Carlos Milla

All Publications

• Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding. bioRxiv : the preprint server for biology Chen, Q., Cai, P., Chang, T. H., Burgener, E., Kratochvil, M. J., Gupta, A., Hargil, A., Secor, P. R., Nielsen, J. E., Barron, A. E., Milla, C., Heilshorn, S. C., Spakowitz, A., Bollyky, P. L. 2024

  Abstract

  Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of morbidity and mortality in patients with cystic fibrosis, necessitating treatment with inhaled antibiotics. Pf phage is a filamentous bacteriophage produced by Pa that has been reported to act as a structural element in Pa biofilms. Pf presence has been associated with resistance to antibiotics and poor outcomes in cystic fibrosis, though the underlying mechanisms are unclear. Here, we have investigated how Pf phages and sputum biopolymers impede antibiotic diffusion using human sputum samples and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf phages and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf phages in sputum reduce the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf phages and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

  View details for DOI 10.1101/2024.03.10.584330

  View details for PubMedID 38496625

• The Inovirus Pf4 Triggers Antiviral Responses and Disrupts the Proliferation of Airway Basal Epithelial Cells. Viruses Popescu, M. C., Haddock, N. L., Burgener, E. B., Rojas-Hernandez, L. S., Kaber, G., Hargil, A., Bollyky, P. L., Milla, C. E. 2024; 16 (1)

  Abstract

  BACKGROUND: The inovirus Pf4 is a lysogenic bacteriophage of Pseudomonas aeruginosa (Pa). People with Cystic Fibrosis (pwCF) experience chronic airway infection with Pa and a significant proportion have high numbers of Pf4 in their airway secretions. Given the known severe damage in the airways of Pa-infected pwCF, we hypothesized a high Pf4 burden can affect airway healing and inflammatory responses. In the airway, basal epithelial cells (BCs) are a multipotent stem cell population critical to epithelium homeostasis and repair. We sought to investigate the transcriptional responses of BCs under conditions that emulate infection with Pa and exposure to high Pf4 burden.METHODS: Primary BCs isolated from pwCF and wild-type (WT) donors were cultured in vitro and exposed to Pf4 or bacterial Lipopolysaccharide (LPS) followed by transcriptomic and functional assays.RESULTS: We found that BCs internalized Pf4 and this elicits a strong antiviral response as well as neutrophil chemokine production. Further, we found that BCs that take up Pf4 demonstrate defective migration and proliferation.CONCLUSIONS: Our findings are highly suggestive of Pf4 playing a role in the pathogenicity of Pa in the airways. These findings provide additional evidence for the ability of inoviruses to interact with mammalian cells and disrupt cell function.

  View details for DOI 10.3390/v16010165

  View details for PubMedID 38275975

• The challenges and promise of sweat sensing. Nature biotechnology Davis, N., Heikenfeld, J., Milla, C., Javey, A. 2024

  Abstract

  The potential of monitoring biomarkers in sweat for health-related applications has spurred rapid growth in the field of wearable sweat sensors over the past decade. Some of the key challenges have been addressed, including measuring sweat-secretion rate and collecting sufficient sample volumes for real-time, continuous molecular analysis without intense exercise. However, except for assessment of cystic fibrosis and regional nerve function, the ability to accurately measure analytes of interest and their physiological relevance to health metrics remain to be determined. Although sweat is not a crystal ball into every aspect of human health, we expect sweat measurements to continue making inroads into niche applications involving active sweating, such as hydration monitoring for athletes and physical laborers and later for medical and casual health monitoring of relevant drugs and hormones.

  View details for DOI 10.1038/s41587-023-02059-1

  View details for PubMedID 38212492

• Clinical and functional spectrum of RAC2-related immunodeficiency. Blood Donkó, A., Sharapova, S. O., Kabat, J., Ganesan, S., Hauck, F., Marois, L., Abbott, J., Moshous, D., Williams, K. W., Campbell, N., Martin, P. L., Lagresle-Peyrou, C., Trojan, T. D., Kuzmenko, N., Deordieva, E., Raykina, E., Abers, M. S., Abolhassani, H., Barlogis, V., Milla, C. C., Hall, G., Mousallem, T., Church, J. A., Kapoor, N., Cros, G., Chapdelaine, H., Franco-Jarava, C., Lopez-Lerma, I., Miano, M., Leiding, J. W., Klein, C., Marie José, S., Fischer, A., Hsiao, K. C., Martelius, T., Seppänen, M. R., Barmettler, S., Walter, J. E., Masmas, T. N., Mukhina, A., Falcone, E. L., Kracker, S., Shcherbina, A., Holland, S. M., Leto, T. L., Hsu, A. P. 2024

  Abstract

  Mutations in the small Rho-family GTPase, RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 RAC2 patients (23 previously reported) from 37 families. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n=5), infantile LAD-like disease (n=5), or CID (n=44). Disease correlated to RAC2 activity: Constitutively-active, RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, while dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration and visible neutrophil macropinosomes. Among 42 CID patients with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, PAK1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction ranging from early-onset severe combined immunodeficiency to later-onset combined immunodeficiencies depending on RAC2 activity.

  View details for DOI 10.1182/blood.2023022098

  View details for PubMedID 38194689

• Situs ambiguus is associated with adverse clinical outcomes in children with primary ciliary dyskinesia. Chest Kaspy, K. R., Dell, S. D., Davis, S. D., Ferkol, T. W., Rosenfeld, M., Sagel, S. D., Milla, C., Olivier, K. N., Barber, A. T., Wee, W., Lin, F. C., Li, L., Rampakakis, E., Zariwala, M. A., Knowles, M. R., Leigh, M. W., Shapiro, A. J. 2023

  Abstract

  Primary ciliary dyskinesia (PCD) is a rare disorder of motile cilia associated with situs abnormalities. At least 12% of patients with PCD have situs ambiguus (SA), including organ laterality defects falling outside normal arrangement [situs solitus (SS)] or mirror image inversion [situs inversus totalis (SIT)].Do patients with PCD and SA have worse clinical outcomes compared to those with SS or SIT?This cross-sectional, multicenter study evaluated participants ≤21 years with PCD. Participants were classified as SA, including heterotaxy, or non-SA (SS or SIT). Markers of disease severity were compared between situs groups, adjusting for age at enrollment and severe CCDC39/CCDC40 genotype, using generalized linear models, logistic and Poisson regression.In 397 participants with PCD (mean age 8.4 years, range 0.1-21), 42 were classified as SA, including 16 (38%) with complex cardiovascular malformations (CVM) or atrial isomerism, 13 (31%) with simple CVM, and 13 (31%) without CVM. Of these, 15 (36%) underwent cardiac surgery, 24 (57%) had an anatomic spleen abnormality, and 7 (17%) had both. The remaining 355 participants were non-SA, including 152 with SIT and 203 with SS. Overall, 70 (17%) participants had the severe CCDC39/CCDC40 genotype. Compared to non-SA participants, those with SA had lower median body mass index Z-scores (p=0.03), lower forced vital capacity Z-scores (p=0.01), more hospitalizations and intravenous antibiotic courses for acute respiratory infections during the 5-years before enrollment (p<0.01). Participants with CVM requiring surgery or anatomic spleen abnormalities had lower median body mass index Z-scores and more hospitalizations and intravenous therapies for respiratory illnesses compared to non-SA participants.Children with PCD and SA have worse nutritional and pulmonary outcomes with more hospitalizations for acute respiratory illnesses than those with SS or SIT combined. Poor nutrition and increased hospitalizations for respiratory infections in SA participants with PCD are associated with CVM requiring cardiac surgery and/or splenic anomalies.gov: NCT02389049, NCT00323167.

  View details for DOI 10.1016/j.chest.2023.12.005

  View details for PubMedID 38072392

• Azithromycin promotes proliferation, and inhibits inflammation in nasal epithelial cells in primary ciliary dyskinesia. Scientific reports Varenyiova, Z., Rojas-Hernandez, L. S., Spano, J., Capek, V., Rosenberg-Hasson, Y., Holmes, T., Milla, C. 2023; 13 (1): 14453

  Abstract

  Primary ciliary dyskinesia (PCD) is a genetic disorder associated with recurrent and chronic respiratory infections due to functional defects of motile cilia. In this study, we aimed to elucidate inflammatory and proliferative responses in PCD respiratory epithelium and evaluate the effect of Azithromycin (AZT) on these responses. Airway basal cells (BCs) were isolated from nasal samples of Wild-type (WT) epitope of healthy donors and PCD donors with bi-allelic mutations in DNAH5, DNAH11 and CCDC39. Cells were expanded in vitro and stimulated with either Lipopolysaccharide (LPS) or vehicle control. Post stimulation, cells were treated with either Azithromycin (AZT) or vehicle control. Cell proliferation was imaged in real-time. Separately, BCs from the same donors were expanded and grown at an air-liquid interface (ALI) to generate a multi-ciliated epithelium (MCE). Once fully mature, cells were stimulated with LPS, AZT, LPS + AZT or vehicle control. Inflammatory profiling was performed on collected media by cytokine Luminex assay. At baseline, there was a significantly higher mean production of pro-inflammatory cytokines by CCDC39 BCs and MCEs when compared to WT, DNAH11 and DNAH5 cells. AZT inhibited production of cytokines induced by LPS in PCD cells. Differences in cell proliferation were noted in PCD and this was also corrected with AZT treatment.

  View details for DOI 10.1038/s41598-023-41577-5

  View details for PubMedID 37660113

  View details for PubMedCentralID PMC10475097

• Treatment of Fetal Cystic Fibrosis With Cystic Fibrosis Transmembrane Conductance Regulator Modulation Therapy. Annals of internal medicine Blumenfeld, Y. J., Hintz, S. R., Aziz, N., Barth, R. A., Spano, J. M., El-Sayed, Y. Y., Milla, C. 2023

  View details for DOI 10.7326/L23-0112

  View details for PubMedID 37307583

• Airway Disease Progression on Chest Computed Tomography in Children With Primary Ciliary Dyskinesia Kinghorn, B., Rosenfeld, M., Caudri, D., Dell, S. D., Ferkol, T. W., Knowles, M. R., Milla, C. E., Mollica, F., Onchiri, F., Sagel, S. D., Shapiro, A. J., Pittman, J. E., Sullivan, E., Sullivan, K., Tiddens, H., Zariwala, M. A., Davis, S. D., Leigh, M. W. AMER THORACIC SOC. 2023

  View details for Web of Science ID 000995814704589

• Primary Ciliary Dyskinesia in Patients With CCNO Variants: A Case Series Despotes, K. A., Leigh, M. W., Davis, S. D., Sagel, S. D., Vece, T. J., Dell, S. D., Milla, C. E., Shapiro, A. J., Ferkol, T. W., Sullivan, K. M., Capps, N. A., Knowles, M. R., Zariwala, M. A. AMER THORACIC SOC. 2023

  View details for Web of Science ID 000995814707507

• Non-Invasive Touch-Based Lithium Monitoring Using an Organohydrogel-Based Sensing Interface ADVANCED MATERIALS TECHNOLOGIES Lin, S., Zhu, J., Yeung, J., Wu, T., Cheng, X., Zhao, Y., Wang, B., Tan, J., Peeters, S., Seroussi, A., Sankararaman, S., Milla, C., Emaminejad, S. 2023

  View details for DOI 10.1002/admt.202202141

  View details for Web of Science ID 000973263500001

• Notch signaling inactivation by small molecule gamma-secretase inhibitors restores the multiciliated cell population in the airway epithelium. American journal of physiology. Lung cellular and molecular physiology Vladar, E. K., Kunimoto, K., Rojas-Hernandez, L. S., Spano, J. M., Sellers, Z. M., Joo, N. S., Cooney, R. A., Axelrod, J. D., Milla, C. E. 2023

  Abstract

  Multiciliated cell loss is a hallmark of airway epithelial remodeling in chronic inflammatory airway diseases including cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease. It disrupts mucociliary clearance, which fuels disease progression. Effective clearance requires an optimal proportion of multiciliated and secretory cells. This is controlled by Notch signaling such that between two adjacent cells the one that activates Notch becomes a secretory cell and the one that avoids Notch activation becomes a multiciliated cell. Consequently, blocking Notch by a small molecule inhibitor of the gamma-secretase enzyme that cleaves the Notch receptor for signal activation directs differentiation towards the multiciliated lineage. Thus, gamma-secretase inhibitor (GSI) treatment may alleviate multiciliated cell loss in lung disease. Here we demonstrate therapeutic restoration of multiciliated cells by the GSI LY450139 (semagacestat). LY450139 increased multiciliated cell numbers in a dose-dependent manner in healthy primary human nasal epithelial cells (HNECs) during differentiation and in mature cultures, but not when applied during early epithelialization of progenitors. LY450139 did not impact stem cell proliferation. Basal and apical administration were equally effective. In healthy adult mice, LY450139 increased multiciliated cell numbers without detectible toxicity. LY450139 also increased multiciliated cells and decreased excess mucus secretory cells in CF HNECs and IL-13 remodeled healthy HNECs. LY450139 normalized multiciliated cell numbers in CF HNECs without interfering with the activity of CFTR modulator compounds. In sum, we demonstrate that GSI administration is a promising therapeutic to restore multiciliated cells and potentially improve epithelial function in a wide range of chronic lung diseases.

  View details for DOI 10.1152/ajplung.00382.2022

  View details for PubMedID 37039381

• The Danish-American Research Exchange (DARE): a cross-sectional study of a binational research education program. BMC medical education Mehta, K. M., Petersen, K. L., Goodman, S., Sorensen, H. T., Bogsted, M., Olesen, J. D., Burks, S., Shaw, R. E., Hove, J. D., Ousager, J., Milla, C., Andersen, V., Ejskjar, N., Brix-Christensen, V., Ghose, S., Kjar, A., Chin-Hong, P. V. 2023; 23 (1): 96

  Abstract

  BACKGROUND: Most medical educational programs emphasize clinical observation or clinical skill acquisition, fewer focus upon research. The Danish-American Research Exchange (DARE) program, sponsored by the Lundbeck Foundation, is unique in that the medical student initiates biomedical research collaboration between Danish and US medical institutions. To achieve this, Danish medical students (DARE students) conduct binational mentored research projects while based in the United States for 10months. In addition, DARE students are introduced to interdisciplinary thinking about how to develop ultra-low-cost healthcare interventions through the '$10 Challenge'.METHODS: We conducted a cross-sectional study of DARE alumni over five consecutive years (2015-2020, n=24). Research metrics included completion of a research project, primary authorship, and co-authorship of publications. The number of publications, prior to and after the DARE program were enumerated. For the first four cohorts, graduation from medical school and acceptance or intention to enter a joint MD-PhD program also were assessed. Two focus groups were conducted using constructivist grounded theory. Discussions were transcribed, redacted, and coded using Dedoose software.RESULTS: DARE Medical students were 31.2years (range 24-35), the majority were women (67%;16/24). The majority (17/24;71%) completed a first author publication in a peer-reviewed journal with a median of 3.9 per DARE alumnus. DARE alumnus reported increased proficiency in biostatistics, epidemiology, coding and public speaking as well as stronger research qualities in creativity, critical thinking, comfort in approaching scientist in both the US and Denmark (p<0.001 for all). Qualitative key themes included: increased confidence, a deepening of research inquiry and linkage to a research network.CONCLUSIONS: Preliminarily, this study suggests that medical students can initiate binational collaboration in medicine. Benefits include research productivity, intention to pursue academic medical careers, as well as positive impacts on motivation. This medical student-initiated research model lays the groundwork for using this model across other country pairs to promote binational collaboration.

  View details for DOI 10.1186/s12909-023-04002-z

  View details for PubMedID 36747167

• SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming. Cell Wu, C., Lidsky, P. V., Xiao, Y., Cheng, R., Lee, I. T., Nakayama, T., Jiang, S., He, W., Demeter, J., Knight, M. G., Turn, R. E., Rojas-Hernandez, L. S., Ye, C., Chiem, K., Shon, J., Martinez-Sobrido, L., Bertozzi, C. R., Nolan, G. P., Nayak, J. V., Milla, C., Andino, R., Jackson, P. K. 2022

  Abstract

  How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

  View details for DOI 10.1016/j.cell.2022.11.030

  View details for PubMedID 36580912

• Airway Disease in Children with Primary Ciliary Dyskinesia: Impact of Ciliary Ultrastructure Defect and Genotype. Annals of the American Thoracic Society Kinghorn, B., Rosenfeld, M., Sullivan, E., Onchiri, F., Ferkol, T. W., Sagel, S. D., Dell, S. D., Milla, C., Shapiro, A. J., Sullivan, K. M., Zariwala, M. A., Pittman, J. E., Mollica, F., Tiddens, H. A., Kemner-van de Corput, M., Knowles, M. R., Davis, S. D., Leigh, M. W. 2022

  Abstract

  Primary ciliary dyskinesia (PCD) is characterized by impaired mucociliary clearance, recurrent respiratory infections, and progressive airway damage and obstructive lung disease. While the association of ciliary ultrastructure defect/genotype with severity of airflow obstruction has been well characterized, their association with airway abnormalities on chest computed tomography (CT) has been minimally evaluated.We sought to delineate the association of ciliary defect class/genotype with chest CT scores in children with PCD.Cross-sectional analysis of children with PCD (N=146) enrolled in a prospective multicenter observational study, stratified by defect type: ODA (outer dynein arm), ODA/IDA (outer and inner dynein arm), IDA/MTD (inner dynein arm and microtubular disorganization), and Normal/Near Normal ultrastructure with associated genotypes. CTs were scored utilizing the Melbourne-Rotterdam Annotated Grid Morphometric Analysis for PCD (MERAGMA-PCD), evaluating airway abnormalities in a hierarchical order: atelectasis, bronchiectasis, bronchial wall thickening, mucus plugging/tree in bud opacities. Volume fraction of each component was expressed as % of total lung volume. %Disease was computed as the sum of all components. Regression analyses were utilized to describe the association between clinical predictors and CT scores.Acceptable chest CTs were obtained in 141 children (71 male): 57 ODA, 20 ODA/IDA, 40 IDA/MTD, 24 Normal/Near Normal. Mean (SD) age was 8.5 (4.6) years, forced expiratory volume in 1 second (FEV1) % predicted was 82.4 (19.5), and %Disease was 4.6 (3.5). Children with IDA/MTD defects had higher %Disease compared to children with ODA defects (2.71% higher (95%CI: 1.37-4.06, p<0.001)), driven by higher %Mucus plugging (2.35% higher (1.43-3.26, p<0.001)). Increasing age, lower BMI, and lower FEV1 were associated with higher %Disease (0.23%, 95%CI: 0.11-0.35, p<0.001; 0.03%, 95%CI: 0.01-0.04, p=0.008; and 0.05%, 95%CI: 0.01-0.08, p=0.011, respectively).Children with IDA/MTD defects had significantly greater airway disease on CT, primarily mucus plugging, compared to children with ODA defects.

  View details for DOI 10.1513/AnnalsATS.202206-524OC

  View details for PubMedID 36442147

• Laterality Defects in Primary Ciliary Dyskinesia: Relationship to Ultrastructural Defect or Genotype. Annals of the American Thoracic Society Barber, A. T., Shapiro, A. J., Davis, S. D., Ferkol, T. W., Atkinson, J. J., Sagel, S. D., Dell, S. D., Olivier, K. N., Milla, C. E., Rosenfeld, M., Li, L., Lin, F., Sullivan, K. M., Capps, N. A., Zariwala, M. A., Knowles, M. R., Leigh, M. W., Genetic Disorders of Mucociliary Clearance Consortium (GDMCC) 2022

  Abstract

  RATIONALE: The association between organ laterality abnormalities and ciliary ultrastructural defect or genotype in primary ciliary dyskinesia is poorly understood.OBJECTIVES: To determine if there is an association between presence/type of laterality abnormality and ciliary ultrastructural defect and genotype.METHODS: Participants with primary ciliary dyskinesia in a multicenter, prospective study were grouped based on ciliary ultrastructural defect or genotype. In a retrospective analysis of this data, the association of ciliary ultrastructural defect or genotype and likelihood of a laterality abnormality was evaluated by logistic regression adjusted for presence of two loss-of-function versus one or more not-loss-of-function variants.RESULTS: Of 559 participants, 286 (51.2%), 215 (38.5%), and 58 (10.4%) were identified as having situs solitus, situs inversus totalis, and situs ambiguus, respectively; heterotaxy, defined as situs ambiguus with complex cardiovascular defects, was present in 14 (2.5%). Compared to group with inner dynein arm defects with microtubular disorganization, laterality defects were more likely in the outer dynein arm defects group (OR 2.07, 95% CI 1.21-3.54, P<0.01) and less likely in the normal/near normal ultrastructure group (OR 0.04, 95% CI 0.013-0.151, P<0.01). Heterotaxy was present in 11 of 242 (4.5%) in the outer dynein arm defects group but zero of 96 in the inner dynein arm defects with microtubular disorganization group (P=0.038).CONCLUSION: In primary ciliary dyskinesia, risk of a laterality abnormality differs by ciliary ultrastructure defect. Pathophysiologic mechanisms underlying these differences require further exploration.

  View details for DOI 10.1513/AnnalsATS.202206-487OC

  View details for PubMedID 36342963

• Autonomous wearable sweat rate monitoring based on digitized microbubble detection. Lab on a chip Lin, H., Yu, W., Suarez, J. E., Athavan, H., Wang, Y., Yeung, C., Lin, S., Sankararaman, S., Milla, C., Emaminejad, S. 2022

  Abstract

  Advancements in wearable bioanalytical microsystems have enabled diurnal and (semi)continuous monitoring of physiologically-relevant indices that are accessible through probing sweat. To deliver an undistorted and physiologically-meaningful interpretation of these readings, tracking the sweat secretion rate is essential, because it allows for calibrating the biomarker readings against variations in sweat secretion and inferring the body's hydration/electrolyte homeostasis status. To realize an autonomous wearable solution with intrinsically high signal-to-noise ratio sweat rate sensing capabilities, here, we devise a digitized microbubble detection mechanism-delivered by a hybrid microfluidic/electronic system with a compact footprint. This mechanism is based on the intermittent generation of microliter-scale bubbles via electrolysis and the instantaneous measurement of their time-of-flight (and thus, velocity) via impedimetric sensing. In this way, we overcome the limitations of previously proposed sweat rate sensing modalities that are inherently susceptible to non-targeted secretion characteristics (pH, conductivity, and temperature), constrained by volume, or lack system integration for autonomous on-body operation. By deploying our solution in human subject trials, we validate the utility of our solution for seamless monitoring of exercise- and iontophoretically-induced sweat secretion profiles.

  View details for DOI 10.1039/d2lc00670g

  View details for PubMedID 36268642

• Wearable microneedle-based electrochemical aptamer biosensing for precision dosing of drugs with narrow therapeutic windows. Science advances Lin, S., Cheng, X., Zhu, J., Wang, B., Jelinek, D., Zhao, Y., Wu, T., Horrillo, A., Tan, J., Yeung, J., Yan, W., Forman, S., Coller, H. A., Milla, C., Emaminejad, S. 2022; 8 (38): eabq4539

  Abstract

  Therapeutic drug monitoring is essential for dosing pharmaceuticals with narrow therapeutic windows. Nevertheless, standard methods are imprecise and involve invasive/resource-intensive procedures with long turnaround times. Overcoming these limitations, we present a microneedle-based electrochemical aptamer biosensing patch (muNEAB-patch) that minimally invasively probes the interstitial fluid (ISF) and renders correlated, continuous, and real-time measurements of the circulating drugs' pharmacokinetics. The muNEAB-patch is created following an introduced low-cost fabrication scheme, which transforms a shortened clinical-grade needle into a high-quality gold nanoparticle-based substrate for robust aptamer immobilization and efficient electrochemical signal retrieval. This enables the reliable in vivo detection of a wide library of ISF analytes-especially those with nonexistent natural recognition elements. Accordingly, we developed muNEABs targeting various drugs, including antibiotics with narrow therapeutic windows (tobramycin and vancomycin). Through in vivo animal studies, we demonstrated the strong correlation between the ISF/circulating drug levels and the device's potential clinical use for timely prediction of total drug exposure.

  View details for DOI 10.1126/sciadv.abq4539

  View details for PubMedID 36149955

• Basal airway epithelial cells from PCD have distinct proliferative and inflammatory responses Varenyiova, Z., Rojas, L., Spano, J., Capek, V., Holmes, T., Milla, C. EUROPEAN RESPIRATORY SOC JOURNALS LTD. 2022

  View details for DOI 10.1183/13993003.congress-2022.2226

  View details for Web of Science ID 000893392403152

• Distinct inflammatory responses and effect of Azithromycin on respiratory cells in PCD Varenyiova, Z., Rojas, L., Spano, J., Holmes, T., Milla, C. EUROPEAN RESPIRATORY SOC JOURNALS LTD. 2022

  View details for DOI 10.1183/13993003.congress-2022.2228

  View details for Web of Science ID 000893392403154

• Biochemical, biophysical, and immunological characterization of respiratory secretions in severe SARS-CoV-2 infections. JCI insight Kratochvil, M. J., Kaber, G., Demirdjian, S., Cai, P. C., Burgener, E. B., Nagy, N., Barlow, G. L., Popescu, M., Nicolls, M. R., Ozawa, M. G., Regula, D. P., Pacheco-Navarro, A. E., Yang, S., de Jesus Perez, V. A., Karmouty-Quintana, H., Peters, A. M., Zhao, B., Buja, M. L., Johnson, P. Y., Vernon, R. B., Wight, T. N., Stanford COVID-19 Biobank Study Group, Milla, C. E., Rogers, A. J., Spakowitz, A. J., Heilshorn, S. C., Bollyky, P. L. 2022; 7 (12)

  Abstract

  Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e., resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percentages of solids and protein content were greatly elevated in COVID-19 compared with heathy control samples and closely resembled levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) were major components of respiratory secretions in COVID-19 and were likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors, with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observed increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor-stimulated gene-6 staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicated that increases in HA and DNA in COVID-19 respiratory secretion samples correlated with enhanced inflammatory burden and suggested that DNA and HA may be viable therapeutic targets in COVID-19 infection.

  View details for DOI 10.1172/jci.insight.152629

  View details for PubMedID 35730564

• Association of Neonatal Hospital Length of Stay with Lung Function in Primary Ciliary Dyskinesia. Annals of the American Thoracic Society Wee, W. B., Leigh, M. W., Davis, S. D., Rosenfeld, M., Sullivan, K. M., Sawras, M. G., Ferkol, T. W., Knowles, M. R., Milla, C., Sagel, S. D., Zariwala, M. A., Pullenayegum, E., Dell, S. D. 2022

  Abstract

  RATIONALE: Primary ciliary dyskinesia(PCD), an inherited lung disease, is characterized by abnormal ciliary function leading to progressive bronchiectasis. There is wide variability in respiratory disease severity at birth and later in life.OBJECTIVE: To evaluate the association between neonatal hospital length of stay(neonatal-LOS) and supplemental oxygen duration(SuppO2) with lung function in pediatric PCD. We hypothesized that longer neonatal-LOS and SuppO2 are associated with worse lung function (i.e., forced expiratory volume in 1-second percent predicted(FEV1pp)).METHODS: Secondary analysis of the 'Genetic Disorders of Mucociliary Clearance Consortium' prospective longitudinal multi-center cohort study. Participants enrolled, during 2006-2011, were <19 years old with a confirmed PCD diagnosis and followed annually for 5 years. The exposure variables were neonatal-LOS and SuppO2, counted in days since birth. The outcome, FEV1pp, was measured annually by spirometry. The associations of neonatal-LOS and SuppO2 with FEV1pp were evaluated with a linear mixed effects model with repeated measures and random intercepts, adjusted for age and ciliary ultrastructural defects(EM-defect).RESULTS: Included were 123 participants (male:47%, mean enrollment age:8.3 years (range:0-18)) with 578 visits (median follow-up:5 years). The median neonatal-LOS was 9 days (range:1-90) and median SuppO2 was 5 days (range:0-180). Neonatal-LOS was associated with worse lung function (-0.27 FEV1pp/day (95%CI:-0.53 to -0.01), p=0.04). SuppO2 was not associated with lung function.CONCLUSIONS: Neonatal-LOS is associated with worse lung function in pediatric PCD, independent of age and EM-defect. Future research on the mechanisms of neonatal respiratory distress and its management may help us understand the variability of lung health outcomes in PCD.

  View details for DOI 10.1513/AnnalsATS.202202-116OC

  View details for PubMedID 35657736

• Oral hymecromone decreases hyaluronan in human study participants. The Journal of clinical investigation Rosser, J. I., Nagy, N., Goel, R., Kaber, G., Demirdjian, S., Saxena, J., Bollyky, J. B., Frymoyer, A. R., Pacheco-Navarro, A. E., Burgener, E. B., Rajadas, J., Wang, Z., Arbach, O., Dunn, C. E., Kalinowski, A., Milla, C. E., Bollyky, P. L. 2022; 132 (9)

  Abstract

  BACKGROUNDHyaluronan (HA), an extracellular matrix glycosaminoglycan, has been implicated in the pathophysiology of COVID-19 infection, pulmonary hypertension, pulmonary fibrosis, and other diseases, but is not targeted by any approved drugs. We asked whether hymecromone (4-methylumbelliferone [4-MU]), an oral drug approved in Europe for biliary spasm treatment that also inhibits HA in vitro and in animal models, could be repurposed as an inhibitor of HA synthesis in humans.METHODSWe conducted an open-label, single-center, dose-response study of hymecromone in healthy adults. Subjects received hymecromone at 1200 (n = 8), 2400 (n = 9), or 3600 (n = 9) mg/d divided into 3 doses daily, administered orally for 4 days. We assessed safety and tolerability of hymecromone and analyzed HA, 4-MU, and 4-methylumbelliferyl glucuronide (4-MUG; the main metabolite of 4-MU) concentrations in sputum and serum.RESULTSHymecromone was well tolerated up to doses of 3600 mg/d. Both sputum and serum drug concentrations increased in a dose-dependent manner, indicating that higher doses lead to greater exposures. Across all dose arms combined, we observed a significant decrease in sputum HA from baseline after 4 days of treatment. We also observed a decrease in serum HA. Additionally, higher baseline sputum HA levels were associated with a greater decrease in sputum HA.CONCLUSIONAfter 4 days of exposure to oral hymecromone, healthy human subjects experienced a significant reduction in sputum HA levels, indicating this oral therapy may have potential in pulmonary diseases where HA is implicated in pathogenesis.TRIAL REGISTRATIONClinicalTrials.gov NCT02780752.FUNDINGStanford Medicine Catalyst, Stanford SPARK, Stanford Innovative Medicines Accelerator program, NIH training grants 5T32AI052073-14 and T32HL129970.

  View details for DOI 10.1172/JCI157983

  View details for PubMedID 35499083

• Relationship Between Genotype/Ultrastructural Defect and Neonatal Respiratory Distress in Primary Ciliary Dyskinesia Barber, A., Shapiro, A. J., Davis, S. D., Ferkol, T. W., Atkinson, J. J., Sagel, S. D., Dell, S. D., Olivier, K. N., Milla, C. E., Rosenfeld, M., Li, L., Lin, F., Sullivan, K. M., Zariwala, M. A., Knowles, M. R., Leigh, M. W. AMER THORACIC SOC. 2022

  View details for Web of Science ID 000792480405654

• Antibody Response to SARS-CoV-2 mRNA Vaccination in Patients with Cystic Fibrosis Lee, L., Milla, C. E., Kim, P. S. AMER THORACIC SOC. 2022

  View details for Web of Science ID 000792480401273

• Filamentous Bacteriophage in Patients with Primary Ciliary Dyskinesia and Chronic Pseudomonas Aeruginosa Endobronchial Infection Burgener, E. B., Spano, J. M., Bollyky, P. L., Milla, C. E. AMER THORACIC SOC. 2022

  View details for Web of Science ID 000792480402682

• Progression of Upper Airway Manifestations in Primary Ciliary Dyskinesia Throughout Childhood Dagher, I., Leigh, M. W., Rosenfeld, M., Ferkol, T. W., Sagel, S. D., Dell, S. D., Milla, C. E., Sullivan, K. M., Li, L., Lin, F., Zariwala, M., Knowles, M. R., Davis, S. D. AMER THORACIC SOC. 2022

  View details for Web of Science ID 000792480405652

• A touch-based multimodal and cryptographic bio-human-machine interface. Proceedings of the National Academy of Sciences of the United States of America Lin, S., Zhu, J., Yu, W., Wang, B., Sabet, K. A., Zhao, Y., Cheng, X., Hojaiji, H., Lin, H., Tan, J., Milla, C., Davis, R. W., Emaminejad, S. 2022; 119 (15): e2201937119

  Abstract

  Significance The awareness of the individuals' biological status is critical for creating interactive environments. Accordingly, we devised a multimodal cryptographic bio-human-machine interface (CB-HMI), which seamlessly translates touch-based entries into encrypted biochemical, biophysical, and biometric indices (i.e., circulating biomarkers levels, heart rate, oxygen saturation level, and fingerprint pattern). As its central component, the CB-HMI features thin hydrogel-coated chemical sensors and a signal interpretation framework to access/interpret biochemical indices, bypassing the challenge of circulating analyte accessibility and the confounding effect of pressing force variability. Upgrading the surrounding objects with CB-HMI, we demonstrated new interactive solutions for driving safety and medication use, where the integrated CB-HMI uniquely enabled one-touch bioauthentication (based on the user's biological state/identity), prior to rendering the intended services.

