Identification of an alveolar type I epithelial cell-specific DNA nuclear import sequence for gene delivery.

The ability to restrict gene delivery and expression to particular cell types is of paramount importance for many types of gene therapy, especially in the lung. The alveolar epithelial type I (ATI) cell, in particular, is an attractive cell type to target, as it comprises 95% of the internal surface area of the lung. We demonstrate, through microinjection of fluorescently labeled plasmids, that a DNA sequence within the rat T1α promoter was able to mediate ATI cell-specific plasmid DNA nuclear import due to the binding of ATI-enriched transcription factors. Promoter deletion analysis and site-directed mutagenesis of specific transcription-factor-binding sites within the +101 to -200 bp region of the T1α promoter identified HNF3 and TTF-1 as critical transcription factors for import. To test for nuclear import in vivo, plasmids expressing GFP from the CMV promoter were delivered into the lungs of mice by electroporation and evaluated immunohistochemically 48 h later. Plasmids carrying the 1.3 kbp T1α sequence resulted in GFP expression almost exclusively in ATI cells. This represents a new and highly efficient way to target a specific lung epithelial cell type both in vitro and in vivo based on the restriction of DNA nuclear import.

ß1-Na(+),K(+)-ATPase gene therapy upregulates tight junctions to rescue lipopolysaccharide-induced acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with diverse disorders and characterized by disruption of the alveolar-capillary barrier, leakage of edema fluid into the lung, and substantial inflammation leading to acute respiratory failure. Gene therapy is a potentially powerful approach to treat ALI/ARDS through repair of alveolar epithelial function. Herein, we show that delivery of a plasmid expressing ß1-subunit of the Na(+),K(+)-ATPase (ß1-Na(+),K(+)-ATPase) alone or in combination with epithelial sodium channel (ENaC) α1-subunit using electroporation not only protected from subsequent lipopolysaccharide (LPS)-mediated lung injury, but also treated injured lungs. However, transfer of α1-subunit of ENaC (α1-ENaC) alone only provided protection benefit rather than treatment benefit although alveolar fluid clearance had been remarkably enhanced. Gene transfer of ß1-Na(+),K(+)-ATPase, but not α1-ENaC, not only enhanced expression of tight junction protein zona occludins-1 (ZO-1) and occludin both in cultured cells and in mouse lungs, but also reduced pre-existing increase of lung permeability in vivo. These results demonstrate that gene transfer of ß1-Na(+),K(+)-ATPase upregulates tight junction formation and therefore treats lungs with existing injury, whereas delivery of α1-ENaC only maintains pre-existing tight junction but not for generation. This indicates that the restoration of epithelial/endothelial barrier function may provide better treatment of ALI/ARDS.

Highly acetylated tubulin permits enhanced interactions with and trafficking of plasmids along microtubules.

Microtubule-based transport is required for plasmid translocation to the nucleus during transfections, and having stable structures could enhance this movement. In previous studies, in which the cytoskeleton was disrupted, we found that populations of microtubules remain that are stable and highly acetylated. By increasing the levels of acetylated tubulin through inhibition of the tubulin deacetylase HDAC6, we observe more rapid plasmid nuclear localization of transfected plasmids and greater levels of gene transfer. In this study, we sought to understand plasmid movement in cells with enhanced tubulin acetylation. Using variations of a microtubule spin-down assay, we found that plasmids bound to hyper-acetylated microtubules to a greater degree than they did to unmodified microtubules. To determine whether microtubule acetylation also affects cytoplasmic trafficking, plasmid movement was evaluated in real time by particle tracking in cells with varying levels of acetylated microtubules. We found that plasmids display greater net rates of movement, spend more time in productive motion and display longer runs of continuous motion in cells with highly acetylated microtubules compared with those with fewer modifications. These results all suggest that plasmid movement is enhanced along highly acetylated microtubules, reducing the time spent in the cytoplasm before nuclear import. Taken together, these findings provide a foundation for determining how modulation of microtubule acetylation can be used as a means to increase intracellular trafficking of plasmids and enhance gene therapy.

