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Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is linked to heterozygous mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development. There are no effective treatments for ACDMPV other than lung transplant, and new pharmacological agents activating FOXF1 signaling are urgently needed.Identify small molecule compounds that stimulate FOXF1 signaling.We used mass spectrometry, immunoprecipitation, and the in vitro ubiquitination assay to identify TanFe, a small molecule compound from the nitrile group, which stabilizes the FOXF1 protein in the cell. The efficacy of TanFe was tested in mouse models of ACDMPV and Acute Lung Injury and in human vascular organoids derived from iPSCs of ACDMPV patient.We identified HECTD1 as an E3 ubiquitin ligase involved in ubiquitination and degradation of the FOXF1 protein. The TanFe compound disrupted FOXF1-HECTD1 protein-protein interactions and decreased ubiquitination of the FOXF1 protein in pulmonary endothelial cells in vitro. TanFe increased protein levels of FOXF1 and its target genes Flk1, Flt1 and Cdh5 in LPS-injured mouse lungs, decreasing endothelial permeability and inhibiting lung inflammation. Treatment of pregnant mice with TanFe increased FOXF1 protein levels in lungs of Foxf1+/- embryos, stimulated neonatal lung angiogenesis, and completely prevented the mortality of Foxf1+/- mice after birth. TanFe increased angiogenesis in human vascular organoids derived from iPSCs of ACDMPV patient with FOXF1 deletion.TanFe is a novel activator of FOXF1, providing a new therapeutic candidate for treatment of ACDMPV and other neonatal pulmonary vascular diseases.

CFTR modulators, especially (elexacaftor/tezacaftor/ivacaftor), have positively impacted the CF population and quickly decreased LTx numbers. However, no study has investigated if this reduction is universal across all races/ethnicities.Using the UNOS Registry, we explored the frequency/proportions of LTx in WNH and NW (Black, non-Hispanic/Hispanic-Latino/Asian-non Hispanic/American Indian-Alaskan Native-non-Hispanic/Native Hawaiian/Other Pacific Islander-non-Hispanic/Multiracial) in children and adults with CF in the US.Between 1990 and 2019, the annual mean (±SD) number of LTxs for children with CF was 23.2 (±7.7) compared to 5 in 2020 (p .001) and in 2021 (p .001). In adults from 1990 to 2019, the mean (±SD) number of LTxs performed was 144.9 (±73.5), which was significantly higher than 2020 (n = 73; p .001) and 2021 (n = 45; p .001). Comparing 1990-2019 to post-2019, the proportion of LTxs performed in both children and adults with CF has decreased from 50.5% (696/1378) to 16.4% (9/55) and from 12.1% (4773/39542) to 2.4% (118/5004), respectively. In WNH pediatric patients, the difference in the percentage of all LTx made up by CF patients between the two eras was 41.2% compared to NW patients where the difference was 11%. Similarly in adults, the difference between the two eras was 10.4% in WNH and 2.4% in NW patients.The recent reduction in LTx for the CF population has had less impact on the NW population in the US, so the continuation of optimal referrals for this group is needed.

BK polyomavirus (BKPyV) infection can lead to nephropathy and hemorrhagic cystitis (HC). We evaluated BKPyV genotypes in two individuals after hematopoietic cell transplant (HCT). The first case developed HC and was infected with genotype Ib-2, while the second did not develop HC and was infected with genotype Ia.

Background Circulating biomarkers reflective of lung disease activity and severity have the potential to improve patient care and accelerate drug development in CF. The objective of this study was to leverage banked specimens to test the hypothesis that blood-based biomarkers discriminate CF children segregated by lung disease severity. Methods Banked serum samples were selected from children who were categorized into two extremes of phenotype associated with lung function (‘mild’ or ‘severe’) based on CF-specific data and were matched on age, gender, CFTR genotype, and P. aeruginosa infection status. Targeted inflammatory proteins, lipids, and discovery metabolite profiles were measured in these serum samples. Results The severe cohort, characterized by a lower CF-specific FEV1 percentile, had significantly higher circulating concentrations of high sensitivity C-reactive protein, serum amyloid A, granulocyte colony stimulating factor, and calprotectin compared to the mild cohort. The mild cohort tended to have higher serum linoleic acid concentrations. The metabolite arabitol was lower in the severe cohort while other CF relevant metabolic pathways showed non-significant differences after adjusting for multiple comparisons. A sensitivity analysis to correct for biased estimates that may result from selecting subjects using an extremes of phenotype approach confirmed the protein biomarker findings. Conclusions Circulating inflammatory proteins differ in CF children segregated by lung function. These findings serve to demonstrate the value of maintaining centralized, high quality patient derived samples for future research, with linkage to clinical information to answer testable hypotheses in biomarker development.