  View details for DOI 10.1073/pnas.2201937119

  View details for PubMedID 35377784

• Biochemical, Biophysical, and Immunological Characterization of Respiratory Secretions in Severe SARS-CoV-2 (COVID-19) Infections. medRxiv : the preprint server for health sciences Kratochvil, M. J., Kaber, G., Demirdjian, S., Cai, P. C., Burgener, E. B., Nagy, N., Barlow, G. L., Popescu, M., Nicolls, M. R., Ozawa, M. G., Regula, D. P., Pacheco-Navarro, A. E., Yang, S., de Jesus Perez, V. A., Karmouty-Quintana, H., Peters, A. M., Zhao, B., Buja, M. L., Johnson, P. Y., Vernon, R. B., Wight, T. N., Stanford COVID-19 Biobank Study Group, Milla, C. E., Rogers, A. J., Spakowitz, A. J., Heilshorn, S. C., Bollyky, P. L. 2022

  Abstract

  Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We find the percent solids and protein content are greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibit heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observe increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factora"stimulated gene-6 (TSG6) staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicate that increases in HA and DNA in COVID-19 respiratory secretion samples correlate with enhanced inflammatory burden and suggest that DNA and HA may be viable therapeutic targets in COVID-19 infection.

  View details for DOI 10.1101/2022.03.28.22272848

  View details for PubMedID 35411348

• Combined agonists act synergistically to increase mucociliary clearance in a cystic fibrosis airway model. Scientific reports Joo, N. S., Cho, H., Shinbashi, M., Choi, J. Y., Milla, C. E., Engelhardt, J. F., Wine, J. J. 2021; 11 (1): 18828

  Abstract

  Mucus clearance, a primary innate defense mechanism of airways, is defective in patients with cystic fibrosis (CF) and CF animals. In previous work, the combination of a low dose of the cholinergic agonist, carbachol with forskolin or a beta adrenergic agonist, isoproterenol synergistically increased mucociliary clearance velocity (MCCV) in ferret tracheas. Importantly, the present study shows that synergistic MCCV can also be produced in CF ferrets, with increases~55% of WT. Synergistic MCCV was also produced in pigs. The combined agonists increased MCCV by increasing surface fluid via multiple mechanisms: increased fluid secretion from submucosal glands, increased anion secretion across surface epithelia and decreased Na+ absorption. To avoid bronchoconstriction, the cAMP agonist was applied 30min before carbachol. This approach to increasing mucus clearance warrants testing for safety and efficacy in humans as a potential therapeutic for muco-obstructive diseases.

  View details for DOI 10.1038/s41598-021-98122-5

  View details for PubMedID 34552115

• Macrophage-derived IL-6 trans-signaling as a novel target in the pathogenesis of bronchopulmonary dysplasia. The European respiratory journal Hirani, D., Alvira, C. M., Danopoulos, S., Milla, C., Donato, M., Tian, L., Mohr, J., Dinger, K., Vohlen, C., Selle, J., Koningsbruggen-Rietschel, S. V., Barbarino, V., Pallasch, C., Rose-John, S., Odenthal, M., Pryhuber, G. S., Mansouri, S., Savai, R., Seeger, W., Khatri, P., Al Alam, D., Dotsch, J., Alejandre Alcazar, M. A. 2021

  Abstract

  RATIONALE: Premature infants exposed to oxygen are at risk for bronchopulmonary dysplasia (BPD), which is characterised by lung growth arrest. Inflammation is important, but the mechanisms remain elusive. Here, we investigated inflammatory pathways and therapeutic targets in severe clinical and experimental BPD.METHODS AND RESULTS: First, transcriptomic analysis with in-silico cellular deconvolution identified a lung-intrinsic M1-like-driven cytokine pattern in newborn mice after hyperoxia. These findings were confirmed by gene expression of macrophage-regulating chemokines (Ccl2, Ccl7, Cxcl5) and markers (Il6, Il17A, Mmp12). Second, hyperoxia-activated IL-6/STAT3 signaling was measured in vivo and related to loss of alveolar epithelial type II cells (ATII) as well as increased mesenchymal marker. Il6 null mice exhibited preserved ATII survival, reduced myofibroblasts and improved elastic fiber assembly, thus enabling lung growth and protecting lung function. Pharmacological inhibition of global IL-6 signaling and IL-6 trans-signaling promoted alveolarisation and ATII survival after hyperoxia. Third, hyperoxia triggered M1-like polarisation, possibly via Klf4; hyperoxia-conditioned medium of macrophages and IL-6 impaired ATII proliferation. Finally, clinical data demonstrate elevated macrophage-related plasma cytokines as potential biomarkers that identify infants receiving oxygen at increased risk of developing BPD. Moreover, macrophage-derived IL6 and active STAT3 were related to loss of epithelial cells in BPD lungs.CONCLUSION: We present a novel IL-6-mediated mechanism by which hyperoxia activates macrophages in immature lungs, impairs ATII homeostasis, and disrupts elastic fiber formation, thereby inhibiting lung growth. The data provide evidence that IL-6 trans-signaling could offer an innovative pharmacological target to enable lung growth in severe neonatal chronic lung disease.

  View details for DOI 10.1183/13993003.02248-2020

  View details for PubMedID 34446466

• Upper Airway Manifestations of Primary Ciliary Dyskinesia During Childhood Dagher, Leigh, M. W., Rosenfeld, M., Ferkol, T. W., Sagel, S. D., Dell, S. M., Milla, C. E., Sullivan, K. M., Dalldorf, D. A., Li, L., Lin, F., Zariwala, M. A., Knowles, M. R., Davis, S. D. AMER THORACIC SOC. 2021

  View details for Web of Science ID 000685468900177

• Relationship Between Genotype and Laterality Defects in Primary Ciliary Dyskinesia Barber, A., Shapiro, A. J., Davis, S. D., Ferkol, T. W., Atkinson, J. J., Sagel, S. D., Dell, S. M., Olivier, K. N., Milla, C. E., Rosenfeld, M., Li, L., Lin, F., Sullivan, K. M., Zariwala, M., Knowles, M. R., Leigh, M. W. AMER THORACIC SOC. 2021

  View details for Web of Science ID 000685468900175

• Therapies Used for Primary Ciliary Dyskinesia in North American Children Gardner, R. A., Davis, S. D., Rosenfeld, M., Ferkol, T. W., Sagel, S. D., Dell, S. D., Milla, C. E., Sullivan, K. M., Zariwala, M. A., Knowles, M. R., Leigh, M. W., Genetic Disorders Mucociliary AMER THORACIC SOC. 2021

  View details for Web of Science ID 000685468903145

• Physical Properties of COVID-19 Acute Respiratory Distress Syndrome (ARDS) Sputum Pacheco-Navarro, A., Kratochvil, M. J., Kaber, G., Roque, J., Blish, C., Yang, S., Nadeau, K. C., Heilshorn, S. C., Milla, C. E., Rogers, A., Bollyky, P. AMER THORACIC SOC. 2021

  View details for Web of Science ID 000685468900294

• Severity of Neonatal Respiratory Distress and Future Lung Function in Children with Primary Ciliary Dyskinesia Wee, W. B., Sullivan, K. M., Sawras, M. G., Leigh, M. W., Davis, S. D., Pullenayegum, E., Ferkol, T. W., Knowles, M. R., Milla, C. E., Rosenfeld, M., Sagel, S. D., Zariwala, M. A., Dell, S. D. AMER THORACIC SOC. 2021

  View details for Web of Science ID 000685468900176

• Long-term safety and efficacy of lumacaftor-ivacaftor therapy in children aged 6-11 years with cystic fibrosis homozygous for the F508del-CFTR mutation: a phase 3, open-label, extension study. The Lancet. Respiratory medicine Chilvers, M. A., Davies, J. C., Milla, C., Tian, S., Han, Z., Cornell, A. G., Owen, C. A., Ratjen, F. 2021

  Abstract

  BACKGROUND: The safety and efficacy of 24 weeks of lumacaftor-ivacaftor combination therapy in children aged 6-11 years with cystic fibrosis homozygous for the F508del-CFTR mutation was previously shown in two phase 3 studies. Here, we report long-term safety and efficacy data.METHODS: In this phase 3, open-label, multicentre, extension study (study 110), we examined the long-term safety, tolerability, and efficacy of lumacaftor-ivacaftor in children pooled from two phase 3 parent studies (open-label study 011B and randomised, placebo-controlled study 109). The study was conducted at 61 clinics in the USA, Australia, Belgium, Canada, Denmark, France, Germany, Sweden, and the UK. Children with cystic fibrosis homozygous for the F508del-CFTR mutation who had received lumacaftor-ivacaftor or placebo in the parent studies were treated with lumacaftor-ivacaftor for up to 96 weeks; those who had received the combination therapy in the parent studies (the treatment-to-treatment group) received up to 120 weeks of treatment in total. Participants aged 6-11 years at the start of the parent study received lumacaftor 200 mg-ivacaftor 250 mg orally once every 12 h; those aged 12 years or older received lumacaftor 400 mg-ivacaftor 250 mg orally once every 12 h. The primary endpoint was safety and tolerability in all children who had received at least one dose of the study drug. Secondary endpoints included change from baseline in lung clearance index 2·5% (LCI2·5), sweat chloride concentration, body-mass index, and Cystic Fibrosis Questionnaire-Revised respiratory domain score. This extension study is registered with ClinicalTrials.gov, NCT02544451, and has been completed.FINDINGS: The extension study ran from Aug 13, 2015, to Aug 17, 2018. Of 239 children who enrolled in the study and received at least one dose of lumacaftor-ivacaftor, 215 (90%) completed 96 weeks of treatment. Most children (236 [99%] of 239 children) had adverse events that were mild (49 [21%] of 239) or moderate (148 [62%] of 239) in severity, and there was a low rate of adverse events leading to treatment discontinuation. The most frequently reported adverse events were common manifestations or complications of cystic fibrosis, such as cough and pulmonary exacerbation, or were consistent with the known safety profile of lumacaftor-ivacaftor in older children and adults. No new safety concerns were identified with extended lumacaftor-ivacaftor treatment. Children in the placebo-to-treatment group had improvements in efficacy endpoints consistent with those observed in the parent studies. Improvements observed in children treated with lumacaftor-ivacaftor in the parent study were generally maintained in the extension study.INTERPRETATION: Lumacaftor-ivacaftor therapy in children homozygous for F508del-CFTR who initiated treatment at age 6-11 years was generally safe and well tolerated, and efficacy was sustained for up to 120 weeks. These data support the long-term use of lumacaftor-ivacaftor to treat children aged 6 years and older who are homozygous for the F508del-CFTR mutation.FUNDING: Vertex Pharmaceuticals Incorporated.

  View details for DOI 10.1016/S2213-2600(20)30517-8

  View details for PubMedID 33516285

• Dynamic light scattering microrheology for soft and living materials. Soft matter Cai, P. C., Krajina, B. A., Kratochvil, M. J., Zou, L., Zhu, A., Burgener, E. B., Bollyky, P. L., Milla, C. E., Webber, M. J., Spakowitz, A. J., Heilshorn, S. C. 2021

  Abstract

  We present a method for using dynamic light scattering in the single-scattering limit to measure the viscoelastic moduli of soft materials. This microrheology technique only requires a small sample volume of 12 muL to measure up to six decades in time of rheological behavior. We demonstrate the use of dynamic light scattering microrheology (DLSmuR) on a variety of soft materials, including dilute polymer solutions, covalently-crosslinked polymer gels, and active, biological fluids. In this work, we detail the procedure for applying the technique to new materials and discuss the critical considerations for implementing the technique, including a custom analysis script for analyzing data output. We focus on the advantages of applying DLSmuR to biologically relevant materials: breast cancer cells encapsulated in a collagen gel and cystic fibrosis sputum. DLSmuR is an easy, efficient, and economical rheological technique that can guide the design of new polymeric materials and facilitate the understanding of the underlying physics governing behavior of naturally derived materials.

  View details for DOI 10.1039/d0sm01597k

  View details for PubMedID 33427280

• Clinical use of shear-wave elastography for detecting liver fibrosis in children and adolescents with cystic fibrosis. Pediatric radiology Levitte, S. n., Lee, L. W., Isaacson, J. n., Zucker, E. J., Milla, C. n., Barth, R. A., Sellers, Z. M. 2021

  Abstract

  Complications from liver cirrhosis are a leading cause of death in children with cystic fibrosis. Identifying children at risk for developing liver cirrhosis and halting its progression are critical to reducing liver-associated mortality.Quantitative US imaging, such as shear-wave elastography (SWE), might improve the detection of liver fibrosis in children with cystic fibrosis (CF) over gray-scale US alone. We incorporated SWE in our pediatric CF liver disease screening program and evaluated its performance using magnetic resonance (MR) elastography.Ninety-four children and adolescents with CF underwent 178 SWE exams, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT) and platelet measurements. Of these, 27 children underwent 34 MR elastography exams. We evaluated SWE performance using 6-MHz and 9-MHZ point SWE, and 9-MHz two-dimensional (2-D) SWE.The 6-MHz point SWE was the only method that correlated with MR elastography (r=0.52; 95% confidence interval [CI] 0.20-0.74; P=0.003). SWE of 1.45 m/s distinguished normal from abnormal MR elastography (79% sensitivity, 100% specificity, 100% positive predictive value [PPV], 55% negative predictive value [NPV], area under the receiver operating characteristic [AUROC] curve 0.94). SWE of 1.84 m/s separated mild-moderate (3.00-4.77 kPa) from severe (>4.77 kPa) MR elastography (88% sensitivity, 86% specificity, 78% PPV, 93% NPV, AUROC 0.79). Elevations of AST, ALT, GGT and thrombocytopenia were associated with higher SWE. AST-to-platelet ratio index of 0.42, fibrosis-4 of 0.29, and GGT-to-platelet ratio of 1.43 all had >95% NPV for SWE >1.84 m/s.Given its correlation with MR elastography, SWE might be a clinically useful predictor of liver fibrosis. We identified imaging criteria delineating the use of SWE to identify increased liver stiffness in children with CF. With multicenter validation, these data might be used to improve the detection and monitoring of liver fibrosis in children with CF.

  View details for DOI 10.1007/s00247-021-05015-w

  View details for PubMedID 33759025

• An autonomous wearable system for diurnal sweat biomarker data acquisition. Lab on a chip Hojaiji, H., Zhao, Y., Gong, M. C., Mallajosyula, M., Tan, J., Lin, H., Hojaiji, A. M., Lin, S., Milla, C., Madni, A. M., Emaminejad, S. 2020

  Abstract

  To track dynamically varying and physiologically relevant biomarker profiles in sweat, autonomous wearable platforms are required to periodically sample and analyze sweat with minimal or no user intervention. Previously reported sweat sensors are functionally limited to capturing biomarker information at one time-point/period, thereby necessitating repeated user intervention to increase the temporal granularity of biomarker data. Accordingly, we present a compact multi-compartment wearable system, where each compartment can be activated to autonomously induce/modulate sweat secretion (via iontophoretic actuation) and analyze sweat at set time points. This system was developed following a hybrid-flex design and a vertical integration scheme-integrating the required functional modules: miniaturized iontophoresis interfaces, adhesive thin film microfluidic-sensing module, and control/readout electronics. The system was deployed in a human subject study to track the diurnal variation of sweat glucose levels in relation to the daily food intake. The demonstrated autonomous operation for diurnal sweat biomarker data acquisition illustrates the system's suitability for large-scale and longitudinal personal health monitoring applications.

  View details for DOI 10.1039/d0lc00820f

  View details for PubMedID 33052990

• RETROSPECTIVE CHARACTERIZATION OF NONTUBERCULOUS MYCOBACTERIA AT A SINGLE CF CENTER Si, X., Qin, F., Milla, C. WILEY. 2020: S175

  View details for Web of Science ID 000575294100350

• PF BACTERIOPHAGE ALTERS IMMUNE PROFILE IN EPITHELIAL CELLS AND SPUTUM FROM PATIENTS WITH CYSTIC FIBROSIS Burgener, E. B., Rojas, L., Secor, P. R., Bollyky, P. L., Milla, C. WILEY. 2020: S172

  View details for Web of Science ID 000575294100342

• IMPROVING THE TRANSITION-OF-CARE PROCESS IN CYSTIC FIBROSIS Alicia, M., Si, X., Helmers, M., Chhatwani, L., Milla, C. WILEY. 2020: S339–S340

  View details for Web of Science ID 000575294100761

• Longitudinal assessment of Lung Clearance Index as a marker of disease progression in a cohort of children with cystic fibrosis and normal spirometry Spano, J., Ryan, S., Milla, C. EUROPEAN RESPIRATORY SOC JOURNALS LTD. 2020

  View details for DOI 10.1183/13993003.congress-2020.4315

  View details for Web of Science ID 000606501407332

• Area Under the Curve Achievement of Once Daily Tobramycin in Children with Cystic Fibrosis during Clinical Care. Pediatric pulmonology Brockmeyer, J. M., Wise, R. T., Burgener, E. B., Milla, C., Frymoyer, A. 2020

  Abstract

  BACKGROUND: The area under the concentration-time curve over 24 hours (AUC24 ) is frequently utilized to monitor tobramycin exposure in children with cystic fibrosis (CF). An understanding of exposure target achievement during clinical implementation of an AUC24 based approach in children is limited.METHODS: A retrospective chart review was performed in children with CF treated with once daily tobramycin and drug concentration monitoring at a pediatric CF center. During clinical care AUC24 was estimated using a traditional log-linear regression approach (LLR). AUC24 was also estimated retrospectively using a pharmacokinetic model-based Bayesian forecasting approach (BF). AUC24 achievement after both approaches were compared.RESULTS: In 77 treatment courses (mean age 12.7 ± 5.0 years), a target AUC24 100-125 mg*h/L was achieved after starting dose in 21 (27%) and after initial dose adjustment in 35 (45%). In the first 7 days of treatment, 24 (32%) required ≥3 dose adjustments, and the mean number of drug concentrations measured was 7.1 ± 3.2. Examination of a BF approach demonstrated adequate prediction of measured tobramycin concentrations (median bias -2.1% [95% CI -3.1 to -1.4]; median precision 7.6% [95% CI 7.1 to 8.2%]). AUC24 estimates utilizing the BF approach were higher than the LLR approach with a mean difference of 6.4 mg*h/L (95% CI 4.8 to 8.0 mg*h/L).CONCLUSIONS: Achievement of a narrow AUC24 target is challenging during clinical care, and dose individualization is needed in most children with CF. Implementing a BF approach for estimating AUC24 in children with CF is supported. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/ppul.25037

  View details for PubMedID 32827334

• Noninvasive wearable electroactive pharmaceutical monitoring for personalized therapeutics. Proceedings of the National Academy of Sciences of the United States of America Lin, S., Yu, W., Wang, B., Zhao, Y., En, K., Zhu, J., Cheng, X., Zhou, C., Lin, H., Wang, Z., Hojaiji, H., Yeung, C., Milla, C., Davis, R. W., Emaminejad, S. 2020

  Abstract

  To achieve the mission of personalized medicine, centering on delivering the right drug to the right patient at the right dose, therapeutic drug monitoring solutions are necessary. In that regard, wearable biosensing technologies, capable of tracking drug pharmacokinetics in noninvasively retrievable biofluids (e.g., sweat), play a critical role, because they can be deployed at a large scale to monitor the individuals' drug transcourse profiles (semi)continuously and longitudinally. To this end, voltammetry-based sensing modalities are suitable, as in principle they can detect and quantify electroactive drugs on the basis of the target's redox signature. However, the target's redox signature in complex biofluid matrices can be confounded by the immediate biofouling effects and distorted/buried by the interfering voltammetric responses of endogenous electroactive species. Here, we devise a wearable voltammetric sensor development strategy-centering on engineering the molecule-surface interactions-to simultaneously mitigate biofouling and create an "undistorted potential window" within which the target drug's voltammetric response is dominant and interference is eliminated. To inform its clinical utility, our strategy was adopted to track the temporal profile of circulating acetaminophen (a widely used analgesic and antipyretic) in saliva and sweat, using a surface-modified boron-doped diamond sensing interface (cross-validated with laboratory-based assays, R 2 0.94). Through integration of the engineered sensing interface within a custom-developed smartwatch, and augmentation with a dedicated analytical framework (for redox peak extraction), we realized a wearable solution to seamlessly render drug readouts with minute-level temporal resolution. Leveraging this solution, we demonstrated the pharmacokinetic correlation and significance of sweat readings.

  View details for DOI 10.1073/pnas.2009979117

  View details for PubMedID 32719130

• Methods for Extraction and Detection of Pf Bacteriophage DNA from the Sputum of Patients with Cystic Fibrosis. PHAGE (New Rochelle, N.Y.) Burgener, E. B., Secor, P. R., Tracy, M. C., Sweere, J. M., Bik, E. M., Milla, C. E., Bollyky, P. L. 2020; 1 (2): 100–108

  Abstract

  Background: There is increasing interest in the pulmonary microbiome's bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains difficult. Materials and Methods: In this study, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with the goal of detecting Pf bacteriophage virion-derived nucleic acid. We evaluate the impact of bead beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum. Results: Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for polymerase chain reaction or sequencing-based studies. Conclusion: These studies demonstrate the assessment of a specific bacteriophage and bacteria in sputum from patients with CF.

  View details for DOI 10.1089/phage.2020.0003

  View details for PubMedID 32626852

  View details for PubMedCentralID PMC7327540

• Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Immunity, and Chronic Infections. Frontiers in immunology Secor, P. R., Burgener, E. B., Kinnersley, M. n., Jennings, L. K., Roman-Cruz, V. n., Popescu, M. n., Van Belleghem, J. D., Haddock, N. n., Copeland, C. n., Michaels, L. A., de Vries, C. R., Chen, Q. n., Pourtois, J. n., Wheeler, T. J., Milla, C. E., Bollyky, P. L. 2020; 11: 244

  Abstract

  Pf bacteriophage are temperate phages that infect the bacterium Pseudomonas aeruginosa, a major cause of chronic lung infections in cystic fibrosis (CF) and other settings. Pf and other temperate phages have evolved complex, mutualistic relationships with their bacterial hosts that impact both bacterial phenotypes and chronic infection. We and others have reported that Pf phages are a virulence factor that promote the pathogenesis of P. aeruginosa infections in animal models and are associated with worse skin and lung infections in humans. Here we review the biology of Pf phage and what is known about its contributions to pathogenesis and clinical disease. First, we review the structure, genetics, and epidemiology of Pf phage. Next, we address the diverse and surprising ways that Pf phages contribute to P. aeruginosa phenotypes including effects on biofilm formation, antibiotic resistance, and motility. Then, we cover data indicating that Pf phages suppress mammalian immunity at sites of bacterial infection. Finally, we discuss recent literature implicating Pf in chronic P. aeruginosa infections in CF and other settings. Together, these reports suggest that Pf bacteriophage have direct effects on P. aeruginosa infections and that temperate phages are an exciting frontier in microbiology, immunology, and human health.

  View details for DOI 10.3389/fimmu.2020.00244

  View details for PubMedID 32153575

  View details for PubMedCentralID PMC7047154

• Defining the Clinical Utility of the Lung Clearance Index: Are We There Yet? American journal of respiratory and critical care medicine Spano, J. n., Milla, C. E. 2020

  View details for DOI 10.1164/rccm.202010-3899ED

  View details for PubMedID 33181036

• Retrospective Categorization of Nontuberculous Mycobacteria at a Single Cystic Fibrosis Center Si, X., Qin, F., Milla, C. E. AMER THORACIC SOC. 2020

  View details for Web of Science ID 000556393502449

• Improving the Transition of Care Process for Cystic Fibrosis Patients Si, X., Mirza, A. A., Helmers, M., Chhatwani, L., Milla, C. E. AMER THORACIC SOC. 2020

  View details for Web of Science ID 000556393502450

• Association of Genotype and Structural Lung Disease in a Cohort of Children with PCD Kinghorn, B., Rosenfeld, M., Davis, S. D., Ferkol, T. W., Sagel, S. D., Dell, S. D., Milla, C. E., Shapiro, A. J., Sullivan, K. M., Zariwala, M. A., Pittman, J. E., Tiddens, H., Kemner-van de Corput, M., Mollica, F., Sullivan, E., Onchiri, F., Knowles, M. R., Leigh, M. W. AMER THORACIC SOC. 2020

  View details for Web of Science ID 000556622800234

• Ciliary Localization of the Intraflagellar Transport Protein IFT88 Is Disrupted in Cystic Fibrosis. American journal of respiratory cell and molecular biology Stevens, E. M., Vladar, E. K., Alanin, M. C., Christensen, S. T., von Buchwald, C., Milla, C. 2020; 62 (1): 120–23

  View details for DOI 10.1165/rcmb.2018-0287LE

  View details for PubMedID 31891309

• Natural Perspiration Sampling and in Situ Electrochemical Analysis with Hydrogel Micropatches for User-Identifiable and Wireless Chemo/Biosensing. ACS sensors Lin, S., Wang, B., Zhao, Y., Shih, R., Cheng, X., Yu, W., Hojaiji, H., Lin, H., Hoffman, C., Ly, D., Tan, J., Chen, Y., Di Carlo, D., Milla, C., Emaminejad, S. 2019

  Abstract

  Recent advances in microelectronics, microfluidics, and electrochemical sensing platforms have enabled the development of an emerging class of fully integrated personal health monitoring devices that exploit sweat to noninvasively access biomarker information. Despite such advances, effective sweat sampling remains a significant challenge for reliable biomarker analysis, with many existing methods requiring active stimulation (e.g., iontophoresis, exercise, heat). Natural perspiration offers a suitable alternative as sweat can be collected with minimal effort on the part of the user. To leverage this phenomenon, we devised a thin hydrogel micropatch (THMP), which simultaneously serves as an interface for sweat sampling and a medium for electrochemical sensing. To characterize the performance of the THMP, caffeine and lactate were selected as two representative target molecules. We demonstrated the suitability of the sampling method to track metabolic patterns, as well as to render sample-to-answer biomarker data for personal monitoring (through coupling with an electrochemical sensing system). To inform its potential application, this biomarker sampling and sensing system is incorporated within a distributed terminal-based sensing network, which uniquely capitalizes on the fingertip as a site for simultaneous biomarker data sampling and user identification.

  View details for DOI 10.1021/acssensors.9b01727

  View details for PubMedID 31786928

• Proof of concept for identifying cystic fibrosis from perspiration samples. Proceedings of the National Academy of Sciences of the United States of America Zhou, Z., Alvarez, D., Milla, C., Zare, R. N. 2019

  Abstract

  The gold standard for cystic fibrosis (CF) diagnosis is the determination of chloride concentration in sweat. Current testing methodology takes up to 3 h to complete and has recognized shortcomings on its diagnostic accuracy. We present an alternative method for the identification of CF by combining desorption electrospray ionization mass spectrometry and a machine-learning algorithm based on gradient boosted decision trees to analyze perspiration samples. This process takes as little as 2 min, and we determined its accuracy to be 98 ± 2% by cross-validation on analyzing 277 perspiration samples. With the introduction of statistical bootstrap, our method can provide a confidence estimate of our prediction, which helps diagnosis decision-making. We also identified important peaks by the feature selection algorithm and assigned the chemical structure of the metabolites by high-resolution and/or tandem mass spectrometry. We inspected the correlation between mild and severe CFTR gene mutation types and lipid profiles, suggesting a possible way to realize personalized medicine with this noninvasive, fast, and accurate method.

  View details for DOI 10.1073/pnas.1909630116

  View details for PubMedID 31740593

• PF BACTERIOPHAGE BURDEN IN AIRWAY IS ASSOCIATED WITH FEV1 CHANGE IN PATIENTS WITH CYSTIC FIBROSIS AND PSEUDOMONAS INFECTION Burgener, F. B., Sweere, J., Bach, M. S., Secor, P. R., Bollyky, P. L., Milla, C. WILEY. 2019: S306

  View details for Web of Science ID 000484544000493

• Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis SCIENCE TRANSLATIONAL MEDICINE Burgener, E. B., Sweere, J. M., Bach, M. S., Secor, P. R., Haddock, N., Jennings, L. K., Marvig, R. L., Johansen, H., Rossi, E., Cao, X., Tian, L., Nedelec, L., Molin, S., Bollyky, P. L., Milla, C. E. 2019; 11 (488)

  View details for DOI 10.1126/scitranslmed.aau9748

  View details for Web of Science ID 000465116400003

• Primary Ciliary Dyskinesia: Longitudinal Study of Lung Disease by Ultrastructure Defect and Genotype AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Davis, S. D., Rosenfeld, M., Lee, H., Ferkol, T. W., Sagel, S. D., Dell, S. D., Milla, C., Pittman, J. E., Shapiro, A. J., Sullivan, K. M., Nykamp, K. R., Krischer, J. P., Zariwala, M. A., Knowles, M. R., Leigh, M. W., Genetic Disorders Mucociliary Cl 2019; 199 (2): 190–98

  Abstract

  In primary ciliary dyskinesia, factors leading to disease heterogeneity are poorly understood.To describe early lung disease progression in primary ciliary dyskinesia and identify associations between ultrastructural defects and genotypes with clinical phenotype.This was a prospective, longitudinal (5 yr), multicenter, observational study. Inclusion criteria were less than 19 years at enrollment and greater than or equal to two annual study visits. Linear mixed effects models including random slope and random intercept were used to evaluate longitudinal associations between the ciliary defect group (or genotype group) and clinical features (percent predicted FEV1 and weight and height z-scores).A total of 137 participants completed 732 visits. The group with absent inner dynein arm, central apparatus defects, and microtubular disorganization (IDA/CA/MTD) (n = 41) were significantly younger at diagnosis and in mixed effects models had significantly lower percent predicted FEV1 and weight and height z-scores than the isolated outer dynein arm defect (n = 55) group. Participants with CCDC39 or CCDC40 mutations (n = 34) had lower percent predicted FEV1 and weight and height z-scores than those with DNAH5 mutations (n = 36). For the entire cohort, percent predicted FEV1 decline was heterogeneous with a mean (SE) decline of 0.57 (0.25) percent predicted/yr. Rate of decline was different from zero only in the IDA/MTD/CA group (mean [SE], -1.11 [0.48] percent predicted/yr; P = 0.02).Participants with IDA/MTD/CA defects, which included individuals with CCDC39 or CCDC40 mutations, had worse lung function and growth indices compared with those with outer dynein arm defects and DNAH5 mutations, respectively. The only group with a significant lung function decline over time were participants with IDA/MTD/CA defects.

  View details for PubMedID 30067075

• Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis. Science translational medicine Burgener, E. B., Sweere, J. M., Bach, M. S., Secor, P. R., Haddock, N. n., Jennings, L. K., Marvig, R. L., Johansen, H. K., Rossi, E. n., Cao, X. n., Tian, L. n., Nedelec, L. n., Molin, S. n., Bollyky, P. L., Milla, C. E. 2019; 11 (488)

  Abstract

  Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% (n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% (n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF.

  View details for PubMedID 30996083

• New Algorithm for the Integration of Ultrasound Into Cystic Fibrosis Liver Disease Screening. Journal of pediatric gastroenterology and nutrition Sellers, Z. M., Lee, L. W., Barth, R. A., Milla, C. n. 2019; 69 (4): 404–10

  Abstract

  Liver nodularity occurs across the spectrum of cystic fibrosis liver disease (CFLD), from regenerative nodules to cirrhosis, and can occur without liver enzyme abnormalities. Our aims were to determine if incorporating abdominal ultrasound (US) with annual laboratory testing improves the detection of CFLD and establish CF-specific thresholds for liver screening labs.CF patients at least 6 years old who were exocrine pancreatic-insufficient had an US with Doppler and shear wave elastography. Patients were divided into Normal, Echogenic, or Nodular groups, based on US findings. Results were compared with aspartate aminotransferase (AST), alanine aminotransferase (ALT), platelets, AST to platelet ratio index (APRI), Fibrosis 4 (FIB-4), and gamma-glutamyl transferase (GGT) to platelet ratio (GPR). Receiver operator curve, sensitivity, specificity, positive predictive value, negative predictive value, and optimal cut-off with Youden Index were calculated.From 82 patients, incorporation of US identified more nodular livers than using labs alone. The Nodular group had significantly greater median AST (44), ALT (48), GGT (46), APRI (0.619), FIB-4 (0.286), GPR (1.431). Optimal cut-offs to detect liver nodularity in CF were AST >33, ALT >45, GGT >21, Platelets <230, APRI >0.367, FIB-4 >0.222, GPR >0.682. Using GGT, APRI, and GPR, we generated an algorithm to direct the use of US in CFLD screening.Using modified serum lab thresholds, addition of liver fibrosis indices, and/or abdominal US can increase detection of liver nodularity in CF. A combination of GGT, GPR, and APRI can help direct which CF children should undergo US evaluation. These tools may improve earlier identification of fibrosis and/or cirrhosis in CF patients.