Impacts of spinosad and λ-cyhalothrin on spider communities in cabbage fields in south Texas.

Spiders are a principal arthropod group that preys on numerous pests of vegetables and other crops. In this study, we determined the effects of the two most commonly used insecticides, spinosad and λ-cyhalothrin, on diversity of spiders on cabbage in south Texas. In two seasons (fall 2008 and spring 2009), we collected a total of 588 spiders belonging to 53 species in 11 families from spinosad and λ-cyhalothrin-treated cabbages and the untreated control plants. A great majority of spiders were collected from the pitfall traps (554) where only a few (34) were collected from the blower/vacuum sampling. In the insecticide-treated plots, there were significantly fewer spider individuals, species and families than in untreated fields. Spinosad had significantly less effect on spiders in total individuals, number of species and families than λ-cyhalothrin. The effects of the two insecticides were further demonstrated by the Shannon-Weiner index (H') and the hierarchical richness index (HRI). Spider diversity in the spinosad-treated plots were not significantly different from that in the untreated fields but were greater than those in λ-cyhalothrin-treated plots in both seasons when measured by H' values. In contrast, the H' values of spider's diversity in the λ-cyhalothrin-treated plots were significantly lower than spinosad-treated and untreated plots. High values of HRI for spider richness in the spinosad-treated plots suggested that spinosad had less effect on spiders than λ-cyhalothrin. We concluded that spinosad was more compatible with spiders on cabbage compared to λ-cyhalothrin and that this information should be used when developing insecticide resistance management strategies.

Transcription factor plasmid binding modulates microtubule interactions and intracellular trafficking during gene transfer.

For non-viral gene delivery to be successful, plasmids must move through the cytoplasm to the nucleus in order to be transcribed. While the cytoskeletal meshwork acts as a barrier to plasmid DNA movement in the cytoplasm, the microtubule network is required for directed plasmid trafficking to the nucleus. We have shown previously that plasmid-microtubule interactions require cytoplasmic adapter proteins such as molecular motors, transcription factors (TFs) and importins. However, not all plasmid sequences support these interactions to allow movement to the nucleus. We now demonstrate that microtubule-DNA interactions can show sequence specificity with promoters containing binding sites for cyclic AMP response-element binding protein (CREB), including the cytomegalovirus immediate early promoter (CMV(iep)). Plasmids containing CREB-binding sites showed stringent interactions in an in vitro microtubule-binding assay. Using microinjection and real-time particle tracking, we show that the inclusion of TF binding sites within plasmids permits cytoplasmic trafficking of plasmids during gene transfer. We found that CREB-binding sites are bound by CREB in the cytoplasm during transfection, and allow for enhanced rates of movement and subsequent nuclear accumulation. Moreover, small interfering RNA knockdown of CREB prevented this enhanced trafficking. Therefore, TF binding sites within plasmids are necessary for interactions with microtubules and enhance movement to the nucleus.

Progress and prospects: nuclear import of nonviral vectors.

UNLABELLED: The nuclear envelope represents a key barrier to successful nonviral transfection and gene therapy both in vitro and in vivo. Although the main purpose of the nuclear envelope is to partition the cell to maintain cytoplasmic components in the cytoplasm and nuclear components, most notably genomic DNA, in the nucleus, this function poses a problem for transfections in which exogenous DNA is delivered into the cytoplasm. After delivery to the cytoplasm, nucleic acids rapidly become complexed with cellular proteins that mediate interactions with the cellular machinery for trafficking. Thus, it is these proteins that, in essence, control the nuclear import of DNA, and we must also understand their activities in cells. In this review, we will discuss the principles of nuclear import of proteins and DNA-protein complexes, as well as the various approaches that investigators have used to improve nuclear targeting of plasmids. These approaches include complexation of plasmids with peptides, native and engineered proteins, ligands and polymers, as well as the inclusion of transcription factor-binding sites for general and cell-specific delivery. KEYWORDS: nonviral gene transfermid R:plasmidmid R:nuclear pore complexmid R:importinmid R:nuclear localization signalmid R:karyopherin.