Purpose To provide a review of the status of biomarkers in cystic fibrosis drug development, including regulatory definitions and considerations, a summary of biomarkers in current use with supportive data, current gaps, and future needs. Methods Biomarkers are considered across several areas of CF drug development, including cystic fibrosis transmembrane conductance regulator modulation, infection, and inflammation. Results Sweat chloride, nasal potential difference, and intestinal current measurements have been standardized and examined in the context of multicenter trials to quantify CFTR function. Detection and quantification of pathogenic bacteria in CF respiratory cultures (e.g.: Pseudomonas aeruginosa ) are commonly used in early phase antimicrobial clinical trials, and to monitor safety of therapeutic interventions. Sputum (e.g.: neutrophil elastase, myeloperoxidase, calprotectin) and blood biomarkers (e.g.: C reactive protein, calprotectin, serum amyloid A) have had variable success in detecting response to inflammatory treatments. Conclusions Biomarkers are used throughout the drug development process in CF, and many have been used in early phase clinical trials to provide proof of concept, detect drug bioactivity, and inform dosing for later-phase studies. Advances in the precision of current biomarkers, and the identification of new biomarkers with ‘omics-based technologies, are needed to accelerate CF drug development.

Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX-809 has been reported to facilitate the folding and trafficking of F508del-CFTR and augment its channel function. The mechanism of action of VX-809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX-809: does it bind CFTR directly in order to exert its action? We synthesized two VX-809 derivatives, ALK-809 and SUL-809, that possess an alkyne group and retain the rescue capacity of VX-809. By using Cu(I) -catalyzed click chemistry, we provide evidence that the VX-809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.

Multidrug resistance protein 4 (MRP4), a member of the ATP binding cassette transporter family, functions as a plasma membrane exporter of cyclic nucleotides. Recently, we demonstrated that fibroblasts lacking the Mrp4 gene migrate faster and contain higher cyclic-nucleotide levels. Here, we show that cAMP accumulation and protein kinase A (PKA) activity are higher and polarized in Mrp4(-/-) fibroblasts, versus Mrp4(+/+) cells. MRP4-containing macromolecular complexes isolated from these fibroblasts contained several proteins, including actin, which play important roles in cell migration. We found that actin interacts with MRP4, predominantly at the plasma membrane, and an intact actin cytoskeleton is required to restrict MRP4 to specific microdomains of the plasma membrane. Our data further indicated that the enhanced accumulation of cAMP in Mrp4(-/-) fibroblasts facilitates cortical actin polymerization in a PKA-dependent manner at the leading edge, which in turn increases the overall rate of cell migration to accelerate the process of wound healing. Disruption of actin polymerization or inhibition of PKA activity abolished the effect of MRP4 on cell migration. Together, our findings suggest a novel cAMP-dependent mechanism for MRP4-mediated regulation of fibroblast migration whereby PKA and actin play critical roles as downstream effectors.

Mtb regulates many aspects of the host immune response, including CD4+ T lymphocyte responses that are essential for protective immunity to Mtb, and Mtb effects on the immune system are paradoxical, having the capacity to inhibit (immune evasion) and to activate (adjuvant effect) immune cells. Mtb regulates CD4+ T cells indirectly (e.g., by manipulation of APC function) and directly, via integrins and TLRs expressed on T cells. We now report that previously uncharacterized Mtb protein Rv2468c/MT2543 can directly regulate human CD4+ T cell activation by delivering costimulatory signals. When combined with TCR stimulation (e.g., anti-CD3), Rv2468c functioned as a direct costimulator for CD4+ T cells, inducing IFN-γ secretion and T cell proliferation. Studies with blocking antibodies and soluble RGD motifs demonstrated that Rv2468c engaged integrin VLA-5 (α5β1) on CD4+ T cells through its FN-like RGD motif. Costimulation by Rv2468c induced phosphorylation of FAKs and Pyk2. These results reveal that by expressing molecules that mimic host protein motifs, Mtb can directly engage receptors on CD4+ T cells and regulate their function. Rv2468c-induced costimulation of CD4+ T cells could have implications for TB immune pathogenesis and Mtb adjuvant effect.

