Oxidation of Cytochrome 605 Is the Rate-Limiting Step when Ferrimicrobium acidiphilum Respires Aerobically on Soluble Iron.

Proteins that oxidize extracellular substrates in Gram-positive bacteria are poorly understood. Ferrimicrobium acidiphilum is an actinobacterium that respires aerobically on extracellular ferrous ions at pH 1.5. In situ absorbance measurements were conducted on turbid suspensions of intact Fm. acidiphilum using an integrating cavity absorption meter designed for that purpose. Initial velocity kinetic studies monitored the appearance of product ferric ions in the presence of catalytic quantities of cells. Cell-catalyzed iron oxidation obeyed the Michaelis-Menten equation with Km and Vmax values of 71 µM and 0.29 fmol/min/cell, respectively. Limited-turnover kinetic studies were conducted with higher concentrations of cells to detect and monitor changes in the absorbance properties of cellular redox proteins when the cells were exposed to limited quantities of soluble reduced iron. A single a-type cytochrome with reduced absorbance peaks at 448 and 605 nm was the only redox-active chromophore that was visible as the cells respired aerobically on iron. The reduced cytochrome 605 exhibited mathematical and correlational properties that were consistent with the hypothesis that oxidation of the cytochrome constituted the rate-limiting step in the aerobic respiratory process, with a turnover number of 35 ± 2 s-1 Genomic and proteomic analyses showed that Fm. acidiphilum could and did express only two a-type heme copper terminal oxidases. Cytochrome 605 was associated with the terminal oxidase gene that is located between nucleotides 31,090 and 33,039, inclusive, in the annotated circular genome of this bacterium.IMPORTANCE The identities and functions of proteins involved in aerobic respiration on extracellular ferrous ions at acidic pH are poorly understood in the four phyla of Gram-positive eukaryotes and archaea where such activities occur. In situ absorbance measurements were conducted on Fm. acidiphilum as it respired on extracellular iron using an integrating cavity absorption meter that permitted accurate optical measurements in turbid suspensions of the intact bacterium under physiological conditions. The significance of these measurements is that they permitted a direct spectrophotometric examination of the extents and rates of biological electron transfer events in situ under noninvasive physiological conditions without disrupting the complexity of the live cellular environment. One thing is certain: one way to understand how a protein functions in an intact organism is to actually observe that protein as it functions in the intact organism. This paper provides an example of just such an observation.

Purification of metal-dependent lysine deacetylases with consistently high activity.

Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations.

In situ Spectroscopy Reveals that Microorganisms in Different Phyla Use Different Electron Transfer Biomolecules to Respire Aerobically on Soluble Iron.

Absorbance spectra were collected on 12 different live microorganisms, representing six phyla, as they respired aerobically on soluble iron at pH 1.5. A novel integrating cavity absorption meter was employed that permitted accurate absorbance measurements in turbid suspensions that scattered light. Illumination of each microorganism yielded a characteristic spectrum of electrochemically reduced colored prosthetic groups. A total of six different patterns of reduced-minus-oxidized difference spectra were observed. Three different spectra were obtained with members of the Gram-negative eubacteria. Acidithiobacillus, representing Proteobacteria, yielded a spectrum in which cytochromes a and c and a blue copper protein were all prominent. Acidihalobacter, also representing the Proteobacteria, yielded a spectrum in which both cytochrome b and a long-wavelength cytochrome a were clearly visible. Two species of Leptospirillum, representing the Nitrospirae, both yielded spectra that were dominated by a cytochrome with a reduced peak at 579 nm. Sulfobacillus and Alicyclobacillus, representing the Gram-positive Firmicutes, both yielded spectra dominated by a-type cytochromes. Acidimicrobium and Ferrimicrobium, representing the Gram-positive Actinobacteria, also yielded spectra dominated by a-type cytochromes. Acidiplasma and Ferroplasma, representing the Euryarchaeota, both yielded spectra dominated by a ba3-type of cytochrome. Metallosphaera and Sulfolobus, representing the Crenarchaeota, both yielded spectra dominated by the same novel cytochrome as that observed in the Nitrospirae and a new, heretofore unrecognized redox-active prosthetic group with a reduced peak at around 485 nm. These observations are consistent with the hypothesis that individual acidophilic microorganisms that respire aerobically on iron utilize one of at least six different types of electron transfer pathways that are characterized by different redox-active prosthetic groups. In situ absorbance spectroscopy is shown to be a useful complement to existing means of investigating the details of energy conservation in intact microorganisms under physiological conditions.