Electroporation- and mechanical ventilation-mediated gene transfer to the lung.

Our laboratory has previously demonstrated that cytoplasmic trafficking and subsequent nuclear entry of nonviral plasmid DNA can be significantly enhanced through the application of cyclic stretch after transfection in vitro. In this study, we show that cyclic stretching of the murine lung using ventilation immediately after endotracheal administration and transthoracic electroporation of plasmid DNA increases exogenous gene expression up to fourfold in mice that were not ventilated after plasmid administration and transfection by electroporation in vivo. This increase is both time and sequence specific (that is, the ventilation must occur immediately after the transfection event). The ventilation-enhanced gene transfer is also amplitude dependent, confirming similar studies completed in vitro, and is mediated, at least in part, through the cytoplasmic tubulin deacetylase, HDAC6. Using immunohistochemistry, we show that this increase in expression is due to an increase in the number of cells expressing the exogenous protein rather than an increase in the amount of protein produced per cell. These studies show the potential mechanical stimulation has in vivo in significantly increasing nonviral DNA gene expression, and may ultimately pave the way for more successful clinical trials using this type of therapy in the future.

The SP-C promoter facilitates alveolar type II epithelial cell-specific plasmid nuclear import and gene expression.

Although nonviral gene therapy has great potential for use in the lung, the relative lack of cell-specific targeting has limited its applications. We have developed a new approach for cell-specific targeting based on selective nuclear import of plasmids in nondividing cells. Using a microinjection and in situ hybridization approach, we tested several potential DNA sequences for the ability to mediate plasmid nuclear import in alveolar type II epithelial (ATII) cells. Of these, only a sequence within the human surfactant protein C (SP-C) promoter was able to mediate nuclear localization of plasmid DNA specifically in ATII cells but not in other cell types. We have mapped the minimal import sequence to the proximal 318 nucleotides of the promoter, and demonstrate that binding sites for nuclear factor I, thyroid transcriptional factor 1, and GATA-binding protein 6 and the proteins themselves are required for import activity. Using intratracheal delivery of DNA followed by electroporation, we demonstrate that the SP-C promoter sequence will enhance gene expression specifically in ATII cells in mouse lung. This represents a new activity for the SP-C promoter, and thus ATII cell-specific nuclear import of DNA may prove to be a safe and effective method for targeted and enhanced gene expression in ATII cells.

Pol I transcription and pre-rRNA processing are coordinated in a transcription-dependent manner in mammalian cells.

Pre-rRNA synthesis and processing are key steps in ribosome biogenesis. Although recent evidence in yeast suggests that these two processes are coupled, the nature of their association is unclear. In this report, we analyze the coordination between rDNA transcription and pre-rRNA processing in mammalian cells. We found that pol I transcription factor UBF interacts with pre-rRNA processing factors as analyzed by immunoprecipitations, and the association depends on active rRNA synthesis. In addition, injections of plasmids containing the human rDNA promoter and varying lengths of 18S rDNA into HeLa nuclei show that pol I transcription machinery can be recruited to rDNA promoters regardless of the product that is transcribed, whereas subgroups of pre-rRNA processing factors are recruited to plasmids only when specific pre-rRNA fragments are produced. Our observations suggest a model for sequential recruitment of pol I transcription factors and pre-rRNA processing factors to elongating pre-rRNA on an as-needed basis rather than corecruitment to sites of active transcription.

Mapping of host-parasite-microbiome interactions reveals metabolic determinants of tropism and tolerance in Chagas disease.

