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1.  Viral metagenomics reveal blooms of anelloviruses in the respiratory tract of lung transplant recipients 
Few studies have examined the lung virome in health and disease. Outcomes of lung transplantation are known to be influenced by several recognized respiratory viruses, but global understanding of the virome of the transplanted lung is incomplete. To define the DNA virome within the respiratory tract following lung transplantation we carried out metagenomic analysis of allograft bronchoalveolar lavage (BAL), and compared to healthy and HIV+ subjects. Viral concentrates were purified from BAL and analyzed by shotgun DNA sequencing. All of the BAL samples contained reads mapping to anelloviruses, with high proportions in lung transplant samples. Anellovirus populations in transplant recipients were complex, with multiple concurrent variants. Q-PCR quantification revealed that anellovirus sequences were 56-fold more abundant in BAL from lung transplant recipients compared with healthy controls or HIV+ subjects (p<0.0001). Anellovirus sequences were also more abundant in upper respiratory tract specimens from lung transplant recipients than controls (p=0.006). Comparison to metagenomic data on bacterial populations showed that high anellovirus loads correlated with dysbiotic bacterial communities in allograft BAL (p=0.00816). Thus the respiratory tracts of lung transplant recipients contain high levels and complex populations of anelloviruses, warranting studies of anellovirus lung infection and transplant outcome.
PMCID: PMC4276431  PMID: 25403800
2.  Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing 
Genome Biology  2014;15(10):487.
Fungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources.
Here, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which are routinely acquired during DNA library preparation, to annotate output sequence data, and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure.
We find increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in subjects with increasingly severe pulmonary and immunologic deficits. We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci, a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0487-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4232682  PMID: 25344286
3.  T cells expressing chimeric antigen receptors can cause anaphylaxis in humans 
T cells can be redirected to overcome tolerance to cancer by engineering with integrating vectors to express a chimeric antigen receptor (CAR). In preclinical models, we have previously demonstrated that transfection of T cells with messenger RNA (mRNA) coding for a CAR is an alternative strategy that has antitumor efficacy and the potential to evaluate the on-target off-tumor toxicity of new CAR targets safely due to transient mRNA CAR expression. Here, we report the safety observed in four patients treated with autologous T cells that had been electroporated with mRNA coding for a CAR derived from a murine antibody to human mesothelin. Due to the transient nature of CAR expression on the T cells, subjects in the clinical study were given repeated infusions of the CAR-T cells in order to assess their safety. One subject developed anaphylaxis and cardiac arrest within minutes of completing the 3rd infusion. Although human anti-mouse IgG antibodies have been known to develop with CAR-transduced T cells, they have been thought to have no adverse clinical consequences. This is the first description of clinical anaphylaxis resulting from CAR-modified T cells, most likely through IgE antibodies specific to the CAR. These results indicate that the potential immunogenicity of CARs derived from murine antibodies may be a safety issue for mRNA CARs, especially when administered using an intermittent dosing schedule.
PMCID: PMC3888798  PMID: 24432303
4.  Malignant Pleural Mesothelioma: Update on Treatment Options with a Focus on Novel Therapies 
Clinics in chest medicine  2013;34(1):99-111.
PMCID: PMC3612173  PMID: 23411061
malignant pleural mesothelioma; radiation therapy; chemotherapy; multimodal therapy; gene therapy
5.  Effectiveness of high dose remifentanil in preventing coughing and laryngospasm in non-paralyzed patients for advanced bronchoscopic procedures 
Annals of Thoracic Medicine  2014;9(1):23-28.
Anesthesia for bronchoscopy presents unique challenges, as constant stimulus due to bronchoscope needs to be obtunded using drugs with a minimal post-procedure residual effect. Remifentanil for maintenance is an ideal choice, but optimal doses are yet to be determined.
Bronchoscopic procedures were prospectively evaluated for 4 months studying the frequency of complications and anesthesia techniques. Anesthesia was maintained on remifentanil/propofol infusion avoiding neuromuscular blockers. Laryngeal mask airway was used for the controlled ventilation (with high oxygen concentration) that also served as a conduit for bronchoscope insertions. Anesthesiologists were blinded to the study (avoiding performance bias) and the Pulmonologist was blinded to the anesthesia technique (to document unbiased procedural satisfaction scores). Procedures were divided into 2 groups based on the dose of remifentanil used for maintenance: Group-H (high dose −0.26 to 0.5 μg/kg/min and Group-NH (non-high dose ≤0.25 μg/kg/min).
Observed 75 procedures were divided into Group-H (42) and Group-NH (33). Number of statistical difference was found in demography, procedural profile, hemodynamic parameters and total phenylephrine used. Chi-square test showed Group-NH had significantly higher frequency of laryngospasm (P = 0.047) and coughing (P = 0.002). The likelihood ratio of patient coughing and developing laryngospasm in Group-NH was found to be 4.56 and 10.97 times respectively. Minimum pulse-oximeter saturation was statistically higher in Group-H (98.80% vs. 96.50% P = 0.009). Pulmonologist satisfaction scores were significantly better in Group-H.
