Pulmonary and Allergy Copyright (c) 2013 University of Massachusetts Medical School All rights reserved. http://escholarship.umassmed.edu/peds_pulmonary Recent documents in Pulmonary and Allergy en-us Wed, 13 Feb 2013 18:04:38 PST 3600 Production and discovery of novel recombinant adeno-associated viral vectors http://escholarship.umassmed.edu/peds_pulmonary/72 http://escholarship.umassmed.edu/peds_pulmonary/72 Tue, 14 Aug 2012 06:21:33 PDT In this unit, we describe the detailed procedure for a three-plasmid transfection method for rAAV production, and discuss its advantages, limitations, and troubleshooting techniques. We further discuss the rAAV purification process using CsCl gradients, as well as subsequent quality control methods using SDS-PAGE and real-time PCR to assess vector purity, packaging efficiency, and viral titer. Finally, we elaborate on a PCR-based strategy that can be used to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer-generation rAAVs with a greater diversity of tissue tropism for clinical gene therapy. Curr. Protoc. Microbiol. 26:14D.1.1-14D.1.21. © 2012 by John Wiley & Sons, Inc.

]]> Christian Mueller et al. Dependovirus Genetic Vectors Gene Therapy Gene transfer in the lung using recombinant adeno-associated virus http://escholarship.umassmed.edu/peds_pulmonary/71 http://escholarship.umassmed.edu/peds_pulmonary/71 Tue, 14 Aug 2012 06:21:31 PDT Adeno-associated virus (AAV) is a small replication-deficient DNA virus belonging to the Parvovirinae family. It has a single-stranded ∼4.7-kb genome. Recombinant AAV (rAAV) is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. The short viral inverted terminal repeats must remain intact for replication and packaging in production, as well as vector genome processing and persistence in the transduction process. The AAV capsid (serotype) determines the tissue tropism of the rAAV vector. In this unit we will discuss serotype selection for lung targeting along with the factors effecting efficient delivery of rAAV vectors to the murine lung. Detailed procedures for lung delivery (intranasal, orotracheal, and surgical tracheal injection), sample collection, and post-mortem tissue processing will be described. Curr. Protoc. Microbiol. 26:14D.2.1-14D.2.17. © 2012 by John Wiley & Sons, Inc.

]]> Alisha Gruntman et al. Dependovirus Genetic Vectors Gene Therapy Transgenes Gene Transfer Techniques Lung A patient with asthma seeks medical advice http://escholarship.umassmed.edu/peds_pulmonary/70 http://escholarship.umassmed.edu/peds_pulmonary/70 Wed, 20 Jun 2012 12:24:28 PDT Comment on A patient with asthma seeks medical advice in 1828, 1928, and 2012. [N Engl J Med. 2012]

]]> Brian P. O'Sullivan et al. Anti-Asthmatic Agents Asthma Female Humans Long-term, efficient inhibition of microRNA function in mice using rAAV vectors http://escholarship.umassmed.edu/peds_pulmonary/69 http://escholarship.umassmed.edu/peds_pulmonary/69 Mon, 30 Apr 2012 11:14:58 PDT Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA 'tough decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuDs depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122 TuD but not anti-let-7 TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High-throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuDs induced miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.

]]> Jun Xie et al. Dependovirus Genetic Vectors MicroRNAs Dyslipidemias A multi-center controlled trial of growth hormone treatment in children with cystic fibrosis http://escholarship.umassmed.edu/peds_pulmonary/68 http://escholarship.umassmed.edu/peds_pulmonary/68 Mon, 30 Apr 2012 10:39:03 PDT OBJECTIVES: We evaluated safety and efficacy of recombinant human growth hormone (rhGH) for improving growth, lean body mass (LBM), pulmonary function, and exercise tolerance in children with cystic fibrosis (CF) and growth restriction.

STUDY DESIGN: Multicenter, open-label, controlled clinical trial comparing outcomes in prepubertal childrenCF, randomized in a 1:1 ratio to receive daily rhGH (Nutropin AQ) or no treatment (control) for 12 months, followed by a 6-month observation (month 18). Safety was monitored at each visit, including assessments of glucose tolerance.