DNA nanoparticles (DNPs) are nonviral gene transfer vectors with excellent in vivo potential. Previously, we reported that cell surface nucleolin directly binds DNPs, and functions as an important receptor for DNPs. However, the fate of the nucleolin–DNP complex following cellular uptake remains elusive. In this study, we examined the role of lipid rafts in the uptake of DNPs, and found that both nucleolin and DNPs are recovered from the low-density raft fractions of the sucrose gradient. Furthermore, nucleolin colocalizes with, and coimmunoprecipitates with a raft protein, flotillin. Disruption of lipid rafts by depleting membrane cholesterol significantly inhibited DNP transfection, while inhibition of other endocytic pathways had little effect. Following the uptake, the nuclear import of the DNPs required microtubules but not F-actin. By coimmunoprecipitation in conjunction with tandem mass spectrometry, we identified glucocorticoid receptor (GCR) as a nucleolin-associated protein, and confirmed this result by western blot. Cortisone or dexamethasone increased nucleolin's association with GCR, and transfection by DNPs. Finally, we detected the expression of nucleolin on the surface of airway epithelia in vivo. Taken together, our findings shed light on important determinants of DNP trafficking in cells and support the notion that nucleolin is a good target for nonviral gene delivery.

DNA nanoparticles (DNPs) formed by compacting DNA with polyethyleneglycolylated poly-L-lysine are a nonviral vector shown to be safe and efficacious in animals and humans. To extend our capabilities of assessing the efficacy and duration of expression achieved by DNPs, we tested the utility of bioluminescent imaging (BLI) of transgene expression in wildtype and cystic fibrosis (CF) mouse models. We tested the effect of route of administration, mouse coat color, anesthesia, dose, and promoter sequence on the level and duration of expression. Furthermore, we investigated the correlation between imaging and direct analysis of luciferase expression in lung homogenates. We found that intratracheal instillation, and the use of deep and prolonged anesthesia with avertin produced significantly higher expression compared with intranasal administration, and the use of lighter anesthesia with isoflurane. Although similar expression was observed for both dark and light coat animals, imaging signal intensity was attenuated in mice with dark fur. Furthermore, good correlation between imaging and direct homogenate analysis was observed for single dose (r = 0.96), and dose response studies in wildtype (r = 0.82) and CF mice (r = 0.87). Finally, we used imaging to track gene expression over a 56-day time course. We found that the human ubiquitin B promoter gives stable transgene expression up to 49 days following nanoparticle administration, while expression with the cytomegalovirus promoter diminished after 2 days and returned to background levels by day 14. Taken together, our results demonstrate that BLI is an effective and useful modality for measuring gene expression conferred by DNPs in the lung. Microsc. Res. Tech. 73:918–928, 2010. © 2010 Wiley-Liss, Inc.

This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line–derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1–3 weeks after injection.

New acid-degradable cationic nanoparticles were synthesized using a monomer-to-polymer approach, which enabled highly flexible nanoparticle fabrication to obtain controlled properties such as size and conjugation with additional functionalities. The nanoparticles were designed to cause swelling and osmotic destabilization of the endosome, while cationic branches holding anionic DNA are cleaved from the polymeric backbone of the nanoparticles and make plasmid DNA accessible for efficient gene expression. Efficient release of plasmid DNA upon hydrolysis of the nanoparticles at the endosomal pH 5.0 and transportation of the released DNA to the nucleus of a cell were shown. In vitro studies showed significantly higher transfection efficiency by the degradable nanoparticles than polyethylenimine (PEI) polyplexes at very low concentrations (i.e., ng/mL). Size-dependent selective transfection of phagocytic cells (e.g., RAW 309 macrophages) and non-phagocytic cells (e.g., NIH 3T3 fibroblasts) was also achieved by using nanoparticles of two different sizes (240 nm and 680 nm in diameter), which implies feasibility of tunable gene therapy and DNA vaccination using the nanoparticle system. Preliminary pulmonary transfection of mice using the degradable nanoparticles demonstrated a remarkably higher expression of firefly luciferase at 70% lower concentration than using naked DNA alone. Implications and further improvement of the nanoparticles to be used in gene therapy are also discussed.