  View details for DOI 10.1097/MPG.0000000000002412

  View details for PubMedID 31181020

• High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia. Cell stem cell Vaidyanathan, S. n., Salahudeen, A. A., Sellers, Z. M., Bravo, D. T., Choi, S. S., Batish, A. n., Le, W. n., Baik, R. n., de la O, S. n., Kaushik, M. P., Galper, N. n., Lee, C. M., Teran, C. A., Yoo, J. H., Bao, G. n., Chang, E. H., Patel, Z. M., Hwang, P. H., Wine, J. J., Milla, C. E., Desai, T. J., Nayak, J. V., Kuo, C. J., Porteus, M. H. 2019

  Abstract

  Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

  View details for DOI 10.1016/j.stem.2019.11.002

  View details for PubMedID 31839569

• Salivary thiocyanate as a biomarker of Cystic Fibrosis Transmembrane Regulator function. Analytical chemistry Malkovskiy, A. V., Yacob, A. A., Dunn, C. E., Zirbes, J. M., Ryan, S. P., Bollyky, P. L., Rajadas, J. n., Milla, C. E. 2019

  Abstract

  Improved methods are needed to reliably assess CFTR function in vivo in light of recent therapeutic developments targeting the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Saliva from patients with cystic fibrosis (CF) and healthy controls (HC) was studied using colorimetry and non-resonant Raman spectroscopy. Colorimetry experiments showed only a 36% decrease in thiocyanate (SCN-) concentration, but a sharp Raman peak at 2068 cm-1, attributable to (SCN-) vibrations, normalized to C-H peak, was on average 18 times higher for HC samples. Samples from patients under-going treatment with CFTR modulators including ivacaftor, lumacaftor and tezacaftor, showed a high normalized peak in response to therapy. The peak intensity was consistent in longitudinal samples from single donors and in stored samples. The Raman peak ratio is a more sensitive, convenient, non-invasive biomarker for assessments of the therapeutic efficacy of drugs targeting CFTR and provides a value that is in much better agreement with theoretical expectations of saliva SCN- concentrations, compared to colorimetry. Moreover, samples from patients can be collected remotely, as they are stable and can be shipped in dry state. This insight may greatly facilitate assessments of CFTR modulator efficacy in individual pa-tients as well as development of new ones.

  View details for DOI 10.1021/acs.analchem.9b01800

  View details for PubMedID 31117414

• Impact of Residential Distance on Lung Function in the Adult Cystic Fibrosis Population: A Single Center Study Marmor, M., Hernandez, C., Dhillon, G. S., Rehkopf, D., Milla, C. E. AMER THORACIC SOC. 2019

  View details for Web of Science ID 000466776700401

• Elafin Treatment Rescues EGFR-Klf4 Signaling and Lung Cell Survival in Ventilated Newborn Mice. American journal of respiratory cell and molecular biology Alejandre Alcazar, M. A., Kaschwich, M., Ertsey, R., Preuss, S., Milla, C., Mujahid, S., Masumi, J., Khan, S., Mokres, L. M., Tian, L., Mohr, J., Hirani, D. V., Rabinovitch, M., Bland, R. D. 2018; 59 (5): 623–34

  Abstract

  Mechanical ventilation with O2-rich gas (MV-O2) inhibits alveologenesis and lung growth. We previously showed that MV-O2 increased elastase activity and apoptosis in lungs of newborn mice, whereas elastase inhibition by elafin suppressed apoptosis and enabled lung growth. Pilot studies suggested that MV-O2 reduces lung expression of prosurvival factors phosphorylated epidermal growth factor receptor (pEGFR) and Kruppel-like factor 4 (Klf4). Here, we sought to determine whether apoptosis and lung growth arrest evoked by MV-O2 reflect disrupted pEGFR-Klf4 signaling, which elafin treatment preserves, and to assess potential biomarkers of bronchopulmonary dysplasia (BPD). Five-day-old mice underwent MV with air or 40% O2 for 8-24 hours with or without elafin treatment. Unventilated pups served as controls. Immunoblots were used to assess lung pEGFR and Klf4 proteins. Cultured MLE-12 cells were exposed to AG1478 (EGFR inhibitor), Klf4 siRNA, or vehicle to assess effects on proliferation, apoptosis, and EGFR regulation of Klf4. Plasma elastase and elafin levels were measured in extremely premature infants. In newborn mice, MV with air or 40% O2 inhibited EGFR phosphorylation and suppressed Klf4 protein content in lungs (vs. unventilated controls), yielding increased apoptosis. Elafin treatment inhibited elastase, preserved lung pEGFR and Klf4, and attenuated the apoptosis observed in lungs of vehicle-treated mice. In MLE-12 studies, pharmacological inhibition of EGFR and siRNA suppression of Klf4 increased apoptosis and reduced proliferation, and EGFR inhibition decreased Klf4. Plasma elastase levels were more than twofold higher, without a compensating increase of plasma elafin, in infants with BPD, compared to infants without BPD. These findings indicate that pEGFR-Klf4 is a novel prosurvival signaling pathway in lung epithelium that MV disrupts. Elafin preserves pEGFR-Klf4 signaling and inhibits apoptosis, thereby enabling lung growth during MV. Together, our animal and human data raise the question: would elastase inhibition prevent BPD in high-risk infants exposed to MV-O2?

  View details for PubMedID 29894205

• Age-related heterogeneity in dental caries and associated risk factors in individuals with cystic fibrosis ages 6-20 years: A pilot study JOURNAL OF CYSTIC FIBROSIS Chi, D. L., Rosenfeld, M., Mancl, L., Chung, W. O., Presland, R. B., Sarvas, E., Rothen, M., Alkhateeb, A., McNamara, S., Genatossio, A., Virella-Lowell, I., Milla, C., Scott, J. 2018; 17 (6): 747–59

  Abstract

  The literature conflicts regarding dental caries risk in cystic fibrosis (CF) relative to controls.Prospective, observational study of age-related heterogeneity in caries rates and potential risk factors in individuals with CF ages 6-20 at a single clinic in Washington state (N=85). Caries rates for enrolled CF participants and historical controls from NHANES were compared using cubic spline regression models. Generalized linear regression models identified correlates of age and caries in CF.Children ages 6-9 with CF had significantly lower caries than controls (Holm's P<0.05). There was no difference for ages 10-20 by CF status (Holm's P>0.05). Various biological/intraoral, medical, and behavioral factors were associated with caries and age in CF.Younger children with CF may be protected from caries, but there is apparent loss of protection in early adolescence associated with multiple risk factors. Additional studies are needed to confirm these findings.

  View details for PubMedID 30005828

• BREVENAL INCREASES CILIARY BEAT FREQUENCY AND PARTICLE DIFFUSION IN AIRWAY SURFACE LIQUID OF CYSTIC FIBROSIS HUMAN BRONCHIAL EPITHELIAL CELLS WITH NONSENSE MUTATIONS Cohen, Milla, C., Salathe, M., Baumlin, N., Chung, S., Bourdelais, A., Baden, D. WILEY. 2018: 253

  View details for Web of Science ID 000443947300371

• DEFINING THE CLINICAL UTILITY OF THE LUNG CLEARANCE INDEX Zirbes, J. M., Alvarez, D., Ryan, S. P., Milla, C. WILEY. 2018: 248

  View details for Web of Science ID 000443947300358

• ABNORMAL BASAL CELLS UNDERLIE EPITHELIAL DSYFUNCTION IN CYSTIC FIBROSIS Vladar, E. K., Milla, C., Axelrod, J. D. WILEY. 2018: 175

  View details for Web of Science ID 000443947300166

• PF BACTERIOPHAGE IN PATIENTS WITH CYSTIC FIBROSIS IS ASSOCIATED WITH CHRONIC PSEUDOMONAS INFECTION Burgener, E. B., Sweere, J. M., Secor, P. R., Bollyky, P. L., Milla, C. WILEY. 2018: 281

  View details for Web of Science ID 000443947300445

• LUNG HEALTH RISK BEHAVIORS AND PERCEPTIONS IN ADOLESCENTS AND YOUNG ADULTS WITH CYSTIC FIBROSIS Hamberger, E., Halpern-Felsher, B., Milla, C. WILEY. 2018: 64–65

  View details for Web of Science ID 000443947300023

• NEW DEVELOPMENTS IN THE ASSESSMENT OF LUNG FUNCTION IN CF PATIENTS Milla, C. WILEY. 2018: 138–40

  View details for Web of Science ID 000443947300086

• Tear Down this Wall: Diversity and Disparities in Cystic fibrosis. American journal of respiratory and critical care medicine Buu, M. C., Milla, C. E. 2018

  View details for PubMedID 30063377

• KB001-A, a novel anti-inflammatory, found to be safe and well-tolerated in cystic fibrosis patients infected with Pseudomonas aeruginosa JOURNAL OF CYSTIC FIBROSIS Jain, R., Beckett, V. V., Konstan, M. W., Accurso, F. J., Burns, J. L., Mayer-Hamblett, N., Milla, C., VanDevanter, D. R., Chmiel, J. F., KB001-A Study Grp 2018; 17 (4): 484–91

  Abstract

  Chronic Pseudomonas aeruginosa (Pa) airways infection, exuberant local inflammation, and progressive lung function loss are hallmarks of cystic fibrosis (CF). KB001-A is an anti-PcrV PEGylated monoclonal antibody fragment to the Type III secretion system of Pa. This 16-week study evaluated KB001-A associated effect on time-to-need for antibiotics for worsening respiratory signs and symptoms, as well as safety, and treatment-associated changes in symptom scores, inflammatory markers, and spirometry.This was a randomized, double-blind, placebo-controlled, repeat-dose study in CF subjects with Pa. Intravenous 10mg/kg KB001-A or placebo infusions were administered at baseline and weeks 2, 4, 8, and 16, with a 4-week follow-up. Sputum inflammatory markers were assessed in a sub-study. Time-to-need for antibiotics was compared between groups by Kaplan Meier analysis and Cox proportional hazards modeling adjusting for randomization strata.Of 182 subjects, 169 received at least one infusion of KB001-A (n=83) or placebo (n=86). KB001-A was generally safe and well-tolerated as compared to placebo, with no significant emergent adverse effects other than one serious adverse event of elevated hepatic enzymes of unclear etiology. Time to need for antibiotics did not differ between groups (HR: 1.00; 95% CI: 0.69, 1.45, p=0.995). A 3.2 increase in ppFEV1 from placebo favoring KB001-A was observed at week 16 (95% CI: 1.12, 5.30, p=0.003). Mean changes from baseline in log10 sputum neutrophil elastase (NE) had a non-significant decrease (-0.27, 95% CI: -0.58,0.04, p=0.084) while IL-8 concentrations at week 16 were significantly lower (-0.27, 95% CI: -0.55,0.00, p=0.048) among KB001-A subjects (n=16) relative to placebo (n=13).KB001-A was safe and well-tolerated and associated with a modest FEV1 benefit and reduction in select sputum inflammatory markers (IL-8). KB001-A was not associated with an increased time to need for antibiotics. The lack of efficacy seen with KB001-A may be due, in part, to the low levels of the type III secretion proteins previously reported in sputum of CF patients chronically infected with Pa.

  View details for PubMedID 29292092

• Ivacaftor restores CFTR-dependent sweat gland fluid secretion in cystic fibrosis subjects with S945L alleles JOURNAL OF CYSTIC FIBROSIS Kim, J., Davies, Z., Dunn, C., Wine, J. J., Milla, C. 2018; 17 (2): 179–85

  Abstract

  To determine in vivo effects of CFTR modulators on mutation S945L.We measured effects of CFTR modulators on CFTR-dependent sweating ('C-sweat') in two pancreatic sufficient cystic fibrosis (CF) subjects. S1 (S945L/G542X) took ivacaftor and S2 (S945L/F508del) took ivacaftor+tezacaftor. Sweating was stimulated pharmacologically to produce sequentially both CFTR-independent (methacholine stimulated) M-sweat and C-sweat; and the ratio of these was compared. Sweat secretion was measured with two methods: real time secretory rate quantitative recording and by optically measuring the growth of sweat bubbles under oil from multiple identified glands.Using the quantitative recorder, we saw zero C-sweat secretion off-drug, but when on-drug the C-sweat responses for both subjects were comparable to those seen in carriers. The on-drug response was further quantified using the sweat bubble method. Each subject again showed robust C-sweat responses, with C-sweat/M-sweat ratios~half of the ratio determined for a cohort of 40 controls tested under identical conditions.These in vivo results, consistent with prior in vitro findings, indicate that the drug treatments restore near-normal function to S945L-CFTR, and support the use of ivacaftor as a treatment for CF patients who carry this allele.

  View details for PubMedID 29279204

• Progress in Definition, Prevention and Treatment of Fungal Infections in Cystic Fibrosis MYCOPATHOLOGIA Schwarz, C., Hartl, D., Eickmeier, O., Hector, A., Benden, C., Durieu, I., Sole, A., Gartner, S., Milla, C. E., Barry, P. 2018; 183 (1): 21–32

  Abstract

  Cystic fibrosis (CF) is a chronic lethal multi-system condition; however, most of the morbidity and mortality is dependent on the status of the respiratory system. Progressive respiratory decline is mediated by chronic infection and inflammation, punctuated by important acute events known as pulmonary exacerbations which can lead to accelerated decline. The main bacterial species causing infections include Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae and Achromobacter xylosoxidans. In addition to bacteria, fungi are detected in a significant number of patients. The impact of fungal colonization of the airways is still not completely elucidated, but an increasing body of evidence suggests an important role for moulds and yeasts. Although fungal infections are rare, fungi can cause severe pneumonia requiring appropriate targeted treatment. The most common fungi in respiratory samples of patients with CF are Aspergillus fumigatus, Aspergillus terreus and Scedosporium species for filamentous fungi, and yeasts such as Candida albicans and Candida glabrata. Therapeutic strategies depend on the detected fungus and the underlying clinical status of the patient. The antifungal therapy can range from a simple monotherapy up to a combination of three different drugs. Treatment course may be indicated in some patients for two weeks and in others for up to six months, and in rare cases even longer. New antifungal drugs have been developed and are being tested in clinical studies offering the hope of therapeutic alternatives to existing drugs. Identifying relevant risk factors and diagnostic criteria for fungal colonization and infection is crucial to enabling an adequate prevention, diagnosis and treatment.

  View details for PubMedID 28762125

• Sweat rate analysis of ivacaftor potentiation of CFTR in non-CF adults. Scientific reports Kim, J. n., Farahmand, M. n., Dunn, C. n., Milla, C. E., Horii, R. I., Thomas, E. A., Moss, R. B., Wine, J. J. 2018; 8 (1): 16233

  Abstract

  To determine if ivacaftor (Kalydeco) influences non-CF human CFTR function in vivo, we measured CFTR-dependent (C-sweat) and CFTR-independent (M-sweat) rates from multiple identified sweat glands in 8 non-CF adults. The two types of sweating were stimulated sequentially with intradermal injections of appropriate reagents; each gland served as its own control via alternating off-on drug tests on both arms, given at weekly intervals with 3 off and 3 on tests per subject. We compared drug effects on C-sweating stimulated by either high or low concentrations of β-adrenergic cocktail, and on methacholine-stimulated M-sweating. For each subject we measured ~700 sweat volumes from ~75 glands per arm (maximum 12 readings per gland), and sweat volumes were log-transformed for statistical analysis. T-tests derived from linear mixed models (LMMs) were more conservative than the familiar paired sample t-tests, and show that ivacaftor significantly increased C-sweating stimulated by both levels of agonist, with a larger effect in the low cocktail condition; ivacaftor did not increase M-sweat. Concurrent sweat chloride tests detected no effect of ivacaftor. We conclude that ivacaftor in vivo increases the open channel probability (PO) of WT CFTR, provided it is not already maximally stimulated.

  View details for PubMedID 30389955

• Primary Ciliary Dyskinesia: Longitudinal Study of Lung Disease Progression by Ultrastructural Defect and Genotype Davis, S. D., Rosenfeld, M., Lee, H., Ferkol, T. W., Sagel, S. D., Dell, S. D., Milla, C. E., Zariwala, M. A., Knowles, M. R., Leigh, M. W., Genetic Disorders Mucociliary AMER THORACIC SOC. 2018

  View details for Web of Science ID 000449978905261

• The Natural History of Declining Pulmonary Function in Children with Duchenne Muscular Dystrophy Withers, A. L., Wilson, A. C., Buu, M., Milla, C. E., Zirbes, J. M., Hall, G. L. AMER THORACIC SOC. 2018

  View details for Web of Science ID 000449980302322

• Diffuse Large Pulmonary Nodules in a Young Child: Is It Always Metastatic? Burgener, E. B., Milla, C. E. AMER THORACIC SOC. 2018

  View details for Web of Science ID 000449980302091

• Home Monitoring of Patients with Cystic Fibrosis to Identify and Treat Acute Pulmonary Exacerbations eICE Study Results AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Lechtzin, N., Mayer-Hamblett, N., West, N. E., Allgood, S., Wilhelm, E., Khan, U., Aitken, M. L., Ramsey, B. W., Boyle, M. P., Mogayzel, P. J., Gibson, R. L., Orenstein, D., Milla, C., Clancy, J. P., Antony, V., Goss, C. H., eICE Study Team 2017; 196 (9): 1144–51

  Abstract

  Individuals with cystic fibrosis (CF) experience frequent acute pulmonary exacerbations, which lead to decreased lung function and reduced quality of life.The goal of this study was to determine if an intervention directed toward early detection of pulmonary exacerbations using home spirometry and symptom monitoring would result in slower decline in lung function than in control subjects.We conducted a multicenter, randomized trial at 14 CF centers with subjects at least 14 years old. The early intervention arm subjects measured home spirometry and symptoms electronically twice per week. Sites were notified if a participant met criteria for an exacerbation and contacted participants to determine if treatment for acute exacerbation was required. Participants in the usual care arm were seen every 3 months and were asked to contact the site if they were concerned about worsening pulmonary symptoms.The primary outcome was the 52-week change in FEV1. Secondary outcomes included time to first exacerbation and subsequent exacerbation, quality of life, and change in weight. A total of 267 patients were randomized, and the study arms were well matched at baseline. There was no significant difference between study arms in 52-week mean change in FEV1 slope (mean slope difference, 0.00 L, 95% confidence interval, -0.07 to 0.07; P = 0.99). The early intervention arm subjects detected exacerbations more frequently than usual care arm subjects (time to first exacerbation hazard ratio, 1.45; 95% confidence interval, 1.09 to 1.93; P = 0.01). Adverse events were not significantly different between treatment arms.An intervention of home monitoring among patients with CF was able to detect more exacerbations than usual care, but this did not result in slower decline in lung function. Clinical trial registered with www.clinicaltrials.gov (NCT01104402).

  View details for PubMedID 28608719

  View details for PubMedCentralID PMC5694835

• PF BACTERIOPHAGE IN PATIENTS WITH CYSTIC FIBROSIS (CF) IS ASSOCIATED WITH INCREASED SPUTUM ELASTASE AND PSEUDOMONAS AERUGINOSA LOAD Burgener, E. B., Sweere, J. M., Bollyky, P. L., Milla, C. WILEY. 2017: S350

  View details for Web of Science ID 000411113700434

• Lung clearance index is sensitive to small airway disease in pediatric lung transplant recipients JOURNAL OF HEART AND LUNG TRANSPLANTATION Kao, J. E., Zirbes, J. M., Conrad, C. K., Milla, C. E. 2017; 36 (9): 980–84

  Abstract

  The principal obstacle to long-term survival after lung transplant is chronic lung allograft dysfunction (CLAD), which primarily affects the small airways. After transplant, patients are monitored with spirometry, which is a generally insensitive detector of small airways obstruction. The lung clearance index (LCI) is a measure obtained during multiple breath washout (MBW) maneuvers. We hypothesized that among lung allograft recipients, LCI would detect small airways disease not detected with spirometry.This study enrolled 15 patients, 5 of whom already had a diagnosis of CLAD. We added MBW as an additional index of peripheral airway function to the established post-transplant routine care protocol.Of trials, 87.9% yielded valid measurements, and single maneuvers were 2-8 minutes. LCI did not yield any false-negative findings-no patients were considered obstructed by forced expiratory volume in 1 second (FEV1) but normal by LCI. At enrollment, 6 patients without CLAD had an elevated LCI, and 4 progressed to CLAD. Only 2 of these 4 patients would have been identified by a decrease in FEV1.LCI identified lung allograft dysfunction in more patients than the use of standardized spirometric measures, including patients with abnormal FEV1. These data suggest that LCI from MBW may be a more sensitive means to detect allograft peripheral airway disease than standard methods for measurement of small airways function.

  View details for PubMedID 28651906

• Efficacy and safety of lumacaftor and ivacaftor in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR: a randomised, placebo-controlled phase 3 trial. The Lancet. Respiratory medicine Ratjen, F., Hug, C., Marigowda, G., Tian, S., Huang, X., Stanojevic, S., Milla, C. E., Robinson, P. D., Waltz, D., Davies, J. C. 2017; 5 (7): 557-567

  Abstract

  Lumacaftor and ivacaftor combination treatment showed efficacy in patients aged 12 years or older with cystic fibrosis homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in placebo-controlled studies and patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR in an open-label study. We report efficacy and safety of lumacaftor and ivacaftor in patients with cystic fibrosis aged 6-11 years homozygous for F508del-CFTR.In this phase 3, randomised, double-blind, placebo-controlled, multicentre study, patients were enrolled at 54 hospitals and medical centres in nine countries (the USA, Australia, Belgium, Canada, Denmark, France, Germany, Sweden, and the UK). Eligible patients weighed at least 15 kg, with a confirmed diagnosis of cystic fibrosis, percent predicted forced expiratory volume in 1 s (FEV1) of 70 or more, and lung clearance index2·5 (LCI2·5) of 7·5 or more at screening (values less than these thresholds were permitted at day 1). All patients were tested for CFTR genotype at screening; eligible patients had to have the F508del-CFTR mutation on both alleles. Exclusion criteria included any comorbidity or laboratory abnormality that might confound the study results or pose additional risk to the patient. Patients were stratified by weight (<25 kg vs ≥25 kg) and ppFEV1 severity (<90 vs ≥90) determined at the screening visit, and randomly assigned 1:1 to treatment using an interactive web response system to receive 200 mg lumacaftor and 250 mg ivacaftor every 12 hours or placebo for 24 weeks. Patients, all site personnel including the investigator and the site monitor, and the study team were blinded, with the exception of site personnel needing this information in the event of medical emergency or pregnancy and patient safety and regulatory affairs personnel to meet serious adverse event reporting requirements. The primary endpoint was the mean absolute change in LCI2·5 from all on-treatment study visits up to and including week 24. All randomly assigned patients who were exposed to any amount of study drug, with treatment assignment as assigned were included in primary and other efficacy analyses. All patients who were exposed to any amount of study drug, with treatment assignment as treated, were included in the safety analysis. This study was registered with ClinicalTrials.gov, number NCT02514473.Between July 23, 2015, and Sept 20, 2016, a total of 206 patients were enrolled and randomly assigned to receive lumacaftor and ivacaftor (n=104) or placebo (n=102). Two randomly assigned patients were never dosed with study drug (one in the placebo arm due to ineligibility arising from a streptococcal throat infection and one in the lumacaftor and ivacaftor arm due to withdrawal based on refusal to provide blood tests) and were not included in the analyses. 103 patients received at least one dose of lumacaftor and ivacaftor and 101 patients received at least one dose of placebo. For the primary endpoint, the average absolute change in LCI2·5 from baseline over all study visits up to and including the week 24 visit, least squares mean difference was -1·09 units (95% CI -1·43 to -0·75, p<0·0001) for lumacaftor and ivacaftor versus placebo. For the key secondary endpoint of sweat chloride concentration, the least squares mean difference versus placebo was -20·8 mmol/L (95% CI -23·4 to -18·2, average absolute change at day 15/week 4; p<0·0001). The least squares mean difference compared with placebo in absolute change in ppFEV1 from all on-treatment study visits until week 24 was 2·4 (95% CI 0·4-4·4, p=0·0182). 196 (96%) of 204 patients reported adverse events, most of which were mild (87 [43%]) or moderate (98 [48%]). Treatment was discontinued due to adverse events in three (3%) of 103 patients in the lumacaftor and ivacaftor group and two (2%) of 101 patients in the placebo group. Serious adverse events were reported in 13 (13%) of 103 patients in the lumacaftor and ivacaftor group and 11 (11%) of 101 patients in the placebo group.Treatment with lumacaftor and ivacaftor was associated with statistically significant improvements in lung function, as measured by LCI2·5 and ppFEV1, versus placebo in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR. The overall safety profile was consistent with previous phase 3 studies of lumacaftor and ivacaftor.Vertex Pharmaceuticals.

  View details for DOI 10.1016/S2213-2600(17)30215-1

  View details for PubMedID 28606620

• Autonomous sweat extraction and analysis applied to cystic fibrosis and glucose monitoring using a fully integrated wearable platform PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Emaminejad, S., Gao, W., Wu, E., Davies, Z. A., Nyein, H. Y., Challa, S., Ryan, S. P., Fahad, H. M., Chen, K., Shahpar, Z., Talebi, S., Milla, C., Javey, A., Davis, R. W. 2017; 114 (18): 4625-4630

  Abstract

  Perspiration-based wearable biosensors facilitate continuous monitoring of individuals' health states with real-time and molecular-level insight. The inherent inaccessibility of sweat in sedentary individuals in large volume (≥10 µL) for on-demand and in situ analysis has limited our ability to capitalize on this noninvasive and rich source of information. A wearable and miniaturized iontophoresis interface is an excellent solution to overcome this barrier. The iontophoresis process involves delivery of stimulating agonists to the sweat glands with the aid of an electrical current. The challenge remains in devising an iontophoresis interface that can extract sufficient amount of sweat for robust sensing, without electrode corrosion and burning/causing discomfort in subjects. Here, we overcame this challenge through realizing an electrochemically enhanced iontophoresis interface, integrated in a wearable sweat analysis platform. This interface can be programmed to induce sweat with various secretion profiles for real-time analysis, a capability which can be exploited to advance our knowledge of the sweat gland physiology and the secretion process. To demonstrate the clinical value of our platform, human subject studies were performed in the context of the cystic fibrosis diagnosis and preliminary investigation of the blood/sweat glucose correlation. With our platform, we detected the elevated sweat electrolyte content of cystic fibrosis patients compared with that of healthy control subjects. Furthermore, our results indicate that oral glucose consumption in the fasting state is followed by increased glucose levels in both sweat and blood. Our solution opens the possibility for a broad range of noninvasive diagnostic and general population health monitoring applications.

  View details for DOI 10.1073/pnas.1701740114

  View details for PubMedID 28416667

• The magnitude of ivacaftor effects on fluid secretion via R117H-CFTR channels: Human in vivo measurements PLOS ONE Char, J. E., Dunn, C., Davies, Z., Milla, C., Moss, R. B., Wine, J. J. 2017; 12 (4)

  Abstract

  We optically measured effects of orally available ivacaftor (Kalydeco®) on sweat rates of identified glands in 3 R117H subjects, each having a unique set of additional mutations, and compared them with 5 healthy control subjects tested contemporaneously. We injected β-adrenergic agonists intradermally to stimulate CFTR-dependent 'C-sweat' and methacholine to stimulate 'M-sweat', which persists in CF subjects. We focused on an R117H-7T/F508del subject who produced quantifiable C-sweat off ivacaftor and was available for 1 blinded, 3 off ivacaftor, and 3 on ivacaftor tests, allowing us to estimate in vivo fold-increase in sweat rates produced by ivacaftor's effect on the open probability (PO) of R117H-CFTR. Measured sweat rates must be corrected for sweat losses. With estimated sweat losses of 0.023 to 0.08 nl·gland-1·min-1, ivacaftor increased the average C-sweat rates 3-7 fold, and estimated function as % of WT were 4.1-12% off ivacaftor and 21.9-32% on ivacaftor (larger values reflect increased loss estimates). Based on single tests, an R117H-7T/ R117H-7T subject showed 6-9% WT function off ivacaftor and 28-43% on ivacaftor. Repeat testing of an R117H-5T/F508del subject detected only trace responding to ivacaftor. We conclude that in vivo, R117H PO is strongly increased by ivacaftor, but channel number, mainly determined by variable deletion of exon 10, has a marked influence on outcomes.

  View details for DOI 10.1371/journal.pone.0175486

  View details for Web of Science ID 000399875200023

  View details for PubMedID 28419121

• Lumacaftor/Ivacaftor in Patients Aged 6-11 Years with Cystic Fibrosis and Homozygous for F508del-CFTR AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Milla, C. E., Ratjen, F., Marigowda, G., Liu, F., Waltz, D., Rosenfeld, M. 2017; 195 (7): 912-920

  Abstract

  Combination lumacaftor/ivacaftor has been shown to improve lung function and other endpoints in patients aged ≥12 years with cystic fibrosis homozygous for F508del-CFTR but has not been assessed in younger patients.This open-label phase 3 trial evaluated the safety, tolerability, pharmacodynamics, and efficacy of lumacaftor/ivacaftor combination therapy in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR.Patients (N = 58) received 200 mg lumacaftor/250 mg ivacaftor orally every 12 hours for 24 weeks in addition to their existing cystic fibrosis medications.Lumacaftor/ivacaftor was well tolerated; the safety profile was generally similar to that observed in larger lumacaftor/ivacaftor trials in older patients. Four patients discontinued (two due to drug-related adverse events: elevated liver transaminases, n=1; rash, n=1). No safety concerns were associated with spirometry. No significant changes in % predicted FEV1 were observed (change from baseline at Week 24, +2.5 percentage points; 95% CI, -0.2 to 5.2; P = 0.0671). At Week 24, significant improvements from baseline were observed in sweat chloride (-24.8 mmol/L; 95% CI, -29.1 to -20.5; P < 0.0001), BMI z score (+0.15; 95% CI, 0.08 to 0.22; P < 0.0001), Cystic Fibrosis Questionnaire-Revised respiratory domain score (+5.4; 95% CI, 1.4 to 9.4; P = 0.0085), and lung clearance index 2.5 (-0.88; 95% CI, -1.40 to -0.37; P = 0.0018).Lumacaftor/ivacaftor was well tolerated in this young population; no new safety concerns were identified. Improvements in lung clearance index, sweat chloride, nutritional status, and health-related quality of life were observed after 24 weeks of treatment. Clinical trial registration available at www.clinicaltrials.gov, ID NCT0189723.

  View details for DOI 10.1164/rccm.201608-1754OC

  View details for Web of Science ID 000398017200014

• Cystic Fibrosis Transmembrane Conductance Regulator-Related Metabolic Syndrome and Cystic Fibrosis Screen Positive, Inconclusive Diagnosis JOURNAL OF PEDIATRICS Ren, C. L., Borowitz, D. S., Gonska, T., Howenstine, M. S., Levy, H., Massie, J., Milla, C., Munck, A., Southern, K. W. 2017; 181: S45-+

  Abstract

  An unintended consequence of cystic fibrosis (CF) newborn screening (NBS) is the identification of infants with a positive NBS test but inconclusive diagnostic testing. These infants are classified as CF transmembrane conductance regulator-related metabolic syndrome (CRMS) in the US and CF screen positive, inconclusive diagnosis (CFSPID) in other countries. Diagnostic and management decisions of these infants are challenges for CF healthcare professionals and stressful situations for families. As CF NBS has become more widespread across the world, increased information about the epidemiology and outcomes of these infants is becoming available. These data were reviewed at the 2015 CF Foundation Diagnosis Consensus Conference, and a harmonized definition of CRMS and CFSPID was developed.At the consensus conference, participants reviewed published and unpublished studies of CRMS/CFSPID and used a modified Delphi methodology to develop a harmonized approach to the definition of CRMS/CFSPID.Several studies of CRMS/CFSPID from populations around the world have been published in the past year. Although the studies vary in the number of infants studied, study design, and outcome measures, there have been some consistent findings. CRMS/CFSPID occurs relatively frequently, with CF:CRMS that ranges from 3 to 5 cases of CF for every 1 case of CRMS/CFSPID in regions where gene sequencing is not used. The incidence varies by NBS protocol used, and in some regions more cases of CRMS/CFSPID are detected than cases of CF. The majority of individuals with CRMS/CFSPID do not develop CF disease or progress to a diagnosis of CF. However, between 10% and 20% of asymptomatic infants can develop clinical features concerning for CF, such as a respiratory culture positive for Pseudomonas aeruginosa. Most studies have only reported short-term outcomes in the first 1-3 years of life; the long-term outcomes of CRMS/CFSPID remain unknown. The European CF Society definition of CFSPID and the CF Foundation definition of CRMS differ only slightly, and the consensus conference was able to create a unified definition of CRMS/CFSPID.CRMS/CFSPID is a relatively common outcome of CF NBS, and clinicians need to be prepared to counsel families whose NBS test falls into this classification. The vast majority of infants with CRMS/CFSPID will remain free from disease manifestations early in life. However, a small proportion may develop clinical features concerning for CF or demonstrate progression to a clinical phenotype compatible with a CF diagnosis, and their long-term outcomes are not known. A consistent international definition of CRMS/CFSPID will allow for better data collection for study of outcomes and result in improved patient care.

