In vivo gene transfer using cetylated polyethylenimine

This report describes gene transfer in vitro as well as in vivo using cetylated low-molecular mass (600 Da) polyethylenimine (28% of amine groups substituted with cetyl moieties), termed CT-PEI. This compound is hydrophobic and has to be incorporated into liposomes in order to be suitable for gene transfer studies. Serum-induced plasmid DNA degradation assay demonstrated that CT-PEI-containing liposomal carriers could protect complexed DNA (probably via condensation). In vitro luciferase gene expression achieved using medium supplemented with 10% serum was comparable to that achieved in serum-reduced medium and was highest for CT-PEI/cholesterol liposomes, followed by CT-PEI/dioleoylphosphatidylcholine liposomes and PEI 600 Da (uncetylated) carrier. In vivo systemic transfer into mice was most efficient when liposome formulations contained CT-PEI and cholesterol. Higher luciferase expression was then observed in lungs than in liver. In conclusion: liposomes containing cetylated polyethylenimine and cholesterol are a suitable vehicle for investigating systemic plasmid DNA transfer into lungs.     

IL-13 regulates Th17 secretion of IL-17A in an IL-10-dependent manner

IL-13 is a central mediator of airway hyperresponsiveness and mucus expression, both hallmarks of asthma. IL-13 is found in the sputum of patients with asthma; therefore, IL-13 is an attractive drug target for treating asthma. We have shown previously that IL-13 inhibits Th17 cell production of IL-17A and IL-21 in vitro. Th17 cells are associated with autoimmune diseases, host immune responses, and severe asthma. In this study, we extend our in vitro findings and determine that IL-13 increases IL-10 production from Th17-polarized cells and that IL-13-induced IL-10 production negatively regulates the secretion of IL-17A and IL-21. To determine if IL-13 negatively regulates lung IL-17A expression via an IL-10-dependent mechanism in vivo, we used a model of respiratory syncytial virus (RSV) strain A2 infection in STAT1 knockout (KO) mice that increases lung IL-17A and IL-13 expression, cytokines not produced during RSV infection in wild-type mice. To test the hypothesis that IL-13 negatively regulates lung IL-17A expression, we created STAT1/IL-13 double KO (DKO) mice. We found that RSV-infected STAT1/IL-13 DKO mice had significantly greater lung IL-17A expression compared with that of STAT1 KO mice and that increased IL-17A expression was abrogated by anti-IL-10 Ab treatment. RSV-infected STAT1/IL-13 DKO mice also had increased neutrophil infiltration compared with that of RSV-infected STAT1 KO mice. Neutralizing IL-10 increased the infiltration of inflammatory cells into the lungs of STAT1 KO mice but not STAT1/IL-13 DKO mice. These findings are vital to understanding the potential side effects of therapeutics targeting IL-13. Inhibiting IL-13 may decrease IL-10 production and increase IL-17A production, thus potentiating IL-17A-associated diseases.     

Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.     

A dominant negative zebrafish Ahr2 partially protects developing zebrafish from dioxin toxicity

The toxicity by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is thought to be caused by activation of the aryl hydrocarbon receptor (AHR). However, our understanding of how AHR activation by TCDD leads to toxic effects is poor. Ideally we would like to manipulate AHR activity in specific tissues and at specific times. One route to this is expressing dominant negative AHRs (dnAHRs). This work describes the construction and characterization of dominant negative forms of the zebrafish Ahr2 in which the C-terminal transactivation domain was either removed, or replaced with the inhibitory domain from the Drosophila engrailed repressor protein. One of these dnAhr2s was selected for expression from the ubiquitously active e2fα promoter in transgenic zebrafish. We found that these transgenic zebrafish expressing dnAhr2 had reduced TCDD induction of the Ahr2 target gene cyp1a, as measured by 7-ethoxyresorufin-O-deethylase activity. Furthermore, the cardiotoxicity produced by TCDD, pericardial edema, heart malformation, and reduced blood flow, were all mitigated in the zebrafish expressing the dnAhr2. These results provide in vivo proof-of-principle results demonstrating the effectiveness of dnAHRs in manipulating AHR activity in vivo, and demonstrating that this approach can be a means for blocking TCDD toxicity.     