Evidence suggests that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, may reduce the risk of Alzheimer’s disease. Statin action in patients with Alzheimer’s disease, as in those with heart disease, is likely to be at least partly independent of the effects of statins on cholesterol. Statins can alter cellular signaling and protein trafficking through inhibition of isoprenylation of Rho, Cdc42, and Rab family GTPases. The effects of statins on protein isoprenylation in vivo, particularly in the central nervous system, are poorly studied. We utilized two-dimensional gel electrophoresis approaches to directly monitor the levels of isoprenylated and non-isoprenylated forms of Rho, and Rab family GTPases. We report that simvastatin significantly inhibit RhoA and Rab4, and Rab6 isoprenylation at doses as low as 50 nM in vitro. We also provide the first in vivo evidence that statins inhibit the isoprenylation of RhoA in the brains of rats and RhoA, Cdc42, and H-Ras in the brains of mice treated with clinically relevant doses of simvastatin.

Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.

Conventional therapy for cystic fibrosis has extended the median survival age, but the disease is still fatal. Gene therapy can correct the primary and secondary defects associated with cystic fibrosis, but limited extent and duration of the corrections as well as concerns about the safety of some current delivery systems have prevented gene therapy from being curative. For viral vectors, the main challenges are access to target cells and host immunity, which prevents efficient re-administration. Masking viral particles from the immune system, the use of alternative serotypes, or retargeting have been employed to address these issues. Non-viral vectors have dramatically improved over the past five years but improvements in efficacy are needed. In lung, naked DNA has been inefficient and lipid-based vectors have only achieved efficient gene transfer at doses that elicit limiting inflammatory responses. Molecular conjugates or polymer-based delivery overcomes some limitations, with good ability to transfect non-dividing cells. Improvements of viral and non-viral vectors continue to advance the construction of stable, safe and efficacious vectors that can be re-administered.

Objective Despite the growing body of evidence showing adverse health effects from inhalation exposure to the trichothecene-producing mold Stachybotrys chartarum, controversy remains. Currently, there are no reliable assays suitable for clinical diagnosis of exposure. We hypothesized that satratoxin G (SG)–albumin adducts may serve as biomarkers of exposure to this fungus. Design We studied the formation of adducts of SG with serum albumin in vitro using Western blots and mass spectrometry (MS) and searched for similar adducts formed in vivo using human and animal serum. Results Samples of purified human serum albumin that had been incubated with increasing concentrations of SG showed concentration-dependent albumin bands in Western blots developed with anti-SG antibodies. MS analysis found that as many as 10 toxin molecules can be bound in vitro to one albumin molecule. The sequencing of albumin-adduct tryptic peptides and the analysis of pronase/aminopeptidase digests demonstrated that lysyl, cysteinyl, and histidyl residues are involved in the formation of these adducts. Serum samples from three patients with documented exposure to S. chartarum similarly revealed lysine–, cysteine–, and histidine–SG adducts after exhaustive digestion, affinity column enrichment, and MS analysis. These adducts were also found in the sera from rats exposed to the spores of S. chartarum in contrast to control human subjects and control animals. Conclusions These data document the occurrence of SG–albumin adducts in both in vitro experiments and in vivo human and animal exposures to S. chartarum. Relevance to clinical practice SG–amino acid adducts may serve as reliable dosimeter biomarkers for detection of exposure to S. chartarum.

Gene transfer complexes containing poly-L-lysine (poly-K) and DNA with ligands directed at the serpin enzyme complex receptor (sec-R) deliver reporter genes to receptor-bearing cells in vivo. Expression lasts for about 30 days, when complexes containing long-chain poly-K are used. Extending the duration of expression would be desirable if correction of genetic defects is the goal. To test whether the mechanism by which expression is extinguished was due to an immune response to the transgene, or the loss of the transgene, we conducted two experiments. In the first, we injected sec-R-targeted lacZ complexes intravenously (i.v.) into mice genetically engineered to express this gene briefly during development. These mice, who should recognize the protein as 'self', also extinguished lacZ expression after 30 days. In a second experiment, we injected immunodeficient animals with sec-R-targeted human factor IX complexes. A similar temporal pattern of expression was observed in Rag-1 -/- mice, in whom expression also extinguished by 40 days. Moreover, factor IX plasmid DNA was detected in the lung and spleen 50 days after injection of complexes, suggesting that not all cells which had taken up the transgene had been destroyed. Thus, the host's immune response to the transgene may not account for the loss of reporter gene expression from these molecular conjugates. We further tested whether repeat administration of sec-R-targeted complexes will be limited by host immune responses. Mice were pre-dosed twice with sec-R-targeted complexes containing lacZ over a 40-day period. We then injected the animals i.v. with sec-R-targeted human factor IX complexes and measured gene expression and antibody production. Although 14 of 36 animals displayed low-titer antibodies to the ligand in targeted complex, expression levels were unaffected compared with virgin dosing. When the complexes were administered three times intranasally (n=10), no antibodies against the complex were detected in blood. Plasma from mice dosed with saline, nontargeted complex or naked DNA did not react with the ligand, ligand-poly K conjugate or targeted complex. All animals exhibiting human factor IX expression developed antibodies to that transgene by 21 days. Thus, at least three repeat administrations of sec-R-directed molecular conjugates are possible, provided that immune responses to the transgene itself are not limiting.