Chagas disease (CD) is a parasitic disease caused by Trypanosoma cruzi protozoa, presenting with cardiomyopathy, megaesophagus, and/or megacolon. To determine the mechanisms of gastrointestinal (GI) CD tissue tropism, we systematically characterized the spatial localization of infection-induced metabolic and microbiome alterations, in a mouse model of CD. Notably, the impact of the transition between acute and persistent infection differed between tissue sites, with sustained large-scale effects of infection in the esophagus and large intestine, providing a potential mechanism for the tropism of CD within the GI tract. Infection affected acylcarnitine metabolism; carnitine supplementation prevented acute-stage CD mortality without affecting parasite burden by mitigating infection-induced metabolic disturbances and reducing cardiac strain. Overall, results identified a previously-unknown mechanism of disease tolerance in CD, with potential for new therapeutic regimen development. More broadly, results highlight the potential of spatially resolved metabolomics to provide insight into disease pathogenesis and infectious disease drug development.

Immunization of Baboons with Schistosoma mansoni Cercariae attenuated by gamma irradiation.

Studies on the efficacy of a vaccine against schistosomiasis in young baboons (Papio anubis) disclosed that immunization with Schistosoma mansoni cercariae attenuated by gamma irradiation induced significant protection against subsequent infection with normal, viable S. mansoni cercariae. Such immunization resulted in reduced worm burdens (70 percent) and egg excretion rates (82 percent). These results support immunization as a potential method for schistosomiasis control.

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences.

Two shortcomings of nonviral gene therapy are a lack of tissue-specific targeting of vectors and low levels of gene transfer. Our laboratory has begun to address these limitations by designing plasmids that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing expression in a controlled manner. We have shown that a 176 bp portion of the smooth muscle gamma-actin (SMGA) promoter can mediate plasmid nuclear import specifically in smooth muscle cells (SMCs). Here, we demonstrate that the binding sites for serum response factor (SRF) and NKX3-1/3-2 within this DNA nuclear targeting sequence (DTS) are required for plasmid nuclear import. Knockdown of these factors with siRNA abrogates plasmid nuclear import, indicating that they are necessary cofactors. In addition, coinjection of recombinant SRF and Nkx3.2 with the vector in TC7 epithelial cells rescues import. Finally, we show that the SRF nuclear localization sequence (NLS) is required for vector nuclear import. We propose that SRF and NKX3-1/3-2 bind the SMGA DTS in the cytoplasm, thus coating the plasmid with NLSs that mediate translocation across the nuclear pore complex. This discovery could aid in the development of more efficient nonviral vectors for gene transfer to SMCs.

Electrical Impedance Spectroscopy Study of Biological Tissues.

The objective of this study was to investigate the electrical impedance properties of rat lung and other tissues ex vivo using Electrical Impedance Spectroscopy. Rat lungs (both electroporated and naïve (untreated)), and mesenteric vessels (naïve) were harvested from male Sprague-Dawley rats; their electrical impedance were measured using a Solartron 1290 impedance analyzer. Mouse lung and heart samples (naïve) were also studied. The resistance (Real Z, ohm) and the reactance (Im Z, negative ohm)) magnitudes and hence the Cole-Cole (Real Z versus Im Z) plots are different for the electroporated lung and the naive lung. The results confirm the close relationship between the structure and the functional characteristic. These also vary for the different biological tissues studied. The impedance values were higher at low frequencies compared to those at high frequencies. This study is of practical interest for biological applications of electrical pulses, such as electroporation, whose efficacy depends on cell type and its electrical impedance characteristics.

Peptide nucleic acids: versatile tools for gene therapy strategies.