Microbes in beach sands: integrating environment,ecology and public health

Beach sand is a habitat that supports many microbes, including viruses, bacteria, fungi and protozoa (micropsammon). The apparently inhospitable conditions of beach sand environments belie the thriving communities found there. Physical factors, such as water availability and protection from insolation; biological factors, such as competition, predation, and biofilm formation; and nutrient availability all contribute to the characteristics of the micropsammon. Sand microbial communities include autochthonous species/phylotypes indigenous to the environment. Allochthonous microbes, including fecal indicator bacteria (FIB) and waterborne pathogens, are deposited via waves, runoff, air, or animals. The fate of these microbes ranges from death, to transient persistence and/or replication, to establishment of thriving populations (naturalization) and integration in the autochthonous community. Transport of the micropsammon within the habitat occurs both horizontally across the beach, and vertically from the sand surface and ground water table, as well as at various scales including interstitial flow within sand pores, sediment transport for particle-associated microbes, and the large-scale processes of wave action and terrestrial runoff. The concept of beach sand as a microbial habitat and reservoir of FIB and pathogens has begun to influence our thinking about human health effects associated with sand exposure and recreational water use. A variety of pathogens have been reported from beach sands, and recent epidemiology studies have found some evidence of health risks associated with sand exposure. Persistent or replicating populations of FIB and enteric pathogens have consequences for watershed/beach management strategies and regulatory standards for safe beaches. This review summarizes our understanding of the community structure, ecology, fate, transport, and public health implications of microbes in beach sand. It concludes with recommendations for future work in this vastly under-studied area.     

SIV Vpx Is Essential for Macrophage Infection but Not for Development of AIDS

Analysis of rhesus macaques infected with a vpx deletion mutant virus of simian immunodeficiency virus mac239 (SIVΔvpx) demonstrates that Vpx is essential for efficient monocyte/macrophage infection in vivo but is not necessary for development of AIDS. To compare myeloid-lineage cell infection in monkeys infected with SIVΔvpx compared to SIVmac239, we analyzed lymphoid and gastrointestinal tissues from SIVΔvpx-infected rhesus (n = 5), SIVmac239-infected rhesus with SIV encephalitis (7 SIV239E), those without encephalitis (4 SIV239noE), and other SIV mutant viruses with low viral loads (4 SIVΔnef, 2 SIVΔ3). SIV+ macrophages and the percentage of total SIV+ cells that were macrophages in spleen and lymph nodes were significantly lower in rhesus infected with SIVΔvpx (2.2%) compared to those infected with SIV239E (22.7%), SIV239noE (8.2%), and SIV mutant viruses (10.1%). In colon, SIVΔvpx monkeys had fewer SIV+ cells, no SIV+ macrophages, and lower percentage of SIV+ cells that were macrophages than the other 3 groups. Only 2 SIVΔvpx monkeys exhibited detectable virus in the colon. We demonstrate that Vpx is essential for efficient macrophage infection in vivo and that simian AIDS and death can occur in the absence of detectable macrophage infection.     

Modeling Dynamics of Culex pipiens Complex Populations and Assessing Abatement Strategies for West Nile Virus