High dose of remifentanil infusion is associated with a lower incidence of coughing and laryngospasms during bronchoscopy. Simultaneously, it improves Pulmonologist's satisfaction and procedural conditions.
PMCID: PMC3912682  PMID: 24551014
High dose remifentanil; laryngospasm during bronchoscopy; remifentanil for bronchoscopy
6.  Lung-enriched Organisms and Aberrant Bacterial and Fungal Respiratory Microbiota after Lung Transplant 
Rationale: Long-term survival after lung transplantation is limited by infectious complications and by bronchiolitis obliterans syndrome (BOS), a form of chronic rejection linked in part to microbial triggers.
Objectives: To define microbial populations in the respiratory tract of transplant patients comprehensively using unbiased high-density sequencing.
Methods: Lung was sampled by bronchoalveolar lavage (BAL) and upper respiratory tract by oropharyngeal wash (OW). Bacterial 16S rDNA and fungal internal transcribed spacer sequencing was used to profile organisms present. Outlier analysis plots defining taxa enriched in lung relative to OW were used to identify bacteria enriched in lung against a background of oropharyngeal carryover.
Measurements and Main Results: Lung transplant recipients had higher bacterial burden in BAL than control subjects, frequent appearance of dominant organisms, greater distance between communities in BAL and OW indicating more distinct populations, and decreased respiratory tract microbial richness and diversity. Fungal populations were typically dominated by Candida in both sites or by Aspergillus in BAL but not OW. 16S outlier analysis identified lung-enriched taxa indicating bacteria replicating in the lower respiratory tract. In some cases this confirmed respiratory cultures but in others revealed enrichment by anaerobic organisms or mixed outgrowth of upper respiratory flora and provided quantitative data on relative abundances of bacteria found by culture.
Conclusions: Respiratory tract microbial communities in lung transplant recipients differ in structure and composition from healthy subjects. Outlier analysis can identify specific bacteria replicating in lung. These findings provide novel approaches to address the relationship between microbial communities and transplant outcome and aid in assessing lung infections.
PMCID: PMC3480531  PMID: 22798321
microbiome; 16S; ITS; bronchiolitis obliterans syndrome
7.  A Trial of Intrapleural Adenoviral-mediated Interferon-α2b Gene Transfer for Malignant Pleural Mesothelioma 
New therapeutic strategies are needed for malignant pleural mesothelioma (MPM). We conducted a single-center, open-label, nonrandomized, pilot and feasibility trial using two intrapleural doses of an adenoviral vector encoding human IFN-α (Ad.IFN-α2b). Nine subjects were enrolled at two dose levels. The first three subjects had very high pleural and systemic IFN-α concentrations resulting in severe “flu-like” symptoms necessitating dose de-escalation. The next six patients had reduced (but still significant) pleural and serum IFN-α levels, but with tolerable symptoms. Repeated vector administration appeared to prolong IFN-α expression levels. Anti-tumor humoral immune responses against mesothelioma cell lines were seen in seven of the eight subjects evaluated. No clinical responses were seen in the four subjects with advanced disease. However, evidence of disease stability or tumor regression was seen in the remaining five patients, including one dramatic example of partial tumor regression at sites not in contiguity with vector infusion. These data show that Ad.IFN-α2b has potential therapeutic benefit in MPM and that it generates anti-tumor immune responses that may induce anatomic and/or metabolic reductions in distant tumor.
Clinical trial registered with (NCT 01212367).
PMCID: PMC3262033  PMID: 21642245
clinical trials; immunotherapy; gene therapy
Clinics in chest medicine  2011;32(4):865-885.
Both advanced stage lung cancer and malignant pleural mesothelioma are associated with a poor prognosis. Although there have been advances in treatment regimens for both diseases, these have had only a modest effect on their progressive course. Gene therapy for thoracic malignancies represents a novel therapeutic approach and has been evaluated in a number of clinical trials over the last two decades. Strategies have included induction of apoptosis, tumor suppressor gene replacement, suicide gene expression, cytokine based therapy, various vaccination approaches, and adoptive transfer of modified immune cells. This review will consider the clinical results, limitations, and future directions of gene therapy trials for thoracic malignancies.
PMCID: PMC3210443  PMID: 22054892
Gene Therapy; Immunotherapy; Lung cancer; Mesothelioma
9.  Topographical Continuity of Bacterial Populations in the Healthy Human Respiratory Tract 
Rationale: Defining the biogeography of bacterial populations in human body habitats is a high priority for understanding microbial–host relationships in health and disease. The healthy lung was traditionally considered sterile, but this notion has been challenged by emerging molecular approaches that enable comprehensive examination of microbial communities. However, studies of the lung are challenging due to difficulties in working with low biomass samples.
