Fast-moving stars around an intermediate-mass black hole in ω Centauri.

Black holes have been found over a wide range of masses, from stellar remnants with masses of 5-150 solar masses (M☉), to those found at the centres of galaxies with M > 105M☉. However, only a few debated candidate black holes exist between 150M☉ and 105M☉. Determining the population of these intermediate-mass black holes is an important step towards understanding supermassive black hole formation in the early universe1,2. Several studies have claimed the detection of a central black hole in ω Centauri, the most massive globular cluster of the Milky Way3-5. However, these studies have been questioned because of the possible mass contribution of stellar mass black holes, their sensitivity to the cluster centre and the lack of fast-moving stars above the escape velocity6-9. Here we report the observations of seven fast-moving stars in the central 3 arcsec (0.08 pc) of ω Centauri. The velocities of the fast-moving stars are significantly higher than the expected central escape velocity of the star cluster, so their presence can be explained only by being bound to a massive black hole. From the velocities alone, we can infer a firm lower limit of the black hole mass of about 8,200M☉, making this a good case for an intermediate-mass black hole in the local universe.

People-Centered and Ecosystem-Based Knowledge Co-Production to Promote Proactive Biodiversity Conservation and Sustainable Development in Namibia.

Growing levels of uncertainty and vulnerability generated by land use conversion and climate change set demands on local communities and national institutions to build synergies between the diverse array of knowledge systems in order to provide policy makers and practitioners with the best available information to decide what urgent actions must be taken. Science policy arenas and agreements such as the Intergovernmental Platform on Biodiversity and Ecosystem Services (IPBES) and the Convention on Biological Diversity (CBD) recognize the importance of different types of knowledge and the need for broad stakeholder involvement, yet the use of indigenous and local knowledge (ILK) in environmental decision-making processes is still underdeveloped. This study involved working with local stakeholders, using the MARISCO method (adaptive MAnagement of vulnerability and RISks at COnservation sites) to carry out a systematic situation analysis of the existing socioenvironmental conditions. The assessments were conducted in the Kavango East Region in northern Namibia with the participation of inhabitants of the Khaudum North Complex, a protected area network covering wooded savannahs belonging to the Northern Kalahari sandveld. General outcomes of the assessments and evaluations made by the local stakeholders concerning the most critical drivers of degradation of the ecosystems appeared to support existing scientific knowledge of the study area, demonstrating that community-based assessments can provide valuable information about socioecological systems where scientific data are scarce. The findings of this study also highlight the importance of power dynamics for the implementation of participatory processes and the interpretation of their outcomes.

Pulmonary Abnormalities in Young, Light-Use Waterpipe (Hookah) Smokers.

RATIONALE: Waterpipes, also called hookahs, are currently used by millions of people worldwide. Despite the increasing use of waterpipe smoking, there is limited data on the health effects of waterpipe smoking and there are no federal regulations regarding its use. OBJECTIVES: To assess the effects of waterpipe smoking on the human lung using clinical and biological parameters in young, light-use waterpipe smokers. METHODS: We assessed young, light-use, waterpipe-only smokers in comparison with lifelong nonsmokers using clinical parameters of cough and sputum scores, lung function, and chest high-resolution computed tomography as well as biological parameters of lung epithelial lining fluid metabolome, small airway epithelial (SAE) cell differential and transcriptome, alveolar macrophage transcriptome, and plasma apoptotic endothelial cell microparticles. MEASUREMENTS AND MAIN RESULTS: Compared with nonsmokers, waterpipe smokers had more cough and sputum as well as a lower lung diffusing capacity, abnormal epithelial lining fluid metabolome profile, increased proportions of SAE secretory and intermediate cells, reduced proportions of SAE ciliated and basal cells, markedly abnormal SAE and alveolar macrophage transcriptomes, and elevated levels of apoptotic endothelial cell microparticles. CONCLUSIONS: Young, light-use, waterpipe-only smokers have a variety of abnormalities in multiple lung-related biological and clinical parameters, suggesting that even limited waterpipe use has broad consequences on human lung biology and health. We suggest that large epidemiological studies should be initiated to investigate the harmful effects of waterpipe smoking.