  View details for PubMedID 28129812

• Implementation of Depression Screening and Global Health Assessment in Pediatric Subspecialty Clinics. The Journal of adolescent health : official publication of the Society for Adolescent Medicine Iturralde, E. n., Adams, R. N., Barley, R. C., Bensen, R. n., Christofferson, M. n., Hanes, S. J., Maahs, D. M., Milla, C. n., Naranjo, D. n., Shah, A. C., Tanenbaum, M. L., Veeravalli, S. n., Park, K. T., Hood, K. K. 2017

  Abstract

  Adolescents with chronic illness face greater risk of psychosocial difficulties, complicating disease management. Despite increased calls to screen for patient-reported outcomes, clinical implementation has lagged. Using quality improvement methods, this study aimed to investigate the feasibility of standardized screening for depression and assessment of global health and to determine recommended behavioral health follow-up, across three pediatric subspecialty clinics.A total of 109 patients aged 12-22 years (median = 16.6) who were attending outpatient visits for treatment of diabetes (80% type 1), inflammatory bowel disease, or cystic fibrosis completed the 9-item Patient Health Questionnaire (PHQ-9) depression and Patient-Reported Outcomes Measurement Information System (PROMIS) Pediatric Global Health measures on electronic tablets. Patients screening positive on the PHQ-9 received same-day behavioral health assessment and regular phone check-ins to facilitate necessary follow-up care.Overall, 89% of 122 identified patients completed screening during a 6-month window. Patients completed measures in a timely manner (within 3 minutes) without disruption to clinic flow, and they rated the process as easy, comfortable, and valuable. Depression scores varied across disease type. Patients rated lower global health relative to a previously assessed validation cohort. Depression and global health related significantly to certain medical outcomes. Fifteen percent of patients screened positive on the PHQ-9, of whom 50% confirmed attending behavioral health appointments within 6 months of screening.A standardized depression and global health assessment protocol implemented across pediatric subspecialties was feasible and effective. Universal behavioral health screening for adolescents and young adults living with chronic disease is necessary to meet programmatic needs in pediatric subspecialty clinics.

  View details for PubMedID 28830798

• Efficacy and safety of lumacaftor and ivacaftor in patients aged 6–11 years with cystic fibrosis homozygous for F508del-CFTR: a randomised, placebo-controlled phase 3 trial Lancet Respiratory Medicine Ratjen, F., Hug, C., Marigowda, G., Tian, S., Huang, X., Stanojevic, S., Milla, C. E., Robinson, P. D., Waltz, D., Davies, J. C. 2017; 5 (7): 557–67

  Abstract

  Lumacaftor and ivacaftor combination treatment showed efficacy in patients aged 12 years or older with cystic fibrosis homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in placebo-controlled studies and patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR in an open-label study. We report efficacy and safety of lumacaftor and ivacaftor in patients with cystic fibrosis aged 6-11 years homozygous for F508del-CFTR.In this phase 3, randomised, double-blind, placebo-controlled, multicentre study, patients were enrolled at 54 hospitals and medical centres in nine countries (the USA, Australia, Belgium, Canada, Denmark, France, Germany, Sweden, and the UK). Eligible patients weighed at least 15 kg, with a confirmed diagnosis of cystic fibrosis, percent predicted forced expiratory volume in 1 s (FEV1) of 70 or more, and lung clearance index2·5 (LCI2·5) of 7·5 or more at screening (values less than these thresholds were permitted at day 1). All patients were tested for CFTR genotype at screening; eligible patients had to have the F508del-CFTR mutation on both alleles. Exclusion criteria included any comorbidity or laboratory abnormality that might confound the study results or pose additional risk to the patient. Patients were stratified by weight (<25 kg vs ≥25 kg) and ppFEV1 severity (<90 vs ≥90) determined at the screening visit, and randomly assigned 1:1 to treatment using an interactive web response system to receive 200 mg lumacaftor and 250 mg ivacaftor every 12 hours or placebo for 24 weeks. Patients, all site personnel including the investigator and the site monitor, and the study team were blinded, with the exception of site personnel needing this information in the event of medical emergency or pregnancy and patient safety and regulatory affairs personnel to meet serious adverse event reporting requirements. The primary endpoint was the mean absolute change in LCI2·5 from all on-treatment study visits up to and including week 24. All randomly assigned patients who were exposed to any amount of study drug, with treatment assignment as assigned were included in primary and other efficacy analyses. All patients who were exposed to any amount of study drug, with treatment assignment as treated, were included in the safety analysis. This study was registered with ClinicalTrials.gov, number NCT02514473.Between July 23, 2015, and Sept 20, 2016, a total of 206 patients were enrolled and randomly assigned to receive lumacaftor and ivacaftor (n=104) or placebo (n=102). Two randomly assigned patients were never dosed with study drug (one in the placebo arm due to ineligibility arising from a streptococcal throat infection and one in the lumacaftor and ivacaftor arm due to withdrawal based on refusal to provide blood tests) and were not included in the analyses. 103 patients received at least one dose of lumacaftor and ivacaftor and 101 patients received at least one dose of placebo. For the primary endpoint, the average absolute change in LCI2·5 from baseline over all study visits up to and including the week 24 visit, least squares mean difference was -1·09 units (95% CI -1·43 to -0·75, p<0·0001) for lumacaftor and ivacaftor versus placebo. For the key secondary endpoint of sweat chloride concentration, the least squares mean difference versus placebo was -20·8 mmol/L (95% CI -23·4 to -18·2, average absolute change at day 15/week 4; p<0·0001). The least squares mean difference compared with placebo in absolute change in ppFEV1 from all on-treatment study visits until week 24 was 2·4 (95% CI 0·4-4·4, p=0·0182). 196 (96%) of 204 patients reported adverse events, most of which were mild (87 [43%]) or moderate (98 [48%]). Treatment was discontinued due to adverse events in three (3%) of 103 patients in the lumacaftor and ivacaftor group and two (2%) of 101 patients in the placebo group. Serious adverse events were reported in 13 (13%) of 103 patients in the lumacaftor and ivacaftor group and 11 (11%) of 101 patients in the placebo group.Treatment with lumacaftor and ivacaftor was associated with statistically significant improvements in lung function, as measured by LCI2·5 and ppFEV1, versus placebo in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR. The overall safety profile was consistent with previous phase 3 studies of lumacaftor and ivacaftor.Vertex Pharmaceuticals.

  View details for DOI 10.1016/S2213-2600(17)30215-1

• Corrections to an ATS Workshop Report on Multiple-Breath Washout Testing for Patients with Cystic Fibrosis. Annals of the American Thoracic Society Subbarao, P., Milla, C. E., Morgan, W. J., Ratjen, F. 2017; 14 (1): 145-?

  View details for DOI 10.1513/AnnalsATS.201610-796LE

  View details for PubMedID 28035881

• Lumacaftor/Ivacaftor in Patients Aged 6-11 Years With Cystic Fibrosis Homozygous for F508del-CFTR. American journal of respiratory and critical care medicine Milla, C. E., Ratjen, F., Marigowda, G., Liu, F., Waltz, D., Rosenfeld, M. 2016: -?

  Abstract

  Combination lumacaftor/ivacaftor has been shown to improve lung function and other endpoints in patients aged ≥12 years with cystic fibrosis homozygous for F508del-CFTR but has not been assessed in younger patients.This open-label phase 3 trial evaluated the safety, tolerability, pharmacodynamics, and efficacy of lumacaftor/ivacaftor combination therapy in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR.Patients (N = 58) received 200 mg lumacaftor/250 mg ivacaftor orally every 12 hours for 24 weeks in addition to their existing cystic fibrosis medications.Lumacaftor/ivacaftor was well tolerated; the safety profile was generally similar to that observed in larger lumacaftor/ivacaftor trials in older patients. Four patients discontinued (two due to drug-related adverse events: elevated liver transaminases, n=1; rash, n=1). No safety concerns were associated with spirometry. No significant changes in % predicted FEV1 were observed (change from baseline at Week 24, +2.5 percentage points; 95% CI, -0.2 to 5.2; P = 0.0671). At Week 24, significant improvements from baseline were observed in sweat chloride (-24.8 mmol/L; 95% CI, -29.1 to -20.5; P < 0.0001), BMI z score (+0.15; 95% CI, 0.08 to 0.22; P < 0.0001), Cystic Fibrosis Questionnaire-Revised respiratory domain score (+5.4; 95% CI, 1.4 to 9.4; P = 0.0085), and lung clearance index 2.5 (-0.88; 95% CI, -1.40 to -0.37; P = 0.0018).Lumacaftor/ivacaftor was well tolerated in this young population; no new safety concerns were identified. Improvements in lung clearance index, sweat chloride, nutritional status, and health-related quality of life were observed after 24 weeks of treatment. Clinical trial registration available at www.clinicaltrials.gov, ID NCT0189723.

  View details for PubMedID 27805836

• PHASE 1 INITIAL RESULTS EVALUATING SAFETY, TOLERABILITY, PK AND BIOMARKER DATA USING PTI-428, A NOVEL CFTR MODULATOR, IN PATIENTS WITH CYSTIC FIBROSIS Mouded, M., Layish, D., Sawicki, G. S., Milla, C., Flume, P. A., Tolle, J., Vansaghi, L., Watson, C., Munoz, B., Bhalla, A., Ivarsson, M., Lee, P. WILEY-BLACKWELL. 2016: 262–63

  View details for Web of Science ID 000384815300264

• Evaluating Outcomes Disparities in the Hispanic Cystic Fibrosis Population A Need for a National Analysis Response CHEST Buu, M. C., Milla, C. E., Wise, P. H. 2016; 150 (3): 753

  View details for PubMedID 27613985

• Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation. JCI insight Vladar, E. K., Nayak, J. V., Milla, C. E., Axelrod, J. D. 2016; 1 (13)

  Abstract

  Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease.

  View details for PubMedID 27570836

• Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation JCI INSIGHT Vladar, E. K., Nayak, J. V., Milla, C. E., Axelrod, J. D. 2016; 1 (13)

  View details for DOI 10.1172/jci.insight.88027

  View details for Web of Science ID 000387119700008

• Sweat chloride testing: controversies and issues. The Lancet. Respiratory medicine Kharrazi, M., Milla, C., Wine, J. 2016; 4 (8): 605-607

  View details for DOI 10.1016/S2213-2600(16)30182-5

  View details for PubMedID 27511631

• Clinical Features and Associated Likelihood of Primary Ciliary Dyskinesia in Children and Adolescents. Annals of the American Thoracic Society Leigh, M. W., Ferkol, T. W., Davis, S. D., Lee, H., Rosenfeld, M., Dell, S. D., Sagel, S. D., Milla, C., Olivier, K. N., Sullivan, K. M., Zariwala, M. A., Pittman, J. E., Shapiro, A. J., Carson, J. L., Krischer, J., Hazucha, M. J., Knowles, M. R. 2016; 13 (8): 1305-1313

  Abstract

  Primary ciliary dyskinesia (PCD), a genetically heterogeneous, recessive disorder of motile cilia, is associated with distinct clinical features. Diagnostic tests, including ultrastructural analysis of cilia, nasal nitric oxide measurements, and molecular testing for mutations in PCD genes, have inherent limitations.To define a statistically valid combination of systematically defined clinical features that strongly associates with PCD in children and adolescents.Investigators at seven North American sites in the Genetic Disorders of Mucociliary Clearance Consortium prospectively and systematically assessed individuals (aged 0-18 yr) referred due to high suspicion for PCD. The investigators defined specific clinical questions for the clinical report form based on expert opinion. Diagnostic testing was performed using standardized protocols and included nasal nitric oxide measurement, ciliary biopsy for ultrastructural analysis of cilia, and molecular genetic testing for PCD-associated genes. Final diagnoses were assigned as "definite PCD" (hallmark ultrastructural defects and/or two mutations in a PCD-associated gene), "probable/possible PCD" (no ultrastructural defect or genetic diagnosis, but compatible clinical features and nasal nitric oxide level in PCD range), and "other diagnosis or undefined." Criteria were developed to define early childhood clinical features on the basis of responses to multiple specific queries. Each defined feature was tested by logistic regression. Sensitivity and specificity analyses were conducted to define the most robust set of clinical features associated with PCD.From 534 participants 18 years of age and younger, 205 were identified as having "definite PCD" (including 164 with two mutations in a PCD-associated gene), 187 were categorized as "other diagnosis or undefined," and 142 were defined as having "probable/possible PCD." Participants with "definite PCD" were compared with the "other diagnosis or undefined" group. Four criteria-defined clinical features were statistically predictive of PCD: laterality defect; unexplained neonatal respiratory distress; early-onset, year-round nasal congestion; and early-onset, year-round wet cough (adjusted odds ratios of 7.7, 6.6, 3.4, and 3.1, respectively). The sensitivity and specificity based on the number of criteria-defined clinical features were four features, 0.21 and 0.99, respectively; three features, 0.50 and 0.96, respectively; and two features, 0.80 and 0.72, respectively.Systematically defined early clinical features could help identify children, including infants, likely to have PCD. Clinical trial registered with ClinicalTrials.gov (NCT00323167).

  View details for DOI 10.1513/AnnalsATS.201511-748OC

  View details for PubMedID 27070726

• RASA1 somatic mutation and variable expressivity in capillary malformation/arteriovenous malformation (CM/AVM) syndrome. American journal of medical genetics. Part A Macmurdo, C. F., Wooderchak-Donahue, W., Bayrak-Toydemir, P., Le, J., Wallenstein, M. B., Milla, C., Teng, J. M., Bernstein, J. A., Stevenson, D. A. 2016; 170 (6): 1450-1454

  Abstract

  Germline mutations in RASA1 are associated with capillary malformation-arteriovenous malformation (CM-AVM) syndrome. CM-AVM syndrome is characterized by multi-focal capillary malformations and arteriovenous malformations. Lymphatic anomalies have been proposed as part of the phenotype. Intrafamilial variability has been reported, suggesting modifiers and somatic events. The objective of the study was to identify somatic RASA1 "second hits" from vascular malformations associated with CM-AVM syndrome, and describe phenotypic variability. Participants were examined and phenotyped. Genomic DNA was extracted from peripheral blood on all participants. Whole-exome sequencing was performed on the proband. Using Sanger sequencing, RASA1 exon 8 was PCR-amplified to track the c.1248T>G, p.Tyr416X germline variant through the family. A skin biopsy of a capillary malformation from the proband's mother was also obtained, and next-generation sequencing was performed on DNA from the affected tissue. A familial germline heterozygous novel pathogenic RASA1 variant, c.1248T>G (p.Tyr416X), was identified in the proband and her mother. The proband had capillary malformations, chylothorax, lymphedema, and overgrowth, while her affected mother had only isolated capillary malformations. Sequence analysis of DNA extracted from a skin biopsy of a capillary malformation of the affected mother showed a second RASA1 somatic mutation (c.2245C>T, p.Arg749X). These results and the extreme variable expressivity support the hypothesis that somatic "second hits" are required for the development of vascular anomalies associated with CM-AVM syndrome. In addition, the phenotypes of the affected individuals further clarify that lymphatic manifestations are also part of the phenotypic spectrum of RASA1-related disorders. © 2016 Wiley Periodicals, Inc.

  View details for DOI 10.1002/ajmg.a.37613

  View details for PubMedID 26969842

• The evolving spectrum of ciliopathies and respiratory disease CURRENT OPINION IN PEDIATRICS Milla, C. E. 2016; 28 (3): 339-347

  Abstract

  Research on the biology of cilia, complex hair-like cellular organelles, has greatly informed our understanding of its crucial role in respiratory health and the pathogenesis of primary ciliary dyskinesia (PCD), including the genetics behind this condition. This review will summarize the current state of the art in the field highlighting its clinical implications.The genetics of PCD have exploded over the past few years as knowledge acquired from model systems has permitted the identification of genes that are key components of the ciliary apparatus and its function. In addition, clinical criteria and diagnostic tools have emerged that permit more clear identification of affected individuals.The rate of progress in the field continues to accelerate through international collaborative efforts and standardization of methods. Although the genetics behind PCD are complex, given the large number of genes associated with disease, as well as the large number of possible mutations even at the individual gene level, this knowledge is rapidly translating in improved diagnostics and hopefully in the near future in the identification of potential therapeutics.

  View details for DOI 10.1097/MOP.0000000000000358

  View details for Web of Science ID 000376387000013

  View details for PubMedID 27070443

• Assessing Differences in Mortality Rates and Risk Factors Between Hispanic and Non-Hispanic Patients With Cystic Fibrosis in California CHEST Buu, M. C., Sanders, L. M., Mayo, J. A., Milla, C. E., Wise, P. H. 2016; 149 (2): 380-389

  Abstract

  Over the past 30 years, therapeutic advances have extended the median life span of patients with cystic fibrosis (CF). Hispanic patients are a vulnerable subpopulation with high of prevalence of risk factors for worse health outcomes. The consequences of these differences on health outcomes have not been well described. The objective of this study is to characterize the difference in health outcomes, including mortality rate, between Hispanic and non-Hispanic patients with CF.Retrospective analysis of CF Foundation patient registry data of California residents with CF, diagnosed during or after 1991, from 1991-2010. Ethnicity was self-reported. Primary outcome was mortality. Hazard ratios were estimated from a Cox regression model, stratified by gender and adjusted for socioeconomic status, clinical risk factors, and year of diagnosis.Of 1719, 485 (28.2%) self-identified as Hispanic. Eighty-five deaths occurred, with an overall mortality rate of 4.9%. Unadjusted mortality rate was higher among Hispanic patients than non-Hispanic patients (9.1% vs. 3.3%, p<0.0001). Compared with non-Hispanic patients, Hispanic patients had lower survival rate 18 years post-diagnosis (75.9% vs. 91.5%, p<0.0001). Adjusted for socioeconomic status and clinical risk factors, Hispanic patients had increased rate of death compared to non-Hispanic patients (HR 2.81, 95% CI 1.70-4.63).Hispanic patients with CF have a higher mortality rate than non-Hispanic patients, even after adjusting for socioeconomic status and clinical severity. Further investigation of mechanism for the measured difference in lung function will help inform interventions and improve the health of all CF patients.

  View details for DOI 10.1378/chest.14-2189

  View details for Web of Science ID 000369660400021

• Diagnosis, monitoring, and treatment of primary ciliary dyskinesia: PCD foundation consensus recommendations based on state of the art review. Pediatric pulmonology Shapiro, A. J., Zariwala, M. A., Ferkol, T., Davis, S. D., Sagel, S. D., Dell, S. D., Rosenfeld, M., Olivier, K. N., Milla, C., Daniel, S. J., Kimple, A. J., Manion, M., Knowles, M. R., Leigh, M. W. 2016; 51 (2): 115-132

  Abstract

  Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, rare lung disease resulting in chronic oto-sino-pulmonary disease in both children and adults. Many physicians incorrectly diagnose PCD or eliminate PCD from their differential diagnosis due to inexperience with diagnostic testing methods. Thus far, all therapies used for PCD are unproven through large clinical trials. This review article outlines consensus recommendations from PCD physicians in North America who have been engaged in a PCD centered research consortium for the last 10 years. These recommendations have been adopted by the governing board of the PCD Foundation to provide guidance for PCD clinical centers for diagnostic testing, monitoring, and appropriate short and long-term therapeutics in PCD patients.

  View details for DOI 10.1002/ppul.23304

  View details for PubMedID 26418604

• Dna Extraction From Cystic Fibrosis Sputum Samples Is Not Method Dependent Tracy, M. C., Yacob, A., Chen, C., Milla, C. E., Cornfield, D. N. AMER THORACIC SOC. 2016

  View details for Web of Science ID 000390749600791

• Comparison Of Lung Clearance Index And Quantitative CT Air Trapping In Infant/toddler/preschool Children Vs. School Age Children With Cystic Fibrosis (cf) Robinson, T. E., Goris, M. L., Milla, C. E., Kongstad, T., Green, K., Nielsen, K. G. AMER THORACIC SOC. 2016

  View details for Web of Science ID 000390749605252

• Evaporimeter and Bubble-Imaging Measures of Sweat Gland Secretion Rates. PloS one Kim, J., Farahmand, M., Dunn, C., Davies, Z., Frisbee, E., Milla, C., Wine, J. J. 2016; 11 (10)

  Abstract

  Beta-adrenergically-stimulated sweat rates determined by evaporimetry or by sweat bubble imaging are useful for measuring CFTR function because they provide a near-linear readout across almost the full range of CFTR function. They differentiate cystic fibrosis (CF) subjects from CF carriers and carriers from controls. However, evaporimetry, unlike bubble imaging, appears to be unable to detect improved levels of CFTR function in G551D subjects taking the CFTR modulator ivacaftor. Here, we quantify the sensitivity of evaporimetry and bubble imaging methods for assessing low levels of CFTR-dependent sweat rates. To establish sensitivity, we did dose-ranging studies using intradermally injected [cAMP]i-elevating cocktails. We reduced isoproterenol/aminophylline levels while maintaining a high level of atropine to block muscarinic elevation of [Ca2+]i. We stimulated the same sets of glands for both assays and recorded responses for 20 min. In response to a 3-log dilution of the stimulating cocktail (0.1%), bubble responses were detected in 12/12 tests (100%), with 49% ± 3% of glands secreting to produce an aggregate volume of 598 nl across the 12, 20-min tests. This was ~5% of the response to full cocktail. Evaporimetry detected responses in 3/12 (25%) tests with an aggregate secretion volume of 175 nl. After stimulation with a still more dilute cocktail (0.03%), bubble imaging detected 15 ± 13% of glands secreting at a rate ~0.9% of the response to full cocktail, while zero responding was seen with evaporimetry. The bubble imaging method detected secretion down to aggregate rates of <0.2 nl/(cm2·min), or ~1/30th of the average basal transepithelial water loss (TEWL) in the test subject of 4 g/m2·hr or 6.7 nl/(cm2·min). The increased sensitivity of bubble imaging may be required to detect small but physiologically important increases in secretion rates produced by CFTR modulators.

  View details for DOI 10.1371/journal.pone.0165254

  View details for PubMedID 27768743

  View details for PubMedCentralID PMC5074501

• Circulating Elastase Confers A High Risk For The Development Of Bronchiolitis Obliterans Syndrome Milla, C. E., Zirbes, J. M., Yacob, A., Tian, L., Zamanian, R. T., Bental-Roof, M., Bland, R., Rabinovitch, M., Dhillon, G., Nicolls, M. AMER THORACIC SOC. 2016

  View details for Web of Science ID 000390749604185

• Clinical Features And Associated Likelihood Of Primary Ciliary Dyskinesia In Adults Sullivan, K. M., Daniels, M., Atkinson, J. J., Ferkol, T. W., Hall, D., Lee, H., Metjian, H., Olivier, K. N., Rosenfeld, M., Milla, C. E., Zariwala, M., Sagel, S. D., Carson, J., Krischer, J., Hazucha, M., Knowles, M., Leigh, M., GDMCC AMER THORACIC SOC. 2016

  View details for Web of Science ID 000390749600764

• Respiratory Microbiology In Primary Ciliary Dyskinesia: Comparisons To A Pediatric Cystic Fibrosis Cohort Tracy, M. C., Zirbes, J. M., Hernandez, C., Cornfield, D. N., Milla, C. E. AMER THORACIC SOC. 2016

  View details for Web of Science ID 000390749606226

• Inhaled ß2-Agonist Therapy Increases Functional Residual Capacity in Mechanically Ventilated Children With Respiratory Failure. Pediatric critical care medicine Ramsi, M. A., Henry, M., Milla, C. E., Cornfield, D. N. 2015; 16 (7): e189-93

  Abstract

  To test the hypothesis that in mechanically ventilated children with respiratory failure, aerosolized albuterol modifies functional residual capacity, lung mechanics, oxygen consumption, and hemodynamics.Prospective, self-control clinical trial.A 24-bed PICU in a quaternary care, academic children's hospital.25 children (age range, 1-18 yr) undergoing mechanical ventilation to treat respiratory failure. Entry criteria included previously prescribed inhaled β2 agonists. Physiologic measurements were performed prior to and 20 minutes after administration of aerosolized albuterol solution. Functional residual capacity was determined via nitrogen washout.Functional residual capacity, oxygen consumption, respiratory mechanics, and vital signs were measured were measured prior to and 20 minutes after administration of aerosolized albuterol solution. Functional residual capacity was determined via nitrogen washout.At baseline, functional residual capacity is only 53% of predicted. After aerosolized albuterol, functional residual capacity increased by 18.3% (p = 0.008). Overall, aerosolized albuterol had no effect on airway resistance. However, in patients with an endotracheal tube size of more than or equal to 4.0 mm, resistance decreased from 33 ± 3 to 25 ± 3 (p < 0.02). Inhaled albuterol administration had no effect on oxygen consumption despite an increase in heart rate from 116 ± 2 to 128 ± 2 beats/min (p < 0.0001).In pediatric patients with respiratory failure, aerosolized albuterol increases functional residual capacity without a decrease in resistance. In infants and children, aerosolized albuterol might favorably enhance pulmonary mechanics and thereby represent a novel strategy for lung recruitment in children with respiratory failure.

  View details for DOI 10.1097/PCC.0000000000000448

  View details for PubMedID 25901546

• Assessing differences in mortality rates and risk factors between Hispanic and non-Hispanic patients with cystic fibrosis in California. Chest Buu, M. C., Sanders, L. M., Mayo, J., Milla, C. E., Wise, P. H. 2015

  Abstract

  Over the past 30 years, therapeutic advances have extended the median life span of patients with cystic fibrosis (CF). Hispanic patients are a vulnerable subpopulation with high of prevalence of risk factors for worse health outcomes. The consequences of these differences on health outcomes have not been well described. The objective of this study is to characterize the difference in health outcomes, including mortality rate, between Hispanic and non-Hispanic patients with CF.Retrospective analysis of CF Foundation patient registry data of California residents with CF, diagnosed during or after 1991, from 1991-2010. Ethnicity was self-reported. Primary outcome was mortality. Hazard ratios were estimated from a Cox regression model, stratified by gender and adjusted for socioeconomic status, clinical risk factors, and year of diagnosis.Of 1719, 485 (28.2%) self-identified as Hispanic. Eighty-five deaths occurred, with an overall mortality rate of 4.9%. Unadjusted mortality rate was higher among Hispanic patients than non-Hispanic patients (9.1% vs. 3.3%, p<0.0001). Compared with non-Hispanic patients, Hispanic patients had lower survival rate 18 years post-diagnosis (75.9% vs. 91.5%, p<0.0001). Adjusted for socioeconomic status and clinical risk factors, Hispanic patients had increased rate of death compared to non-Hispanic patients (HR 2.81, 95% CI 1.70-4.63).Hispanic patients with CF have a higher mortality rate than non-Hispanic patients, even after adjusting for socioeconomic status and clinical severity. Further investigation of mechanism for the measured difference in lung function will help inform interventions and improve the health of all CF patients.

  View details for DOI 10.1378/chest.14-2189

  View details for PubMedID 26086984

• Recent advances in cystic fibrosis CURRENT OPINION IN PEDIATRICS Milla, C. E., Moss, R. B. 2015; 27 (3): 317-324

  Abstract

  The field of cystic fibrosis (CF) continues to evolve at a fast pace thanks to novel observations that have enabled deeper understanding of the disease pathophysiology. Parallel groundbreaking developments in innovative therapies permit, for the first time, distinct disease modification.This review highlights important discoveries in fluid homeostasis and mucus secretion in CF that further informs the pathophysiology of the airway disease that characterizes CF. In addition, current concepts and novel paradigms, such as 'theratypes' and 'CF transmembrane conductance regulator chaperome', which will be important for the continued development of disease modifying therapies, are reviewed.The rate of progress in the field continues to accelerate with new knowledge informing the development of innovative therapies. This has already led to tangible substantial and unprecedented clinical benefit for selected subsets of the CF patient population. In the years ahead, further knowledge acquisition may motivate the extension of these benefits to the larger population of people with CF.

  View details for DOI 10.1097/MOP.0000000000000226

  View details for Web of Science ID 000354214800009

  View details for PubMedID 25888148

• Multiple-Breath Washout as a Lung Function Test in Cystic Fibrosis. A Cystic Fibrosis Foundation Workshop Report. Annals of the American Thoracic Society Subbarao, P., Milla, C., Aurora, P., Davies, J. C., Davis, S. D., Hall, G. L., Heltshe, S., Latzin, P., Lindblad, A., Pittman, J. E., Robinson, P. D., Rosenfeld, M., Singer, F., Starner, T. D., Ratjen, F., Morgan, W. 2015; 12 (6): 932-939

  Abstract

  The lung clearance index (LCI) is a lung function parameter derived from the multiple-breath washout (MBW) test. Although first developed 60 years ago, the technique was not widely used for many years. Recent technological advances in equipment design have produced gains in popularity for this test among cystic fibrosis (CF) researchers and clinicians, particularly for testing preschool-aged children. LCI has been shown to be feasible and sensitive to early CF lung disease in patients of all ages from infancy to adulthood. A workshop was convened in January 2014 by the North American Cystic Fibrosis Foundation to determine the readiness of the LCI for use in multicenter clinical trials as well as clinical care. The workshop concluded that the MBW text is a valuable potential outcome measure for CF clinical trials in preschool-aged patients and in older patients with FEV1 in the normal range. However, gaps in knowledge about the choice of device, gas, and standardization across systems are key issues precluding its use as a clinical trial end point in infants. Based on the current evidence, there are insufficient data to support the use of LCI or MBW parameters in the routine clinical management of patients with CF.

  View details for DOI 10.1513/AnnalsATS.201501-021FR

  View details for PubMedID 26075554

• Lung matrix and vascular remodeling in mechanically ventilated elastin haploinsufficient newborn mice. American journal of physiology. Lung cellular and molecular physiology Hilgendorff, A., Parai, K., Ertsey, R., Navarro, E., Jain, N., Carandang, F., Peterson, J., Mokres, L., Milla, C., Preuss, S., Alcazar, M. A., Khan, S., Masumi, J., Ferreira-Tojais, N., Mujahid, S., Starcher, B., Rabinovitch, M., Bland, R. 2015; 308 (5): L464-78

  Abstract

  Elastin plays a pivotal role in lung development. We therefore queried if elastin haploinsufficient newborn mice (Eln(+/-)) would exhibit abnormal lung structure and function related to modified extracellular matrix (ECM) composition. Because mechanical ventilation (MV) has been linked to dysregulated elastic fiber formation in the newborn lung, we also asked if elastin haploinsufficiency would accentuate lung growth arrest seen after prolonged MV of neonatal mice. We studied 5-day-old wild-type (Eln(+/+)) and Eln(+/-) littermates at baseline and after MV with air for 8-24 h. Lungs of unventilated Eln(+/-) mice contained ∼50% less elastin and ∼100% more collagen-1 and lysyl oxidase compared with Eln(+/+) pups. Eln(+/-) lungs contained fewer capillaries than Eln(+/+) lungs, without discernible differences in alveolar structure. In response to MV, lung tropoelastin and elastase activity increased in Eln(+/+) neonates, whereas tropoelastin decreased and elastase activity was unchanged in Eln(+/-) mice. Fibrillin-1 protein increased in lungs of both groups during MV, more in Eln(+/-) than in Eln(+/+) pups. In both groups, MV caused capillary loss, with larger and fewer alveoli compared with unventilated controls. Respiratory system elastance, which was less in unventilated Eln(+/-) compared with Eln(+/+) mice, was similar in both groups after MV. These results suggest that elastin haploinsufficiency adversely impacts pulmonary angiogenesis and that MV dysregulates elastic fiber integrity, with further loss of lung capillaries, lung growth arrest, and impaired respiratory function in both Eln(+/+) and Eln(+/-) mice. Paucity of lung capillaries in Eln(+/-) newborns might help explain subsequent development of pulmonary hypertension previously reported in adult Eln(+/-) mice.

  View details for DOI 10.1152/ajplung.00278.2014

  View details for PubMedID 25539853

  View details for PubMedCentralID PMC4346771

• Clinical features of childhood primary ciliary dyskinesia by genotype and ultrastructural phenotype. American journal of respiratory and critical care medicine Davis, S. D., Ferkol, T. W., Rosenfeld, M., Lee, H., Dell, S. D., Sagel, S. D., Milla, C., Zariwala, M. A., Pittman, J. E., Shapiro, A. J., Carson, J. L., Krischer, J. P., Hazucha, M. J., Cooper, M. L., Knowles, M. R., Leigh, M. W. 2015; 191 (3): 316-324

  Abstract

  The relationship between clinical phenotype of childhood primary ciliary dyskinesia (PCD) and ultrastructural defects and genotype is poorly defined.To delineate clinical features of childhood PCD and their associations with ultrastructural defects and genotype.A total of 118 participants younger than 19 years old with PCD were evaluated prospectively at six centers in North America using standardized procedures for diagnostic testing, spirometry, chest computed tomography, respiratory cultures, and clinical phenotyping.Clinical features included neonatal respiratory distress (82%), chronic cough (99%), and chronic nasal congestion (97%). There were no differences in clinical features or respiratory pathogens in subjects with outer dynein arm (ODA) defects (ODA alone; n = 54) and ODA plus inner dynein arm (IDA) defects (ODA + IDA; n = 18) versus subjects with IDA and central apparatus defects with microtubular disorganization (IDA/CA/MTD; n = 40). Median FEV1 was worse in the IDA/CA/MTD group (72% predicted) versus the combined ODA groups (92% predicted; P = 0.003). Median body mass index was lower in the IDA/CA/MTD group (46th percentile) versus the ODA groups (70th percentile; P = 0.003). For all 118 subjects, median number of lobes with bronchiectasis was three and alveolar consolidation was two. However, the 5- to 11-year-old IDA/CA/MTD group had more lobes of bronchiectasis (median, 5; P = 0.0008) and consolidation (median, 3; P = 0.0001) compared with the ODA groups (median, 3 and 2, respectively). Similar findings were observed when limited to participants with biallelic mutations.Lung disease was heterogeneous across all ultrastructural and genotype groups, but worse in those with IDA/CA/MTD ultrastructural defects, most of whom had biallelic mutations in CCDC39 or CCDC40.