Nanoparticles containing DNA compacted with poly-l-lysine modified on an N-terminal cysteine with polyethylene glycol can effectively transfect cells of the airway epithelium when applied by the luminal route. To evaluate the toxicity of these nanoparticles, we administered 10 and 100 microg DNA compacted into nanoparticles suspended in normal saline by the intranasal route to mice and determined the pulmonary and systemic responses to this challenge, compared to administration of saline alone, and in some experiments, compared to administration of naked DNA, Escherichia coli genomic DNA, or lipofectin-complexed naked DNA. There was no systemic response to either dose of nanoparticles in serum chemistries, hematologic parameters, serum complement, IL-6, or MIP-2 levels or in the activity, growth, and grooming of the mice. Nanoparticles containing 10 microg DNA induced responses comparable to saline in all measures, including BAL cell counts and differentials and cytokine levels and histology. However, mice dosed with 100 microg DNA in nanoparticles had modest increases in BAL neutrophils 48 and 72 h after dosing, modest increases in BAL IL-6 and KC beginning 24 and 48 h, respectively, after dosing, and, on histology of the lung, a trace to 1+ mononuclear cell infiltrates about the pulmonary veins at 48 h, which were markedly reduced by 10 days and gone by 28 days after dosing. BAL neutrophil and cytokine responses were no greater than those entrained by naked DNA for up to 24 h. However, compared to administration of only 10 microg E. coli genomic DNA, the response to compacted DNA was much less. A low dose of lipofectin-complexed DNA (5 microg DNA) induced the same response as 20-fold higher doses of DNA nanoparticles. These data indicate that DNA nanoparticles have no measurable toxic effect at a dose of 10 microg and a very modest effect, which is not limiting, at a dose of 100 microg, which gives maximal gene expression. This favorable toxicity profile encourages development of stabilized compacted DNA for airway administration.

Non-viral methods of gene transfer are being investigated to treat cystic fibrosis (CF) and include naked DNA, lipid-DNA complexes and complexes of DNA with polycations such as poly-L-lysine (poly K) or polyethylenimine (PEI), all of which can carry the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most recent promising strategy is the use of polycation-DNA complexes, particularly those prepared with poly-K and substituted with polyethylene glycol. These complexes produced partial correction of the CF defect in a mouse model with minimal toxicity, and have advanced to clinical trial. Improvements in this and other non-viral methods are in process and include i). targeting the complexes to the desired cells using receptor ligands, ii). lessening toxicity by changing the mix of lipids or adding protective molecules to polycations, iii). modifying the plasmid DNA to reduce inflammatory CpG sequences and enhance intensity, duration and tissue specificity of expression, and iv). modification of the complexes to improve nuclear access.

The airway epithelium has been an important target for gene therapy in the last decade. This route of administration is readily accessible and the airway is affected by a number of disorders for which gene therapy may be useful. Viral vectors were first used to transfer genes to the airway of cystic fibrosis patients a decade ago. However, in vivo results have been disappointing, with substantial inflammation and toxicity occurring at doses that do not produce significant gene transfer. More recently researchers have used plasmid DNA based technologies as an alternative. DNA alone can transfect cells in culture and in vivo, though it is inefficient. Complexing DNA with cationic molecules improves efficiency. Nonviral gene transfer with lipid-DNA complexes has showed promise in cell culture and animals, but is much less efficacious in humans, and inflammation occurs at doses below those required for therapeutic effect. Trans-nuclear membrane delivery appears to be the limiting factor of current liposome-DNA complex efficiency in vivo. DNA compacted with polycations has also been used with good success in animal models, with minimal toxicity and is currently being tested in human trials. In this review, we will discuss the advances and limitations of these nonviral vectors in gene delivery, particularly to airway epithelial cells.