Persistence of circulating endothelial microparticles in COPD despite smoking cessation.

INTRODUCTION: Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation. METHODS: Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b-CD31+), pulmonary capillary EMP (CD42b-CD31+ACE+) and apoptotic EMP (CD42b-CD62E+/CD42b-CD31+) levels were quantified by flow cytometry. RESULTS: Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit. CONCLUSIONS: Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation. TRIAL REGISTRATION NUMBER: NCT00974064; NCT01776398.

Low sphingosine-1-phosphate impairs lung dendritic cells in cystic fibrosis.

Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) leads to chronic inflammation and infection of the respiratory tract. The role of CFTR for cells of the pulmonary immune system is only partly understood. The present study analyzes the phenotype and immune stimulatory capacity of lung dendritic cells (DCs) from CFTR knockout (CF) mice. Total numbers of conventional DCs, plasmacytoid DCs, and CD103-positive DCs were lower in CF mice compared with wild-type (WT) control mice, as was the expression of major histocompatibility complex class II molecules (MHCII), CD40, and CD86. After pulmonary infection with respiratory syncytial virus, DC numbers increased in WT mice but not in CF mice, and the T cell-stimulatory capacity of CF DCs was impaired. The culture of CF lung DCs with bronchoalveolar lavage fluid (BALF) from WT mice increased the expression of MHCII, CD40, and CD86. The supplementation of CF BALF with sphingosine-1-phosphate (S1P), a mediator of immune cell migration and activation that is decreased in CF BALF, rescued the reduced expression of MHCII and CD40 in WT lung DCs and human blood DCs. These findings suggest that DCs are impaired in the CF lung, and that altered S1P affects lung DC function. These findings provide a novel link between defective CFTR and pulmonary innate immune dysfunction in CF.

Circulating endothelial microparticles as a measure of early lung destruction in cigarette smokers.

RATIONALE: There is increasing evidence that emphysema is associated with primary loss of pulmonary capillary endothelium. Plasma levels of endothelial microparticles (EMPs), small vesicles released from activated or apoptotic endothelial cells, are elevated in vascular-related disorders. OBJECTIVES: To evaluate whether plasma EMP levels are elevated in smokers with early lung destruction as assessed by normal spirometry but reduced diffusing capacity of the lung for carbon monoxide (Dl(co)). METHODS: Lung health was assessed by pulmonary function tests (PFTs: spirometry, total lung capacity, Dl(co)) and chest X-ray; smoking status was assessed by urine nicotine and cotinine. EMP levels (CD42b(-)CD31(+) microparticles) were quantified as activated or apoptotic. The initial cohort (n = 92) included healthy nonsmokers (normal PFTs), healthy smokers (normal PFTs), and smokers with early evidence of lung destruction (normal spirometry, low Dl(co)). Two prospective cohorts were then tested: a group similar to the initial cohort and an HIV1(+) cohort. MEASUREMENTS AND MAIN RESULTS: Healthy smokers had mildly increased levels of EMPs. Strikingly, 95% of smokers with normal spirometry, low Dl(co) had increased EMPs, with reduced CD62(+)/CD31(+) ratios (P < 10(-4)) and elevated CD42b(-)CD31(+) annexin V(+) EMPs (P < 10(-4)), suggesting derivation from endothelial apoptosis. Most elevated EMPs were angiotensin-converting enzyme positive, suggesting derivation from pulmonary capillaries. Both prospective cohorts confirmed the initial cohort data. CONCLUSIONS: Plasma EMPs with apoptotic characteristics are elevated in smokers with normal spirometry but reduced Dl(co), consistent with the concept that emphysema is associated, in part, with capillary endothelium apoptosis, suggesting that the early development of emphysema might be monitored with plasma EMP levels.

Absence of vaccine-enhanced RSV disease and changes in pulmonary dendritic cells with adenovirus-based RSV vaccine.