  View details for DOI 10.1164/rccm.201409-1672OC

  View details for PubMedID 25493340

• Pulmonary nocardiosis in an immunocompetent patient with cystic fibrosis. Case reports in pulmonology Schoen, L., Santoro, J. D., Milla, C., Bhargava, S. 2015; 2015: 984171-?

  Abstract

  Nocardia spp. are bacteria of low virulence that cause infection classically in immunocompromised hosts with the lungs as the primary site of infection in the majority of cases. Patients with cystic fibrosis have pulmonary disease characterized by frequent and progressive bacterial infections. Reports of Nocardia spp. isolation in CF are rare in the literature and may represent colonization or active infection, the significance and optimal treatment of which are unknown. We report the second case to date of Nocardia transvalensis pulmonary infection in an immunocompetent patient with CF and the first in a child under the age of eighteen.

  View details for DOI 10.1155/2015/984171

  View details for PubMedID 25960909

  View details for PubMedCentralID PMC4414227

• Prevalence Of Airway Microbial Flora In Primary Ciliary Dyskinesia Chang, H., Adjemian, J., Dell, S. M., Ferkol, T. W., Leigh, M. W., Milla, C. E., Rosenfeld, M., Sagel, S. D., Knowles, M. R., Olivier, K. N. AMER THORACIC SOC. 2015

  View details for Web of Science ID 000377582801197

• Laterality defects other than situs inversus totalis in primary ciliary dyskinesia: insights into situs ambiguus and heterotaxy. Chest Shapiro, A. J., Davis, S. D., Ferkol, T., Dell, S. D., Rosenfeld, M., Olivier, K. N., Sagel, S. D., Milla, C., Zariwala, M. A., Wolf, W., Carson, J. L., Hazucha, M. J., Burns, K., Robinson, B., Knowles, M. R., Leigh, M. W. 2014; 146 (5): 1176-1186

  Abstract

  Motile cilia dysfunction causes primary ciliary dyskinesia (PCD), situs inversus totalis (SI), and a spectrum of laterality defects, yet the prevalence of laterality defects other than SI in PCD has not been prospectively studied.In this prospective study, participants with suspected PCD were referred to our multisite consortium. We measured nasal nitric oxide (nNO) level, examined cilia with electron microscopy, and analyzed PCD-causing gene mutations. Situs was classified as (1) situs solitus (SS), (2) SI, or (3) situs ambiguus (SA), including heterotaxy. Participants with hallmark electron microscopic defects, biallelic gene mutations, or both were considered to have classic PCD.Of 767 participants (median age, 8.1 years, range, 0.1-58 years), classic PCD was defined in 305, including 143 (46.9%), 125 (41.0%), and 37 (12.1%) with SS, SI, and SA, respectively. A spectrum of laterality defects was identified with classic PCD, including 2.6% and 2.3% with SA plus complex or simple cardiac defects, respectively; 4.6% with SA but no cardiac defect; and 2.6% with an isolated possible laterality defect. Participants with SA and classic PCD had a higher prevalence of PCD-associated respiratory symptoms vs SA control participants (year-round wet cough, P < .001; year-round nasal congestion, P = .015; neonatal respiratory distress, P = .009; digital clubbing, P = .021) and lower nNO levels (median, 12 nL/min vs 252 nL/min; P < .001).At least 12.1% of patients with classic PCD have SA and laterality defects ranging from classic heterotaxy to subtle laterality defects. Specific clinical features of PCD and low nNO levels help to identify PCD in patients with laterality defects.ClinicalTrials.gov; No.: NCT00323167; URL: www.clinicaltrials.gov.

  View details for DOI 10.1378/chest.13-1704

  View details for PubMedID 24577564

  View details for PubMedCentralID PMC4219335

• ESTIMATING IVACAFTOR EFFECTS ON R117H CFTR OPEN PROBABILITY: HUMAN IN VIVO MEASUREMENTS Char, J. E., Dunn, C. E., Davies, Z., Milla, C. E., Wine, J. J., Moss, R. B. WILEY-BLACKWELL. 2014: 291

  View details for Web of Science ID 000342926000298

• LONGITUDINAL ASSESSMENT OF SWEAT CHLORIDE CONCENTRATION AMONG INFANTS POSITIVE TO CYSTIC FIBROSIS NEWBORN SCREENING (CF NBS) IN CALIFORNIA Zirbes, J. M., Hardy, K., Sudhakar, R., Salinas, D. B., Saeed, M., Marty, K., Milla, C. E. WILEY-BLACKWELL. 2014: 388

  View details for Web of Science ID 000342926000557

• Anti-PcrV Antibody in Cystic Fibrosis: A Novel Approach Targeting Pseudomonas aeruginosa Airway Infection PEDIATRIC PULMONOLOGY Milla, C. E., Chmiel, J. F., Accurso, F. J., VanDevanter, D. R., Konstan, M. W., Yarranton, G., Geller, D. E. 2014; 49 (7): 650-658

  Abstract

  Pseudomonas aeruginosa (Pa) airway infection is associated with increased morbidity and mortality in cystic fibrosis (CF). The type III secretion system is one of the factors responsible for the increased virulence and pro-inflammatory effects of Pa. KB001 is a PEGylated, recombinant, anti-Pseudomonas-PcrV antibody Fab' fragment that blocks the function of Pa TTSS. We studied the safety, pharmacokinetic (PK), and pharmacodynamic properties of KB001 in CF subjects with chronic Pa infection. Twenty-seven eligible CF subjects (≥12 years of age, FEV1 ≥40% of predicted, and sputum Pa density >10(5)  CFU/g) received a single intravenous dose of KB001 (3 mg/kg or 10 mg/kg) or placebo. Safety, PK, Pa density, clinical outcomes, and inflammatory markers were assessed. KB001 had an acceptable safety profile and a mean serum half-life of 11.9 days. All subjects had Pa TTSS expression in sputum. There were no significant differences between KB001 and placebo for changes in Pa density, symptoms, or spirometry after a single dose. However, compared to baseline, at Day 28 there was a trend towards a dose-dependent reduction in sputum myeloperoxidase, IL-1, and IL-8, and there were significant overall differences in change in sputum neutrophil elastase and neutrophil counts favoring the KB001 10 mg/kg group versus placebo (-0.61 log(10) and -0.63 log(10) , respectively; P < 0.05). These results support targeting Pa TTSS with KB001 as a nonantibiotic strategy to reduce airway inflammation and damage in CF patients with chronic Pa infection. Repeat-dosing studies are necessary to evaluate the durability of the anti-inflammatory effects and how that may translate into clinical benefit. (NCT00638365).

  View details for DOI 10.1002/ppul.22890

  View details for Web of Science ID 000338006700005

  View details for PubMedID 24019259

  View details for PubMedCentralID PMC4079258

• Mutations in RSPH1 Cause Primary Ciliary Dyskinesia with a Unique Clinical and Ciliary Phenotype. American journal of respiratory and critical care medicine Knowles, M. R., Ostrowski, L. E., Leigh, M. W., Sears, P. R., Davis, S. D., Wolf, W. E., Hazucha, M. J., Carson, J. L., Olivier, K. N., Sagel, S. D., Rosenfeld, M., Ferkol, T. W., Dell, S. D., Milla, C. E., Randell, S. H., Yin, W., Sannuti, A., Metjian, H. M., Noone, P. G., Noone, P. J., Olson, C. A., Patrone, M. V., Dang, H., Lee, H., Hurd, T. W., Gee, H. Y., Otto, E. A., Halbritter, J., Kohl, S., Kircher, M., Krischer, J., Bamshad, M. J., Nickerson, D. A., Hildebrandt, F., Shendure, J., Zariwala, M. A. 2014; 189 (6): 707-717

  Abstract

  Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia, but the genetic cause is not defined for all patients with PCD.To identify disease-causing mutations in novel genes, we performed exome sequencing, follow-up characterization, mutation scanning, and genotype-phenotype studies in patients with PCD.Whole-exome sequencing was performed using NimbleGen capture and Illumina HiSeq sequencing. Sanger-based sequencing was used for mutation scanning, validation, and segregation analysis.We performed exome sequencing on an affected sib-pair with normal ultrastructure in more than 85% of cilia. A homozygous splice-site mutation was detected in RSPH1 in both siblings; parents were carriers. Screening RSPH1 in 413 unrelated probands, including 325 with PCD and 88 with idiopathic bronchiectasis, revealed biallelic loss-of-function mutations in nine additional probands. Five affected siblings of probands in RSPH1 families harbored the familial mutations. The 16 individuals with RSPH1 mutations had some features of PCD; however, nasal nitric oxide levels were higher than in patients with PCD with other gene mutations (98.3 vs. 20.7 nl/min; P < 0.0003). Additionally, individuals with RSPH1 mutations had a lower prevalence (8 of 16) of neonatal respiratory distress, and later onset of daily wet cough than typical for PCD, and better lung function (FEV1), compared with 75 age- and sex-matched PCD cases (73.0 vs. 61.8, FEV1 % predicted; P = 0.043). Cilia from individuals with RSPH1 mutations had normal beat frequency (6.1 ± Hz at 25°C), but an abnormal, circular beat pattern.The milder clinical disease and higher nasal nitric oxide in individuals with biallelic mutations in RSPH1 provides evidence of a unique genotype-phenotype relationship in PCD, and suggests that mutations in RSPH1 may be associated with residual ciliary function.

  View details for DOI 10.1164/rccm.201311-2047OC

  View details for PubMedID 24568568

  View details for PubMedCentralID PMC3983840

• A Little CFTR Goes a Long Way: CFTR-Dependent Sweat Secretion from G551D and R117H-5T Cystic Fibrosis Subjects Taking Ivacaftor PLOS ONE Char, J. E., Wolfe, M. H., Cho, H., Park, I., Jeong, J. H., Frisbee, E., Dunn, C., Davies, Z., Milla, C., Moss, R. B., Thomas, E. A., Wine, J. J. 2014; 9 (2)

  Abstract

  To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (-) ivacaftor, 3 only (+) ivacaftor and 3 (+/-) ivacaftor (1-5 tests per condition). The total number of gland measurements was 852 (-) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor  = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

  View details for DOI 10.1371/journal.pone.0088564

  View details for Web of Science ID 000331254600090

  View details for PubMedID 24520399

  View details for PubMedCentralID PMC3919757

• A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor. PloS one Char, J. E., Wolfe, M. H., Cho, H., Park, I., Jeong, J. H., Frisbee, E., Dunn, C., Davies, Z., Milla, C., Moss, R. B., Thomas, E. A., Wine, J. J. 2014; 9 (2)

  Abstract

  To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (-) ivacaftor, 3 only (+) ivacaftor and 3 (+/-) ivacaftor (1-5 tests per condition). The total number of gland measurements was 852 (-) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor  = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

  View details for DOI 10.1371/journal.pone.0088564

  View details for PubMedID 24520399

• LONGITUDINAL PLASMA ENDOTHELIN-1 LEVELS IN PREMATURE INFANTS WITH AND WITHOUT BRONCHOPULMONARY DYSPLASIA Johnson, C., Chitkara, R., McCarthy, E., Fineman, J. R., Sun, C., Kim, L., Hintz, S. R., Van Meurs, K. P., Punn, R., Milla, C. E., Feinstein, J. A. LIPPINCOTT WILLIAMS & WILKINS. 2014: 179–80

  View details for Web of Science ID 000336284900134

• Standardizing nasal nitric oxide measurement as a test for primary ciliary dyskinesia. Annals of the American Thoracic Society Leigh, M. W., Hazucha, M. J., Chawla, K. K., Baker, B. R., Shapiro, A. J., Brown, D. E., LaVange, L. M., Horton, B. J., Qaqish, B., Carson, J. L., Davis, S. D., Dell, S. D., Ferkol, T. W., Atkinson, J. J., Olivier, K. N., Sagel, S. D., Rosenfeld, M., Milla, C., Lee, H., Krischer, J., Zariwala, M. A., Knowles, M. R. 2013; 10 (6): 574-581

  Abstract

  Several studies suggest that nasal nitric oxide (nNO) measurement could be a test for primary ciliary dyskinesia (PCD), but the procedure and interpretation have not been standardized.To use a standard protocol for measuring nNO to establish a disease-specific cutoff value at one site, and then validate at six other sites.At the lead site, nNO was prospectively measured in individuals later confirmed to have PCD by ciliary ultrastructural defects (n = 143) or DNAH11 mutations (n = 6); and in 78 healthy and 146 disease control subjects, including individuals with asthma (n = 37), cystic fibrosis (n = 77), and chronic obstructive pulmonary disease (n = 32). A disease-specific cutoff value was determined, using generalized estimating equations (GEEs). Six other sites prospectively measured nNO in 155 consecutive individuals enrolled for evaluation for possible PCD. Measurements and MainAt the lead site, nNO values in PCD (mean ± standard deviation, 20.7 ± 24.1 nl/min; range, 1.5-207.3 nl/min) only rarely overlapped with the nNO values of healthy control subjects (304.6 ± 118.8; 125.5-867.0 nl/min), asthma (267.8 ± 103.2; 125.0-589.7 nl/min), or chronic obstructive pulmonary disease (223.7 ± 87.1; 109.7-449.1 nl/min); however, there was overlap with cystic fibrosis (134.0 ± 73.5; 15.6-386.1 nl/min). The disease-specific nNO cutoff value was defined at 77 nl/minute (sensitivity, 0.98; specificity, >0.999). At six other sites, this cutoff identified 70 of the 71 (98.6%) participants with confirmed PCD.Using a standardized protocol in multicenter studies, nNO measurement accurately identifies individuals with PCD, and supports its usefulness as a test to support the clinical diagnosis of PCD.

  View details for DOI 10.1513/AnnalsATS.201305-110OC

  View details for PubMedID 24024753

• In Vivo Readout of CFTR Function: Ratiometric Measurement of CFTR-Dependent Secretion by Individual, Identifiable Human Sweat Glands PLOS ONE Wine, J. J., Char, J. E., Chen, J., Cho, H., Dunn, C., Frisbee, E., Joo, N. S., Milla, C., Modlin, S. E., Park, I., Thomas, E. A., Tran, K. V., Verma, R., Wolfe, M. H. 2013; 8 (10)

  Abstract

  To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (∼50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ∼0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.

  View details for DOI 10.1371/journal.pone.0077114

  View details for Web of Science ID 000326152300015

  View details for PubMedID 24204751

  View details for PubMedCentralID PMC3811985

• Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia AMERICAN JOURNAL OF HUMAN GENETICS Austin-Tse, C., Halbritter, J., Zariwala, M. A., Gilberti, R. M., Gee, H. Y., Hellman, N., Pathak, N., Liu, Y., Panizzi, J. R., Patel-King, R. S., Tritschler, D., Bower, R., O'Toole, E., Porath, J. D., Hurd, T. W., Chaki, M., Diaz, K. A., Kohl, S., Lovric, S., Hwang, D., Braun, D. A., Schueler, M., Airik, R., Otto, E. A., Leigh, M. W., Noone, P. G., Carson, J. L., Davis, S. D., Pittman, J. E., Ferkol, T. W., Atkinson, J. J., Olivier, K. N., Sagel, S. D., Dell, S. D., Rosenfeld, M., Milla, C. E., Loges, N. T., Omran, H., Porter, M. E., King, S. M., Knowles, M. R., Drummond, I. A., Hildebrandt, F. 2013; 93 (4): 672-686

  Abstract

  Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.

  View details for DOI 10.1016/j.ajhg.2013.08.015

  View details for Web of Science ID 000326305600009

  View details for PubMedID 24094744

  View details for PubMedCentralID PMC3791264

• Assessment of clinical response to ivacaftor with lung clearance index in cystic fibrosis patients with a G551D-CFTR mutation and preserved spirometry: a randomised controlled trial LANCET RESPIRATORY MEDICINE Davies, J., Sheridan, H., Bell, N., Cunningham, S., Davis, S. D., Elborn, J. S., Milla, C. E., Starner, T. D., Weiner, D. J., Lee, P., Ratjen, F. 2013; 1 (8): 630-638

  Abstract

  Ivacaftor has shown a clinical benefit in patients with cystic fibrosis who have the G551D-CFTR mutation and reduced lung function. Lung clearance index (LCI) using multiple-breath washout might be an alternative to and more sensitive method than forced expiratory volume in 1 s (FEV1) to assess treatment response in the growing number of children and young adults with cystic fibrosis who have normal spirometry. The aim of the study was to assess the treatment effects of ivacaftor on LCI in patients with cystic fibrosis, a G551D-CFTR mutation, and an FEV1 >90% predicted.This phase 2, multicentre, placebo-controlled, double-blind 2×2 crossover study of ivacaftor treatment was conducted in patients with cystic fibrosis, at least one G551D-CFTR allele, and an FEV1 >90% predicted. Patients also had to have an LCI higher than 7·4 at screening, age of 6 years or older, and a weight higher than or equal to 15 kg. Eligible patients were randomly allocated to receive one of two treatment sequences (placebo first followed by ivacaftor 150 mg twice daily [sequence 1] or ivacaftor 150 mg twice daily first followed by placebo [sequence 2]) of 28 days' treatment in each period, with a 28-day washout between the two treatment periods. Randomisation (ratio 1:1) was done with block sizes of 4, and all site personnel including the investigator, the study monitor, and the Vertex study team were masked to treatment assignment. The primary outcome measure was change from baseline in LCI. The study is registered at ClinicalTrials.gov, NCT01262352.Between February and November, 2011, 21 patients were enrolled, of which 11 were assigned to the sequence 1 group, and 10 to the sequence 2 group. 20 of these patients received treatment and 17 completed the trial (eight in sequence 1 group and 9 in sequence 2 group). Treatment with ivacaftor led to significant improvements compared with placebo in LCI (difference between groups in the average of mean changes from baseline at days 15 and 29 was -2·16 [95% CI -2·88 to -1·44]; p<0·0001). Adverse events experienced by study participants were similar between treatment groups; at least one adverse event was reported by 15 (79%) of 19 patients who received placebo and 13 (72%) of 18 patients who received ivacaftor. No deaths occurred during study period.In patients with cystic fibrosis aged 6 years or older who have at least one G551D-CFTR allele, ivacaftor led to improvements in LCI. LCI might be a more sensitive alternative to FEV1 in detecting response to intervention in these patients with mild lung disease.Vertex Pharmaceuticals Incorporated.

  View details for DOI 10.1016/S2213-2600(13)70182-6

  View details for Web of Science ID 000342690300017

  View details for PubMedID 24461666

• Novel CFTR Variants Identified during the First 3 Years of Cystic Fibrosis Newborn Screening in California JOURNAL OF MOLECULAR DIAGNOSTICS Prach, L., Koepke, R., Kharrazi, M., Keiles, S., Salinas, D. B., Reyes, M. C., Pian, M., Opsimos, H., Otsuka, K. N., Hardy, K. A., Milla, C. E., Zirbes, J. M., Chipps, B., O'Bra, S., Saeed, M. M., Sudhakar, R., Lehto, S., Nielson, D., Shay, G. F., Seastrand, M., Jhawar, S., Nickerson, B., Landon, C., Thompson, A., Nussbaum, E., Chin, T., Wojtczak, H. 2013; 15 (5): 710-722

  Abstract

  California uses a unique method to screen newborns for cystic fibrosis (CF) that includes gene scanning and DNA sequencing after only one California-40 cystic fibrosis transmembrane conductance regulator (CFTR) panel mutation has been identified in hypertrypsinogenemic specimens. Newborns found by sequencing to have one or more additional mutations or variants (including novel variants) in the CFTR gene are systematically followed, allowing for prospective assessment of the pathogenic potential of these variants. During the first 3 years of screening, 55 novel variants were identified. Six of these novel variants were discovered in five screen-negative participants and three were identified in multiple unrelated participants. Ten novel variants (c.2554_2555insT, p.F1107L, c.-152G>C, p.L323P, p.L32M, c.2883_2886dupGTCA, c.2349_2350insT, p.K114del, c.-602A>T, and c.2822delT) were associated with a CF phenotype (42% of participants were diagnosed at 4 to 25 months of age), whereas 26 were associated with CFTR-related metabolic syndrome to date. Associations with the remaining novel variants were confounded by the presence of other diseases or other mutations in cis or by inadequate follow-up. These findings have implications for how CF newborn screening and follow-up is conducted and will help guide which genotypes should, and which should not, be considered screen positive for CF in California and elsewhere.

  View details for DOI 10.1016/j.jmoldx.2013.05.006

  View details for Web of Science ID 000323870900019

  View details for PubMedID 23810505

• Polyvinylpyrrolidone microneedles enable delivery of intact proteins for diagnostic and therapeutic applications ACTA BIOMATERIALIA Sun, W., Araci, Z., Inayathullah, M., Manickam, S., Zhang, X., Bruce, M. A., Marinkovich, M. P., Lane, A. T., Milla, C., Rajadas, J., Butte, M. J. 2013; 9 (8): 7767-7774

  Abstract

  We present a method of fabricating microneedles from polyvinylpyrrolidone (PVP) that enables delivery of intact proteins (or peptides) to the dermal layers of the skin. PVP is known to self-assemble into branched hollow fibers in aqueous and alcoholic solutions; we utilized this property to develop dissolvable patches of microneedles. Proteins were dissolved in concentrated PVP solution in both alcohol and water, poured into polydimethylsiloxane templates shaped as microneedles and, upon evaporation of solvent, formed into concentric, fibrous, layered structures. This approach of making PVP microneedles overcomes problems in dosage, uniform delivery and stability of protein formulation as compared to protein-coated metallic microneedles or photopolymerized PVP microneedles. Here we characterize the PVP microneedles and measure the delivery of proteins into skin. We show that our method of fabrication preserves the protein conformation. These microneedles can serve as a broadly useful platform for delivering protein antigens and therapeutic proteins to the skin, for example for allergen skin testing or immunotherapy.

  View details for DOI 10.1016/j.actbio.2013.04.045

  View details for PubMedID 23648574

• Study design considerations for evaluating the efficacy and safety of pancreatic enzyme replacement therapy in patients with cystic fibrosis. Clinical investigation Konstan, M. W., Borowitz, D., Mayer-Hamblett, N., Milla, C., Hendeles, L., Murray, S., Kronmal, R. A., Casey, S., Rose, L. M., Morgan, W. J., Ramsey, B. W. 2013; 3 (8): 731-741

  Abstract

  In 2006, the US FDA issued a 'Guidance for Industry' regarding submission of New Drug Applications for pancreatic enzyme replacement therapy (PERT) products. Five oral delayed-release PERT products have been approved by the FDA, and several others are under development and/ or evaluation for New Drug Application submission. We present in this paper recommendations of the Cystic Fibrosis Foundation's Cystic Fibrosis (CF) Therapeutics Development Network and Data Safety Monitoring Board regarding study design considerations for evaluating PERT products in patients with CF. Careful attention to study design and accuracy of the outcome measures has confirmed our understanding of the efficacy and safety of PERT for the treatment of exocrine pancreatic insufficiency of CF.

  View details for DOI 10.4155/cli.13.63

  View details for PubMedID 25132954

  View details for PubMedCentralID PMC4131768

• Cystic fibrosis in the era of genomic medicine. Current opinion in pediatrics Milla, C. E. 2013; 25 (3): 323-328

  Abstract

  The field of cystic fibrosis (CF) is changing dramatically as the scientific knowledge accumulated since the cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is being translated into effective therapies to correct the basic defect and provide better disease models and in-depth understanding of the basic mechanisms of disease.This review focuses on three main aspects of the recent advances in the field: understanding the lung disease pathophysiology (in particular, the early events that condition its onset), better definition of the complex microbiology of the CF airway, and therapeutic developments. Although the most recently developed therapies, whether approved or under study, do not constitute a definitive cure, the benefit to patients is already becoming clearly apparent.As the field continues to change rapidly and new therapies are being identified, CF has become a paradigm for the application of concepts such as translational medicine, genomic medicine, and personalized care, with measurable clinical benefit for the patients affected by this disease.

  View details for DOI 10.1097/MOP.0b013e328360dbf5

  View details for PubMedID 23652683

• Exome Sequencing Identifies Mutations in CCDC114 as a Cause of Primary Ciliary Dyskinesia AMERICAN JOURNAL OF HUMAN GENETICS Knowles, M. R., Leigh, M. W., Ostrowski, L. E., Huang, L., Carson, J. L., Hazucha, M. J., Yin, W., Berg, J. S., Davis, S. D., Dell, S. D., Ferkol, T. W., Rosenfeld, M., Sagel, S. D., Milla, C. E., Olivier, K. N., Turner, E. H., Lewis, A. P., Bamshad, M. J., Nickerson, D. A., Shendure, J., Zariwala, M. A. 2013; 92 (1): 99-106

  Abstract

  Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ~60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.

  View details for DOI 10.1016/j.ajhg.2012.11.003

  View details for Web of Science ID 000313759000010

  View details for PubMedID 23261302

  View details for PubMedCentralID PMC3542458

• In vivo readout of CFTR function: ratiometric measurement of CFTR-dependent secretion by individual, identifiable human sweat glands. PloS one Wine, J. J., Char, J. E., Chen, J., Cho, H., Dunn, C., Frisbee, E., Joo, N. S., Milla, C., Modlin, S. E., Park, I., Thomas, E. A., Tran, K. V., Verma, R., Wolfe, M. H. 2013; 8 (10)

  Abstract

  To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (∼50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ∼0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.

  View details for DOI 10.1371/journal.pone.0077114

  View details for PubMedID 24204751

• Quantitative Analysis of the Human Airway Microbial Ecology Reveals a Pervasive Signature for Cystic Fibrosis SCIENCE TRANSLATIONAL MEDICINE Blainey, P. C., Milla, C. E., Cornfield, D. N., Quake, S. R. 2012; 4 (153)

  Abstract

  Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the CF transmembrane conductance regulator. Disruption of electrolyte homeostasis at mucosal surfaces leads to severe lung, pancreatic, intestinal, hepatic, and reproductive abnormalities. Loss of lung function as a result of chronic lung disease is the primary cause of death from CF. Using high-throughput sequencing to survey microbes in the sputum of 16 CF patients and 9 control individuals, we identified diverse microbial communities in the healthy samples, contravening conventional wisdom that healthy airways are not significantly colonized. Comparing these communities with those from the CF patients revealed significant differences in microbial ecology, including differential representation of uncultivated phylotypes. Despite patient-specific differences, our analysis revealed a focal microbial profile characteristic of CF. The profile differentiated case and control groups even when classically recognized CF pathogens were excluded. As a control, lung explant tissues were also processed from a group of patients with pulmonary disease. The findings in lung tissue corroborated the presence of taxa identified in the sputum samples. Comparing the sequencing results with clinical data indicated that diminished microbial diversity is associated with severity of pulmonary inflammation within our adult CF cohort.

  View details for DOI 10.1126/scitranslmed.3004458

  View details for Web of Science ID 000309525600003

  View details for PubMedID 23019655

• PATTERNS OF HEALTH CARE UTILIZATION IN CHILDREN WITH CYSTIC FIBROSIS ENROLLED IN A STATE PROGRAM FOR CHILDREN WITH SPECIAL HEALTH CARE NEEDS Buu, M. C., Milla, C. E., Sundaram, V., Goldstein, B., Wise, P. H. WILEY-BLACKWELL. 2012: 381–381

  View details for Web of Science ID 000308882000518

• Dyspnea in a patient with raynaud's phenomenon: The uncovering of interstitial lung disease PEDIATRIC PULMONOLOGY Coates, A., Meehan, R., Milla, C. 2012; 47 (9): 926-927

  Abstract

  Interstitial lung disease (ILD) can develop in patients with connective tissue disease (CTD) in the context of progressive multiorgan involvement, but ILD can also be the predominant manifestation of active CTD. A high index of suspicion for CTD in patients presenting with pulmonary disease might facilitate timely, accurate diagnosis and management.

  View details for DOI 10.1002/ppul.22549

  View details for Web of Science ID 000307547600012

  View details for PubMedID 22467473

• IMPROVING PATIENT AND FAMILY EDUCATION AND QUALITY OF LIFE THROUGH AN INDIVIDUALIZED CF ACTION PLAN AND ORGANIZATIONAL TOOL Coates, A., Helmers, M., Matel, J., Shelton, K., Martins, L., Huerta, M., Souza, C., Cornfield, D., Milla, C. WILEY-BLACKWELL. 2012: 390–391

  View details for Web of Science ID 000308882000543

• Effect of Endoscopic Sinus Surgery on Pulmonary Status of Adults with Cystic Fibrosis OTOLARYNGOLOGY-HEAD AND NECK SURGERY Kempainen, R. R., Sajan, J. A., Pylkas, A. M., Dunitz, J. M., Rimell, F. L., Milla, C. E. 2012; 147 (3): 557-562

  Abstract

  Functional endoscopic sinus surgery (FESS) provides symptomatic relief of sinus disease in patients with cystic fibrosis (CF), but it is unclear whether it has beneficial effects on lung disease in this population. This study assessed the effect of FESS on the respiratory status of adult patients with CF.Retrospective chart review.Tertiary medical center.Thirty-two adult patients with CF who underwent 45 operative cases.Clinical information retrieved for the 12-month periods preceding and following to determine the effect of FESS on the rate of decline in lung function, as well as intravenous antibiotic use and hospitalization for pulmonary exacerbation.The rate of decline in forced expiratory volume in 1 second and forced vital capacity was not significantly different in the 12 months before and after FESS. Functional endoscopic sinus surgery did not reduce days hospitalized or days on intravenous antibiotics for a respiratory exacerbation in the pre- vs postoperative period. Limiting the analysis to the 30 surgeries that were performed in patients with concomitant respiratory symptoms (ie, excluding the 15 surgeries performed for sinus symptoms alone) did not significantly alter the results. Covariates of importance in CF, including CFTR genotype, gender, or microbiology, did not affect the study results.These results did not demonstrate an effect of FESS on progression of lung disease in patients with CF, but further research is needed because low statistical power has made some of the negative findings inconclusive.

  View details for DOI 10.1177/0194599812444247

  View details for Web of Science ID 000314281300027

  View details for PubMedID 22517014

• Pulmonary Complications of Endocrine and Metabolic Disorders PAEDIATRIC RESPIRATORY REVIEWS Milla, C. E., Zirbes, J. 2012; 13 (1): 23-28

  Abstract

  There are many important respiratory manifestations of endocrine and metabolic diseases in children. Acute and chronic pulmonary infections are the most common respiratory abnormalities in patients with diabetes mellitus, although cardiogenic and non-cardiogenic pulmonary oedema are also possible. Pseudohypoaldosteronism type 1 may be indistinguishable from cystic fibrosis (CF) unless serum aldosterone, plasma renin activity, and urinary electrolytes are measured and mutation analysis rules out CF. Hypo- and hyperthyroidism may alter lung function and affect the central respiratory drive. The thyroid hormone plays an essential role in lung development, surfactant synthesis, and lung defence. Complications of hypoparathyroidism are largely due to hypocalcaemia. Laryngospasm can lead to stridor and airway obstruction. Ovarian tumours, benign or malignant, may present with unilateral or bilateral pleural effusions. Metabolic storage disorders, primarily as a consequence of lysosomal dysfunction from enzymatic deficiencies, constitute a diverse group of rare conditions that can have profound effects on the respiratory system.

