Production of lentiviral vectors with enhanced efficiency to target dendritic cells by attenuating mannosidase activity of mammalian cells

<p>Abstract</p> <p>Background</p> <p>Dendritic cells (DCs) are antigen-presenting immune cells that interact with T cells and have been widely studied for vaccine applications. To achieve this, DCs can be manipulated by lentiviral vectors (LVs) to express antigens to stimulate the desired antigen-specific T cell response, which gives this approach great potential to fight diseases such as cancers, HIV, and autoimmune diseases. Previously we showed that LVs enveloped with an engineered Sindbis virus glycoprotein (SVGmu) could target DCs through a specific interaction with DC-SIGN, a surface molecule predominantly expressed by DCs. We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that an increase in high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit mannosidase, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production.</p> <p>Results</p> <p>Through western blot analysis and binding tests, we were able to infer that binding of SVGmu to DC-SIGN is directly related to amount of high-mannose structures on SVGmu. We also found that the titer for the LV (FUGW/SVGmu) produced with DMJ against 293T.DCSIGN, a human cell line expressing the human DC-SIGN atnibody, was over four times higher than that of vector produced without DMJ. In addition, transduction of a human DC cell line, MUTZ-3, yielded a higher transduction efficiency for the LV produced with DMJ.</p> <p>Conclusion</p> <p>We conclude that LVs produced under conditions with inhibited mannosidase activity can effectively modify cells displaying the DC-specific marker DC-SIGN. This study offers evidence to support the utilization of DMJ in producing LVs that are enhanced carriers for the development of DC-directed vaccines.</p

Potential for Development of an Escherichia coli—Based Biosensor for Assessing Bioavailable Methionine: A Review

Methionine is an essential amino acid for animals and is typically considered one of the first limiting amino acids in animal feed formulations. Methionine deficiency or excess in animal diets can lead to sub-optimal animal performance and increased environmental pollution, which necessitates its accurate quantification and proper dosage in animal rations. Animal bioassays are the current industry standard to quantify methionine bioavailability. However, animal-based assays are not only time consuming, but expensive and are becoming more scrutinized by governmental regulations. In addition, a variety of artifacts can hinder the variability and time efficacy of these assays. Microbiological assays, which are based on a microbial response to external supplementation of a particular nutrient such as methionine, appear to be attractive potential alternatives to the already established standards. They are rapid and inexpensive in vitro assays which are characterized with relatively accurate and consistent estimation of digestible methionine in feeds and feed ingredients. The current review discusses the potential to develop Escherichia coli-based microbial biosensors for methionine bioavailability quantification. Methionine biosynthesis and regulation pathways are overviewed in relation to genetic manipulation required for the generation of a respective methionine auxotroph that could be practical for a routine bioassay. A prospective utilization of Escherichia coli methionine biosensor would allow for inexpensive and rapid methionine quantification and ultimately enable timely assessment of nutritional profiles of feedstuffs

Virus-Receptor Mediated Transduction of Dendritic Cells by Lentiviruses Enveloped with Glycoproteins Derived from Semliki Forest Virus

Lentiviruses have recently attracted considerable interest for their potential as a genetic modification tool for dendritic cells (DCs). In this study, we explore the ability of lentiviruses enveloped with alphaviral envelope glycoproteins derived from Semliki Forest virus (SFV) to mediate transduction of DCs. We found that SFV glycoprotein (SFV-G)-pseudotyped lentiviruses use C-type lectins (DC-SIGN and L-SIGN) as attachment factors for transduction of DCs. Importantly, SFV-G pseudotypes appear to have enhanced transduction towards C-type lectin-expressing cells when produced under conditions limiting glycosylation to simple high-mannose, N-linked glycans. These results, in addition to the natural DC tropism of SFV-G, offer evidence to support the use of SFV-G-bearing lentiviruses to genetically modify DCs for the study of DC biology and DC-based immunotherapy

How a Diverse Research Ecosystem Has Generated New Rehabilitation Technologies: Review of NIDILRR’s Rehabilitation Engineering Research Centers

Over 50 million United States citizens (1 in 6 people in the US) have a developmental, acquired, or degenerative disability. The average US citizen can expect to live 20% of his or her life with a disability. Rehabilitation technologies play a major role in improving the quality of life for people with a disability, yet widespread and highly challenging needs remain. Within the US, a major effort aimed at the creation and evaluation of rehabilitation technology has been the Rehabilitation Engineering Research Centers (RERCs) sponsored by the National Institute on Disability, Independent Living, and Rehabilitation Research. As envisioned at their conception by a panel of the National Academy of Science in 1970, these centers were intended to take a “total approach to rehabilitation”, combining medicine, engineering, and related science, to improve the quality of life of individuals with a disability. Here, we review the scope, achievements, and ongoing projects of an unbiased sample of 19 currently active or recently terminated RERCs. Specifically, for each center, we briefly explain the needs it targets, summarize key historical advances, identify emerging innovations, and consider future directions. Our assessment from this review is that the RERC program indeed involves a multidisciplinary approach, with 36 professional fields involved, although 70% of research and development staff are in engineering fields, 23% in clinical fields, and only 7% in basic science fields; significantly, 11% of the professional staff have a disability related to their research. We observe that the RERC program has substantially diversified the scope of its work since the 1970’s, addressing more types of disabilities using more technologies, and, in particular, often now focusing on information technologies. RERC work also now often views users as integrated into an interdependent society through technologies that both people with and without disabilities co-use (such as the internet, wireless communication, and architecture). In addition, RERC research has evolved to view users as able at improving outcomes through learning, exercise, and plasticity (rather than being static), which can be optimally timed. We provide examples of rehabilitation technology innovation produced by the RERCs that illustrate this increasingly diversifying scope and evolving perspective. We conclude by discussing growth opportunities and possible future directions of the RERC program