Peptide nucleic acids, or PNAs, are oligonucleotide analogs in which the phosphodiester backbone is replaced with a polyamide structure. First synthesized less than 10 years ago, they have received great attention due to their several favorable properties, including resistance to nuclease and protease digestion, stability in serum and cell extracts, and their high affinity for RNA and single and double-stranded DNA targets. Although initially designed and demonstrated to function as antisense and antigene reagents that inhibit both transcription and translation by steric hindrance, more recent applications have included gene activation by synthetic promoter formation and mutagenesis of chromosomal targets. Most notably for gene delivery, they have been used to specifically label plasmids and act as adapters to link synthetic peptides or ligands to the DNA. Thus, their great potential lies in the ability to attach specific targeting peptides to plasmids to circumvent such barriers to gene transfer as cell-targeting or nuclear localization, thereby increasing the efficacy of gene therapy.

Serine/threonine protein phosphatase 5 (PP5) participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling.

BACKGROUND: In most cells glucocorticoid receptors (GR) reside predominantly in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GR-complex regulates the transcription of GR-responsive genes. Serine/threonine protein phosphatase type 5 (PP5) associates with the GR-heat-shock protein-90 complex, and the suppression of PP5 expression with ISIS 15534 stimulates the activity of GR-responsive reporter plasmids, without affecting the binding of hormone to the GR. RESULTS: To further characterize the mechanism by which PP5 affects GR-induced gene expression, we employed immunofluorescence microscopy to track the movement of a GR-green fluorescent fusion protein (GR-GFP) that retained hormone binding, nuclear translocation activity and specific DNA binding activity, but is incapable of transactivation. In the absence of glucocorticoids, GR-GFP localized mainly in the cytoplasm. Treatment with dexamethasone results in the efficient translocation of GR-GFPs into the nucleus. The nuclear accumulation of GR-GFP, without the addition of glucocorticoids, was also observed when the expression of PP5 was suppressed by treatment with ISIS 15534. In contrast, ISIS 15534 treatment had no apparent effect on calcium induced nuclear translocation of NFAT-GFP. CONCLUSION: These studies suggest that PP5 participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling, and that the GR-induced transcriptional activity observed when the expression of PP5 is suppressed by treatment with ISIS 15534 results from the nuclear accumulation of GR in a form that is capable of binding DNA yet still requires agonist to elicit maximal transcriptional activation.

Improved assay for monocyte chemotaxis using frozen stored responder cells.

Frozen stored human mononuclear cells were compared with non-frozen cells as responder cells in the monocyte chemotaxis assay. Frozen stored cells were found to inhibit lower background migration, resulting in greater experimental-to-control ratios. The use of frozen cells also greatly improved the speed, standardization of conditions and day-to-day reproducibility of the assay procedure. Frozen cells were at least as sensitive as fresh cells for detecting low chemotactic factor concentrations and were superior for detecting differences in concentration.

Identification of Schistosoma mansoni antigens recognized by spleen cells of C57B1/6 mice immunized with ultraviolet-irradiated cercariae.

Spleen cells of C57B1/6 mice immunized twice with Schistosoma mansoni cercariae attenuated by ultraviolet irradiation proliferated and produced interleukin-(II)-2 and/or II-4 in response to both soluble schistosomular and adult worm antigens of 72-68, 60-62, 50, 45, 29.5 and 28 kDa. All of these bands, except the 45 kDa, were also recognized by serum antibodies in Western blotting.

Identification and characterization of Schistosoma mansoni antigens recognized by T and B lymphocytes of humans with early active intestinal and/or urinary schistosomiasis.