The primary mosquito species associated with underground stormwater systems in the United States are the Culex pipiens complex species. This group represents important vectors of West Nile virus (WNV) throughout regions of the continental U.S. In this study, we designed a mathematical model and compared it with surveillance data for the Cx. pipiens complex collected in Beaufort County, South Carolina. Based on the best fit of the model to the data, we estimated parameters associated with the effectiveness of public health insecticide (adulticide) treatments (primarily pyrethrin products) as well as the birth, maturation, and death rates of immature and adult Cx. pipiens complex mosquitoes. We used these estimates for modeling the spread of WNV to obtain more reliable disease outbreak predictions and performed numerical simulations to test various mosquito abatement strategies. We demonstrated that insecticide treatments produced significant reductions in the Cx. pipiens complex populations. However, abatement efforts were effective for approximately one day and the vector mosquitoes rebounded until the next treatment. These results suggest that frequent insecticide applications are necessary to control these mosquitoes. We derived the basic reproductive number (ℜ₀) to predict the conditions under which disease outbreaks are likely to occur and to evaluate mosquito abatement strategies. We concluded that enhancing the mosquito death rate results in lower values of ℜ₀, and if ℜ₀<1, then an epidemic will not occur. Our modeling results provide insights about control strategies of the vector populations and, consequently, a potential decrease in the risk of a WNV outbreak.     

Genotype‐by‐sequencing of the plant‐pathogenic fungi Pyrenophora teres and Sphaerulina musiva utilizing Ion Torrent sequence technology

Genetic and genomics tools to characterize host–pathogen interactions are disproportionately directed to the host because of the focus on resistance. However, understanding the genetics of pathogen virulence is equally important and has been limited by the high cost of de novo genotyping of species with limited marker data. Non‐resource‐prohibitive methods that overcome the limitation of genotyping are now available through genotype‐by‐sequencing (GBS). The use of a two‐enzyme restriction‐associated DNA (RAD)‐GBS method adapted for Ion Torrent sequencing technology provided robust and reproducible high‐density genotyping of several fungal species. A total of 5783 and 2373 unique loci, ‘sequence tags’, containing 16 441 and 9992 single nucleotide polymorphisms (SNPs) were identified and characterized from natural populations of Pyrenophora teres f. maculata and Sphaerulina musiva, respectively. The data generated from the P. teres f. maculata natural population were used in association mapping analysis to map the mating‐type gene to high resolution. To further validate the methodology, a biparental population of P. teres f. teres, previously used to develop a genetic map utilizing simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers, was re‐analysed using the SNP markers generated from this protocol. A robust genetic map containing 1393 SNPs on 997 sequence tags spread across 15 linkage groups with anchored reference markers was generated from the P. teres f. teres biparental population. The robust high‐density markers generated using this protocol will allow positional cloning in biparental fungal populations, association mapping of natural fungal populations and population genetics studies.     

Distribution of small phytoflagellates along an Arctic fjord transect

Phytoflagellates < 10 μm substantially contribute to the abundance, biomass and primary production in polar waters, but information on the distribution of specific groups is scarce. We applied catalysed reporter deposition‐fluorescence in situ hybridization to investigate the distribution of total phytoflagellates and of eight specific groups along a 100 km transect west off Kongsfjorden (Spitsbergen) from 29 to 31 July 2010. Phytoflagellates contributed to > 75% of the depth‐integrated abundance and biomass of total eukaryotes < 10 μm at all stations. Their depth‐integrated abundance and biomass decreased along the transect from 1.5 × 10¹² cells m^?2 (6.6 × 10¹² pgC m^?2) at the outermost station to 1.7 × 10¹⁰ cells m^?2 (4.7 × 10¹⁰ pgC m^?2) at the innermost station. Chlorophytes contributed to the total abundance of phytoflagellates with a range from 13% to 87% (0.7–30.5 × 10³ cells ml^?1), and predominated in open waters. The contribution of haptophytes was < 1–38% (10–4500 cells ml^?1). The other groups represented < 10%. The temperature and salinity positively correlated with the total abundance of phytoflagellates, chlorophytes, haptophytes, bolidophytes and pelagophytes. Cryptophytes, pedinellids and pavlovophytes were negatively associated with the nutrient concentrations. The community composition of phytoflagellates changed along the transect, which could have implications on food web dynamics and biogeochemical cycles between the open ocean environment and Kongsfjorden investigated here.