Objectives: Our goal was to use molecular methods to define the bacterial microbiota present in the lungs of healthy individuals and assess its relationship to upper airway populations.
Methods: We sampled respiratory flora intensively at multiple sites in six healthy individuals. The upper tract was sampled by oral wash and oro-/nasopharyngeal swabs. Two bronchoscopes were used to collect samples up to the glottis, followed by serial bronchoalveolar lavage and lower airway protected brush. Bacterial abundance and composition were analyzed by 16S rDNA Q-PCR and deep sequencing.
Measurements and Main Results: Bacterial communities from the lung displayed composition indistinguishable from the upper airways, but were 2 to 4 logs lower in biomass. Lung-specific sequences were rare and not shared among individuals. There was no unique lung microbiome.
Conclusions: In contrast to other organ systems, the respiratory tract harbors a homogenous microbiota that decreases in biomass from upper to lower tract. The healthy lung does not contain a consistent distinct microbiome, but instead contains low levels of bacterial sequences largely indistinguishable from upper respiratory flora. These findings establish baseline data for healthy subjects and sampling approaches for sequence-based analysis of diseases.
PMCID: PMC3208663  PMID: 21680950
healthy lung colonization; microbiome; 16S rDNA; pyrosequencing
10.  Advances in Diagnostic Bronchoscopy 
Diagnostic bronchoscopy has undergone two major paradigm shifts in the last 40 years. First, the advent of flexible bronchoscopy gave chest physicians improved access to the tracheobronchial tree with a rapid learning curve and greater patient comfort compared with rigid bronchoscopy. The second paradigm shift has evolved over the last 5 years with the proliferation of new technologies that have significantly enhanced the diagnostic capabilities of flexible bronchoscopy compared with traditional methods. At the forefront of these new technologies is endobronchial ultrasound. In its various forms, endobronchial ultrasound has improved diagnostic yield for pulmonary masses, nodules, intrathoracic adenopathy, and disease extent, thereby reducing the need for more invasive surgical interventions. Various navigational bronchoscopy systems have become available to increase flexible bronchoscope access to small peripheral pulmonary lesions. Furthermore, various modalities of airway assessment, including optical microscopic imaging technologies, may play significant roles in the diagnosis of a variety of pulmonary diseases in the future. Finally, the combination of new diagnostic bronchoscopy technologies and novel approaches in molecular analysis and biomarker assessment hold promise for enhanced diagnosis and personalized management of many pulmonary disorders. In this review, we provide a contemporary review of diagnostic bronchoscopy developments over the past decade.
PMCID: PMC3159074  PMID: 20378726
endobronchial ultrasound; lung cancer; autofluorescence; tomography; confocal
11.  Systemic Blockade of Transforming Growth Factor-β (TGF-β) Signaling Augments the Efficacy of Immunogene Therapy 
Cancer research  2008;68(24):10247-10256.
Locally-produced TGF-β promotes tumor-induced immunosuppression and contributes to resistance to immunotherapy. This paper explores the potential for increased efficacy when combining immunotherapies with TGF-β suppression using the TGF-β type I receptor kinase inhibitor, SM16. Adenovirus expressing IFNβ (Ad.IFNβ) was injected intratumorally once in established subcutaneous AB12 (mesothelioma) and LKR (lung cancer) tumors or intratracheally in a K-ras orthotopic lung tumor model. Mice bearing TC1 (lung cancer) tumors were vaccinated with two injections of adenovirus expressing HPV-E7 (Ad.E7). SM16 was administered orally in formulated chow. Tumor growth was assessed and cytokine-expression and cell populations were measured in tumors and spleens by real time-PCR and flow cytometry. SM16 potentiated the efficacy of both immunotherapies in each of the models and caused changes in the tumor microenvironment. The combination of SM16 and Ad.INFβ increased the number of intratumoral leukocytes (including macrophages, NK cells, and CD8+ cells) and increased the percentage of T-cells expressing the activation marker CD25. SM16 also augmented the anti-tumor effects of Ad.E7 in the TC1 flank tumor model. The combination did not increase HPV-E7 tetramer-positive CD8+ T cells in the spleens, but did induce a marked increase in the tumors. Tumors from SM16-treated mice showed increased mRNA and protein for immunostimulatory cytokines and chemokines, as well as endothelial adhesion molecules, suggesting a mechanism for the increased intratumoral leukocyte trafficking. Blockade of the TGF-β signaling pathway augments the anti-tumor effects of Ad.INFβ immune-activating or Ad.E7 vaccination therapy. The addition of TGF-β blocking agents in clinical trials of immunotherapies may increase efficacy.
PMCID: PMC2637471  PMID: 19074893
tumor immunology; immunosuppression; TGFβ; tumor associated macrophages; cytokines; lung cancer; mesothelioma; tumor vaccine; interferon-β

Results 1-11 (11)