RESULTS: Sixty-eight subjects were randomized (control n = 32; rhGH n = 36). Mean height standard deviation score (SDS) in the rhGH group increased by 0.5 ± 0.4 at 12 months (mean ± SD, P < 0.001); the control group height SDS remained unchanged. Weight increased by 3.8 ± 1.8 versus 2.8 ± 1.5 kg, (mean ± SD, P = 0.0356) and LBM increased by 3.8 ± 1.8 versus 2.1 ± 1.4 kg (P = 0.0002) in the rhGH group versus controls, respectively. Forced vital capacity increased by 325 ± 319 in the rhGH group compared with 178 ± 152 ml in controls (mean ± SD, P = 0.032). Forced expiratory volume in 1 sec improved in both groups with a significant difference between groups after adjustment for baseline severity (LS mean ± SE: rhGH, 224 ± 37, vs. controls, 108 ± 40 ml; P = 0.04). There was no difference between groups in exercise tolerance (6-min walk distance) at 1 year. Changes in glucose tolerance for the two groups were similar over the 12-month study period, with three subjects developing IGT and one CFRD in each group. One rhGH-treated patient developed increased intracranial pressure.

CONCLUSIONS: Treatment with rhGH in prepubertal children with CF was effective in promoting growth, weight, LBM, lung volume, and lung flows, and had an acceptable safety profile.

]]> Michael S. Stalvey et al. Cystic Fibrosis Human Growth Hormone Cystic fibrosis transmembrane conductance regulator deficiency exacerbates islet cell dysfunction after beta-cell injury http://escholarship.umassmed.edu/peds_pulmonary/67 http://escholarship.umassmed.edu/peds_pulmonary/67 Mon, 05 Mar 2012 13:28:06 PST The cause of cystic fibrosis-related diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTR(-/-)), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced beta-cell injury. Fasting blood glucose levels were similar among age-matched non-STZ-administered animals, but they were significantly higher in CFTR(-/-) mice 4 weeks after STZ administration (288.4 +/- 97.4, 168.4 +/- 35.9, and 188.0 +/- 42.3 mg/dl for CFTR(-/-), C57BL/6J, and FVB/NJ, respectively; P < 0.05). After intraperitoneal glucose administration, elevated blood glucose levels were also observed in STZ-administered CFTR(-/-) mice. STZ reduced islets among all strains; however, only CFTR(-/-) mice demonstrated a negative correlation between islet number and fasting blood glucose (P = 0.02). To determine whether a second alteration associated with cystic fibrosis (i.e., airway inflammation) could impact glucose control, animals were challenged with Aspergillus fumigatus. The A. fumigatus-sensitized CFTR(-/-) mice demonstrated similar fasting and stimulated glucose responses in comparison to nonsensitized animals. These studies suggest metabolic derangements in CFRD originate from an islet dysfunction inherent to the CFTR(-/-) state.

]]> Michael S. Stalvey et al. Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Diabetes Mellitus, Experimental Inflammation Islets of Langerhans RNA, Messenger Reverse Transcriptase Polymerase Chain Reaction Enhanced IgE allergic response to Aspergillus fumigatus in CFTR-/- mice http://escholarship.umassmed.edu/peds_pulmonary/66 http://escholarship.umassmed.edu/peds_pulmonary/66 Mon, 05 Mar 2012 13:28:03 PST To gain insight into aberrant cytokine regulation in cystic fibrosis (CF), we compared the phenotypic manifestations of allergen challenge in gut-corrected CFTR-deficient mice with background-matched C57Bl6 (B6) mice. Aspergillus fumigatus (Af) antigen was used to mimic allergic bronchopulmonary aspergillosis, a peculiar hyper-IgE syndrome with a high prevalence in CF patients. CFTR-/-, C57BL/6 and FVB/NJ mice were sensitized with Af antigen by serial intraperitoneal injections. Control mice were mock sensitized with PBS. Challenges were performed by inhalation of Af antigen aerosol. After Af antigen challenge, histologic analysis showed goblet cell hyperplasia and lymphocytic infiltration in both strains. However, total serum IgE levels were markedly elevated in CF mice. Sensitized CF mice showed a five-fold greater IgE response to sensitization as compared with B6- and FVB-sensitized controls. Additional littermate controls to fully normalize for B6-FVB admixture in the strain background confirmed the role of CFTR mutation in the hyper-IgE syndrome. Cytokine mRNA levels of IL-5 and GM-CSF in the bronchoalveolar lavage (BAL) fluid, and BAL cell differentials indicated that CFTR mutation caused a shift from an IL-5-predominant to an IL-4-predominant cytokine profile. This system models a very specific type of airway inflammation in CF and could provide insights into pathogenesis and treatment of the disease.

]]> Christian Mueller et al. Aspergillus fumigatus Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Enzyme-Linked Immunosorbent Assay Immunoglobulin E Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector http://escholarship.umassmed.edu/peds_pulmonary/65 http://escholarship.umassmed.edu/peds_pulmonary/65 Mon, 05 Mar 2012 13:28:00 PST Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/beta-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors.