Schistosome antigens selected as vaccine candidates should induce in the majority of humans T and B cell-mediated immunity that results in protection against infection. As a first step towards the identification of such antigens, we attempted to define and characterize the soluble adult Schistosoma mansoni worm antigen (SAWA) bands that are recognized by serum antibodies and/or peripheral blood mononuclear cells of Egyptian children with early active S. mansoni and/or S. haematobium infection. Considerable inter-subject variation was observed in the SAWA bands recognized by antibodies and T lymphocytes, as demonstrated by Western blotting and T cell Western assays, respectively. The humoral response rate for the separated SAWA bands varied between 0% and 88% of infected subjects. The bands of 153, 144, 38 and 32 kDa reacted with the sera of 60 to 88% of infected subjects but not with the sera of uninfected controls. The bands of 144, 38, 32 and 18 kDa elicited proliferative responses in the lymphocytes of 42-63% of infected subjects. It was thus concluded that the SAWA bands of 144, 38 and 32 kDa are likely to carry T and B cell epitopes that could stimulate immune responses in a majority of individuals. The selected bands (144, 38 and 32 kDa) were found to include glycoproteins containing D-mannopyranosyl or glycosyl residues, and respectively 62.5, 46 and 55% amino acids by weight. The amino acid molar ratios of these bands were completely different.

Inhaled nitric oxide is not a negative inotropic agent in a porcine model of pulmonary hypertension.

BACKGROUND: Reports of pulmonary edema complicating inhaled nitric oxide therapy in patients with chronic heart failure and pulmonary hypertension have raised the concern that inhaled nitric oxide may have negative inotropic effects. METHODS AND RESULTS: We investigated the effect of multiple doses of inhaled nitric oxide (20, 40 and 80 ppm) on left ventricular contractile state in 10 open-chest pigs. Pressure-volume loops were generated during transient preload reduction to determine the end-systolic pressure-volume relationship and the stroke work-end-diastolic volume relation. Inhaled nitric oxide had no effect on systemic vascular resistance, cardiac output, end-systolic pressure volume relationship or stroke work-end-diastolic volume relation under normal conditions. After induction of pulmonary hypertension (intravenous thromboxane A2 analog), inhalation of nitric oxide (80 ppm) resulted in a reduction in pulmonary vascular resistance (mean +/- standard error of the mean) from 10.4 +/- 3 to 6.5 +/- 2 Wood units (p < 0.001) and in pulmonary artery pressure from 44 +/- 4 to 33 +/- 4 mm Hg (p < 0.05). Left ventricular end-diastolic volume rose from 53 +/- 9 ml to 57 +/- 10 ml (p = 0.02). No statistically significant change in cardiac output or systemic vascular resistance was observed. Inhaled nitric oxide had no effect on end-systolic pressure-volume relationship or stroke work-end-diastolic volume relation. CONCLUSIONS: In a porcine model of pulmonary hypertension, inhaled nitric oxide does not impair left ventricular contractile function. Therefore the cause of pulmonary edema observed in some patients receiving inhaled nitric oxide is not due to a negative inotropic action of this therapy.

Reversal of iatrogenic myocardial edema and related abnormalities of diastolic properties in the pig left ventricle.

OBJECTIVE: This study examines the resolution of iatrogenic edema and related changes in systolic and diastolic properties in the intact pig left ventricle. METHODS: The coronary arteries were perfused for 50 to 60 seconds with diluted blood (hematocrit value 10% +/- 1%, edema group, n = 5) or whole blood (hematocrit value 28% +/- 1%, control group, n = 6) infused into the aortic root during aortic crossclamping in conditioned, anesthetized pigs. After whole blood reperfusion, preload reduction by vena caval occlusion was used to define systolic and diastolic properties at 15-minute intervals. Left ventricular pressure and conductance, aortic flow, and two-dimensional echocardiography were recorded. RESULTS: Left ventricular mass (wall volume) in the edema group increased significantly compared with that in control pigs after crossclamp removal. Mass returned to preperfusion levels after 45 minutes. The ventricular stiffness constant (beta) increased significantly in the edema group versus the control group, returning to baseline by 30 minutes. The diastolic relaxation constant (tau) and base constant (alpha) did not differ between groups. There was no significant change in contractility. CONCLUSION: Increases in left ventricular mass and diastolic stiffness induced by coronary perfusion with hemodiluted blood resolve after 45 minutes of whole blood perfusion in pigs. This study defines physiologic effects of edema in the normal heart while eliminating most common confounding experimental errors.