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant.

Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm(2) in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s(-1). The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment.

Cystic fibrosis transmembrane conductance regulator recruitment to phagosomes in neutrophils.

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.

Response of Differentiated Human Airway Epithelia to Alcohol Exposure and Klebsiella Pneumoniae Challenge.

Alcohol abuse has been associated with increased susceptibility to pulmonary infection. It is not fully defined how alcohol contributes to the host defense compromise. Here primary human airway epithelial cells were cultured at an air-liquid interface to form a differentiated and polarized epithelium. This unique culture model allowed us to closely mimic lung infection in the context of alcohol abuse by basolateral alcohol exposure and apical live bacterial challenge. Application of clinically relevant concentrations of alcohol for 24 hours did not significantly alter epithelial integrity or barrier function. When apically challenged with viable Klebsiella pneumoniae, the cultured epithelia had an enhanced tightness which was unaffected by alcohol. Further, alcohol enhanced apical bacterial growth, but not bacterial binding to the cells. The cultured epithelium in the absence of any treatment or stimulation had a base-level IL-6 and IL-8 secretion. Apical bacterial challenge significantly elevated the basolateral secretion of inflammatory cytokines including IL-2, IL-4, IL-6, IL-8, IFN-γ, GM-CSF, and TNF-α. However, alcohol suppressed the observed cytokine burst in response to infection. Addition of adenosine receptor agonists negated the suppression of IL-6 and TNF-α. Thus, acute alcohol alters the epithelial cytokine response to infection, which can be partially mitigated by adenosine receptor agonists.

Chloride transport in functionally active phagosomes isolated from Human neutrophils.

Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC-dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport properties, and multiple chloride channels including CFTR are responsible for chloride supply to neutrophil phagosomes.

Comparisons of resistance of CF and non-CF pathogens to hydrogen peroxide and hypochlorous acid oxidants in vitro.

BACKGROUND: Cystic fibrosis (CF) lung disease has a unique profile of pathogens predominated by Pseudomonas aeruginosa (PsA) and Staphylococcus aureus (SA). These microorganisms must overcome host immune defense to colonize the CF lungs. Polymorphonuclear neutrophils are a major component of the host defense against bacterial infection. A crucial microbicidal mechanism is the production of oxidants including hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) by neutrophils to achieve efficient bacterial killing. To determine to what degrees various CF pathogens resist the oxidants relative to non-CF pathogens, we compared the susceptibility of PsA, SA, Burkholderia cepacia (BC), Klebsiella pneumoniae (KP), and Escherichia coli (EC) to various concentrations of H2O2 or HOCl, in vitro. The comparative oxidant-resistant profiles were established. Oxidant-induced damage to ATP production and cell membrane integrity of the microbes were quantitatively assessed. Correlation of membrane permeability and ATP levels with bacterial viability was statistically evaluated. RESULTS: PsA was relatively resistant to both H2O2 (LD50 = 1.5 mM) and HOCl (LD50 = 0.035 mM). SA was susceptible to H2O2 (LD50 = 0.1 mM) but resistant to HOCl (LD50 = 0.035 mM). Interestingly, KP was extremely resistant to high doses of H2O2 (LD50 = 2.5-5.0 mM) but was very sensitive to low doses of HOCl (LD50 = 0.015 mM). BC was intermediate to resist both oxidants: H2O2 (LD50 = 0.3-0.4 mM) and HOCl (LD50 = 0.025 mM). EC displayed the least resistance to H2O2 (LD50 = 0.2-0.3 mM) and HOCl (LD50 = 0.015 mM). The identified profile of H2O2-resistance was KP > PsA > BC > EC > SA and the profile of HOCl-resistance PsA > SA > BC > EC > KP. Moreover, both oxidants affected ATP production and membrane integrity of the cells. However, the effects varied among the tested organisms and, the oxidant-mediated damage correlated differentially with the bacterial viability. CONCLUSIONS: The order of HOCl-resistance identified herein best fits the clinical profile of CF infections. Even though oxidants are able to disrupt ATP production and cell membrane integrity, the degrees of damage vary among the organisms and correlate differentially with their viability.