Induced pseudoscalar coupling of the proton weak interaction

The induced pseudoscalar coupling $g_p$ is the least well known of the weak coupling constants of the proton's charged--current interaction. Its size is dictated by chiral symmetry arguments, and its measurement represents an important test of quantum chromodynamics at low energies. During the past decade a large body of new data relevant to the coupling $g_p$ has been accumulated. This data includes measurements of radiative and non radiative muon capture on targets ranging from hydrogen and few--nucleon systems to complex nuclei. Herein the authors review the theoretical underpinnings of $g_p$, the experimental studies of $g_p$, and the procedures and uncertainties in extracting the coupling from data. Current puzzles are highlighted and future opportunities are discussed.Comment: 58 pages, Latex, Revtex4, prepared for Reviews of Modern Physic

Escherichia coli, an Intestinal Microorganism, as a Biosensor for Quantification of Amino Acid Bioavailability

In animal diets optimal amino acid quantities and balance among amino acids is of great nutritional importance. Essential amino acid deficiencies have negative impacts on animal physiology, most often expressed in sub-optimal body weight gains. Over supplementation of diets with amino acids is costly and can increase the nitrogen emissions from animals. Although in vivo animal assays for quantification of amino acid bioavailability are well established, Escherichia coli-based bioassays are viable potential alternatives in terms of accuracy, cost, and time input. E. coli inhabits the gastrointestinal tract and although more abundant in colon, a relatively high titer of E. coli can also be isolated from the small intestine, where primary absorption of amino acids and peptides occur. After feed proteins are digested, liberated amino acids and small peptides are assimilated by both the small intestine and E. coli. The similar pattern of uptake is a necessary prerequisite to establish E. coli cells as accurate amino acid biosensors. In fact, amino acid transporters in both intestinal and E. coli cells are stereospecific, delivering only the respective biological l-forms. The presence of free amino- and carboxyl groups is critical for amino acid and dipeptide transport in both biological subjects. Di-, tri- and tetrapeptides can enter enterocytes; likewise only di-, tri- and tetrapeptides support E. coli growth. These similarities in addition to the well known bacterial genetics make E. coli an optimal bioassay microorganism for the assessment of nutritionally available amino acids in feeds

Virus-producing constructs used to make pseudotyped lentiviruses.

<p>(A) Schematic diagrams of constructs encoding the lentiviral backbone FUGW and envelope glycoproteins. Ubi: the human ubiquitin-C promoter; GFP: enhanced green fluorescence protein; WRE: the woodchuck hepatitis virus posttranscriptional regulatory element (WRE) to increase the level of transcription; ΔU3: deleted U3 region that results in the transcriptional activation of the integrated viral LTR promoter; pA: polyadenylation signal; E1, E2, 6k, E3: SFV-glycoprotein (E1 for fusion, E2 for receptor binding, 6k a linker, and E3 is a signal sequence). The VSV-G expressing plasmid contains the rabbit β-globin intron and poly(A) signal. (B) Viral supernatants harvested from virus-producing cells transiently transfected with GFP-vpr, SFV-G, or VSV-G, and other necessary packaging constructs, were coated to a poly-lysine containing coverslip by centrifugation. The resulting coverslips were then rinsed and immunostained with an anti-SFV-G antibody (red) to label the glycoproteins and imaged using a laser confocal microscope.</p

Effects of DC-SIGN or L-SIGN expression.

<p>(A) Expression of L-SIGN and DC-SIGN in 3T3 (solid fill), 3T3.DCSIGN (open fill) and 3T3.LSIGN (gray fill), respectively, was detected using cross reactive DC-SIGN/L-SIGN antibody and quantified by flow cytometry. (B) Effects of DC-SIGN or L-SIGN expression on the infectivity of pseudotyped lentiviruses. SFV-G- and VSV-G-pseudotyped lentiviruses were normalized by p24 and spin-inoculated with LSIGN- or DCSIGN-expressing 3T3 cells; the parental 3T3 cells were included as controls. Three days later, the transduction efficiency was measured by analyzing GFP expression. Fold increase in percentage of GFP-positive cells is shown based on 3T3 cells where FUGW/SFVG transduced 6.6±0.7% and FUGW/VSVG transduced 72.2±1.0%. (C) Specificity of binding to DC-SIGN. [³⁵S]-methionine-labeled FUGW/SFVG or FUGW/VSVG were incubated with 3T3 or DC-SIGN/L-SIGN-expressing cells at 4°C. Cells were washed and ³⁵S radioactivity of the resuspended cells was quantitated with a liquid scintillation counter. Fold increase in [³⁵S] bound viral particles is shown based on 3T3 cells with 3.59±0.27% and 25.23±1.25% of the total CPM of virus bound for FUGW/SFVG and FUGW/VSVG, respectively, where values are given as the mean of triplicates ± S.E.</p