A photoprecursor Pc 227 is covalently bound onto gold nanoparticles (Au NPs) to produce the known photodynamic therapy (PDT) drug Pc 4 upon 660 nm photoirradiation. The photochemical formation of the photoproduct Pc 4 is identified by spectroscopy, chromatography, and mass spectrometry and its PDT efficacy is equal to Pc 4 when administered non-covalently by Au NPs, with the added benefit of improved covalent delivery and targeted NIR-triggered release from the covalent Pc 227-Au NP conjugate, while during transport the attached Pc 227 is quenched by the Au NP and PDT inactivated.

The homeostatic balance between oxidants and antioxidants in biological systems is known as redox balance, and is regulated by complex processes. Redox balance regulates many of the known cellular pathways and disease processes. The dysregulation of redox balance can lead to acute or long-term oxidative or reductive stresses that are associated with many of the abnormalities observed in cystic fibrosis (CF). Over the past 5 decades researchers have examined contributors to redox dysregulation, their molecular products, and their impact on ion transport, cell proliferation, inflammation, bacterial killing, and the metabolism of nucleic acids, proteins, and lipids in CF. CF patients exhibit elevated markers of oxidative stress when compared to non-CF healthy controls; however, whether the reported redox imbalance is sufficient to produce pathology has been controversial. In addition, comparisons between CF and non-CF disease controls have been lacking. To better understand the mechanisms which mediate the generation of oxidants and antioxidants in CF and the importance of their balance in effecting oxidative or reductive stress, we will review the determinants of redox balance in the blood, lumen, and cellular compartments. From the perspective of methodological application, we will focus on the approaches most often used to study oxidant and antioxidants in CF, including biochemical, proteomic, metabolomic, and lipidomic studies, with a discussion of the few transcriptomic analyses that predict changes in the expression of regulators of redox. Finally, we will discuss the utility of oxidants and antioxidants as biomarkers of disease and the use of antioxidant therapy in CF.

Several viral and nonviral methods have introduced functional genes into the lungs. An alternative strategy, receptor-mediated gene transfer, exploits the ability of receptors on the surface of cells to bind and internalize DNA complexes and could potentially be used to deliver genes to specific cells in the lung. The gene encoding human alpha1-antitrypsin (A1AT) was delivered to macrophages in vitro and in vivo by targeting the mannose receptor with mannose-terminal molecular conjugates. The human A1AT transcript was detected 2 d after transfection of macrophages in culture, but transgene expression was transient. Human A1AT protein was secreted into the culture medium, and Western blot hybridization revealed the mature human antiprotease. In addition, Sprague-Dawley rats underwent intravenous injections of increasing doses of plasmid DNA (0.2 mg, 1.0 mg, and 2.0 mg) complexed to the molecular conjugate. Four days after transfection, human A1AT mRNA was found in lungs from six of the 13 rats (46%) that received the higher doses of plasmid. Transgene expression was limited to cells in perivascular and alveolar regions, which conformed to the distribution of pulmonary macrophages. Human A1AT was measured in the epithelial lining fluid of rats treated with transfection complexes. Animals that received 1.0 mg of plasmid had human A1AT levels of 7.4 +/- 3.4 pM, which was significantly different from nontransfected and mock-transfected controls. Thus the mannose receptor permitted direct delivery of genes to pulmonary macrophages, though transgene expression was detected in the lung only at low levels.

The serpin enzyme complex receptor (SECR) expressed on hepatocytes binds to a conserved sequence in alpha 1-antitrypain (alpha 1-AT) and other serpins. A molecular conjugate consisting of a synthetic peptide (C1315) based on the SECR binding motif of human alpha 1-AT covalently coupled to poly-L-lysine was used to introduce reporter genes into hepatoma cell lines in culture. This conjugate condensed DNA into spheroidal particles 18-25 nm in diameter. When transfected with the SECR-directed complex containing pGL3, Hep G2 cells that express the receptor, but not Hep G2 cells that do not, expressed a peak luciferase activity of 538,731 +/- 144,346 integrated light units/mg protein 4 days after transfection. Free peptide inhibited uptake and expression in a dose-dependent manner. Complexes of DNA condensed with polylysine or LC-sulfo-N-succinimidyl-3-(2-pyridyldithio)propionate-substituted polylysine were ineffective. Transfection with a plasmid encoding human factor IX produced expression in Hep G2 (high) and HuH7 cells that express SECR but not Hep G2 (low) cells that lack the receptor. Fluorescein-labeled C1315 peptide labeled 9-31% of Hep G2 (high), 10-14% of HuH7, and 0.6-3.4% of Hep G2 (low) cells, and when the lac Z gene was transfected, only these cells expressed beta-galactosidase. SECR-mediated gene transfer gives efficient, specific uptake and high-level expression of three reporter genes, and the system merits further study for gene therapy.

