Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.
25 Related JoVE Articles!
Intra-lymph Node Injection of Biodegradable Polymer Particles
Institutions: University of Maryland, College Park.
Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.
size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.
Bioengineering, Issue 83, biomaterial, immunology, microparticle, nanoparticle, vaccine, adjuvant, lymph node, targeting, polymer
PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS
Institutions: Barrow Neurological Institute.
Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g.
poly(vinyl alcohol), PVA) or solvents (e.g.
Chemistry, Issue 82, Nanoparticles, Microparticles, PLGA, TPGS, drug delivery, scanning electron microscopy, emulsion, polymers
Intralymphatic Immunotherapy and Vaccination in Mice
Institutions: University Hospital Zurich.
Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.
intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 μl of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.
Immunology, Issue 84, Vaccination, Immunization, intralymphatic immunotherapy, Lymph node injection, vaccines, adjuvants, surgery, anesthesia
Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System
Institutions: Icahn School of Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai.
The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.
Infection, Issue 81, Influenza A virus, Orthomyxoviridae Infections, Influenza, Human, Influenza in Birds, Influenza Vaccines, hemagglutinin, neuraminidase, H7N9, baculovirus, insect cells, recombinant protein expression
Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria
Institutions: University of Greifswald.
Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1
. Streptococcus pneumoniae
is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo
role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae
pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.
Infection, Issue 84, Gram-Positive Bacteria, Streptococcus pneumoniae, Pneumonia, Bacterial, Respiratory Tract Infections, animal models, community-acquired pneumonia, invasive pneumococcal diseases, Pneumococci, bioimaging, virulence factor, dissemination, bioluminescence, IVIS Spectrum
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface
Institutions: University of Southern California.
Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ
regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.
Bioengineering, Issue 89, Enamel, Amelogenin, Chitosan hydrogel, Apatite, Biomimetic, Erosion, Superficial enamel reconstruction, Dense interface
Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation
Institutions: Université de Montréal, Université de Montréal, Université de Montréal.
Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-γ production by T lymphocytes in response to in vitro
stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens.
Immunology, Issue 89, Varicella zoster virus, cell-mediated immunity, T cells, interferon gamma, ELISpot, umbilical cord blood transplantation
Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Institutions: University of Manitoba, University of Manitoba.
Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo
flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.
Medicine, Issue 89, mucosal, immunology, FGT, lavage, cervical, CMC
Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections
Institutions: Universidad de la República, Trinity College Dublin.
Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung’s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages.
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae
inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs’ immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.
Medicine, Issue 90, Sublingual immunotherapy, Pneumonia, Streptococcus pneumoniae, Lungs, Flagellin, TLR5, NLRC4
Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection
Institutions: University of Maine, University of Maine.
Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo
, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans
, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans
. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.
Immunology, Issue 93, Zebrafish, mucosal candidiasis, mucosal infection, epithelial barrier, epithelial cells, innate immunity, swimbladder, Candida albicans, in vivo.
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Institutions: Harvard Medical School, MGH for Children, Massachusetts General Hospital.
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro
models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro
model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa
(PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli
(MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro
model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro
coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Infection, Issue 83, Cellular Biology, Epithelium, Neutrophils, Pseudomonas aeruginosa, Respiratory Tract Diseases, Neutrophils, epithelial barriers, pathogens, transmigration
Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation
Institutions: Mount Sinai School of Medicine .
Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1
. Detection of pathogenic antigen by naïve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3
. Thus, DCs link the innate and adaptive immune systems.
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8
. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12
. Electroporation has been used with mixed results13,14,15
, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16
. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFNβ
. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17
, at both the mRNA and protein levels.
Immunology, Issue 53, Dendritic cells, nucleofection, high-throughput, siRNA, interferon signaling
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
Institutions: Northeastern University.
More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1
. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression.
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5
. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention
. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7
. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9
. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10
. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.