]]> Jeffrey Sirninger et al. Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Gene Expression Gene Therapy Genetic Vectors Pseudomonas aeruginosa Effects of CFTR, interleukin-10, and Pseudomonas aeruginosa on gene expression profiles in a CF bronchial epithelial cell Line http://escholarship.umassmed.edu/peds_pulmonary/64 http://escholarship.umassmed.edu/peds_pulmonary/64 Mon, 05 Mar 2012 13:27:57 PST Mutations in CFTR lead to a complex phenotype that includes increased susceptibility to Pseudomonas infections, a functional deficiency of IL-10, and an exaggerated proinflammatory cytokine response. We examined the effects of CFTR gene correction on the gene expression profile of a CF bronchial epithelial cell line (IB3-1) and determined which CF-related gene expression changes could be reversed by IL-10 expression. We performed microarray experiments to monitor the gene expression profile of three cell lines over a time course of exposure to Pseudomonas. At baseline, we identified 843 genes with statistically different levels of expression in CFTR-corrected (S9) cells compared to the IB3-1 line or the IL-10-expressing line. K-means clustering and functional group analysis revealed a primary up-regulation of ubiquitination enzymes and TNF pathway components and a primary down-regulation of protease inhibitors and protein glycosylation enzymes in CF. Key gene expression changes were confirmed by real-time RT-PCR. Massive reprogramming of gene expression occurred 3 h after Pseudomonas exposure. Changes specific to CF included exaggerated activation of cytokines, blunted activation of anti-proteases, and repression of protein glycosylation enzymes. In conclusion, the CFTR genotype changes the expression of multiple genes at baseline and in response to bacterial challenge, and only a subset of these changes is secondary to IL-10 deficiency.

]]> Isabel Virella-Lowell et al. Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Gene Expression Profiling Interleukin-10 Pseudomonas aeruginosa Using hyperpolarized 3He MRI to evaluate treatment efficacy in cystic fibrosis patients http://escholarship.umassmed.edu/peds_pulmonary/63 http://escholarship.umassmed.edu/peds_pulmonary/63 Tue, 17 Jan 2012 11:54:58 PST PURPOSE: To use hyperpolarized (HP) (3)He MR imaging to assess functional lung ventilation in subjects with cystic fibrosis (CF) before and after treatment.

MATERIALS AND METHODS: We performed HP (3)He static ventilation MRI scans on three subjects, using a Philips 3.0 Tesla (T) Achieva MRI scanner, before and after 11 days of in-patient treatment with combined intravenous and inhaled therapies for pulmonary exacerbations of CF. We also collected spirometry data. We quantified pulmonary ventilation volume measured with HP (3)He MRI using an advanced semi-automated analysis technique.

RESULTS: Following 11 days of treatment with intravenous antibiotics, hypertonic saline, and rhDNase, HP (3)He MR images in one subject displayed a 25% increase in total ventilation volume. Total ventilation volume in the other two subjects slightly decreased. All three subjects showed increases in FEV(1) and FVC following treatment.

CONCLUSION: In all subjects, the HP (3)He MR images provided detailed information on precisely where in the lungs gas was reaching. These data provide additional support for the conclusion that HP noble gas MRI can be a powerful tool for evaluating lung ventilation in patients with cystic fibrosis, but also raise important questions about the correlation between spirometry and HP gas MRI measurements.

]]> Yanping Sun et al. Cystic Fibrosis Magnetic Resonance Imaging No immune responses by the expression of the yeast Ndi1 protein in rats http://escholarship.umassmed.edu/peds_pulmonary/62 http://escholarship.umassmed.edu/peds_pulmonary/62 Tue, 17 Jan 2012 11:54:56 PST BACKGROUND: The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the host's immune system.

METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol.

CONCLUSIONS/SIGNIFICANCE: We demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign proteins that are directed to subcellular organelle such as mitochondria.

]]> Mathieu Marella et al. Saccharomyces cerevisiae Proteins Electron Transport Complex I Immunity Gene Therapy Induction of Group IVC Phospholipase A2 in Allergic Asthma: Transcriptional Regulation by TNF-α in Bronchoepithelial Cells http://escholarship.umassmed.edu/peds_pulmonary/61 http://escholarship.umassmed.edu/peds_pulmonary/61 Tue, 17 Jan 2012 08:15:43 PST Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus, Aspergillus fumigatus (Af), in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC phospholipase A2 (cPLA2γ or PLA2G4C). Our results infer that Af extract can induce cPLA2γ levels directly in eosinophils while induction in lung epithelial cells is most likely a consequence of TNF-α secretion by Af-activated macrophages. The mechanism of TNF-α-dependent induction of cPLA2γ gene expression was elucidated through a combination of promoter deletions, ChIP and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE, NF-κB and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, c-Jun/ATF-2, p65/p65 and USF1/USF2 complexes are recruited to the cPLA2γ enhancer/promoter in response to TNF-α with overexpression and dominant negative studies implying a strong level of cooperation and interplay between these factors. Overall, our data link cytokine-mediated alterations in cPLA2γ gene expression with allergic asthma and outline a complex regulatory mechanism.