RNA interference against CFTR affects HL60-derived neutrophil microbicidal function.

Biosynthesis of hypochlorous acid, a potent antimicrobial oxidant, in phagosomes is one of the chief mechanisms employed by polymorphonuclear neutrophils to combat infections. This reaction, catalyzed by myeloperoxidase, requires chloride anion (Cl(-)) as a substrate. Thus, Cl(-) availability is a rate-limiting factor that affects neutrophil microbicidal function. Our previous research demonstrated that defective CFTR, a cAMP-activated chloride channel, present in cystic fibrosis (CF) patients leads to deficient chloride transport to neutrophil phagosomes and impaired bacterial killing. To confirm this finding, here we used RNA interference against this chloride channel to abate CFTR expression in the neutrophil-like cells derived from HL60 cells, a promyelocytic leukemia cell line, with dimethyl sulfoxide. The resultant CFTR deficiency in the phagocytes compromised their bactericidal capability, thereby recapitulating the phenotype seen in CF patient cells. The results provide further evidence suggesting that CFTR plays an important role in phagocytic host defense.

Ethanol upregulates glucocorticoid-induced leucine zipper expression and modulates cellular inflammatory responses in lung epithelial cells.

Alcohol abuse is associated with immunosuppressive and infectious sequelae. Particularly, alcoholics are more susceptible to pulmonary infections. In this report, gene transcriptional profiles of primary human airway epithelial cells exposed to varying doses of alcohol (0, 50, and 100 mM) were obtained. Comparison of gene transcription levels in 0 mM alcohol treatments with those in 50 mM alcohol treatments resulted in 2 genes being upregulated and 16 genes downregulated by at least 2-fold. Moreover, 0 mM and 100 mM alcohol exposure led to the upregulation of 14 genes and downregulation of 157 genes. Among the upregulated genes, glucocorticoid-induced leucine zipper (GILZ) responded to alcohol in a dose-dependent manner. Moreover, GILZ protein levels also correlated with this transcriptional pattern. Lentiviral expression of GILZ small interfering RNA in human airway epithelial cells diminished the alcohol-induced upregulation, confirming that GILZ is indeed an alcohol-responsive gene. Gene silencing of GILZ in A549 cells resulted in secretion of significantly higher amounts of inflammatory cytokines in response to IL-1beta stimulation. The GILZ-silenced cells were more resistant to alcohol-mediated suppression of cytokine secretion. Further data demonstrated that the glucocorticoid receptor is involved in the regulation of GILZ by alcohol. Because GILZ is a key glucocorticoid-responsive factor mediating the anti-inflammatory and immunosuppressive actions of steroids, we propose that similar signaling pathways may play a role in the anti-inflammatory and immunosuppressive effects of alcohol.

CFTR-mediated halide transport in phagosomes of human neutrophils.

Chloride serves as a critical component of innate host defense against infection, providing the substrate for MPO-catalyzed production of HOCl in the phagosome of human neutrophils. Here, we used halide-specific fluorescent sensors covalently coupled to zymosan particles to investigate the kinetics of chloride and iodide transport in phagosomes of human neutrophils. Using the self-ratioable fluorescent probe specific for chloride anion, we measured chloride dynamics within phagosomes in response to extracellular chloride changes by quantitative fluorescence microscopy. Under the experimental conditions used, normal neutrophils showed rapid phagosomal chloride uptake with an initial influx rate of 0.31 +/- 0.04 mM/s (n=5). GlyH-101, a CFTR(inh), decreased the rate of uptake in a dose-dependent manner. Neutrophils isolated from CF patients showed a significantly slower rate of chloride uptake by phagosomes, having an initial influx rate of 0.043 +/- 0.012 mM/s (n=5). Interestingly, the steady-state level of chloride in CF phagosomes was approximately 26 mM, significantly lower than that of the control ( approximately 68 mM). As CFTR transports chloride as well as other halides, we conjugated an iodide-sensitive probe as an independent approach to confirm the results. The dynamics of iodide uptake by neutrophil phagosomes were monitored by flow cytometry. CFTR(inh)172 blocked 40-50% of the overall iodide uptake by phagosomes in normal neutrophils. In a parallel manner, the level of iodide uptake by CF phagosomes was only 20-30% of that of the control. Taken together, these results implicate CFTR in transporting halides into the phagosomal lumen.