  View details for DOI 10.1016/j.prrv.2011.01.004

  View details for Web of Science ID 000299582300005

  View details for PubMedID 22208790

• ANTIBODY-BASED ANTIBACTERIAL AGENTS: AN EMERGING OPTION DRUGS OF THE FUTURE Milla, C. E. 2012; 37 (1): 33-43

  View details for DOI 10.1358/dof.2012.37.1.1688497

  View details for Web of Science ID 000304727800005

• METABOLIC CONDITIONING AND REPROGRAMMING ARE HALLMARKS OF NEUTROPHILIC INFLAMMATION IN CYSTIC FIBROSIS Tirouvanziam, R., Laval, J., Gudiputi, L., Touhami, J., Battini, J., Conrad, C., Milla, C., Moss, R., Sitbon, M., Herzenberg, L. BIRKHAUSER VERLAG AG. 2011: 78

  View details for Web of Science ID 000291358900199

• Diagnostic Yield of Nasal Scrape Biopsies in Primary Ciliary Dyskinesia: A Multicenter Experience PEDIATRIC PULMONOLOGY Olin, J. T., Burns, K., Carson, J. L., Metjian, H., Atkinson, J. J., Davis, S. D., Dell, S. D., Ferkol, T. W., Milla, C. E., Olivier, K. N., Rosenfeld, M., Baker, B., Leigh, M. W., Knowles, M. R., Sagel, S. D. 2011; 46 (5): 483-488

  View details for DOI 10.1002/ppul.21402

  View details for Web of Science ID 000289510500010

• CFTR transcription defects in pancreatic sufficient cystic fibrosis patients with only one mutation in the coding region of CFTR JOURNAL OF MEDICAL GENETICS Sheridan, M. B., Hefferon, T. W., Wang, N., Merlo, C., Milla, C., Borowitz, D., Green, E. D., Mogayzel, P. J., Cutting, G. R. 2011; 48 (4): 235–41

  Abstract

  Patients with cystic fibrosis (CF) manifest a multisystem disease due to deleterious mutations in each gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). However, the role of dysfunctional CFTR is uncertain in individuals with mild forms of CF (ie, pancreatic sufficiency) and mutation in only one CFTR gene.Eleven pancreatic sufficient (PS) CF patients with only one CFTR mutation identified after mutation screening (three patients), mutation scanning (four patients) or DNA sequencing (four patients) were studied. Bi-directional sequencing of the coding region of CFTR was performed in patients who had mutation screening or scanning. If a second CFTR mutation was not identified, CFTR mRNA transcripts from nasal epithelial cells were analysed to determine if any PS-CF patients harboured a second CFTR mutation that altered RNA expression.Sequencing of the coding regions of CFTR identified a second deleterious mutation in five of the seven patients who previously had mutation screening or mutation scanning. Five of the remaining six patients with only one deleterious mutation identified in the coding region of one CFTR gene had a pathologic reduction in the amount of RNA transcribed from their other CFTR gene (8.4-16% of wild type).These results show that sequencing of the coding region of CFTR followed by analysis of CFTR transcription could be a useful diagnostic approach to confirm that patients with mild forms of CF harbour deleterious alterations in both CFTR genes.

  View details for DOI 10.1136/jmg.2010.083287

  View details for Web of Science ID 000288784300004

  View details for PubMedID 21097845

  View details for PubMedCentralID PMC3065505

• Diagnostic yield of nasal scrape biopsies in primary ciliary dyskinesia: A multicenter experience. Pediatric pulmonology Olin, J. T., Burns, K., Carson, J. L., Metjian, H., Atkinson, J. J., Davis, S. D., Dell, S. D., Ferkol, T. W., Milla, C. E., Olivier, K. N., Rosenfeld, M., Baker, B., Leigh, M. W., Knowles, M. R., Sagel, S. D. 2011

  Abstract

  Examination of ciliary ultrastructure remains the cornerstone diagnostic test for primary ciliary dyskinesia (PCD), a disease of abnormal ciliary structure and/or function. Obtaining a biopsy with sufficient interpretable cilia and producing quality transmission electron micrographs (TEM) is challenging. Methods for processing tissues for optimal preservation of axonemal structures are not standardized. This study describes our experience using a standard operating procedure (SOP) for collecting nasal scrape biopsies and processing TEMs in a centralized laboratory. We enrolled patients with suspected PCD at research sites of the Genetic Disorders of Mucociliary Clearance Consortium. Biopsies were performed according to a SOP whereby curettes were used to scrape the inferior surface of the inferior turbinate, with samples placed in fixative. Specimens were shipped to a central laboratory where TEMs were prepared and blindly reviewed. Four hundred forty-eight specimens were obtained from 107 young children (0-5 years), 189 older children (5-18 years), and 152 adults (> 18 years), and 88% were adequate for formal interpretation. The proportion of adequate specimens was higher in adults than in children. Fifty percent of the adequate TEMs showed normal ciliary ultrastructure, 39% showed hallmark ultrastructural changes of PCD, and 11% had indeterminate findings. Among specimens without clearly normal ultrastructure, 72% had defects of the outer and/or inner dynein arms (IDA), while 7% had central apparatus defects with or without IDA defects. In summary, nasal scrape biopsies can be performed in the outpatient setting and yield interpretable samples, when performed by individuals with adequate training and experience according to an SOP. Pediatr. Pulmonol. © 2010 Wiley-Liss, Inc.

  View details for DOI 10.1002/ppul.21402

  View details for PubMedID 21284095

• METABOLITE PROFILING OF CF AIRWAY FLUID SUGGESTS A ROLE FOR CATECHOLAMINES IN EARLY AND CHRONIC DISEASE Gudiputi, L., Aronov, P., Makam, M., Zirbes, J., Conrad, C., Milla, C., Herzenberg, L., Moss, R., Tirouvanziam, R. WILEY-BLACKWELL. 2011: 240–240

  View details for Web of Science ID 000296071800163

• LONGITUDINAL ASSESSMENT OF SWEAT CHLORIDE VALUES IN INFANTS WITH 2 CFTR MUTATIONS IDENTIFIED BY NEWBORN SCREENING Zirbes, J. M., Hardy, K., Kharrazi, M., Milla, C. WILEY-BLACKWELL. 2011: 370–71

  View details for Web of Science ID 000296071800516

• Characteristics of gastroesophageal reflux in adults with cystic fibrosis JOURNAL OF CYSTIC FIBROSIS Sabati, A. A., Kempainen, R. R., Milla, C. E., Ireland, M., Schwarzenberg, S. J., Dunitz, J. M., Khan, K. M. 2010; 9 (5): 365-370

  Abstract

  Gastroesophageal reflux (GER) in adults with cystic fibrosis (CF) is poorly characterized. This study examines the frequency and predictors of GER symptoms and their relationship to lung function in adults with CF.Cross-sectional study of adults at the University of Minnesota CF Clinic using two validated self report surveys: The Mayo GER questionnaire and the GERD Symptom Assessment Scale (GSAS).Of 274 invited patients, 201 (73%) completed the surveys and 173 performed spirometry at the same visit. Frequent symptoms (at least weekly) were reported by 24% of the patients and an additional 39% experienced occasional symptoms. Heartburn, acid regurgitation and dysphagia were the most common symptoms and 18% reported that GER symptoms worsened their respiratory condition. Females and patients reporting weight loss had more symptoms (mean GSAS symptom score 4.9 vs. 4.0, p=0.025 and 5.3 vs. 4.2, p=0.04) and more severe symptoms (mean GSAS distress score 5.6 vs. 3.8, p=0.005 and 6.8 vs. 4.0, p=0.01) compared to males and those who did not report weight loss. Patients on acid suppression (n=122, 61%) continued to report heartburn (n=80, 66%) and acid regurgitation (n=47, 23%). GER symptoms and severity of symptoms were not predictive of FEV(1) or FVC.GER symptoms were present in a majority of patients. Females and patients with weight loss require special attention to their GER symptoms. Many patients on acid suppression continued to be report symptoms.

  View details for DOI 10.1016/j.jcf.2010.06.004

  View details for Web of Science ID 000283411500011

  View details for PubMedID 20674518

• Pre-transplant risk factors affecting outcome in Hurler syndrome BONE MARROW TRANSPLANTATION Orchard, P. J., Milla, C., Braunlin, E., DeFor, T., Bjoraker, K., Blazar, B. R., Peters, C., Wagner, J., Tolar, J. 2010; 45 (7): 1239-1246

  Abstract

  Allogeneic transplantation remains the standard of care for patients with Hurler syndrome. As enzyme replacement therapy (ERT) has become available, controversy has emerged in regards to whether the use of enzyme in the peri-transplant period is appropriate. An analysis was performed on 74 patients with Hurler syndrome transplanted at the University of Minnesota between 1990 and 2003, before our use of ERT associated with transplant, with the intention of determining if patients at higher risk during the transplant can be identified based on evaluations and events before transplantation. Age, the presence of hydrocephalus, a history of cardiovascular issues or upper airway obstruction before transplant was not associated with significant differences in survival. In contrast, patients who had a history of lower airway disease, including reactive airway disease or bronchiolitis, or a history of pneumonia, had a significantly inferior outcome based on OS. The risk for serious respiratory complications was also assessed by evaluating the incidence of intubation. Overall, 31% of these patients were intubated. The risk of intubation was higher in older patients and in those with a history of lower airway disease. These findings have implications for the care of patients with high-risk features.

  View details for DOI 10.1038/bmt.2009.319

  View details for Web of Science ID 000279614900016

  View details for PubMedID 19898501

• Comparison of Settings Used for High-Frequency Chest-Wall Compression in Cystic Fibrosis RESPIRATORY CARE Kempainen, R. R., Milla, C., Dunitz, J., Savik, K., Hazelwood, A., Williams, C., Rubin, B. K., Billings, J. L. 2010; 55 (6): 695-701

  Abstract

  Cystic fibrosis (CF) patients commonly use a high-frequency chest-wall compression (HFCWC) device for airway clearance that generates oscillatory flow with a sine-wave configuration. Typical HFCWC settings combine a lower Vest inflation pressure setting (eg, 5 on the Vest's arbitrary 1-10 scale for the setting that controls the background pressure of the inflatable vest) with mid-range frequency (14-16 Hz) (lower-pressure/mid-frequency HFCWC).To determine whether HFCWC with higher pressure settings (6-10 on the Hill-Rom Vest's arbitrary 1-10 scale) combined with variable mid-frequencies (8, 9, and 10 Hz, plus 18, 19, and 20 Hz) (higher-pressure/variable-frequency HFCWC) results in greater sputum expectoration than lower-pressure/mid-frequency HFCWC.This was a controlled randomized crossover study. Sixteen clinically stable, adult CF patients participated. Patients performed airway clearance with HFCWC, once each with lower-pressure/mid-frequency HFCWC and higher-pressure/variable-frequency HFCWC, on separate occasions. All sputum produced during each session was collected. Patients completed pulmonary function tests before and after each session.Median sputum wet weight was greater with higher-pressure/variable-frequency HFCWC than with lower-pressure/mid-frequency HFCWC (6.4 g, range 0.49-22.0 g, versus 4.8 g, range 0.24-15.0 g, P = .02). Dry sputum weight differences did not reach statistical significance (higher-pressure/variable-frequency HFCWC 0.20 g, range 0.009-0.62 g, lower-pressure/mid-frequency HFCWC 0.12 g, range 0.0001-1.0 g, P = .23). Higher-pressure/variable-frequency HFCWC and lower-pressure/mid-frequency HFCWC resulted in similar increases in FEV(1) (70 mL vs 90 mL, P = .21) and forced vital capacity (80 mL vs 80 mL, P = .94). Post-therapy sputum viscoelastic properties did not differ. Patients perceived the 2 regimens as equally comfortable and effective (P = .35 and P = .35, respectively).In adult CF patients, single-session higher-pressure/variable-frequency HFCWC resulted in greater sputum expectoration by wet weight, but not other differences, compared to the commonly used lower-pressure/mid-frequency settings. Longer-term comparisons are needed in a larger, more diverse population to determine whether sustained use of the higher-pressure/variable-frequency settings results in clinically important differences in outcomes.

  View details for Web of Science ID 000279232200003

  View details for PubMedID 20507651

• HOSPITALIZATION AND DEATH RATES OF HISPANIC CYSTIC FIBROSIS PEDIATRIC PATIENTS IN CALIFORNIA Buu, M. C., Milla, C., Chan, J., Wise, P. H., Cornfield, D. N. WILEY-BLACKWELL. 2010: 390–391

  View details for Web of Science ID 000282988800558

• Lung Density And Quantitative Air Trapping In Infants/Toddlers With Cystic Fibrosis Utilizing Controlled Ventilation Infant CT (CViCT) Scanning Robinson, T. E., Raman, P., Milla, C. E., Waltz, D. A., Goris, M. L. AMER THORACIC SOC. 2010

  View details for Web of Science ID 000208771001039

• Nutrition in Cystic Fibrosis SEMINARS IN RESPIRATORY AND CRITICAL CARE MEDICINE Matel, J. L., Milla, C. E. 2009; 30 (5): 579-586

  Abstract

  Cystic fibrosis (CF) is mostly recognized for its pulmonary morbidity, but the earliest manifestations of the disease are related to its gastrointestinal and nutritional derangements. Destruction of acinar pancreatic tissue, pancreatic ductular obstruction, and lack of enzymatic activity lead to malabsorption (particularly of fats), diarrhea, and failure to thrive. A minority of CF patients carrying milder CF transmembrane conductance regulator (CFTR) mutations have preserved pancreatic secretory activity and are free from significant malabsorption early in life. However, these patients are at risk for losing pancreatic function over time. Nutritional status plays an important role in the progression of the pulmonary disease in CF. Further, CF patients with better nutritional status have a survival advantage. Several factors contribute to impaired nutritional status in CF (e.g., pancreatic insufficiency, chronic malabsorption, recurrent sinopulmonary infections, chronic inflammation, increased energy expenditure, suboptimal intake). Progressive lung disease further increases calorie requirements by increasing the work of breathing. Treatment programs that place an emphasis on higher caloric intake and more aggressive nutritional management in CF patients report better outcomes. Basic tenets of nutritional repletion in CF include the use of pancreatic enzyme replacement therapy and following a high calorie, high protein, unrestricted diet. At the Stanford Cystic Fibrosis Center, nutritional status is assessed on an ongoing basis through anthropometric parameters and annual assessment of body composition, bone density, glucose tolerance, and various biochemical and micronutrient levels. Based on the anthropometric data obtained on routine clinical encounters, patients are categorized as to their nutritional risk. This proactive approach for the early identification of nutritional risk has become a major theme within the network of US CF centers. Aggressive nutritional support with adequate pancreatic replacement management should lead to both normal growth and lung function preservation. In addition, nutritional status has to be monitored closely during routine encounters to allow for early intervention once derangements are noted. This will include increasing calories in the early stages of lung disease and being vigilant of gastrointestinal symptomatology and complications.

  View details for DOI 10.1055/s-0029-1238916

  View details for Web of Science ID 000270175900009

  View details for PubMedID 19760545

• Cystic fibrosis related diabetes PAEDIATRIC RESPIRATORY REVIEWS Zirbes, J., Milla, C. E. 2009; 10 (3): 118-123

  Abstract

  Diabetes is a frequent complication seen in cystic fibrosis patients as they reach adulthood. Cystic fibrosis related diabetes (CFRD) is distinguished as a separate entity with features that include progressive loss of islet beta cell mass and insulin deficiency, as well as insulin resistance. Abnormalities in glucose tolerance may be detectable for many years prior to the development of overt diabetes. Therefore oral glucose tolerance testing is the preferred screening method for the identification of those patients at the highest risk for progression to diabetes. Progression to diabetes has been linked to poor outcomes in CF including loss of pulmonary function and increased mortality among females. Given the role that insulin deficiency plays in CFRD, insulin replacement therapy remains the only recommended intervention. In the absence of definitive supportive data, the use of oral antidiabetic agents is not considered standard therapy and needs further study. As with other forms of diabetes, CFRD patients also experience microvascular complications and should be periodically evaluated for manifestations.

  View details for DOI 10.1016/j.prrv.2009.04.004

  View details for Web of Science ID 000279268600007

  View details for PubMedID 19651382

• Longitudinal Assessment of Lung Function From Infancy to Childhood in Patients With Cystic Fibrosis 20th Annual North American Cystic Fibrosis Conference Harrison, A. N., Regelmann, W. E., Zirbes, J. M., Milla, C. E. WILEY-LISS. 2009: 330–39

  Abstract

  Infant pulmonary function testing (IPFT) has become an important clinical tool for the evaluation of lung function in infants with Cystic Fibrosis (CF); however, it is still unclear whether lung function in infancy is predictive of lung function later in life. We hypothesized that measures of airflow obstruction by IPFT would correlate strongly with lung function by conventional spirometry later in childhood. STUDY DESIGN AND METHODOLOGY: A retrospective analysis was performed of all CF infants studied with IPFT at the University of Minnesota Children's Hospital between September 1994 and March 2003. A total of 41 patients underwent IPFT and had valid spirometry results available at age 6 or later. IPFT values, such as I:E ratio, respiratory rate, tidal volume, and T(ptef)/T(e), were calculated from tidal breathing loops. Passive respiratory system mechanics, which included C(rs), R(rs), and tau(rs), were measured by the single breath end-inspiratory occlusion technique. Forced expiratory flows, including V(max)FRC, FVC, FEF(50), and FEF(75), were obtained by rapid thoracic compression and included a full vital capacity maneuver by the multiple inflation method. FRC measurements were calculated from data obtained via nitrogen washout in a subset of patients. In addition, information on age at diagnosis and results of oropharyngeal (OP) cultures at diagnosis and on subsequent visits was recorded. Standard spirometry was performed in all patients starting at age 5. The first valid flow-volume loop after age six was selected for analysis.Significant correlations were observed for the R(rs) and the FEF(50) by IPFT and the FEV(1) and the FEF(25-75) by standard spirometry (r > 0.4 and P < 0.03 for all correlations). These correlations were the strongest for those IPFT measurements obtained within 1 month of diagnosis and when R(rs) was expressed as sG(rs). The correlations observed were independent of the effects of age at diagnosis, gender and presence of Pseudomonas in oropharyngeal cultures at the time of diagnosis. Mean R(rs) declined from 0.050 to 0.027 cm H(2)O/ml/sec with treatment (P < 0.0001). There were no other significant associations found between other IPFT values measured and FEV(1) by spirometry.Measures of airflow obstruction on IPFT, specifically R(rs), sG(rs), and FEF(50), were strongly correlated with future lung function. IPFT measurement of R(rs) in addition to forced expiratory flows may help select patients at the greatest risk of early lung function decline. This study supports the use of R(rs) as a surrogate variable to help assess the impact of early therapies in CF.

  View details for DOI 10.1002/ppul.20994

  View details for Web of Science ID 000264965400005

  View details for PubMedID 19274621

• Associations of Psychosocial Factors With Health Outcomes Among Youth With Cystic Fibrosis PEDIATRIC PULMONOLOGY Patterson, J. M., Wall, M., Berge, J., Milla, C. 2009; 44 (1): 46-53

  Abstract

  The purpose of this study was to examine the relationship of strains, resources, feelings, and behaviors about treatment adherence reported by youth with cystic fibrosis (CF) with repeated clinic measures of their pulmonary function and nutritional status.Linear mixed models, stratified by gender, adjusting for age, were used to examine the effects of strains, resources, and adherence behaviors on repeated pulmonary function and nutritional status measures. All 10-21 years old with CF at the Minnesota Cystic Fibrosis Center were invited by mail to participate. Of these 177 youth, 51% (43 boys, 47 girls) returned surveys. Forced expiratory volume in 1 sec and predicted weight-for-height were extracted from participants' clinic records for the 18 months following receipt of the survey.Females showed significantly greater variability in repeated measures of pulmonary function and nutritional status compared to males. Parent-youth strains, physical strains, activity limitations, and cough suppression had significant effects on the 18-month mean of pulmonary function measures for females, but only physical strains had a significant effect for males.Compared to males, females experienced more strains and poorer treatment adherence, which may be factors associated with declines in pulmonary function observed among females with CF during the adolescent years.

  View details for DOI 10.1002/ppul.20925

  View details for Web of Science ID 000262386100006

  View details for PubMedID 19085923

• ANTI-INFLAMMATORY EFFECT OF KB001, AN ANTI-PCRV ANTIBODY FRAGMENT, IN CF PATIENTS CHRONICALLY INFECTED WITH PSEUDOMONAS AERUGINOSA Milla, C., Chmiel, J. F., Accurso, F. J., McCoy, K. S., Billings, J. L., Atkinson, J. J., Clancy, J. P., Liou, T. G., Acton, J. D., Lynch, S., Slusher, N., Burns, J. L., Hamblett, N. M., Harris, J. K., Patel, R., Tremblay, T. M., Parli, T. J. WILEY-BLACKWELL. 2009: 341–341

  View details for Web of Science ID 000270703400448

• Steroid-sparing effect of Omalizumab for allergic bronchopulmonary aspergillosis and cystic fibrosis PEDIATRIC PULMONOLOGY Zirbes, J. M., Milla, C. E. 2008; 43 (6): 607-610

  Abstract

  Allergic bronchopulmonary aspergillosis (ABPA) is a complication commonly encountered in patients with CF that produces significant respiratory morbidity. Chronic airway colonization with Aspergillus induces strong inflammatory responses with high IgE levels. Current guidelines for therapy include prolonged courses of systemic corticosteroids as the main therapeutic strategy. However this has the potential to induce significant detrimental side effects in children. Omalizumab is a humanized monoclonal antibody directed against IgE that prevents its binding to high- and low-affinity receptors on effector cells. It has been shown to be effective in improving asthma control in patients with a strong allergic component. We present our long term experience with the use of Anti-IgE therapy in three children with CF and ABPA (mean age at start of therapy 14.2 years) who were steroid dependent. All three were already experiencing significant side effects from chronic steroid therapy. After the start of Omalizumab these children have experienced significant and sustained clinical improvements at the same time that they were discontinued from chronic systemic steroids. Our experience suggests that IgE blockade has tremendous potential as a strategy to control this disease in steroid dependent patients.

  View details for DOI 10.1002/ppul.20804

  View details for Web of Science ID 000256744200012

  View details for PubMedID 18433040

• Gender differences in treatment adherence among youth with cystic fibrosis: Development of a new questionnaire JOURNAL OF CYSTIC FIBROSIS Patterson, J. M., Wall, M., Berge, J., Milla, C. 2008; 7 (2): 154-164

  Abstract

  Some prior studies have reported that girls with cystic fibrosis (CF) experience higher morbidity and mortality compared to boys. In this study, the authors compared boys' and girls' perceptions of disease-related strains and resources associated with living with CF, and the relationship of these factors to CF treatment feelings and behaviors.All 10-21 year olds with CF at the Minnesota Cystic Fibrosis Center were invited by mail to complete a new self-report survey (Living with CF Questionnaire--LCFQ). Of these 177 youth, 58% (49 boys and 54 girls) returned surveys.Exploratory and confirmatory factor analyses revealed nine factors in the LCFQ. Partial support was found for hypothesized gender differences in these factors. Compared to boys, girls reported significantly more illness-related strains and worries, including emotional strains, greater treatment discouragement, lower self-esteem, and lower adherence to some aspects of the CF treatment regimen (coughing, eating high-fat foods, taking meds/pills).Living with CF appears to have a greater emotional impact on adolescent girls compared to boys. These gender differences may contribute to the poorer pulmonary function observed among girls with cystic fibrosis during the adolescent years.

  View details for DOI 10.1016/j.jcf.2007.07.008

  View details for Web of Science ID 000255299400011

  View details for PubMedID 17719857

• Comparison of high-frequency chest wall oscillation with differing waveforms for airway clearance in cystic fibrosis CHEST Kempainen, R. R., Williams, C. B., Hazelwood, A., Rubin, B. K., Milla, C. E. 2007; 132 (4): 1227-1232

  Abstract

  High-frequency chest wall oscillation (HFCWO) is commonly used by cystic fibrosis (CF) patients for airway clearance. The primary objective of this study was to determine whether the use of a newer HFCWO device that generates oscillations with a triangular waveform results in greater sputum production than a commonly used device that generates oscillations with a sine waveform.This was a controlled, randomized, double-blind, crossover study. Fifteen clinically stable, adult CF patients participated. Patients performed airway clearance with each device once and at matched oscillation frequencies and pressures. All sputum produced during each session was collected. Patients completed pulmonary function tests before and after each session, and rated the comfort of the two devices.Mean sputum wet and dry weight produced during sine waveform and triangular waveform HFCWO sessions did not differ (p = 0.11 and p = 0.2, respectively). Mean changes in FEV(1) and FVC following HFCWO therapy were also comparable (p = 0.21 and p = 0.56, respectively). However, there was a significant reduction in air trapping by residual volume/total lung capacity ratio following triangular waveform HFCWO (p = 0.01). In addition, in vitro cough transportability was 10.6% greater following therapy with the triangular waveform device (p = 0.05). Patients perceived the two devices as equally comfortable (p = 0.8).Single-session sputum production is comparable with sine and triangular waveform HFCWO devices. Longer term comparisons are needed to determine whether sustained use of the devices results in clinically important differences in outcomes.

  View details for DOI 10.1378/chest.07-1078

  View details for Web of Science ID 000250254000022

  View details for PubMedID 17890465

• Hepatolithiasis and Cholangiocarcinoma in cystic fibrosis: A case series and review of the literature DIGESTIVE DISEASES AND SCIENCES Perdue, D. G., Cass, O. W., Milla, C., Dunitz, J., Jessurun, J., Sharp, H. L., Schwarzenberg, S. J. 2007; 52 (10): 2638-2642

  View details for DOI 10.1007/s10620-006-9259-1

  View details for Web of Science ID 000249300100027

  View details for PubMedID 17443409

• Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: A Randomized placebo-controlled phase 2B trial HUMAN GENE THERAPY Moss, R. B., Milla, C., Colombo, J., Accurso, F., Zeitlin, P. L., Clancy, J. P., Spencer, L. T., Pilewski, J., Waltz, D. A., Dorkin, H. L., Ferkol, T., Pian, M., Ramsey, B., Carter, B. J., Martin, D. B., Heald, A. E. 2007; 18 (8): 726-732

  Abstract

  Previous studies have demonstrated that delivery of a recombinant adeno-associated virus (AAV) vector encoding the complete human cystic fibrosis transmembrane regulator (CFTR) cDNA (tgAAVCF) to the nose, sinus, and lungs of subjects with cystic fibrosis (CF) was safe and well tolerated. In a small randomized, double-blind study of three doses of aerosolized tgAAVCF or placebo at 30-day intervals, encouraging but non-significant trends in pulmonary function and induced sputum interleukin 8 (IL-8) levels were seen at early time points. This larger study was conducted to verify these trends. One hundred and two subjects aged 12 years and older with mild-to-moderate cystic fibrosis (forced expiratory flow in 1 sec [FEV1]:60% predicted) were randomized to two aerosolized doses of 1x10(13)DNase-resistant particles of tgAAVCF (n=51) or matching placebo (n=51) administered 30 days apart. Although tgAAVCF was well tolerated, the study did not meet its primary efficacy end point of statistically significant improvement in FEV1 30 days after initial administration of tgAAVCF compared with placebo. There were no significant differences in spirometric lung function over time, induced sputum biologic markers, or days of antibiotic use in either treatment group. Thus repeated doses of aerosolized tgAAVCF were safe and well tolerated, but did not result in significant improvement in lung function over time. Because gene transfer is the simplest, most basic way to correct the underlying genetic defect that leads to disease in CF, further research is warranted to develop an effective gene transfer agent for the treatment of CF.

  View details for DOI 10.1089/hum.2007.022

  View details for Web of Science ID 000249124900005

  View details for PubMedID 17685853

• Nutrition and lung disease in cystic fibrosis CLINICS IN CHEST MEDICINE Milla, C. E. 2007; 28 (2): 319-?

  Abstract

  Among patients who have cystic fibrosis (CF), lung disease is a significant contributor to morbidity. From a clinical perspective, the link between malnutrition and lung dysfunction in CF is well established; however, the causal relationship remains unclear. Nutritional intervention for CF patients is predicated on the hypothesis that improved nutritional status improves pulmonary function. Which interventions will be of most value and have sustained gains is not completely clear from the available data. Taking into account that several factors condition the deficits that lead to malnutrition in CF, multidisciplinary interventions are likely to give the best results. More research is needed to better dissect the nutritional factors involved in lung disease and to identify effective and safe interventions through systematic controlled trials.

  View details for DOI 10.1016/j.ccm.2007.02.006

  View details for Web of Science ID 000246780300007

  View details for PubMedID 17467551

• Microvascular complications in cystic fibrosis-related diabetes DIABETES CARE Schwarzenberg, S. J., Walk, D., Thomas, W., Milla, C., Olsen, T. W., Moran, A., Grover, T. 2007; 30 (5): 1056-1061

  Abstract

  The incidence of cystic fibrosis-related diabetes (CFRD) and the prevalence of diabetic microvascular complications were determined at the University of Minnesota.Cystic fibrosis patients have undergone annual oral glucose tolerance testing since 1990. Database review was performed to determine diabetes duration and the results of annual urine albumin-to-creatinine ratio (U(alb:Cr)) screening and dilated retinal exams. In addition, 59 individuals underwent detailed retinopathy, nephropathy, neuropathy, and gastroenteropathy screening.During 1990-2005, 775 patients aged > or = 6 years were followed. CFRD was diagnosed by an oral glucose tolerance test or fasting hyperglycemia in 285 subjects (52% female), 64% of whom had fasting hyperglycemia. Most patients with CFRD without fasting hyperglycemia progressed to CFRD with fasting hyperglycemia over time. No subject with CFRD without fasting hyperglycemia had retinopathy or abnormal U(alb:Cr). In CFRD subjects with fasting hyperglycemia and diabetes for > or = 10 years, 14% had microalbuminuria and 16% had retinopathy. Autonomic neuropathy and gastrointestinal symptoms each were seen in 52% and somatic abnormalities in 22% of patients with or without fasting hyperglycemia.Diabetic microvascular complications occur in CFRD, although the prevalence of retinopathy and nephropathy appears to be less than that found in other forms of diabetes. Annual complication screening should occur after known diabetes duration of 5 years in patients with CFRD with fasting hyperglycemia.

  View details for DOI 10.2337/dc06-1576

  View details for Web of Science ID 000246291400004

  View details for PubMedID 17322485

• Microbiology, safety, and pharmacokinetics of aztreonam lysinate for inhalation in patients with cystic fibrosis 18th Annual North American Cystic Fibrosis Conference Gibson, R. L., Retsch-Bogart, G. Z., Oermann, C., Milla, C., Pilewski, J., Daines, C., Ahrens, R., Leon, K., Cohen, M., McNamara, S., Callahan, T. L., Markus, R., Burns, J. L. WILEY-LISS. 2006: 656–65

  Abstract

  Aztreonam lysinate for inhalation (AI) is a novel monobactam formulation being investigated for pulmonary Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF).Pre-clinical studies investigated the pre- and post-nebulization activity of AI and its activity in the presence of CF sputum. A double-blind, placebo-controlled, dose-escalation trial determined pharmacokinetics and tolerability of AI in subjects with CF. Single daily escalating doses of AI 75, 150, or 225 mg, or placebo were self-administered using an eFlow Electronic Nebulizer. Sputum samples were collected up to 4 hr and blood samples up to 8 hr post-dose.AI activity against multiple CF isolates was retained after nebulization via eFlow, and activity was not inhibited by CF sputum. All 12 adult subjects and 11/12 adolescents tolerated all AI doses. One patient had an asymptomatic FEV1 decrease > 20% with the 150 mg dose. Median aztreonam sputum concentrations in adults 10 min after AI 75, 150, and 225 mg were 383, 879, and 985 microg/g, respectively. Median sputum concentrations in adolescents 10 min after AI 75, 150, and 225 mg were 324, 387, and 260 microg/g, respectively. Systemic exposure to AI was low. Plasma pharmacokinetics in adults receiving AI 75 mg were Cmax = 419 ng/g, Tmax = 0.99 hr, t1/2 = 2.1 hr. Aztreonam concentrations in sputum were at or above the MIC50 for at least 4 hr post-dose.These data support the continued development of AI for treatment of pulmonary infections in patients with CF.

  View details for DOI 10.1002/ppul.20429

  View details for Web of Science ID 000238663200009

  View details for PubMedID 16703579

• Different frequencies should be prescribed for different high frequency chest compression machines. Biomedical instrumentation & technology Milla, C. E., Hansen, L. G., Warwick, W. J. 2006; 40 (4): 319-324

  Abstract

  High frequency chest compression (HFCC) is used for treatment and prevention of the lung diseases characterized by impaired mucus clearance and/or cough, where patients are at risk for acquiring acute bronchitis or pneumonia. The HFCC treatment frequencies may be prescribed according to the manufacturers' generic guidelines or may be determined for each individual patient by a "tuning" method that measures, at the mouth, the air volume displacement and the associated airflows produced at each frequency. Tuning is performed while the patient is breathing normally during the HFCC system operation. After measurements for several breaths at one frequency have been collected, the program randomly selects and measures another frequency until the entire frequency range of the machine being tuned has been sampled. Frequencies range from 6 to 21 Hz for the sine waveform machines and from 6 to 25 Hz for the square waveform machines. Each group of flow signals is digitized and analyzed by the program. For each frequency, the HFCC flow velocities and volumes are computed and averaged. These average flows and volumes are rank ordered; the three frequencies with the highest flows and the three frequencies producing the largest volumes are selected for prescription. If the same frequency is selected as one of the three best frequencies for both flow and volume, the next ranked frequency is selected randomly for flow or volume. Significant differences exist between patients and HFCC machines. In a series of 100 cystic fibrosis (CF) patients with varying degrees of lung disease, we found that the best-ranked frequencies varied from patient to patient and did not correlate with patients' age, gender, height, weight, or spirometry parameters. With the sine waveform, the highest HFCC airflows were between 13 and 20 Hz 82% of the time and the largest HFCC volumes were between 6 and 10 Hz 83% of the time. With the square waveform, both the highest average HFCC flow rates and the largest volume average HFCC displacements were between 6 and 14 Hz. Nevertheless, in this sample of 100 consecutive tunings, every frequency from 6 and 20 Hz was a best frequency for at least one patient. These findings provide the basis for recommending a tuning protocol to be used for prescribing frequencies with the various HFCC machines, because they are different from one another. If a patient's tuning cannot be done, it may be useful to prescribe the best frequencies based on the waveform machine he or she uses.