]]> Justin S. Bickford et al. Aspergillus fumigatus Asthma Group IV Phospholipases A2 Tumor Necrosis Factor-alpha The role of gene and cell therapy in the era of health care reform http://escholarship.umassmed.edu/peds_pulmonary/60 http://escholarship.umassmed.edu/peds_pulmonary/60 Tue, 17 Jan 2012 08:15:41 PST Walter H. Ettinger et al. Gene Therapy Cell Transplantation Health Care Reform Delivery of Health Care Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results http://escholarship.umassmed.edu/peds_pulmonary/59 http://escholarship.umassmed.edu/peds_pulmonary/59 Tue, 17 Jan 2012 08:15:39 PST Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

]]> Terence R. Flotte et al. Dependovirus Gene Therapy Genetic Vectors Simplexvirus Transfection alpha 1-Antitrypsin alpha 1-Antitrypsin Deficiency Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles http://escholarship.umassmed.edu/peds_pulmonary/58 http://escholarship.umassmed.edu/peds_pulmonary/58 Tue, 17 Jan 2012 08:15:37 PST Alpha-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

]]> Christian Mueller et al. Dependovirus Genetic Vectors MicroRNAs Transgenes alpha 1-Antitrypsin Deficiency Liver Diseases Neurodegenerative Diseases Lack of cystic fibrosis transmembrane conductance regulator in CD3+ lymphocytes leads to aberrant cytokine secretion and hyperinflammatory adaptive immune responses http://escholarship.umassmed.edu/peds_pulmonary/57 http://escholarship.umassmed.edu/peds_pulmonary/57 Fri, 13 Jan 2012 13:14:03 PST Cystic fibrosis (CF), the most common fatal monogenic disease in the United States, results from mutations in CF transmembrane conductance regulator (CFTR), a chloride channel. The mechanisms by which CFTR mutations cause lung disease in CF are not fully defined but may include altered ion and water transport across the airway epithelium and aberrant inflammatory and immune responses to pathogens within the airways. We have shown that Cftr(-/-) mice mount an exaggerated IgE response toward Aspergillus fumigatus, with higher levels of IL-13 and IL-4, mimicking both the T helper cell type 2-biased immune responses seen in patients with CF. Herein, we demonstrate that these aberrations are primarily due to Cftr deficiency in lymphocytes rather than in the epithelium. Adoptive transfer experiments with CF splenocytes confer a higher IgE response to Aspergillus fumigatus compared with hosts receiving wild-type splenocytes. The predilection of Cftr-deficient lymphocytes to mount T helper cell type 2 responses with high IL-13 and IL-4 was confirmed by in vitro antigen recall experiments. Conclusive data on this phenomenon were obtained with conditional Cftr knockout mice, where mice lacking Cftr in T cell lineages developed higher IgE than their wild-type control littermates. Further analysis of Cftr-deficient lymphocytes revealed an enhanced intracellular Ca(2+) flux in response to T cell receptor activation. This was accompanied by an increase in nuclear localization of the calcium-sensitive transcription factor, nuclear factor of activated T cell, which could drive the IL-13 response. In summary, our data identified that CFTR dysfunction in T cells can lead directly to aberrant immune responses. These findings implicate the lymphocyte population as a potentially important target for CF therapeutics.

]]> Christian Mueller et al. Animals Antigens, CD3 Aspergillus Calcium Cystic Fibrosis Transmembrane Conductance Regulator Cytokines Hypersensitivity Immune System Immunoglobulin E Lymphocytes Mice Mice, Inbred C57BL Mice, Knockout Mice, Transgenic Mutation NFATC Transcription Factors Spleen Asthma http://escholarship.umassmed.edu/peds_pulmonary/56 http://escholarship.umassmed.edu/peds_pulmonary/56 Fri, 13 Jan 2012 13:14:01 PST Chapter opening: Asthma is a disease characterized by (1) reversible airway obstruction; (2) airway inflammation; and (3) increased airway responsiveness to a variety of stimuli (hyperreactive airways). It is estimated that 10 million persons in the United States have asthma and that 5–10% of children suffer from this disease. The prevalence of asthma may be even greater if children with mild symptoms such as chronic cough, recurrent croup, and exercise-induced asthma (EIA) are included.