The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils.

Chloride anion is essential for myeloperoxidase (MPO) to produce hypochlorous acid (HOCl) in polymorphonuclear neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN-mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride-deficient medium killed PsA poorly. However, as the chloride level was raised, the killing efficiency increased in a dose-dependent manner. By using specific inhibitors to selectively block NADPH oxidase, MPO, and cystic fibrosis transmembrane conductance regulator (CFTR) functions, neutrophil-mediated killing of PsA could be attributed to three distinct mechanisms: CFTR-dependent and oxidant-dependent; chloride-dependent but not CFTR- and oxidant-dependent; and independent of any of the tested factors. Therefore, chloride anion is involved in oxidant- and nonoxidant-mediated bacterial killing. We previously reported that neutrophils from CF patients are defective in chlorination of ingested bacteria, suggesting that the chloride channel defect might impair the MPO-hydrogen peroxide-chloride microbicidal function. Here, we compared the competence of killing PsA by neutrophils from normal donors and CF patients. The data demonstrate that the killing rate by CF neutrophils was significantly lower than that by normal neutrophils. CF neutrophils in a chloride-deficient environment had only one-third of the bactericidal capacity of normal neutrophils in a physiological chloride environment. These results suggest that CFTR-dependent chloride anion transport contributes significantly to killing PsA by normal neutrophils and when defective as in CF, may compromise the ability to clear PsA.

Functional expression of N-formyl peptide receptors in human bone marrow-derived mesenchymal stem cells.

Tissue injury enhances homing and engraftment of mesenchymal stem cells (MSCs). However, the mechanisms by which MSCs sense the signals released by injured tissues and migrate toward injury sites have not been fully defined. In the current report, we investigated whether human MSCs express the N-formyl peptide receptor (FPR) and the formyl peptide receptor-like-1 (FPRL1). These receptors bind to N-formylated peptides by which phagocytes migrate to inflammatory sites and fibroblasts repopulate wounds to remodel the damaged tissues. Reverse-transcription polymerase chain reaction (PCR) demonstrated that MSCs express both FPR and FPRL1 at the transcriptional level. Flow cytometric analyses revealed expression of both receptors at the protein level. Fusion of the enhanced green fluorescence protein (eGFP) to the C terminus of each receptor showed localization to the cell surface. Moreover, MSCs responded to stimulation by N-formyl methionyl leucyl phenylalanine (fMLP), a prototypic N-formyl peptide, demonstrating rapid intracellular calcium mobilization that can be blocked by pertussis toxin or cyclosporin H. It is noteworthy that the fMLP-stimulated MSCs had an enhanced adhesion to extracellular matrix protein-coated surfaces. In addition, MSCs migrated toward gradients of increasing fMLP concentration, indicating that the receptors were functionally involved in positive chemotaxis to formylated peptides. Therefore, the N-formyl peptide receptors present in MSCs may play an important role in signaling stem cell adhesion, migration, and homing to injured and inflamed tissue for repair. Such a mechanism could potentially be exploited to direct the stem cells to target specific tissue sites, such as cystic fibrosis lungs, for therapy. Disclosure of potential conflicts of interest is found at the end of this article.

CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis.

Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to a limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR expresssion when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC-MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically prelabeled with [(13)C]-l-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides evidence which suggests that CFTR channel expression in neutrophils and its dysfunction affect neutrophil chlorination of phagocytosed bacteria.

Direct measurement of free chloride concentrations in the phagolysosomes of human neutrophils.

