (BECs) to express ISGs. Our findings demonstrate that RSV strain may impact patterns of types I and III IFN expression and the magnitude of the ISG response by DCs and BECs.


Subject(s)
Dendritic Cells/immunology , Interferon-alpha/metabolism , Interferon-beta/metabolism , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus, Human/immunology , Adult , Bronchi/cytology , Cells, Cultured , Cytokines/metabolism , Dendritic Cells/virology , Epithelial Cells/cytology , Humans , Inflammation/immunology , Lung/immunology , Lung/pathology , Lung/virology , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology , Respiratory Mucosa/virology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/metabolism
2.
J Vis Exp ; (97)2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25867042

ABSTRACT

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.


Subject(s)
Interferons/biosynthesis , Interferons/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , High-Throughput Screening Assays/methods , Humans , Interferon Type I/biosynthesis , Interferon Type I/genetics , Transcriptome
3.
J Biomater Sci Polym Ed ; 24(8): 912-26, 2013.
Article in English | MEDLINE | ID: mdl-23647248

ABSTRACT

Thiolated polymers containing disulfide linkages are commonly researched in gene delivery with the assumption that the thiolated complexes form disulfide bonds. This study investigates the extent of disulfide linking in a thiol-containing polymer and determines the impact that free thiols have on the polymer's delivery potential. A fluorescent cationic polymer containing thiol pendant chains was prepared from poly(allylamine) and 2-iminothiolate (Traut's reagent). Polymer fluorescence was determined by UV plate readings and fluorescent microscopy. Transfection efficiency and cytotoxicity were assessed in MCF-7 breast cancer cells. Results show that thiolated polymers exhibited fluorescence at ex/em ∼595/620. Fluorescent measurements, microscopy imaging, and DNA electrophoresis show that thiolated polymers are not internalized by cells in a culture, yet, they bind to the cell surface, perhaps valuable for applications requiring cell adhesion.


Subject(s)
Dithionitrobenzoic Acid/chemistry , Gene Transfer Techniques , Imidoesters/chemistry , Polyamines/chemistry , Cell Count , Cell Line , Cell Membrane/metabolism , DNA/chemistry , DNA/genetics , Humans , Protein Binding , Transfection
4.
Biomacromolecules ; 13(5): 1331-9, 2012 May 14.
Article in English | MEDLINE | ID: mdl-22515194

ABSTRACT

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.


Subject(s)
DNA/chemistry , Gene Transfer Techniques , Genetic Vectors/chemistry , Polyamines/chemistry , Sulfhydryl Compounds/chemistry , Binding Sites , Plasmids/chemistry
5.
Immunol Cell Biol ; 90(8): 774-83, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22249201

ABSTRACT

Recent genome-wide association studies suggest distinct roles for 12 human interferon-alpha (IFN-α) and 3 IFN-λ subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides. We found that in response to poly I:C and LPS, monocytes, MDM and MDDC express a subtype pattern restricted primarily to IFN-ß and IFN-λ1. In addition, while CpG elicited expression of all type I IFN subtypes by pDC, imiquimod did not. Furthermore, MDM and mDC highly express IFN-λ, and the subtypes of IFN-λ are expressed hierarchically in the order IFN-λ1 followed by IFN-λ2, and then IFN-λ3. These data support a model of coordinated cell- and ligand-specific expression of types I and III IFN. Defining IFN subtype expression profiles in a variety of contexts may elucidate specific roles for IFN subtypes as protective, therapeutic or pathogenic mediators.


Subject(s)
Gene Expression Profiling , Interferon-alpha/genetics , Interleukins/genetics , Animals , DNA Probes/metabolism , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Gene Expression Regulation/drug effects , Humans , Interferon-alpha/metabolism , Interferons , Interleukins/metabolism , Ligands , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mice , Myeloid Cells/drug effects , Myeloid Cells/metabolism , Oligodeoxyribonucleotides/pharmacology , Organ Specificity/drug effects , Organ Specificity/genetics , Poly I-C/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Toll-Like Receptors/metabolism
6.
J Biomol Tech ; 19(5): 342-7, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19183798

ABSTRACT

To standardize the amount of biological material between samples (e.g., number of cells or amount of tissue) for quantitative real-time reverse transcriptase PCR (qRT-PCR), the cycle of the target gene at which expression is detected (the cycle threshold, or Ct) is divided by the Ct of a gene either thought to be unaffected by experimental conditions or similarly expressed among donors. Genes that maintain cellular structure or homeostasis, referred to as housekeeping genes, or 18S ribosomal RNA are often used for this purpose. Although unstable or inconsistent housekeeping gene expression will misrepresent experimental effects on target gene expression, housekeeping genes are often chosen arbitrarily rather than systematically. We designed a simple and systematic approach towards selection of housekeeping genes based on Ct variance (as reflected by the standard deviation) and normality of distribution. We validated this approach by comparing stability and consistency of expression of 11 housekeeping genes across different types of cells, experimental treatments, and human donors. Finally, we demonstrated the consequences of inconsistent housekeeping gene expression on the calculation of target gene expression, and conclude that validation of stability of housekeeping gene expression by considering both distribution normality and standard deviation is straightforward and critical for proper experimental design.