The development of a vaccine against respiratory syncytial virus (RSV) has been hampered by the risk for vaccine-enhanced RSV pulmonary disease induced by immunization with formalin-inactivated RSV (FIRSV). This study focuses on the evaluation of vaccine-enhanced pulmonary disease following immunization with AdF.RGD, an integrin-targeted adenovirus vector that expresses the RSV F protein and includes an RGD (Arg-Gly-Asp) motif. Immunization of BALB/c mice with AdF.RGD, resulted in anti-RSV protective immunity and induced increased RSV-specific IFN-γ T cell responses compared to FIRSV. RSV infection 5 wk after immunization with FIRSV induced pulmonary inflammatory responses in the lung, that was not observed with AdF.RGD. Additionally, In the FIRSV-immunized mice following infection with RSV, pulmonary DC increased and Tregs decreased. This suggests that distinct responses of pulmonary DC and Tregs are a features of vaccine-enhanced RSV disease and that immunization with an RGD-modified Ad vaccine does not trigger vaccine-enhanced disease.

Smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease.

When exposed to a specific microenvironment, macrophages acquire either M1- or M2-polarized phenotypes associated with inflammation and tissue remodeling, respectively. Alveolar macrophages (AM) directly interact with environmental stimuli such as cigarette smoke, the major risk factor for chronic obstructive pulmonary disease (COPD), a disease characterized by lung inflammation and remodeling. Transcriptional profiling of AM obtained by bronchoalveolar lavage of 24 healthy nonsmokers, 34 healthy smokers, and 12 COPD smokers was performed to test the hypothesis whether smoking alters AM polarization, resulting in a disease-relevant activation phenotype. The analysis revealed that AM of healthy smokers exhibited a unique polarization pattern characterized by substantial suppression of M1-related inflammatory/immune genes and induction of genes associated with various M2-polarization programs relevant to tissue remodeling and immunoregulation. Such reciprocal changes progressed with the development of COPD, with M1-related gene expression being most dramatically down-regulated (p < 0.0001 vs healthy nonsmokers, p < 0.002 vs healthy smokers). Results were confirmed with TaqMan real-time PCR and flow cytometry. Among progressively down-regulated M1-related genes were those encoding type I chemokines CXCL9, CXCL10, CXCL11, and CCL5. Progressive activation of M2-related program was characterized by induction of tissue remodeling and immunoregulatory genes such as matrix metalloproteinase (MMP)2, MMP7, and adenosine A3 receptor (ADORA3). Principal component analysis revealed that differential expression of polarization-related genes has substantial contribution to global AM phenotypes associated with smoking and COPD. In summary, the data provide transcriptome-based evidence that AM likely contribute to COPD pathogenesis in a noninflammatory manner due to their smoking-induced reprogramming toward M1-deactivated, partially M2-polarized macrophages.

Trans-splicing repair of CD40 ligand deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked immunodeficiency.

X-linked immunodeficiency with hyper-IgM (HIGM1), characterized by failure of immunoglobulin isotype switching, is caused by mutations of the CD40 ligand (CD40L), which is normally expressed on activated CD4(+) T cells. As constitutive expression of CD40L induces lymphomas, we corrected the mutation while preserving the natural regulation of CD40L using pre-mRNA trans-splicing. Bone marrow from mice lacking CD40L was modified with a lentivirus trans-splicer encoding the normal CD40L exons 2-5 and was administered to syngenic CD40L-knockout mice. Recipient mice had corrected CD40L mRNA, antigen-specific IgG1 responses to keyhole limpet hemocyanin immunization, regulated CD4(+) T-cell CD40L expression after CD3 stimulation in primary and secondary transplanted mice, attenuation of Pneumocystis carinii pneumonia, and no evidence of lymphoproliferative disease over 1 year. Thus, HIGM1 can be corrected by CD40L trans-splicing, leading to functional correction of the genetic defect without the adverse consequences of unregulated expression of the CD40L gene.

Overexpression of sonic Hedgehog in the lung mimics the effect of lung injury and compensatory lung growth on pulmonary Sca-1 and CD34 positive cells.