Human neutrophils synthesize hypochlorous acid, a nonradical oxidant, as one of the antimicrobial agents to kill phagocytosed pathogens within phagolysosomes. The production of HOCl is catalyzed by myeloperoxidase using chloride anions (Cl-). Even though various approaches have been documented to measure cytosolic Cl- concentrations, direct detection of phagolysosomal free Cl- in a real-time fashion is not available. Here we report the development of a new dual-labeled fluorescent probe by conjugation of the chloride-sensitive 6-methoxyquinoline and chloride-insensitive tetramethylrhodamine to the zymosan particles. On the surface of the zymosan particles, the chloride-sensitive fluorophore responded linearly to chloride concentrations when plotted according to the Stern-Volmer collisional quenching equation and the chloride-insensitive fluorophore served as an internal reference. After phagocytosis into neutrophils, the probe was able to monitor intraphagosomal chloride levels authentically. Human neutrophils in the medium containing 122 mM chloride had a phagolysosomal chloride level of 73.3 +/- 12.2 mM (N = 5). In contrast, the neutrophils in a chloride-free isoosmotic medium had a chloride level of 6.6 +/- 1.9 mM (N = 5) in the phagolysosomal compartment. These data clearly demonstrated that the extracellular chloride levels affect intraphagolysosomal chloride levels in human neutrophils. Moreover, when the extracellular chloride concentration was switched from high level (122 mM) to low level (0 mM) or vice versa, the probe demonstrated complete reversibility. The data indicate that the new zymosan-conjugated chloride probe described here can be used as an indicator for measurement of the free chloride level within the phagolysosomes of neutrophils and other phagocytic cell types.

Conditional expression of a suicide gene by the telomere reverse transcriptase promoter for potential post-therapeutic deletion of tumorigenesis.

Integration of a therapeutic gene into the host cell genome permits stable expression of the gene product in the target cells and its progeny. However, non-directional integration of any given gene can pose the risk of activating tumor genes or silencing tumor suppressor genes. Therefore, including a safety-control element into integrating vector systems is an important advance towards safer human gene therapy. Here, we report on a gene expression cassette that can be potentially exploited in integrating vector systems to eliminate post-therapeutic tumorigenesis. The Herpes simplex virus thymidine kinase (hsvTK) gene under the transcriptional control of the human telomere reverse transcriptase promoter (hTERTp) was incorporated into a self-inactivating HIV-based lentiviral vector. The hTERT promoter is silent in normal somatic cells and re-activated in tumor cells. Therefore, normal gene-corrected cells should not express hsvTK from the promoter. However, if some gene-corrected cells subsequently become tumorigenic and the hTERT promoter is re-activated, application of ganciclovir (GCV), a clinically used antiviral drug, will achieve selective deletion of the cancerous cells. Our experimental data indicated that the hTERTp-hsvTK cassette in the lentiviral vector was sufficient to differentiate between tumor cells and normal cells, thus eradicating tumor cells selectively in vitro and in vivo. These results proved the principle of using the element in integrating vectors for safer gene delivery.

Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: potential therapy for cystic fibrosis.

Cystic fibrosis (CF), the most prevalent, fatal genetic disorder in the Caucasian population, is caused by mutations of CF transmembrane conductance regulator (CFTR). The mutations of this chloride channel alter the transport of chloride and associated liquid and thereby impair lung defenses. Patients typically succumb to chronic bacterial infections and respiratory failure. Restoration of the abnormal CFTR function to CF airway epithelium is considered the most direct way to treat the disease. In this report, we explore the potential of adult stem cells from bone marrow, referred to as mesenchymal or marrow stromal stem cells (MSCs), to provide a therapy for CF. We found that MSCs possess the capacity of differentiating into airway epithelia. MSCs from CF patients are amenable to CFTR gene correction, and expression of CFTR does not influence the pluripotency of MSCs. Moreover, the CFTR-corrected MSCs from CF patients are able to contribute to apical Cl(-) secretion in response to cAMP agonist stimulation, suggesting the possibility of developing cell-based therapy for CF. The ex vivo coculture system established in this report offers an invaluable approach for selection of stem-cell populations that may have greater potency in lung differentiation.