  View details for PubMedID 16941931

• Inflammatory cytokines and the development of pulmonary complications after allogeneic hematopoietic cell transplantation in patients with inherited metabolic storage disorders BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Kbarbanda, S., Panoskaltsis-Mortari, A., Haddad, I. Y., Blazar, B. R., Orchard, P. J., Cornfield, D. N., Grewal, S. S., Peters, C., Regelmann, W. E., Milla, C. E., Baker, K. S. 2006; 12 (4): 430-437

  Abstract

  Patients with inherited metabolic storage disorders are at a higher risk of developing pulmonary complications after hematopoietic cell transplantation (HCT). This single-center prospective study of 48 consecutive inherited metabolic storage disorder patients was performed to identify risk factors for the development of pulmonary complications after HCT. Before HCT, subjects underwent bronchoalveolar lavage (BAL) for cell count, culture, nitrite levels, and analysis of proinflammatory cytokines and chemokines. The overall incidence of pulmonary complications was 52% (infectious, 23%; noninfectious, 29%) over a period of 4 years. Diffuse alveolar hemorrhage was the most frequent noninfectious complication and occurred in 19% of patients, all of whom had a diagnosis of mucopolysaccharidosis (Hurler and Maroteaux-Lamy syndromes). Levels of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor alpha, macrophage inflammatory protein 1alpha, and granulocyte colony-stimulating factor in BAL fluid samples obtained before HCT were higher in patients with mucopolysaccharidoses than in patients with leukodystrophies. In addition, levels of IL-1beta, IL-6, IL-8, and granulocyte colony-stimulating factor were increased in the BAL fluid of patients who developed noninfectious pulmonary complications compared with those who did not develop pulmonary complications. It is interesting to note that most noninfectious pulmonary complications occurred in patients with mucopolysaccharidoses, especially diffuse alveolar hemorrhage, which occurred exclusively in patients with mucopolysaccharidoses. Higher levels of bronchial proinflammatory cytokines and chemokines may be predictive of the development of subsequent posttransplantation noninfectious complications in patients with mucopolysaccharidoses, especially those with Hurler syndrome. Larger studies will be required to further elucidate etiologic mechanisms and predictive factors.

  View details for DOI 10.1016/j.bbmt.2005.12.026

  View details for Web of Science ID 000236494600006

  View details for PubMedID 16545727

• Cystic fibrosis pulmonary exacerbations JOURNAL OF PEDIATRICS Ferkol, T., Rosenfeld, M., Milla, C. E. 2006; 148 (2): 259-264

  View details for DOI 10.1016/j.jpeds.2005.10.019

  View details for Web of Science ID 000235929600025

  View details for PubMedID 16492439

• Diabetes is associated with dramatically decreased survival in female but not male subjects with cystic fibrosis DIABETES CARE Milla, C. E., Billings, J., Moran, A. 2005; 28 (9): 2141-2144

  Abstract

  Survival analysis was performed on a prospectively followed cohort of patients with cystic fibrosis (CF) to determine the impact of the development of diabetes on survival.Clinical data were retrieved for patients diagnosed with CF-related diabetes (CFRD) at the Minnesota CF Center in 1987-2002. Kaplan-Meier survival analysis was performed to estimate median survival. Data were analyzed by Cox regression to evaluate the influence of clinical characteristics at the time of CFRD diagnosis on mortality.Clinical information was reviewed from 1,081 CF patients. A total of 123 patients with CFRD with fasting hyperglycemia were identified (58 males). Median survival was 49.5 years for male subjects without diabetes, 47.4 years for male subjects with diabetes, 47.0 years for female subjects without diabetes, and 30.7 years for female subjects with diabetes. Only female sex and forced expiratory volume in 1 s at the time of CFRD diagnosis were significant predictors of the subsequent risk of death (P < 0.001). This strong association was not confounded by CFTR genotype, BMI, steroid use, respiratory pathogens, HbA1c, or pregnancy.Female subjects with CFRD have a remarkably poorer prognosis compared with all male subjects with CF and female subjects with CF but without diabetes. The etiology of this sex difference is not clear. We speculate it might involve the interaction of female hormones and diabetes on promotion of a proinflammatory state or that androgens might protect male subjects from the catabolic effects of insulin deficiency. Alternatively, the appearance of frank diabetes in female subjects with CF may simply be a marker for some other biological difference that is not immediately apparent.

  View details for Web of Science ID 000231525700009

  View details for PubMedID 16123480

• Absence of host tumor necrosis factor receptor 1 attenuates manifestations of idiopathic pneumonia syndrome AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Shukla, M., Yang, S. X., Milla, C., Panoskaltsis-Mortari, A., Blazar, B. R., Haddad, I. Y. 2005; 288 (5): L942-L949

  Abstract

  The interaction of TNF-alpha with TNF receptor 1 (TNFR1) activates several signal transduction pathways that lead to apoptosis or NF-kappa B-dependent inflammation and immunity. We hypothesized that host TNFR1 expression contributes to noninfectious lung injury and inflammation commonly observed after bone marrow transplantation (BMT), termed idiopathic pneumonia syndrome (IPS). C57BL/6 TNFR1-sufficient (TNFR1(+/+)) and -deficient (TNFR1(-/-)) mice were total body irradiated with or without cyclophosphamide conditioning and were given bone marrow plus IPS-inducing donor spleen T cells from B10.BR wild-type mice. TNFR1(-/-) recipient mice exhibited improved early post-BMT survival associated with decreased permeability edema. In addition, the low lung compliance measured in anesthetized, ventilated TNFR1(+/+) mice on day 7 after BMT was restored to baseline during TNFR1 deficiency. Importantly, bronchoalveolar lavage fluid (BALF) inflammatory cells from TNFR1(-/-) vs. TNFR1(+/+) mice generated less nitric oxide (.NO) and nitrating species and exhibited suppressed programmed cell death as assessed using flow cytometry. However, cellular infiltration and levels of proinflammatory cytokines and chemokines were generally higher in BALF collected on day 7 after BMT from TNFR1(-/-) compared with TNFR1(+/+) recipient mice. Our results support a major role of host TNFR1 in regulation of .NO production and lung dysfunction after allogeneic BMT.

  View details for DOI 10.1152/ajplung.00260.2004

  View details for Web of Science ID 000228265300022

  View details for PubMedID 15608149

• Safety and tolerability of denufosol tetrasodium inhalation solution, a novel P2Y(2) receptor agonist: Results of a phase 1/phase 2 multicenter study in mild to moderate cystic fibrosis Annual North American Cystic Fibrosis Conference Deterding, R., Retsch-Bogart, G., Milgram, L., Gibson, R., Daines, C., Zeitlin, P. L., Milla, C., Marshall, B., LaVange, L., Engels, J., Mathews, D., Gorden, J., Schaberg, A., Williams, J., Ramsey, B. WILEY-LISS. 2005: 339–48

  Abstract

  Denufosol tetrasodium (INS37217) is a selective P2Y(2) agonist that stimulates ciliary beat frequency and Cl(-) secretion in normal and cystic fibrosis (CF) airway epithelia, and is being investigated as an inhaled treatment for CF. The Cl(-) secretory response is mediated via a non-CFTR pathway, and the driving force for Cl(-) secretion is enhanced by the effect of P2Y(2) activation to also inhibit epithelial Na(+) transport. Denufosol is metabolically more stable and better tolerated, and may enhance mucociliary clearance for a longer period of time than previously investigated P2Y(2) agonists. The goal of this phase 1/phase 2 study was to assess the safety and tolerability of single and repeated doses of aerosolized denufosol in subjects with CF. The study was a double-blind, placebo-controlled, multicenter comparison of ascending single doses of denufosol (10, 20, 40, and 60 mg, administered by inhalation via the Pari LC Star nebulizer) vs. placebo (normal saline), followed by a comparison of twice-daily administration of the maximum tolerated dose (MTD) of denufosol or placebo for 5 days. Thirty-seven adult (18 years of age or older) and 24 pediatric (5-17 years of age) subjects with CF were evaluated in five cohorts. Subjects were randomized in a 3:1 ratio to receive either denufosol or placebo within each cohort. The percent of subjects experiencing adverse events was similar between the denufosol and placebo groups. The most common adverse event in subjects receiving denufosol was chest tightness in adult subjects (39%) and cough in pediatric subjects (56%). Three (7%) subjects receiving denufosol and one (7%) subject receiving placebo experienced a serious adverse event. Forced expiratory volume in 1 sec (FEV(1)) profiles following dosing were similar across treatment groups, with some acute, reversible decline seen in both groups, most notably in subjects with lower lung function at baseline. In conclusion, doses up to 60 mg of denufosol inhalation solution were well-tolerated in most subjects. Some intolerability was noted among subjects with lower baseline lung function. Based on the results of this phase 1/phase 2 study, the Therapeutics Development Network (TDN) of the Cystic Fibrosis Foundation (CFF) and Inspire Pharmaceuticals, Inc., recently completed a multicenter, 28-day, phase 2 safety and efficacy clinical trial of denufosol inhalation solution in CF subjects with mild lung disease.

  View details for DOI 10.1002/ppul.20192

  View details for Web of Science ID 000227814300010

  View details for PubMedID 15704203

• Altered airway responsiveness in CD38-deficient mice AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Deshpande, D. A., White, T. A., Guedes, A. G., Milla, C., Walseth, T. F., Lund, F. E., Kannan, M. S. 2005; 32 (2): 149-156

  Abstract

  Cyclic ADP-ribose (cADPR) mobilizes calcium from intracellular stores and contributes to agonist-induced intracellular calcium elevation in airway smooth muscle (ASM). In this study we determined the functional role of CD38/cADPR signaling in the regulation of airway tone using CD38 deficient (cd38(-/-)) mice. The responsiveness to different doses of methacholine, as determined by changes in lung resistance and dynamic compliance, was significantly (P < or = 0.05) lower in cd38(-/-) mice compared with wild-type controls. To determine the mechanism responsible for the reduced responsiveness, we measured the intracellular calcium responses to contractile agonists in ASM cells. In ASM cells isolated from cd38(-/-) mice, the intracellular calcium responses to acetylcholine and endothelin-1 were significantly lower than in controls. Pretreatment of ASM cells with a cADPR antagonist resulted in attenuated intracellular calcium responses to endothelin-1 in cells isolated from wild-type mice, but not in those isolated from the cd38(-/-) mice. Very low cADPR levels and no detectable ADP-ribosyl cyclase activity were observed in lung tissue from cd38(-/-) mice, suggesting that CD38 is a critical source for cADPR synthesis. The results of the present study demonstrate that CD38/cADPR contributes to airway smooth muscle tone and responsiveness through its effects on agonist-induced elevation of intracellular calcium in ASM cells.

  View details for DOI 10.1165/rcmb.2004-02430C

  View details for Web of Science ID 000226782700010

  View details for PubMedID 15557017

• Natural history of pulmonary complications in children after bone marrow transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Eikenberry, M., Bartakova, H., DeFor, T., Haddad, I. Y., Ramsay, N. K., Blazar, B. R., Milla, C. E., Cornfield, D. N. 2005; 11 (1): 56-64

  Abstract

  We sought, in children after bone marrow transplantation (BMT), (1) to determine the natural history and incidence of pulmonary complications, (2) to evaluate the diagnostic yield of fiberoptic bronchoscopy and bronchoalveolar lavage (BAL); and (3) to determine the effect of bronchoscopy with lavage on patient outcome. The study design was a retrospective review in a tertiary care university hospital of all children undergoing BMT over a 5-year period. Patients were separated into 2 study groups: children with and without pulmonary complications. Pulmonary complications were defined as new or persistent pulmonary infiltrates on chest radiograph or chest computed tomography scan, respiratory symptoms, hypoxemia, or hemoptysis. Three hundred sixty-three pediatric patients underwent BMT between January 1, 1995, and December 31, 1999. Ninety patients (25%) developed pulmonary complications and were evaluated with bronchoscopy and BAL. Patients with pulmonary complications had a higher mortality (65% versus 44%; P < .01). The median posttransplantation survival for children with pulmonary complications was 258 days, compared with 1572 days in patients without pulmonary complications. The incidence of pulmonary complications was increased in patients with allogeneic BMT (P < .01). The time-dependent onset of severe (grade III to IV) graft-versus-host disease increased the relative risk of pulmonary complications by 2.0 (95% confidence interval, 1.1-3.7; P = .02). Pulmonary complications increased the time-dependent relative risk of mortality by 3.5 (95% confidence interval, 2.5-4.8). The diagnostic yield of bronchoscopy with lavage was 46% in patients undergoing BAL. Diagnostic bronchoscopy did not enhance either 30- or 100-day survival. Pathogen identification did not decrease mortality (P = .45). Pulmonary complications occur in 25% of children undergoing BMT and increase the risk of death in the first year after BMT. Although pathogen identification does not confer a survival advantage, rigorous, prospective screening may allow for earlier identification of pathogens and thereby provide a benefit to this uniquely vulnerable population.

  View details for DOI 10.1016/j.bbmt.2004.09.008

  View details for Web of Science ID 000226450300007

  View details for PubMedID 15625545

• Insulin regulation of free fatty acid kinetics in adult cystic fibrosis patients with impaired glucose tolerance METABOLISM-CLINICAL AND EXPERIMENTAL Moran, A., Basu, R., Milla, C., Jensen, M. D. 2004; 53 (11): 1467-1472

  Abstract

  Cystic fibrosis (CF) patients are insulin-resistant with regards to suppression of hepatic glucose production and proteolysis, but the effect of insulin on adipose free fatty acid (FFA) release has not been studied. [9,10-(3)H]palmitate kinetics were measured in 11 stable adult CF patients with impaired glucose tolerance (IGT) and 9 normal control subjects. Baseline plasma palmitate concentrations [CF = 99 +/- 13 (median 74, range 65 to 187); control = 88 +/- 9 (88, 46 to 138) micromol/L, P = .9] and palmitate flux [CF = 114 +/- 11 (100, 72 to 171); control = 105 +/- 12 (106, 54 to 182) micromol/min, P = 0.9] were not different between CF patients and controls. During a euglycemic clamp with infusion of insulin to physiologic postprandial levels, however, palmitate concentrations tended to be higher in CF patients: CF = 18 +/- 3 (13, 10 to 47), control = 12 +/- 1 (11, 8 to 18) micromol/L, P = 0.08. The higher palmitate concentrations during hyperinsulinemia appeared to be due to reduced suppression of adipose tissue palmitate release, because mean palmitate flux was 33% greater in CF subjects [32 +/- 5 (26, 17 to 66) micromol/min] than controls: [24 +/- 2 (23, 17 to 34) micromol/min], P = .20. There was considerably greater heterogeneity in insulin-induced suppression of plasma palmitate concentration and flux in CF patients compared to normal control subjects. In summary, a defect in insulin suppression of lipolysis was seen in clinically stable CF patients with IGT, similar to what has been described in CF for amino acid and glucose metabolism. This quantitative difference in lipolysis may account for inadequate insulin-induced suppression of hepatic glucose production in CF, and may be a metabolic adaptation to increased energy needs.

  View details for DOI 10.1016/j.metabol.2004.06.015

  View details for Web of Science ID 000225070400014

  View details for PubMedID 15536603

• Association of nutritional status and pulmonary function in children with cystic fibrosis CURRENT OPINION IN PULMONARY MEDICINE Milla, C. E. 2004; 10 (6): 505-509

  Abstract

  Multiple studies have shown that nutritional status is a strong predictor of morbidity and mortality in patients with cystic fibrosis (CF). Since CF is characterized by progressive lung disease, it could be argued that the underlying lung disease is what determines the nutritional failure seen in most patients. This review will summarize the data available from studies that have attempted to better define this relation and also present a review of the possible mechanisms involved taken from both observational and interventional studies.Longitudinal studies with sufficiently large follow-up times have demonstrated that young underweight patients have worst pulmonary function outcomes. More importantly, these studies concur in that the yearly change in growth parameters has a significant effect on the rate at which pulmonary function develops. Although the mechanisms behind this important association are yet unclear, there is some suggestion from interventional studies that the accrual of lean body mass is the factor that is involved in the preservation of lung function.Nutritional status strongly influences pulmonary health among CF patients. Therefore, aggressive nutritional support aiming at achieving normal growth patterns should lead to adequate development of lung function and maintenance of pulmonary health. However, more research is required with long-term longitudinal studies to better identify the most critical nutritional characteristics influencing this process as well as the most effective nutritional interventions.

  View details for Web of Science ID 000224924300009

  View details for PubMedID 15510058

• Myeloperoxidase deficiency enhances inflammation after allogeneic marrow transplantation AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Milla, C., Yang, S. X., Cornfield, D. N., Brennan, M. L., Hazen, S. L., Panoskaltsis-Mortari, A., Blazar, B. R., Haddad, I. Y. 2004; 287 (4): L706-L714

  Abstract

  Myeloperoxidase (MPO)-derived oxidants participate in the respiratory antimicrobial defense system but are also implicated in oxidant-mediated acute lung injury. We hypothesized that MPO contributes to lung injury commonly observed after bone marrow transplantation (BMT). MPO-sufficient (MPO+/+) and -deficient (MPO-/-) mice were given cyclophosphamide and lethally irradiated followed by infusion of inflammation-inducing donor spleen T cells at time of BMT. Despite suppressed generation of nitrative stress, MPO-/- recipient mice unexpectedly exhibited accelerated weight loss and increased markers of lung dysfunction compared with MPO+/+ mice. The increased lung injury during MPO deficiency was a result of donor T cell-dependent inflammatory responses because bronchoalveolar lavage fluids (BALF) from MPO-/- mice contained increased numbers of inflammatory cells and higher levels of the proinflammatory cytokine TNF-alpha and the monocyte chemoattractant protein-1 compared with wild-type mice. Enhanced inflammation in MPO-/- mice was associated with suppressed apoptosis of BALF inflammatory cells. The inflammatory process in MPO-/- recipients was also associated with enhanced necrosis of freshly isolated alveolar type II cells, critical for preventing capillary leak. We conclude that suppressed MPO-derived oxidative/nitrative stress is associated with enhanced lung inflammation and persistent alveolar epithelial injury.

  View details for DOI 10.1152/ajplung.00015.2004

  View details for Web of Science ID 000223762200011

  View details for PubMedID 15020295

• Peroxidase activity within circulating neutrophils correlates with pulmonary phenotype in cystic fibrosis JOURNAL OF LABORATORY AND CLINICAL MEDICINE Garner, H. P., PHILLIPS, J. R., Herron, J. G., Severson, S. J., Milla, C. E., Regelmann, W. E. 2004; 144 (3): 127-133

  Abstract

  Excess neutrophils are present in the airways of patients with cystic fibrosis (CF). Myeloperoxidase (MPO) activity of acid extracts of sputum is directly correlated with airflow obstruction in CF patients. We hypothesized that the sputum MPO was derived from the MPO of neutrophils that entered the airways from the circulation. Active MPO without protease activity injures airways. If MPO activity from circulating neutrophils that emigrate into the airways of these patients causes increased airway epithelial permeability and mucus-gland secretion, then (1) those patients with greater MPO activity per circulating neutrophil would be more likely to produce sputum and (2) the MPO activity per circulating neutrophil would positively correlate with airflow obstruction. We determined the MPO activity for both circulating and sputum neutrophils. Spirometry and respiratory cultures were obtained simultaneously with blood and sputum samples. CF patients with more MPO activity within their circulating neutrophils were more likely to produce sputum ( P =.001, chi 2 test), and the MPO activity per circulating neutrophil was positively correlated with airflow obstruction as measured on the basis of the ratio of 1-second forced expiratory volume to forced vital capacity ( P <. 03, Kruskal-Wallace test). These associations were independent of age, sex, the results of respiratory-tract culture, or protease activity in the circulating neutrophils. MPO activity in circulating neutrophils from CF patients homozygotic for the deletion of phenylalanine at position 508 in the CF transmembrane regulator protein is directly related to the severity of these patients' pulmonary disease. Our results are consistent with the hypothesis that circulating neutrophils deliver active MPO to the airway, producing airway injury and airflow obstruction in homozygotic delF508 CF patients.

  View details for Web of Science ID 000224363900003

  View details for PubMedID 15454881

• Methodologic advancements in the study of airway smooth muscle. journal of allergy and clinical immunology Kotlikoff, M. I., Kannan, M. S., Solway, J., Deng, K., Deshpande, D. A., Dowell, M., Feldman, M., Green, K. S., Ji, G., Johnston, R., Lakser, O., Lee, J., Lund, F. E., Milla, C., Mitchell, R. W., Nakai, J., Rishniw, M., Walseth, T. F., White, T. A., Wilson, J., Xin, H., Woodruff, P. G. 2004; 114 (2): S18-31

  Abstract

  The study of isolated airway myocytes has provided important information relative to specific processes that regulate contraction, proliferation, and synthetic properties of airway smooth muscle (ASM). To place this information in physiological context, however, improved methods to examine airway biology in vivo are needed. Advances in genetic, biochemical, and optical methods provide unprecedented opportunities to improve our understanding of in vivo physiology and pathophysiology. This article describes 4 important methodologic advances in the study of ASM: (1) the development of transgenic mice that could be used to investigate ASM proliferation and phenotype switching during the development of hypersensitivity, and to investigate excitation-contraction coupling; (2) the use of CD38-deficient mice to confirm the role of CD38-dependent, cyclic adenosine diphosphate-ribose-mediated calcium release in airway responsiveness; (3) investigation of the role of actin filament length and p38 mitogen-activated protein kinase activity in regulating the mechanical plasticity-elasticity balance in contracted ASM; and (d) the use of bronchial biopsies to study ASM structure and phenotype in respiratory science.

  View details for PubMedID 15309016

• High-frequency chest compression: effect of the third generation compression waveform. Biomedical instrumentation & technology Milla, C. E., Hansen, L. G., Weber, A., Warwick, W. J. 2004; 38 (4): 322-328

  Abstract

  High-frequency chest compression (HFCC) therapy has become the prevailing form of airway clearance for patients with cystic fibrosis (CF) in the United States. The original square waveform was replaced in 1995 with a sine waveform without published evidence of an equality of effectiveness. The recent development of a triangle waveform for HFCC provided the opportunity to compare the functional and therapeutic effects of different waveforms. Clinical testing was done in patients at home with therapy times recorded with all sputum collected in preweighed sealable vials. The eight study patients with CF were regular users of a sine waveform device. They produced sputum consistently and were clinically stable. They used their optimum frequencies for therapy for each waveform and, for one week for each waveform, collected all sputum during their twice-daily timed HFCC therapies. After collection, these vials were reweighed, desiccated, and reweighed to calculate wet and dry weights of sputum per minute of therapy time. Frequency associated vest pressures transmitted to the mouth, and induced airflows at the mouth were measured in healthy volunteers. The pressure waveforms produced in the vest were, in shape, faithfully demonstrable at the mouth. In the healthy subject the transmission occurred in 2 ms and was attenuated to about 75% of the vest pressure for the triangle waveform and 60% for the sine waveform. All patients produced more sputum with the triangle waveform than with the sine waveform. The mean increase was 20%+ range of 4% to 41%. P value was <.001. Future studies of HFCC should investigate the other effects of the sine and triangle waveforms, as well as the neglected square waveform, on mucus clearance and determine the best frequencies for each waveform, disease, and patient.

  View details for PubMedID 15338841

• Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis - A multicenter, double-blind, placebo-controlled trial CHEST Moss, R. B., Rochman, D., Spencer, L. T., Aitken, M. L., Zeitlhi, P. L., Waltz, D., Milla, C., Brody, A. S., Clancy, J. P., Ramsey, B., Hamblett, N., Heald, A. E. 2004; 125 (2): 509-521

  Abstract

  The primary objective was to determine the safety and tolerability of repeated doses of aerosolized adeno-associated serotype 2 vector containing cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA (cDNA) [tgAAVCF], an adeno-associated virus (AAV) vector encoding the complete human CFTR cDNA. Secondary objectives included evaluation of pulmonary function assessed by spirometry, lung abnormalities by high-resolution CT (HRCT), airway cytokines, vector shedding, serum neutralizing antibody to AAV serotype 2 (AAV2), and gene transfer and expression in a subset of subjects undergoing bronchoscopy with bronchial brushings.Randomized, double-blind, placebo-controlled, phase II trial.Eight cystic fibrosis (CF) centers in the United States.CF patients with mild lung disease, defined as FEV(1) > or =60% predicted.Subjects were randomized to inhale three aerosolized doses of 1 x 10(13) deoxyribonuclease-resistant particles of tgAAVCF or matching placebo at 30-day intervals using the Pari LC Plus nebulizer (PARI; Richmond, VA). Measurements and results: Of 42 subjects randomized, 20 subjects received at least one dose of tgAAVCF and 17 subjects received placebo. No difference in the pattern of adverse events or laboratory abnormalities was noted between the two treatment groups. Improvements in induced-sputum interleukin-8 (p = 0.03) and FEV(1) (p = 0.04) were observed at day 14 and day 30, respectively, in the group receiving tgAAVCF when compared to those receiving placebo. No significant differences in HRCT scans were noted. Vector shedding in sputum was observed at low levels up to 90 days after the third dose of vector. All subjects receiving tgAAVCF exhibited an increase (by at least fourfold) in serum AAV2-neutralizing antibodies and detectable levels in BAL fluid from five of six treated subjects undergoing BAL. Gene transfer but not gene expression was detected in a subset of six tgAAVCF subjects who underwent bronchoscopy.Repeat doses of aerosolized tgAAVCF were safe and well tolerated, and resulted in encouraging trends in improvement in pulmonary function in patients with CF and mild lung disease.

  View details for Web of Science ID 000188978400026

  View details for PubMedID 14769732

• Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Haddad, I. Y., Milla, C., Yang, S. X., Panoskaltsis-Mortari, A., Hawgood, S., Lacey, D. L., Blazar, B. R. 2003; 285 (3): L602-L610

  Abstract

  We reported an association between the ability of recombinant human keratinocyte growth factor (rHuKGF) to upregulate the expression of surfactant protein A (SP-A) and to downregulate pulmonary inflammation that occurs after allogeneic bone marrow transplantation (BMT). To establish a causal relationship, rHuKGF (5 mg/kg) was administered subcutaneously for three consecutive days before irradiation to SP-A-sufficient and -deficient [SP-A(+/+) and SP-A(-/-), respectively] mice given inflammation-inducing allogeneic spleen T cells at the time of BMT. In contrast with SP-A(+/+) mice, rHuKGF failed to suppress the high levels of TNF-alpha, IFN-gamma, and nitric oxide contained in bronchoalveolar lavage fluids collected on day 7 after BMT from SP-A(-/-) mice. Early post-BMT weight loss was attenuated by rHuKGF in both SP-A(+/+) and SP-A(-/-) recipients. In the absence of supportive respiratory care, however, SP-A deficiency eventually abolished the ability of rHuKGF to prevent weight loss and to improve survival monitored for 1 mo after allogeneic BMT. In further experiments, the addition of cyclophosphamide (which is known to cause severe injury to the alveolar epithelium in donor T cell-recipient mice) to the conditioning regimen prevented rHuKGF-induced upregulation of SP-A and suppression of lung inflammation in both SP-A(+/+) and SP-A(-/-) mice. We conclude that endogenous baseline SP-A levels and optimal upregulation of SP-A are required for the anti-inflammatory protective effects of KGF after allogeneic transplantation.

  View details for DOI 10.1152/ajplung.00088.2003

  View details for Web of Science ID 000184565600013

  View details for PubMedID 12740217

• Longitudinal changes in growth parameters are correlated with changes in pulmonary function in children with cystic fibrosis PEDIATRICS Peterson, M. L., Jacobs, D. R., Milla, C. E. 2003; 112 (3): 588-592

  Abstract

  Nutritional status is associated with pulmonary health and survival in children with cystic fibrosis (CF). This study evaluated the weight gain pattern of children with CF in relation to the longitudinal trends of their pulmonary function. Our hypothesis was that children who experience continuous weight gain at a given rate will have better average forced expiratory volume in 1 second (FEV(1)) and change in FEV(1) than children who have weight gain patterns that deviate from this rate, even when total weight gain seems adequate.Prospectively collected data were examined in 319 children, aged 6 to 8, who were routinely followed at the Minnesota Cystic Fibrosis Center. One to 67 measurements of weight (kg), height (cm), and FEV(1) (mL) were taken per child during this 2-year period. The data were analyzed by repeated measure regression analysis and by growth pattern analysis.At baseline, a 1-kg higher initial weight was associated with a 55-mL higher average FEV(1). During the follow-up period, a 1-kg gain in weight was associated with an increase in FEV(1) by 32 mL. Children who had a steady weight gain tended to experience greater increases in FEV(1) than children who experienced periodic losses in weight.We established that children who weigh more and who gain weight at an appropriate and uninterrupted rate have a better FEV(1) trajectory. Aggressive nutritional support to maintain growth in these children may therefore improve FEV(1), which can be taken as a surrogate for better lung health, and may ultimately lead to better survival.

  View details for Web of Science ID 000185035100033

  View details for PubMedID 12949289

• Safety of inhaled nitric oxide after lung transplantation JOURNAL OF HEART AND LUNG TRANSPLANTATION Cornfield, D. N., Milla, C. E., Haddad, I. Y., Barbato, J. E., Park, S. J. 2003; 22 (8): 903-907

  Abstract

  The present study tests the hypothesis that therapy with inhaled nitric oxide (iNO) at the time of lung transplantation in patients undergoing bilateral angle lung transplantation: (i) is safe; and (ii) does not increase either the duration of mechanical ventilation or the incidence of acute graft dysfunction.We conducted a prospective, non-randomized trial of iNO at 20 parts per million. The treatment group was comprised of 14 patients (10 females, 4 males) undergoing lung transplantation to address severe end-stage lung disease and pulmonary hypertension (mean pulmonary artery pressure > 30 mmHg). Clinical and histologic parameters were compared with 22 historical control subjects who were matched with the study population for age, diagnosis and disease severity (17 females, 5 males) and had undergone lung transplantation in the preceding 2-year time period. No significant differences were noted between the 2 study groups at baseline.No toxic effect of iNO treatment was evident. Although the incidence of acute graft dysfunction was the same in both groups, the occurrence of acute graft rejection in the initial 4 weeks after transplant was less frequent in the iNO group than in the control group (7% vs 32%, p = 0.05). Fifty percent of the treatment group, as compared with 22% of the control group, were discharged from the hospital within 2 weeks of the procedure (p = 0.05).Early initiation of iNO in lung transplant patients with pulmonary hypertension is safe and may decrease the incidence of acute graft rejection. We speculate that iNO may exert an immunomodulatory effect.

  View details for DOI 10.1016/S1053-2498(02)00809-4

  View details for Web of Science ID 000184634300011

  View details for PubMedID 12909471

• Insulin glargine improves hemoglobin A1c in children and adolescents with poorly controlled type 1 diabetes. Pediatric diabetes Jackson, A., Ternand, C., Brunzell, C., Kleinschmidt, T., Dew, D., Milla, C., Moran, A. 2003; 4 (2): 64-69

  Abstract

  The pediatric diabetes team at the University of Minnesota made a clinical decision to switch patients with type 1 diabetes with a hemoglobin A1c level greater than 8.0% to insulin glargine in an effort to improve glycemic control. Retrospective chart analysis was performed on 37 patients 6 months after the switch to insulin glargine therapy.After 6 months, the average hemoglobin A1c level in the entire cohort dropped from 10.1 +/- 2.0 to 8.9 +/- 1.6% (p = 0.001). Thirty patients responded with an average hemoglobin A1c drop of 1.7 +/- 1.5%, from 10.3 +/- 2.2 to 8.6 +/- 1.5% (p < 0.001). Seven patients did not respond to insulin glargine therapy, with an average hemoglobin A1c rise of 1.0 +/- 0.8% from a baseline of 9.5 +/- 1.0% to 10.4 +/- 1.4% (p = 0.01). The greatest response was seen in children with an A1c > 12.0%, who dropped their hemoglobin A1c by 3.5 +/- 1.9%. Compared with responders, non-responders had significantly less contact with the diabetes team in the form of clinic visits and telephone conversations both before and after initiation of glargine therapy. Sixty-two per cent of patients received insulin glargine at lunchtime, when injections could be supervised at school. Three episodes of severe hypoglycemia occurred after initiation of insulin glargine therapy.Insulin glargine substantially improved glycemic control in children and adolescents with poorly controlled type 1 diabetes. This response was most remarkable in those with a baseline hemoglobin A1c level > 12.0%, and may have been related to increased supervision of injections.