Cystic fibrosis (CF) is characterized by inflammatory lung disease that significantly contributes to morbidity and mortality. Airway epithelial cells play a role in the inflammatory signaling in CF and have been reported to exhibit a number of dysfunctions in signaling cascades that modulate inflammation. Previously, we reported that the activity of nuclear factor erythroid-derived-like 2 (Nrf2), a transcription factor that regulates antioxidant and cytoprotective protein expression, is diminished in CF epithelia ( 7 ). In this report, we examined the mechanism of Nrf2 dysregulation in vitro in human airway epithelial cell lines and primary cells and in vivo in nasal epithelia excised from ΔF508 CF mutant mice. We found that cAMP-mediated signaling markedly reduces Nrf2 activity in CF vs. non-CF cells. Rp-cAMPS, a cAMP competitor, significantly corrected Nrf2 activity in CF cells, predominantly by increasing the nuclear accumulation of the transcription factor. Furthermore, we found that Rp-cAMPS significantly decreased NF-κB activation following inflammatory stimulation of CF cells. Further investigation revealed that Nrf2 and NF-κB compete for the transcriptional coactivator cAMP responsive element-binding protein (CREB) binding protein (CBP) and that Rp-cAMPS shifts CBP association in favor of Nrf2. Thus our findings provide a link between feedback to CF transmembrane regulator dysfunction and dysregulation of an inflammatory signaling pathway that modulates the coordinated activities of Nrf2 and NF-κB. Furthermore, our studies suggest that strategies that shift CBP association away from NF-κB and toward Nrf2 could have potential therapeutic efficacy for reducing inflammation in patients with CF.

Cystic fibrosis (CF), a lethal genetic disease, is characterized by substantial clinical heterogeneity. Work over the past decade has established that much of the variation is genetically conferred, and recent studies have begun to identify chromosomal locations that identify specific genes as contributing to this variation. Transcriptomic and proteomic data, sampling hundreds and thousands of genes and their products, point to pathways that are altered in the cells and tissues of CF patients. Genetic studies have examined more than half a million polymorphic sites and have identified regions, and probably genes, that contribute to the clinical heterogeneity. The combination of these approaches has great potential because genetic profiling identifies putative disease-modifying processes, and transcript and protein profiling is shedding light on the biology involved. Such studies are providing new insights into the disease, such as altered apoptotic responses, oxidative stress dysregulation, and neuronal involvement, all of which may open new therapeutic avenues to exploration.

In this study, we used bioluminescence imaging (BLI) to track long-term transgene activity following the transfection of brain cells using a nonviral gene therapy technique. Formulations of deoxyribonucleic acid (DNA) combined with 30-mer lysine polymers (substituted with 10 kDa polyethylene glycol) form nanoparticles that transfect brain cells in vivo and produce transgene activity. Here we show that a single intracerebral injection of these DNA nanoparticles (DNPs) into the rat cortex, striatum, or substantia nigra results in long-term and persistent luciferase transgene activity over an 8- to 11-week period as evaluated by in vivo BLI analysis, and single injections of DNPs into the mouse striatum showed stable luciferase transgene activity for 1 year. Compacted DNPs produced in vivo signals 7- to 34-fold higher than DNA alone. In contrast, ex vivo BLI analysis, which is subject to less signal quenching from surrounding tissues, demonstrated a DNP to DNA alone ratio of 76- to 280-fold. Moreover, the ex vivo BLI analysis confirmed that signals originated from the targeted brain structures. In summary, BLI permits serial analysis of luciferase transgene activity at multiple brain locations following gene transfer with DNPs. Ex vivo analysis may permit more accurate determination of relative activities of gene transfer vectors.

Cystic fibrosis is characterized by excessive pulmonary inflammation, which presents early in life and becomes self-sustaining, eventually leading to the destruction of the lung. Treating inflammation is one of the most pressing needs in CF therapy and has been shown to slow lung function deterioration. However, it remains unclear whether excessive inflammation is a direct result of CFTR dysfunction, and thus innate, or develops in response to early stimulation of inflammatory pathways. Here, we will discuss clinically relevant studies and the methods employed by them. We will focus on investigations in cell and animal models as well as patients. Our discussion will describe the character of pulmonary inflammation in CF and present potential therapeutic approaches that can ameliorate excessive responses and improve disease prognosis.