Citation: O'Sullivan BP. Asthma. In: "Manual of Clinical Problems in Pediatrics", Roberts K, ed. Little, Brown and Co., Boston; 4th edition, pg.275-81; 1995 and 5th edition, pg.271-77; 2001.

]]> Brian P. O'Sullivan Asthma Cystic Fibrosis: Diagnosis, Sweat Testing, and Newborn Screening http://escholarship.umassmed.edu/peds_pulmonary/55 http://escholarship.umassmed.edu/peds_pulmonary/55 Fri, 13 Jan 2012 12:44:15 PST Chapter opening: Making the diagnosis of cystic fibrosis (CF) has lifelong implications for affected individuals and their families, for both medical and insurance reasons. Therefore, it is imperative that the clinician approach this problem with sensitivity and seriousness of purpose ...

Citation: O’Sullivan B. Cystic Fibrosis: Diagnosis, Sweat Testing, and Newborn Screening. Lung Biology in Health and Disease 2010;242:90-102.

]]> Brian P. O'Sullivan Cystic Fibrosis Pulmonary Manifestations of Systemic Vasculitis http://escholarship.umassmed.edu/peds_pulmonary/54 http://escholarship.umassmed.edu/peds_pulmonary/54 Fri, 13 Jan 2012 10:25:52 PST Summary: The pulmonary vasculitides are a heterogeneous group of primary or secondary diseases, in which there is inflammation that may lead to progressive destruction of the pulmonary microvasculature. Pulmonary involvement may develop because the lung has an extensive vascular and microvascular network, sensitizing antigens caneasily reach the lung, and there are large numbers of vasoactive and activated immune cells in the lung. Primary or isolated pulmonary vasculitis is extremely rare in children. Pulmonary vasculitis is an unusual condition in children and is almost always seen in conjunction with a systemic vasculitis syndrome. This chapter reviews common systemic vasculitides associated with pulmonary disease and discusses presentation, diagnosis, and therapy.

Citation: O’Sullivan BP and Kremer T: Pulmonary Manifestations of Systemic Vasculitis. Chapter 11. In Eds: Turcios N and Fink RJ: Pulmonary Manifestations of Pediatric Diseases. Philadelphia, PA: Saunders/Elsevier, 2009, pp 241-255.

]]> Brian P. O'Sullivan et al. Systemic Vasculitis Lung Diseases IL-10 delivery by AAV5 vector attenuates inflammation in mice with Pseudomonas pneumonia http://escholarship.umassmed.edu/peds_pulmonary/53 http://escholarship.umassmed.edu/peds_pulmonary/53 Wed, 11 Jan 2012 11:31:49 PST Lung infections with Pseudomonas aeruginosa and other pathogens in cystic fibrosis (CF) cause progressive airway obstruction and tissue damage, the predominant cause of morbidity and mortality in CF. We investigated whether a recombinant adeno-associated virus type 5 (AAV5) vector expressing murine interleukin (IL)-10 (AAV5.Cbeta-mIL-10), a regulatory/anti-inflammatory cytokine, could decrease airway inflammation in IL-10 knockout mice chronically infected with mucoid P. aeruginosa. Mice that received AAV5.Cbeta-mIL10 through intratracheal inoculation produced IL-10 at an average of 25 000 pg/ml in the epithelial lining fluid (ELF) and 12 000 pg/g-lung tissue 6 weeks post-vector delivery, significantly higher levels than in placebo-treated mice. At 3 days post-infection, proinflammatory cytokines (IL-1beta, tumor necrosis factor (TNF)-alpha, macrophage inhibitory protein (MIP)-1alpha and (KC) in the ELF and lung homogenate were decreased (1-9 folds) in the AAV5.Cbeta-mIL10-treated mice accompanied by less pronounced and more localized neutrophil infiltration in lung sections, when compared with placebo-treated mice. These results suggest that AAV5.Cbeta-mIL10 induces IL-10 levels in the lungs mediating a significant anti-inflammatory response and making AAV-IL-10 gene transfer a potentially useful therapy in the treatment of CF lung disease.

]]> S. M. Buff et al. Animals Cystic Fibrosis Dependovirus Gene Therapy Genetic Vectors Interleukin-10 Intubation, Intratracheal Mice Mice, Knockout Neutrophils Pneumonia, Bacterial Pseudomonas Infections *Pseudomonas aeruginosa