Cells localized in the bronchioalveolar duct junction of the murine lung have been identified as potential bronchioalveolar stem cells. Based on the surface marker expression, two main phenotypes have been proposed: Sca-1(+), CD34(+), CD45(-), Pecam(-) and Sca-1(low), CD34(-) CD45(-), Pecam(-) cells. An increase in the number of Sca-1(+), CD34(+) CD45(-), Pecam(-) cells and activation of the sonic hedgehog (Shh) pathway was observed following unilateral pneumonectomy and naphthalene-induced airway injury. Overexpression of Shh in the respiratory tract also resulted in an increase of this cell population. Syngeneic transplantation of beta-galactosidase-expressing bone marrow cells demonstrated that the increase of Sca-1(+), CD34(+), CD45(-), Pecam(-) cells in the lung was a result of local proliferation. Intratracheal administration of purified Shh-stimulated Sca-1(+), CD45(-), Pecam(-) cells coexpressing CD34 to syngeneic mice following pneumonectomy resulted in engraftment of these cells predominantly in the airways for up to 3 months, whereas Sca-1(-), CD45(-), Pecam(-) cells did not engraft. This study suggests that local Sca-1(+), CD34(+), CD45(-), Pecam(-) cells are stimulated during compensatory lung growth, following airway injury and overexpression of Shh and have some potential to engraft in the airways, without showing clonal properties in vivo.

Recent developments for Pseudomonas vaccines.

Infections with Pseudomonas aeruginosa are a major health problem for immune-compromised patients and individuals with cystic fibrosis. A vaccine against: P. aeruginosa has long been sought after, but is so far not available. Several vaccine candidates have been assessed in experimental animals and humans, which include sub-cellular fractions, capsule components, purified and recombinant proteins. Unique characteristics of the host and the pathogen have complicated the vaccine development. This review summarizes the current state of vaccine development for this ubiquitous pathogen, in particular to provide mucosal immunity against infections of the respiratory tract in susceptible individuals with cystic fibrosis.

Sonic hedgehog expands diaphyseal trabecular bone altering bone marrow niche and lymphocyte compartment.

Bone marrow contains distinct microenvironments that regulate hematopoietic stem cells (HSCs). The endosteal HSC niche includes osteoblasts, mineral, and extracellular matrix proteins that interact through various molecular signals to control HSCs. Sonic hedgehog (Shh) is a morphogen involved in the regulation of skeletal development and hematopoiesis, but the effects of Shh on bone in relation to the HSC niche are not well understood. We demonstrate that systemic overexpression of Shh in mice increases osteoblast number with the resultant formation of new trabeculae in the femoral diaphysis. Suggestive of a functional change in the hematopoietic niche, numbers of Lin(-) Sca-1(+) c-Kit(+) cells with hematopoietic progenitor function expand, although cells with in vivo repopulating capacity in the wild-type environment do not increase. Instead, Shh mediates a decrease in number of bone marrow lymphocytes accompanied by a decreased expression of stromal-derived growth factor 1 (SDF-1) and a decrease in Flk2-expressing Lin(-) Sca-1(+) c-Kit(+) cells, indicating a modulation of early lymphopoiesis. This is caused by a microenvironment-induced mechanism as Shh treatment of bone marrow recipients, but not donors, results in a dramatic depletion of lymphocytes. Together, these data suggest that Shh mediates alterations in the bone marrow hematopoietic niche affecting the early lymphoid differentiation.

Dysfunctional glycogen storage in a mouse model of alpha1-antitrypsin deficiency.