Cystic fibrosis is characterized by excessive pulmonary inflammation, which presents early in life and becomes self-sustaining, eventually leading to the destruction of the lung. Treating inflammation is one of the most pressing needs in CF therapy and has been shown to slow lung function deterioration. However, it remains unclear whether excessive inflammation is a direct result of CFTR dysfunction, and thus innate, or develops in response to early stimulation of inflammatory pathways. Here, we will discuss clinically relevant studies and the methods employed by them. We will focus on investigations in cell and animal models as well as patients. Our discussion will describe the character of pulmonary inflammation in CF and present potential therapeutic approaches that can ameliorate excessive responses and improve disease prognosis.

The success of Mycobacterium tuberculosis as a pathogen relies on its ability to regulate the host immune response. M. tuberculosis can manipulate adaptive T cell responses indirectly by modulating antigen-presenting cell (APC) function or by directly interacting with T cells. Little is known about the role of M. tuberculosis molecules in direct regulation of T cell function. Using a biochemical approach, we identified lipoproteins LprG and LpqH as major molecules in M. tuberculosis lysate responsible for costimulation of primary human CD4 + T cells. In the absence of APCs, activation of memory CD4 + T cells with LprG or LpqH in combination with anti-CD3 antibody induces Th1 cytokine secretion and cellular proliferation. Lipoprotein-induced T cell costimulation was inhibited by blocking antibodies to Toll-like receptor 2 (TLR2) and TLR1, indicating that human CD4 + T cells can use TLR2/TLR1 heterodimers to directly respond to M. tuberculosis products. M. tuberculosis lipoproteins induced NF-κB activation in CD4 + T cells in the absence of TCR co-engagement. Thus, TLR2/TLR1 engagement alone by M. tuberculosis lipoprotein triggered intracellular signaling, but upregulation of cytokine production and proliferation required co-engagement of the TCR. In conclusion, our results demonstrate that M. tuberculosis lipoproteins LprG and LpqH participate in the regulation of adaptive immunity not only by inducing cytokine secretion and costimulatory molecules in innate immune cells but also through directly regulating the activation of memory T lymphocytes.

Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9( 11 )-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-κB activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease.

The construction of safe, efficient, and modifiable synthetic DNA nanoparticles is an emerging technology that has achieved important milestones of success in the past 5 years. Advances in chemical conjugation, purification, and controlled synthesis have allowed researchers to produce uniform and stable particles, whose physical characteristics can be well characterized and monitored. As a result of these improvements, DNA nanoparticles have now been cleared for clinical testing, and show good potential for human gene therapy. A very important recent development in the study of DNA nanoparticles is the use of small-animal imaging. Real-time imaging has become a valuable technique for tracking particle biodistribution and gene transfer efficacy. In this chapter, we discuss how bioluminescent, positron emission tomography, and magnetic resonance imaging can be used separately or in concert to study particle delivery, localization, and magnitude of gene expression in vivo.

The recent introduction of electrospray ionization techniques that are suitable for peptides and whole proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for proteins. The advantages gained by these approaches over traditional Edman Degradation sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the protein. This powerful technique has been instrumental for the study of proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of protein expression in cystic fibrosis as an example.

The recent introduction of electrospray ionization techniques that are suitable for peptides and whole proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for proteins. The advantages gained by these approaches over traditional Edman Degradation sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the protein. This powerful technique has been instrumental for the study of proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of protein expression in cystic fibrosis as an example.

The construction of safe, efficient, and modifiable synthetic DNA nanoparticles is an emerging technology that has achieved important milestones of success in the past 5 years. Advances in chemical conjugation, purification, and controlled synthesis have allowed researchers to produce uniform and stable particles, whose physical characteristics can be well characterized and monitored. As a result of these improvements, DNA nanoparticles have now been cleared for clinical testing, and show good potential for human gene therapy. A very important recent development in the study of DNA nanoparticles is the use of small-animal imaging. Real-time imaging has become a valuable technique for tracking particle biodistribution and gene transfer efficacy. In this chapter, we discuss how bioluminescent, positron emission tomography, and magnetic resonance imaging can be used separately or in concert to study particle delivery, localization, and magnitude of gene expression in vivo.

Infection and inflammation are critical in the progression of cystic fibrosis (CF) lung disease, and are the cause of death of most patients. Therefore, understanding their origins and mechanisms i