  View details for PubMedID 14655261

• Abnormal glucose tolerance in cystic fibrosis: Why should patients be screened? JOURNAL OF PEDIATRICS Moran, A., Milla, C. 2003; 142 (2): 97-99

  View details for DOI 10.1067/mpd.2003.90

  View details for Web of Science ID 000181144400005

  View details for PubMedID 12584525

• Continuous propofol infusion in 142 critically ill children PEDIATRICS Cornfield, D. N., Tegtmeyer, K., Nelson, M. D., Milla, C. E., Sweeney, M. 2002; 110 (6): 1177-1181

  Abstract

  In recent years, continuous intravenous propofol infusion has been widely used in pediatric intensive care units. Several case reports have raised concerns about its safety. The objective of this study was to report our experience with continuous intravenous propofol in consecutive patients during an 18-month period.The study design was a retrospective review of a case series. Case was defined as a critically ill child who was treated with continuous intravenous propofol. The attending physician staff agreed to prescribe propofol via continuous intravenous infusion at a dose not to exceed 50 microg/kg/min. The protocol allowed for each patient to receive an additional intravenous bolus of propofol at a dose of 1 mg/kg no more than once per hour. The study entailed data collection from consecutive patients who were prescribed a continuous infusion of propofol in either the pediatric intensive care unit or bone marrow transplant unit.Data from 142 patients were analyzed. Each patient enrolled was adequately sedated. Administration of propofol via continuous intravenous infusion was not associated with metabolic acidosis or hemodynamic compromise. No patient in the study group was inadvertently extubated or had a central venous catheter accidentally discontinued.Propofol can be safely and effectively used to provide sedation to critically ill infants and children. We speculate that continuous infusion of propofol for extended periods of time should not exceed 67 microg/kg/min.

  View details for PubMedID 12456916

• Surfactant protein A decreases lung injury and mortality after murine marrow transplantation AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Yang, S. X., Milla, C., Panoskaltsis-Mortari, A., Hawgood, S., Blazar, B. R., Haddad, I. Y. 2002; 27 (3): 297-305

  Abstract

  Surfactant protein A (SP-A), a collectin associated with surfactant lipids, can have immune modulatory effects. We hypothesized that exogenous and basal endogenous SP-A can function to suppress donor T-cell-dependent inflammation that occurs during the generation of idiopathic pneumonia syndrome after bone marrow transplantation (BMT). Wild-type and SP-A-deficient mice were conditioned with cyclophosphamide and lethal irradiation and then given allogeneic donor bone marrow plus inflammation-inducing spleen T cells. On Day 7 after BMT, bronchoalveolar lavage fluids from SP-A-deficient mice contained increased numbers of inflammatory cells and higher levels of proinflammatory mediators tumor necrosis factor-alpha, interferon-gamma, and nitric oxide than wild-type mice. Exaggerated inflammation in SP-A-deficient mice was associated with decreased dynamic lung compliance and increased donor T-cell-dependent mortality (P = 0.0007, n = 10). Nitrative stress in alveolar macrophages from SP-A(-/-)-conditioned BMT recipients was higher than for SP-A(+/+) mice. Similarly, mice treated with transtracheal human SP-A (50 micro g), instilled on Day 4 after BMT during a time of in vivo donor T cell activation, exhibited decreased inflammation and improved early survival compared with buffer-instilled mice. We concluded that basal endogenous SP-A and enhanced alveolar SP-A level modulate donor T-cell-dependent immune responses and prolong survival after allogeneic BMT.

  View details for DOI 10.1165/rcmb.2002-0035OC

  View details for Web of Science ID 000177806500004

  View details for PubMedID 12204891

• Abnormal lipid concentrations in cystic fibrosis AMERICAN JOURNAL OF CLINICAL NUTRITION FIGUEROA, V., Milla, C., Parks, E. J., Schwarzenberg, S. J., Moran, A. 2002; 75 (6): 1005-1011

  Abstract

  Concentrations of cholesterol and triacylglycerol are commonly believed to be low in persons with cystic fibrosis and thus not of concern.The goal was to determine whether concentrations of cholesterol and triacylglycerol are related to glucose tolerance or nutritional status in patients with cystic fibrosis.Fasting lipid profiles were measured in 192 patients ( +/- SD age: 21 +/- 11 y) in conjunction with an oral-glucose-tolerance test.Cystic fibrosis patients in all age groups had higher triacylglycerol (1.51 +/- 0.95 mmol/L) and lower cholesterol (3.57 +/- 0.96 mmol/L) concentrations than US population means. Thirty patients (16%) had hypertriglyceridemia (3.22 +/- 1.22 mmol/L), and 8 patients (4%) had elevated cholesterol (6.05 +/- 1.32 mmol/L). In most cases, hypertriglyceridemia was isolated; only 3 subjects had elevation of both cholesterol and triacylglycerol. Lipid concentrations were not related to body mass index, weight, glucose tolerance, the areas under the curve for glucose or insulin, or glycated hemoglobin. Lipid concentrations also did not correlate with cystic fibrosis genotype, use of systemic steroids, blood pressure, liver enzymes, C-reactive protein, or pulmonary function.Isolated hypertriglyceridemia appears to be common in cystic fibrosis, whereas cholesterol concentrations are generally low. Hypertriglyceridemia may be related to chronic low-grade inflammation or to a dietary macronutrient imbalance with excessive simple carbohydrate absorption relative to fat absorption. Whether it is associated with a risk of cardiovascular disease in this population is uncertain, but the clinical significance of triacylglycerol elevation may become important as survival improves.

  View details for Web of Science ID 000175783200008

  View details for PubMedID 12036806

• Cystic fibrosis: Soon to be a geriatric problem 25th European Cystic Fibrosis Conference Warwick, W. J., Milla, C. E. MEDIMOND S R L. 2002: 209–211

  View details for Web of Science ID 000180264800037

• Effects of oxidant stress on inflammation and survival of iNOS knockout mice after marrow transplantation AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Yang, S. X., Porter, V. A., Cornfield, D. N., Milla, C., Panoskaltsis-Mortari, A., Blazar, B. R., Haddad, I. Y. 2001; 281 (4): L922-L930

  Abstract

  In a model of idiopathic pneumonia syndrome after bone marrow transplantation (BMT), injection of allogeneic T cells induces nitric oxide (.NO), and the addition of cyclophosphamide (Cy) generates superoxide (O.) and a tissue-damaging nitrating oxidant. We hypothesized that.NO and O. balance are major determinants of post-BMT survival and inflammation. Inducible nitric oxide synthase (iNOS) deletional mutant mice (-/-) given donor bone marrow and spleen T cells (BMS) exhibited improved survival compared with matched BMS controls. Bronchoalveolar lavage fluids obtained on day 7 post-BMT from iNOS(-/-) BMS mice contained less tumor necrosis factor-alpha and interferon-gamma, indicating that.NO stimulated the production of proinflammatory cytokines. However, despite suppressed inflammation and decreased nitrotyrosine staining, iNOS(-/-) mice given both donor T cells and Cy (BMS + Cy) died earlier than iNOS-sufficient BMS + Cy mice. Alveolar macrophages from iNOS(-/-) BMS + Cy mice did not produce.NO but persisted to generate strong oxidants as assessed by the oxidation of the intracellular fluorescent probe 2',7'-dichlorofluorescin. We concluded that.NO amplifies T cell-dependent inflammation and addition of Cy exacerbates.NO-dependent mortality. However, the lack of.NO during Cy-induced oxidant stress decreases survival of T cell-recipient mice, most likely by generation of.NO-independent toxic oxidants.

  View details for Web of Science ID 000171020400019

  View details for PubMedID 11557596

• Insulin and glucose excursion following premeal insulin lispro or repaglinide in cystic fibrosis-related diabetes DIABETES CARE Moran, A., Phillips, J., Milla, C. 2001; 24 (10): 1706-1710

  Abstract

  Insulin and glucose levels in response to premeal insulin lispro or repaglinide were evaluated in adult patients with cystic fibrosis-related diabetes (CFRD) without fasting hyperglycemia.Seven patients with CFRD were fed 1,000-kcal liquid mixed meals. Three study conditions were administered in random order on separate mornings: 1) no premeal diabetes medication, 2) insulin lispro, 0.1 unit/kg body wt premeal and 3) repaglinide 1 mg premeal. Glucose and insulin levels were measured every 20 min for 5 h.Fasting insulin and glucose levels were normal in patients with CFRD, but the peak glucose level was elevated. Insulin lispro significantly decreased the peak glucose level (P = 0.0004) and the 2-h (P = 0.001) and 5-h (P < 0.0001) glucose area under the curve (AUC). Repaglinide significantly decreased the 5-h glucose AUC (P = 0.03). Neither drug completely normalized cystic fibrosis glucose excursion at the doses used for this study. Insulin lispro significantly increased the 5-h insulin AUC (P = 0.04).In response to subcutaneous insulin lispro, postprandial glucose excursion was significantly diminished and insulin secretion was enhanced compared with a control meal in which no medication was given to patients with CFRD. The oral agent repaglinide resulted in lesser corrections in these parameters. Neither drug completely normalized glucose or insulin levels, suggesting that the doses chosen for this study were suboptimal. Placebo-controlled longitudinal studies comparing the effectiveness of repaglinide and insulin on glucose metabolic control as well as overall nutrition and body weight are needed to help determine optimal medical treatment of CFRD.

  View details for Web of Science ID 000171321600002

  View details for PubMedID 11574430

• Protein metabolism in clinically stable adult cystic fibrosis patients with abnormal glucose tolerance DIABETES Moran, A., Milla, C., Ducret, R., Nair, K. S. 2001; 50 (6): 1336-1343

  Abstract

  Cystic fibrosis (CF) patients are reported to experience chronic protein catabolism. Since diabetes or impaired glucose tolerance (IGT) is common in CF, we hypothesized that their protein catabolic state is related to reduced insulin secretion or reduced insulin action. A total of 12 clinically stable adult CF patients with abnormal glucose tolerance and 12 age-, sex-, and lean body mass-matched healthy control subjects underwent protein turnover studies using L-[1-(13)C]leucine, L-[(15)N]phenylalanine, and L-[(2)H(4)]tyrosine, with and without exogenous insulin infusion. In the baseline fasting state, protein metabolism was entirely normal in CF patients, with no evidence of increased protein catabolism. In contrast, striking abnormalities were seen in CF patients when insulin was infused, since they did not experience normal suppression of the appearance rates of leucine, phenylalanine, or tyrosine (indexes of protein breakdown). At an insulin concentration of 45 +/- 2 microU/ml, normal control subjects suppressed the leucine appearance rate by 19 +/- 5% (P < 0.01), ketoisocaproate appearance rate by 10 +/- 3% (P = 0.03), tyrosine appearance rate by 11 +/- 2% (P = 0.03), and phenylalanine appearance rate by 6 +/- 3% (P = 0.07). Phenylalanine conversion to tyrosine decreased by 22 +/- 7% (P = 0.03). At a similar insulin concentration of 44 +/- 3 microU/ml, normal suppression of amino acid appearance did not occur in CF. The leucine appearance rate decreased by 4 +/- 2% (P = 0.65), ketoisocaproate appearance rate by 1 +/- 2% (P = 0.94), tyrosine appearance rate by 0 +/- 6% (P = 0.56), phenylalanine appearance rate by 5 +/- 6% (P = 0.34), and phenylalanine conversion to tyrosine by 5 +/- 6% (P = 0.95). Poor suppression of the amino acid appearance rate in CF was not related to previously documented glucose tolerance status (IGT or CF-related diabetes without fasting hyperglycemia), fasting insulin levels, the acute insulin response, insulin sensitivity, cytokine or counterregulatory hormone levels, resting energy expenditure, caloric intake, pulmonary function, or clinical status. Protein synthesis was not significantly affected by insulin infusion in either normal control subjects or CF patients. In conclusion, clinically stable adult CF patients have normal indexes of protein breakdown and synthesis in the fasting state. In contrast, elevation of plasma insulin to physiological postprandial levels fails to normally suppress indexes of protein breakdown. It is therefore likely that inability to spare protein during the postprandial state is the cause of protein catabolism in these patients.

  View details for Web of Science ID 000168961900014

  View details for PubMedID 11375334

• Human surfactant protein A suppresses T cell-dependent inflammation and attenuates the manifestations of idiopathic pneumonia syndrome in mice AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Yang, S. X., Milla, C., Panoskaltsis-Mortari, A., Ingbar, D. H., Blazar, B. R., Haddad, I. Y. 2001; 24 (5): 527-536

  Abstract

  We have previously shown an association between growth factor-induced upregulation of surfactant protein (SP)-A and suppression of alveolar inflammation in our murine model of donor T cell-dependent lung dysfunction after bone-marrow transplantation, referred to as idiopathic pneumonia syndrome (IPS). We hypothesized that SP-A protects the lung in vivo from IPS injury by downregulation of alveolar inflammation. Human SP-A (100 microg), purified by n-butanol extraction or preparative isoelectric focusing, was transtracheally instilled on Day 4 after BMT during a time of in vivo donor T-cell activation. At 48 h after treatment, immunohistochemical staining of lung sections showed that SP-A did not alter T cell- dependent cellular infiltration. However, macrophages from SP-A-instilled mice were less injured and spontaneously produced less tumor necrosis factor-alpha than did cells from buffer-instilled mice. Although exogenous SP-A did not significantly alter bronchoalveolar lavage fluid (BALF) high levels of total protein (TP), an inverse correlation between BALF SP-A and TP concentrations (r = -0.65; P = 0.02) was observed in SP-A-treated but not in buffer-instilled mice. The only difference between the effects of the two sources of SP-A was that butanol-extracted SP-A, but not isoelectric focusing-purified SP-A, suppressed the interferon-gamma/nitric oxide pathway. We conclude that SP-A downregulates T cell-dependent alveolar inflammation by multiple pathways leading to decreased IPS injury.

  View details for Web of Science ID 000168913300004

  View details for PubMedID 11350821

• Trends in pulmonary function in patients with cystic fibrosis correlate with the degree of glucose intolerance at baseline 12th Annual North American Cystic Fibrosis Conference Milla, C. E., Warwick, W. J., Moran, A. AMER THORACIC SOC. 2000: 891–95

  Abstract

  In patients with cystic fibrosis, CF-related diabetes mellitus (CFRD) has been associated with increased morbidity and mortality. Whether glucose intolerance is also associated with poor outcomes is unclear. To better define these relationships we prospectively followed a group of 152 patients with CF without diabetes for 4 yr. Patients were classified as having normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or CFRD without fasting hyperglycemia (CFRD-No FH). FEV(1), FVC, and body mass index (BMI) were measured at baseline and quarterly. At baseline 45% of the patients had NGT, 38.8% had IGT, and 15.8% had CFRD-No FH. FEV(1), FVC, and BMI at baseline were comparable among these groups (all p > 0.1). After 4 yr an overall decline in FEV(1) and FVC occurred, with no change in BMI. The rates of decline for FEV(1) and FVC correlated with the glucose tolerance groups, with the highest rates of decline occurring among the CFRD-No FH group. In addition, patients in the lowest quartile for insulin production at baseline experienced the highest rates of pulmonary function decline over time, suggesting a relationship between insulin deficiency and clinical deterioration. We conclude that the degree of glucose intolerance is a strong determinant of future lung function decline in patients with CF.

  View details for Web of Science ID 000089363500024

  View details for PubMedID 10988101

• Randomized, controlled trial of low-dose inhaled nitric oxide in the treatment of term and near-term infants with respiratory failure and pulmonary hypertension 66th Annual Meeting of the Society-for-Pediatric-Research Cornfield, D. N., Maynard, R. C., deRegnier, R. A., Guiang, S. F., Barbato, J. E., Milla, C. E. AMER ACAD PEDIATRICS. 1999: 1089–94

  Abstract

  Recent reports indicate that inhaled nitric oxide (iNO) causes selective pulmonary vasodilation, increases arterial oxygen tension, and may decrease the use of extracorporeal membrane oxygenation (ECMO) in infants with persistent pulmonary hypertension of the newborn (PPHN). Despite these reports, the optimal dose and timing of iNO administration in PPHN remains unclear.To test the hypotheses that in PPHN 1) iNO at 2 parts per million (ppm) is effective at acutely increasing oxygenation as measured by oxygenation index (OI); 2) early use of 2 ppm of iNO is more effective than control (0 ppm) in preventing clinical deterioration and need for iNO at 20 ppm; and 3) for those infants who fail the initial treatment protocol (0 or 2 ppm) iNO at 20 ppm is effective at acutely decreasing OI.A randomized, controlled trial of iNO in 3 nurseries in a single metropolitan area. Thirty-eight children, average gestational age of 37.3 weeks and average age <1 day were enrolled. Thirty-five of 38 infants had echocardiographic evidence of pulmonary hypertension. On enrollment, median OI in the control group, iNO at 0 ppm, (n = 23) was 33.1, compared with 36.9 in the 2-ppm iNO group (n = 15).Initial treatment with iNO at 2 ppm for an average of 1 hour was not associated with a significant decrease in OI. Twenty of 23 (87%) control patients and 14 of 15 (92%) of the low-dose iNO group demonstrated clinical deterioration and were treated with iNO at 20 ppm. In the control group, treatment with iNO at 20 ppm decreased the median OI from 42.6 to 23.8, whereas in the 2-ppm iNO group with a change in iNO from 2 to 20 ppm, the median OI did not change (42.6 to 42.0). Five of 15 patients in the low-dose nitric oxide group required ECMO and 2 died, compared with 7 of 23 requiring ECMO and 5 deaths in the control group.In infants with PPHN, iNO 1): at 2 ppm does not acutely improve oxygenation or prevent clinical deterioration, but does attenuate the rate of clinical deterioration; and 2) at 20 ppm acutely improves oxygenation in infants initially treated with 0 ppm, but not in infants previously treated with iNO at 2 ppm. Initial treatment with a subtherapeutic dose of iNO may diminish the clinical response to 20 ppm of iNO and have adverse clinical sequelae.

  View details for Web of Science ID 000083448000007

  View details for PubMedID 10545552

• Recombinant human DNase in cystic fibrosis LANCET Milla, C. E. 1999; 354 (9176): 428-428

  View details for Web of Science ID 000081764900061

  View details for PubMedID 10437902

• NO causes perinatal pulmonary vasodilation through K+-channel activation and intracellular Ca2+ release AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Saqueton, C. B., Miller, R. B., Porter, V. A., Milla, C. E., Cornfield, D. N. 1999; 276 (6): L925-L932

  Abstract

  Evidence suggests that nitric oxide (NO) causes perinatal pulmonary vasodilation through K+-channel activation. We hypothesized that this effect worked through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channel that requires release of intracellular Ca2+ from a ryanodine-sensitive store. We studied the effects of 1) K+-channel blockade with tetraethylammonium, 4-aminopyridine, a voltage-dependent K+-channel blocker, or glibenclamide, an ATP-sensitive K+-channel blocker; 2) cyclic nucleotide-sensitive kinase blockade with either KT-5823, a guanylate-sensitive kinase blocker, or H-89, an adenylate-sensitive kinase blocker; and 3) blockade of intracellular Ca2+ release with ryanodine on NO-induced pulmonary vasodilation in acutely prepared late-gestation fetal lambs. N-nitro-L-arginine, a competitive inhibitor of endothelium-derived NO synthase, was infused into the left pulmonary artery, and tracheotomy was placed. The animals were ventilated with 100% oxygen for 20 min, followed by ventilation with 100% oxygen and inhaled NO at 20 parts/million (ppm) for 20 min. This represents the control period. In separate protocols, the animals received an intrapulmonary infusion of the different blockers and were ventilated as above. Tetraethylammonium (n = 6 animals) and KT-5823 (n = 4 animals) attenuated the response, whereas ryanodine (n = 5 animals) blocked NO-induced perinatal pulmonary vasodilation. 4-Aminopyridine (n = 5 animals), glibenclamide (n = 5 animals), and H-89 (n = 4 animals) did not affect NO-induced pulmonary vasodilation. We conclude that NO causes perinatal pulmonary vasodilation through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channels and release of Ca2+ from ryanodine-sensitive stores.

  View details for Web of Science ID 000080822700005

  View details for PubMedID 10362716

• High levels of peroxynitrite are generated in the lungs of irradiated mice given cyclophosphamide and allogeneic T cells - A potential mechanism of injury after marrow transplantation AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Haddad, I. Y., Panoskaltsis-Mortari, A., Ingbar, D. H., Yang, S. X., Milla, C. E., Blazar, B. R. 1999; 20 (6): 1125-1135

  Abstract

  In a murine bone-marrow transplant (BMT) model designed to determine risk factors for lung dysfunction in irradiated mice, we reported that cyclophosphamide (Cy)-induced injury and lethality depended on the infusion of donor spleen T cells. In the study reported here, we hypothesized that alveolar macrophage (AM)-derived reactive oxygen/nitrogen species are associated with lung dysfunction caused by allogeneic T cells, which stimulate nitric oxide (.NO) production, and by Cy, which stimulates superoxide production.NO reacts with superoxide to form peroxynitrite, a tissue-damaging oxidant. On Day 7 after allogeneic BMT, bronchoalveolar lavage fluid (BALF) obtained from mice injected with T cells contained increased levels of nitrite, which was associated with increased lactate dehydrogenase and protein levels, both of which are indices of lung injury. The injury was most severe in mice receiving both T cells and Cy. Messenger RNA (mRNA) for inducible nitric oxide synthase was detected only in murine lungs injected with T cells +/- Cy. AMs obtained on Day 7 after BMT from mice receiving T cells +/- Cy spontaneously generated between 20 and 40 microM nitrite in culture, versus < 2 microM generated by macrophages obtained from mice undergoing BMT but not receiving T cells. The level of 3-nitrotyrosine, the stable byproduct of the reaction of peroxynitrite with tyrosine residues, was increased in the BALF proteins of mice injected with both T cells and Cy. We conclude that allogeneic T cells stimulate macrophage-derived.NO, and that the addition of Cy favors peroxynitrite formation. Peroxynitrite generation clarifies the dependence of Cy-induced lung injury and lethality on the presence of allogeneic T cells.

  View details for Web of Science ID 000080955600006

  View details for PubMedID 10340931

• Allergic bronchopulmonary aspergillosis and cystic fibrosis PEDIATRIC PULMONOLOGY Milla, C. E. 1999; 27 (2): 71-73

  View details for Web of Science ID 000078797500001

  View details for PubMedID 10088928

• Effects of inhaled nitric oxide and oxygen in high-altitude pulmonary edema 70th Scientific Session of the American-Heart-Association Anand, I. S., Prasad, B. A., Chugh, S. S., Rao, K. R., Cornfield, D. N., Milla, C. E., Singh, N., Singh, S., Selvamurthy, W. LIPPINCOTT WILLIAMS & WILKINS. 1998: 2441–45

  Abstract

  High-altitude pulmonary edema (HAPE) is characterized by pulmonary hypertension, increased pulmonary capillary permeability, and hypoxemia. Treatment is limited to descent to lower altitude and administration of oxygen.We studied the acute effects of inhaled nitric oxide (NO), 50% oxygen, and a mixture of NO plus 50% oxygen on hemodynamics and gas exchange in 14 patients with HAPE. Each gas mixture was given in random order for 30 minutes followed by 30 minutes washout with room air. All patients had severe HAPE as judged by Lake Louise score (6.4+/-0.7), PaO2 (35+/-3. 1 mm Hg), and alveolar to arterial oxygen tension difference (AaDO2) (26+/-3 mm Hg). NO had a selective effect on the pulmonary vasculature and did not alter systemic hemodynamics. Compared with room air, pulmonary vascular resistance fell 36% with NO (P<0.001), 23% with oxygen (P<0.001 versus air, P<0.05 versus NO alone), and 54% with NO plus 50% oxygen (P<0.001 versus air, P<0.005 versus oxygen and versus NO). NO alone improved PaO2 (+14%) and AaDO2 (-31%). Compared with 50% oxygen alone, NO plus 50% oxygen had a greater effect on AaDO2 (-18%) and PaO2 (+21%).Inhaled NO may have a therapeutic role in the management of HAPE. The combined use of inhaled NO and oxygen has additive effects on pulmonary hemodynamics and even greater effects on gas exchange. These findings indicate that oxygen and NO may act on separate but interactive mechanisms in the pulmonary vasculature.

  View details for Web of Science ID 000077278100015

  View details for PubMedID 9832490

• Long term effects of aerosolised rhDNase on pulmonary disease progression in patients with cystic fibrosis THORAX Milla, C. E. 1998; 53 (12): 1014-1017

  Abstract

  After multiple studies, including clinical trials, suggested some mild clinical benefits from the use of rhDNase by patients with cystic fibrosis, a widespread acceptance of the drug has followed. However, long-term effects, specifically on lung disease progression, have not been demonstrated. Experience with the use of this drug in a single cystic fibrosis centre is presented and compared with the trends seen in the patient population of the centre before the introduction of the drug.Patients with cystic fibrosis routinely followed at the University of Minnesota Cystic Fibrosis Center and prescribed rhDNase for at least two years were included in this retrospective study. Data on spirometric parameters (FEV1 and FEV1/FVC), allometric index, and admissions to hospital were retrieved from the centre's database for the two years preceding the prescription of rhDNase and the two years that followed. Trends in pulmonary function and allometric index were analysed by mixed linear modelling, and hospital admission rates for both periods were calculated and compared.One hundred and ninety patients met the inclusion criteria for the study. In the two years preceding the prescription of rhDNase the trends noted were those of a mild decline in FEV1, a stable FEV1/FVC, and a mild improvement in allometric index. In the two years that followed the prescription of rhDNase a mild decline in all these parameters occurred which was a significant change from the previous period (all p < 0.009). There was no difference between females and males in the trends experienced after the start of rhDNase. By logistic regression analysis only the presence of malnutrition at the time of prescription was associated with a positive trend after the introduction of rhDNase. No significant change in the hospital admission rates occurred, with rates of 0.52 (0.16) and 0.56 (0.21) admissions/patient/year for the periods before and after the prescription of rhDNase, respectively.The introduction of rhDNase to the regimen of patients with cystic fibrosis cared for at this centre has not been followed by a positive trend in lung function and nutritional parameters. There are some differences between this patient population and those who participated in previous studies which may help to explain the contrasting findings of this study. However, it is also possible that factors other than mucus clearance need to be improved to achieve a favourable response in disease progression. Patients on this treatment should be followed closely and the benefit judged on an individual basis. More studies are needed to define better the specific indications and use of this form of treatment.

  View details for Web of Science ID 000077712800005

  View details for PubMedID 10195070

• Risk of death in cystic fibrosis patients with severely compromised lung function 1994 International Conference of the American-Thoracic-Society Milla, C. E., Warwick, W. J. AMER COLL CHEST PHYSICIANS. 1998: 1230–34

  Abstract

  Lung disease accounts for most of the mortality in patients with cystic fibrosis (CF). Lung transplantation is an option for patients severely impaired, being recommended when life expectancy is estimated to be <2 years. Our objectives were to evaluate in our patient population the validity of currently accepted criteria for low life expectancy and to identify other potentially useful criteria.Data were retrieved from CF patients followed up at our center who reached and kept an FEV1 <30% predicted. A life table was created and stratified according to characteristics believed to be of importance. In addition, the rate of decline in percent predicted FEV1 was analyzed. These characteristics were evaluated as predictors of risk of death.The median survival was 3.9 years (95% confidence interval, 2.88 to 4.12 years), with no significant differences according to gender, nutritional status, presence of diabetes, or decade in which the patient was cared for. Only by age was there a significant difference in the median survival (p<0.05). By proportional hazards regression, only the rate of decline in percent predicted FEV1 was a significant predictor of the risk of death, with a borderline effect from younger age (p=0.06).In our patient population, a cutoff value of FEV1 of < 30% predicted is not a reliable predictor of high risk of death within 2 years. The yearly rate of decline of percent predicted FEV1 is a better parameter to identify those patients at high risk for death.

  View details for Web of Science ID 000073591500019

  View details for PubMedID 9596299

• PCR ribotyping and endonuclease subtyping in the epidemiology of Burkholderia cepacio infection AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Shreve, M. R., Johnson, S. J., Milla, C. E., Wielinski, C. L., Regelmann, W. E. 1997; 155 (3): 984-989

  Abstract

  Because of conflicting data about hospital-based transmission of Burkholderia (Pseudomonas) cepacia, an important respiratory pathogen in cystic fibrosis (CF), we compared strains found in sputum, lung, or blood of 29 CF patients in our center from 1988 to 1994, studying the relationship between strain and hospital exposure of incident and that of prevalent cases. Exposure was defined as a concurrent hospital stay between a prevalent and an incident case. B. cepacia strains were determined by polymerase chain reaction (PCR) ribotyping and endonuclease subtyping. The 16S to 23S spacer regions of the bacterial ribosomal RNA (rRNA) genes were amplified by PCR, and the product-size patterns used to type each B. cepacia isolate. Endonuclease digestion of the PCR products provided length polymorphisms for subtyping. There were 17 incident events during the period from 1988 to 1994, 16 of which involved a single ribotype. These 16 ribotypes could be divided into five subtypes by endonuclease mapping. Four patients grew B. cepacia from the blood, with the organism being the same strain as found in the lung in each case. Case controls were obtained to evaluate risk factors for B. cepacia acquisition. Concurrent hospitalization with a prevalent case significantly increased the risk of acquisition. There was no association between length of hospitalization, length of exposure, or FEV1 and the risk of B. cepacia acquisition.

  View details for Web of Science ID A1997WN85700032

  View details for PubMedID 9117036

• Glycemic response to dietary supplements in cystic fibrosis is dependent on the carbohydrate content of the formula JOURNAL OF PARENTERAL AND ENTERAL NUTRITION Milla, C., Doherty, L., Raatz, S., Schwarzenberg, S. J., Regelmann, W., Moran, A. 1996; 20 (3): 182-186

  Abstract

  Enteral formula feedings are frequently prescribed to cystic fibrosis (CF) patients to boost caloric intake. A substantial number of these patients are glucose intolerant and have severe respiratory compromise.To determine the effect of the carbohydrate content on glucose tolerance and respiratory function in glucose-intolerant CF patients with poor lung function, we examined the response to bolus feedings of five dietary supplements; a high-fat formula developed in our Clinical Research Center (CRC), Pulmocare, a high-carbohydrate formula developed in our CRC, Ensure Plus, and sugar-free Scandishake.Glucose excursion in response to the formulas with the lowest carbohydrate content was significantly less than that found in response to formulas with higher carbohydrate content. Insulin levels were also markedly lower in response to the low-carbohydrate high-fat formulas. Glucose excursion, expressed as a percent of the response to the CRC high-fat formula, was 111% +/- 12% for Pulmocare (p = NS), 202% +/- 34% for Ensure Plus (p < 01), 227% +/- 37% for CRC high carbohydrate (p = .001), and 357% +/- 33% for sugar-free Scandishake (p < .001). CO2 production, O2 consumption, minute ventilation, and respiratory rate increased modestly but not significantly in response to all formulas. No significant differences were found between the formulas in regards to these parameters. There were no subjective complaints of dyspnea during any of the five studies.The carbohydrate content of liquid dietary supplements appears to be an important determinant of hyperglycemia in glucose-intolerant adult CF patients.

  View details for Web of Science ID A1996UM63800003

  View details for PubMedID 8776690

• Clinical significance of the recovery of Aspergillus species from the respiratory secretions of cystic fibrosis patients PEDIATRIC PULMONOLOGY Milla, C. E., Wielinski, C. L., Regelmann, W. E. 1996; 21 (1): 6-10

  Abstract

  The frequent recovery of Aspergillus species from the respiratory tract secretions of cystic fibrosis (CF) patients is well recognized, and the presence of the fungus in the airways may trigger an inflammatory response that can manifest as the clinical entity known as allergic bronchopulmonary aspergillosis (ABPA). In our CF patient population we studied the clinical characteristics of those who had Aspergillus sp. recovered from their respiratory tract secretions (n = 45) and compared them with the characteristics seen, during the same time period, in those patients who were culture negative for Aspergillus sp. (n = 167). There were no differences in peripheral blood eosinophil count (P = 0.9) or serum immunoglobulin E levels (P = 0.61). By logistic regression analysis there seemed to be an increased risk for more advanced lung disease, both radiographically (defined by a Brasfield chest radiograph score < 18) and by lung function parameters in those who were culture positive. However, after appropriate adjustment, almost all the increased risk was associated with age and gender, but not with the presence of Aspergillus sp. in respiratory secretions. Additionally, increasing age was strongly correlated with the risk of Aspergillus sp. being cultured from respiratory secretions (P = 0.0025). The presence of Aspergillus sp. in respiratory secretions was not associated with two indicators of atopy in our CF patient population. We do not have evidence that the culture of Aspergillus sp. from CF respiratory secretions is independently associated with an increased risk for more advanced lung disease.

  View details for Web of Science ID A1996TX83900003

  View details for PubMedID 8776259

• HIGH-STRENGTH PANCREATIC-ENZYMES LANCET Milla, C. E., Wielinski, C. L., Warwick, W. J. 1994; 343 (8897): 599-599

  View details for Web of Science ID A1994MY84300041

  View details for PubMedID 7906348