Subject(s)
Gene Expression , Reverse Transcriptase Polymerase Chain Reaction/methods , Biotechnology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , Cell Line , Gene Expression/drug effects , Humans , In Vitro Techniques , Ionomycin/pharmacology , Lipopolysaccharides/pharmacology , Monocytes/drug effects , Monocytes/metabolism , Tetradecanoylphorbol Acetate/pharmacology
7.
Mol Ther ; 15(4): 732-40, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17285138

ABSTRACT

Helper-dependent adenoviral vectors (HDAds) are attractive for liver-directed gene therapy because they can mediate long-term, high-level transgene expression without chronic toxicity. However, systemic delivery requires high vector doses for efficient hepatic transduction, resulting in dose-dependent acute toxicity. Clearly, strategies to improve hepatic transduction with low vector doses are needed. In this regard, we have previously shown that hydrodynamic injection of helper-dependent adenoviral vectors into mice results in increased hepatic transduction, reduced systemic vector dissemination, and reduced pro-inflammatory cytokines compared with conventional injection and thus has the potential to improve dramatically the therapeutic index of helper-dependent adenoviral vectors. Unfortunately, the rapid, large-volume injection used in this method cannot be applied to larger animals. Therefore, we have developed a novel balloon occlusion catheter-based method to mimic hydrodynamic injection of helper-dependent adenoviral vectors into non-human primates that does not require rapid, large-volume injection. Using a low, clinically relevant vector dose, this minimally invasive method results in high-efficiency hepatic transduction with minimal toxicity and stable long-term transgene expression for at least 413 days.


Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Genetic Vectors , Liver/metabolism , Animals , Gene Expression , Helper Viruses/genetics , Injections , Male , Mice , Mice, Inbred C57BL , Papio , Time Factors
8.
Mol Ther ; 15(2): 386-92, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17235318

ABSTRACT

Dendritic cells (DCs) are essential for initiating and directing antigen-specific T-cell responses. Genetic modification of DC is under study for cancer immunotherapy, vaccine development, and antigen-targeted immunosuppression. Adenovirus (Ad) type 5 (Ad5)-mediated gene transfer to mouse bone marrow DCs and human monocyte-derived DCs is inefficient because neither express the cognate high-affinity Ads receptor. We show that co-precipitating adenoviral vectors with calcium phosphate (CaPi) increased gene expression (2000-fold) and transduction efficiency (50-fold) in mouse DC, primarily owing to receptor-independent viral uptake. Moreover, Ad5:CaPi-treated DCs were activated to express the maturation surface molecules CD40 and CD86, and to secrete proinflammatory cytokines tumor necrosis factor-alpha and interleukin 6. However, neither DC transduction nor maturation was dependent on viral protein interactions with cell surface integrin. Ad5:CaPi also transduced human DC more efficiently than Ad5 alone, similar to a genetically modified vector (Ad5f35) targeted to the CD46 receptor. As such, this approach combines the efficiency of adenoviral-mediated endosomal escape and nuclear trafficking with the receptor independence of nonviral gene delivery. Importantly, CaPi co-precipitation could be used to functionally modify DC to activate and expand cytomegalovirus-specific memory cytotoxic T lymphocytes. This study identifies a simple technique to improve the efficacy of current Ad5 gene transfer, in support of clinical adoptive immunotherapy.


Subject(s)
Adenoviridae/genetics , Calcium Phosphates/pharmacology , Dendritic Cells/metabolism , Transfection/methods , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , Dendritic Cells/drug effects , Female , Flow Cytometry , Genetic Vectors/genetics , Humans , Integrins/metabolism , Mice , Mice, Inbred BALB C , Protein Binding , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism
9.
Proc Natl Acad Sci U S A ; 102(11): 3930-5, 2005 Mar 15.
Article in English | MEDLINE | ID: mdl-15753292

ABSTRACT

Crigler-Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1. Current therapy relies on phototherapy to prevent kernicterus, but liver transplantation presently is the only permanent cure. Gene therapy is a potential alternative, and recent work has shown that helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. We used a HD-Ad vector to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg/dl to <<1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 x 10(12) viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after one injection of vector, and i.v. injection of bilirubins IIIalpha and XIIIalpha in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6 x 10(11) viral particles per kg. We conclude that complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity.


Subject(s)
Adenoviridae , Genetic Therapy , Genetic Vectors , Glucuronosyltransferase/genetics , Hyperbilirubinemia/drug therapy , Alanine Transaminase/blood , Animals , Bilirubin/blood , DNA/pharmacology , Genetic Vectors/toxicity , Glucuronosyltransferase/metabolism , Humans , RNA, Messenger/metabolism , Rats , Rats, Gunn
SELECTION OF CITATIONS
SEARCH DETAIL
...