Bioengineering, Issue 59, Gelatin Nanoparticle, Gene Therapy, Targeted Delivery, Pancreatic Cancer, Epidermal Growth Factor Receptor, EGFR
Bridging the Bio-Electronic Interface with Biofabrication
Institutions: University of Maryland , University of Maryland , University of Maryland .
Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3
, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5
. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7
,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8
. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7
. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10
. Alginate films and their entrapment of live cells will also be addressed 11
. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission.
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13
. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.
Bioengineering, Issue 64, Biomedical Engineering, electrodeposition, biofabrication, chitosan, alginate, lab-on-a-chip, microfluidic, DTRA
Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK
Institutions: Saint Louis University.
Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.
Infection, Issue 70, Immunology, Virology, Medicine, Infectious Diseases, Ophthalmology, Herpes, herpetic stromal keratitis, HSK, keratitis, pathogenesis, clinical evaluation, virus, eye, mouse, animal model
Isolation of Lymphocytes from Mouse Genital Tract Mucosa
Institutions: University of California, Los Angeles , California NanoSystems.
Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1
. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2
, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.
Immunology, Issue 67, Mucosal immunity, sexually transmitted diseases, genital tract lymphocytes, lymphocyte isolation, flow cytometry, FACS
In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus
Institutions: University of Texas Medical Branch .
Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo
imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.
Virology, Issue 70, Immunology, Medicine, Neuroscience, Molecular Biology, Pathology, IVIS, in vivo modeling, VEE, CNS, Neuroinvasion, Hume’s 3Rs, Encephalitis, bioluminescence, luciferase, virus
Skin Tattooing As A Novel Approach For DNA Vaccine Delivery
Institutions: New York University School of Medicine, New York University School of Medicine, Veterans Affairs New York Harbor.
Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.
Bioengineering, Issue 68, Biomedical Engineering, Genetics, Medicine, DNA, vaccine, immunization method, skin tattooing, intradermal delivery, GFP
Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry
Institutions: Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine.
Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1
and as a technology for regenerative medicine2
. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3
. Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4
that are hydrolytically degradable5,6
and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7
, mouse mammary epithelial cells8
, human brain cancer cells9
and macrovascular (human umbilical vein, HUVECs) endothelial cells10
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11
. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.
Biomedical Engineering, Issue 73, Bioengineering, Tissue Engineering, Cellular Biology, Medicine, Genetics, Biocompatible Materials, Biopolymers, Drug Delivery Systems, Nanotechnology, bioengineering (general), Therapeutics, Nanoparticle, poly(beta-amino ester), high-throughput, transfection, nanoparticle tracking analysis, biomaterial, gene delivery, flow cytometry
Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages
Institutions: Kingsborough Community College, University of Texas at Austin, Kean University.
The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae
. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages.
We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.
Infection, Issue 75, Medicine, Immunology, Infectious Diseases, Microbiology, Molecular Biology, Cellular Biology, Biochemistry, Bioengineering, Bacterial Infections and Mycoses, Mucosal immunity, oleic acid, linoleic acid, linolenic acid, cholera toxin, cholera, fatty acids, tissue culture, MTT assay, mouse, animal model
Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
Institutions: Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg.
HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.
Medicine, Issue 16, HIV, Cell Biology, Recombinase, provirus, HeLa Cells
Titration of Human Coronaviruses Using an Immunoperoxidase Assay
Institutions: INRS-Institut Armand-Frappier.
Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.
Microbiology, Issue 14, Springer Protocols, Human coronavirus, HCoV-229E, HCoV-OC43, cell and tissue sample, titration, immunoperoxidase assay, TCID50
Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation
Institutions: La Jolla Institute for Allergy and Immunology.
Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens. Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells. The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells. Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy. Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.
Immunology, Issue 10, Natural Killer T cells, NKT cells, CD1 Tetramers, antigen presentation, glycolipid antigens, CD1d, Mucosal Immunity, Translational Research