Autophagy is an intracellular pathway that contributes to the degradation and recycling of unfolded proteins. Based on the knowledge that autophagy affects glycogen metabolism and that alpha(1)-antitrypsin (AAT) deficiency is associated with an autophagic response in the liver, we hypothesized that the conformational abnormalities of the Z-AAT protein interfere with hepatocyte glycogen storage and/or metabolism. Compared with wild-type mice (WT), the Z-AAT mice had lower liver glycogen stores (P < 0.001) and abnormal activities of glycogen-related enzymes, including acid alpha-glucosidase (P < 0.05) and the total glycogen synthase (P < 0.05). As metabolic consequences, PiZ mice demonstrated lower blood glucose levels (P < 0.05), lower body weights (P < 0.001), and lower fat pad weights (P < 0.001) compared with WT. After the stress of fasting or partial hepatectomy, PiZ mice had further reduced liver glycogen and lower blood glucose levels (both P < 0.05 compared WT). Finally, PiZ mice exhibited decreased survival after partial hepatectomy (P < 0.01 compared with WT), but this was normalized with postoperative dextrose supplementation. In conclusion, these observations are consistent with the general concept that abnormal protein conformation and degradation affects other cellular functions, suggesting that diseases in the liver might benefit from metabolic compensation if glycogen metabolism is affected.

Expression of B-cell activating factor enhances protective immunity of a vaccine against Pseudomonas aeruginosa.

B-cell activating factor (BAFF), a member of the TNF family, is a potent cytokine with stimulatory effects on B and T cells. To evaluate the potential of transient overexpression of BAFF to enhance vaccine immunogenicity, a replication-deficient adenovirus expressing full-length murine BAFF (AdBAFF) was tested in a mouse vaccine model against Pseudomonas aeruginosa. When coadministered with heat-killed P. aeruginosa, AdBAFF mediated a significant increase in anti-P. aeruginosa-specific serum and lung mucosal antibodies and resulted in improved protection against a lethal respiratory challenge with P. aeruginosa. This effect was independent of the site of administration of AdBAFF and was observed both when AdBAFF was given simultaneously with heat-killed P. aeruginosa as well as when AdBAFF was administered 4 weeks after immunization with heat-killed P. aeruginosa. These data demonstrate that a temporal increase in systemic BAFF levels is able to augment a P. aeruginosa-specific immune response upon immunization with heat-killed P. aeruginosa, suggesting that the immune-stimulatory effects of BAFF may be exploited as a molecular adjuvant for genetic vaccines.

Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells.

BACKGROUND: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC) are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown. METHODS: This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT) mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection. RESULTS: CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP), a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3beta-hydroxysterol-Delta7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC. CONCLUSION: These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.

Overexpression of apoptotic cell removal receptor MERTK in alveolar macrophages of cigarette smokers.

Mononuclear phagocytes play an important role in the removal of apoptotic cells by expressing cell surface receptors that recognize and remove apoptotic cells. Based on the knowledge that cigarette smoking is associated with increased lung cell turnover, we hypothesized that alveolar macrophages (AMs) of normal cigarette smokers may exhibit enhanced expression of apoptotic cell removal receptor genes. AMs obtained by bronchoalveolar lavage of normal nonsmokers (n = 11) and phenotypic normal smokers (n = 13; 36 +/- 6 pack-years) were screened for mRNA expression of all known apoptotic cell removal receptors using Affymetrix HG-U133 Plus 2.0 microarray chips with TaqMan RT-PCR confirmation. Of the 14 known apoptotic receptors expressed, only MER tyrosine kinase (MERTK), a transmembrane tyrosine kinase receptor, was significantly up-regulated in smokers. MERTK expression was then assessed in AMs of smokers versus nonsmokers by TaqMan RT-PCR, immunocytochemistry, Western analysis, and flow analysis. Smoker AMs had up-regulation of MERTK mRNA levels (smoker vs. nonsmoker: 3.6-fold by microarray, P < 0.003; 9.5-fold by TaqMan RT-PCR, P < 0.02). Immunocytochemistry demonstrated a qualitative increase in MERTK protein expression on AMs of smokers. Increased protein expression of MERTK on AMs of smokers was confirmed by Western and flow analyses (P < 0.007 and P < 0.0002, respectively). MERTK, a cell surface receptor that recognizes apoptotic cells, is expressed on human AMs, and its expression is up-regulated in AMs of cigarette smokers. This up-regulation of MERTK may reflect an increased demand for removal of apoptotic cells in smokers, an observation with implications for the development of chronic obstructive pulmonary disease, a disorder associated with dysregulated apoptosis of lung parenchymal cells.

Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid.

Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

Protective immunity to pseudomonas aeruginosa induced with a capsid-modified adenovirus expressing P. aeruginosa OprF.

This study focuses on the development of a new clinical vaccine candidate (AdOprF.RGD.Epi8) against Pseudomonas aeruginosa using an E1(-) E3(-) adenovirus (Ad) vector expressing OprF (AdOprF.RGD.Epi8) and modifications of the Ad genome providing two capsid changes: (i) modification of the Ad hexon gene to incorporate an immune-dominant OprF epitope (Epi8) into loop 1 of the hexon, enabling repeat administration to boost the anti-OprF immune response, and (ii) modification of the fiber gene to incorporate an integrin-binding RGD sequence to enhance gene delivery to antigen-presenting cells. Western analysis confirmed that AdOprF.RGD.Epi8 expresses OprF, contains Epi8 in the hexon protein, and enhances gene transfer to dendritic cells compared to AdOprF, a comparable Ad vector expressing OprF with an unmodified capsid. Intramuscular immunization of C57BL/6 mice with AdOprF.RGD.Epi8 resulted in the generation of anti-OprF antibodies at comparable levels to those induced following immunization with AdOprF, but immunization with AdOprF.RGD.Epi8 was associated with increased CD4 and CD8 gamma interferon T-cell responses against OprF as well as increased survival against lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeat administration of AdOprF.RGD.Epi8 resulted in boosting of the humoral anti-OprF response as well as increased protection, whereas no boosting could be achieved with repeat administration of AdOprF. This suggests that the capsid-modified AdOprF.RGD.Epi8 vector is a more effective immunogen compared to a comparable wild-type Ad capsid, making it a good candidate for an anti-P. aeruginosa vaccine.

Ablation of tumor-derived stem cells transplanted to the central nervous system by genetic modification of embryonic stem cells with a suicide gene.

Embryonic stem cell (ESC)-based therapies open new possibilities as regenerative medicine for the treatment of human disease, but the presence of small numbers of undifferentiated ESCs within the transplant could lead to the development of tumors. The safety of ESC transplants would be enhanced if uncontrolled cell growth could be suppressed, using external stimuli. A lentiviral vector carrying the herpes simplex virus thymidine kinase (HSVtk) and green fluorescent protein (GFP) genes was used to genetically modify murine ESCs (HSVtk+GFP+ ESCs). In the presence of ganciclovir (GCV), 100% of HSVtk+GFP+ ESCs were killed in vitro, and 100% of flank tumors derived from HSVtk+GFP+ ESCs were eliminated. When CNS tumors were produced by the HSVtk+GFP+ ESCs, the tumor mass was completely eliminated on GCV treatment for 1 week. After GCV treatment for 3 weeks, histologic analysis showed no residual tumor cells and TaqMan realtime polymerase chain reaction analysis showed no genomic HSVtk copies or HSVtk mRNA. These data demonstrate that it is possible to use ex vivo gene transfer to modify ESCs with conditional genetic elements that can be activated in vivo to control undifferentiated ESC outgrowth and to eliminate transduced ESCs that have escaped growth control after ESC-mediated therapy to the CNS.

Frataxin deficiency in pancreatic islets causes diabetes due to loss of beta cell mass.

Diabetes is caused by an absolute (type 1) or relative (type 2) deficiency of insulin-producing beta cells. We have disrupted expression of the mitochondrial protein frataxin selectively in pancreatic beta cells. Mice were born healthy but subsequently developed impaired glucose tolerance progressing to overt diabetes mellitus. These observations were explained by impairment of insulin secretion due to a loss of beta cell mass in knockout animals. This phenotype was preceded by elevated levels of reactive oxygen species in knockout islets, an increased frequency of apoptosis, and a decreased number of proliferating beta cells. Hence, disruption of the frataxin gene in pancreatic beta cells causes diabetes following cellular growth arrest and apoptosis, paralleled by an increase in reactive oxygen species in islets. These observations might provide insight into the deterioration of beta cell function observed in different subtypes of diabetes in humans.