Lynn M. Rose

Effect of Azithromycin on Pulmonary Function in Patients With Cystic Fibrosis Uninfected With Pseudomonas aeruginosa: A Randomized Controlled Trial

CONTEXT Azithromycin is recommended as therapy for cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa infection, but there has not been sufficient evidence to support the benefit of azithromycin in other patients with CF. OBJECTIVE To determine if azithromycin treatment improves lung function and reduces pulmonary exacerbations in pediatric CF patients uninfected with P. aeruginosa. DESIGN, SETTING, AND PARTICIPANTS A multicenter, randomized, double-blind placebo-controlled trial was conducted from February 2007 to July 2009 at 40 CF care centers in the United States and Canada. Of the 324 participants screened, 260 were randomized and received study drug. Eligibility criteria included age of 6 to 18 years, a forced expiratory volume in the first second of expiration (FEV(1)) of at least 50% predicted, and negative respiratory tract cultures for P. aeruginosa for at least 1 year. Randomization was stratified by age of 6 to 12 years vs 13 to 18 years and by CF center. INTERVENTION The active group (n = 131) received 250 mg (weight 18-35.9 kg) or 500 mg (weight > or = 36 kg) of azithromycin 3 days per week (Monday, Wednesday, and Friday) for 168 days. The placebo group (n = 129) received identically packaged placebo tablets on the same schedule. MAIN OUTCOME MEASURES The primary outcome was change in FEV(1). Exploratory outcomes included additional pulmonary function end points, pulmonary exacerbations, changes in weight and height, new use of antibiotics, and hospitalizations. Changes in microbiology and adverse events were monitored. RESULTS The mean (SD) age of participants was 10.7 (3.17) years. The mean (SD) FEV(1) at baseline and 168 days were 2.13 (0.85) L and 2.22 (0.86) L for the azithromycin group and 2.12 (0.85) L and 2.20 (0.88) L for the placebo group. The difference in the change in FEV(1) between the azithromycin and placebo groups was 0.02 L (95% confidence interval [CI], -0.05 to 0.08; P = .61). None of the exploratory pulmonary function end points were statistically significant. Pulmonary exacerbations occurred in 21% of the azithromycin group and 39% of the placebo group. Participants in the azithromycin group had a 50% reduction in exacerbations (95% CI, 31%-79%) and an increase in body weight of 0.58 kg (95% CI, 0.14-1.02) compared with placebo participants. There were no significant differences between groups in height, use of intravenous or inhaled antibiotics, or hospitalizations. Participants in the azithromycin group had no increased risk of adverse events, but had less cough (-23% treatment difference; 95% CI, -33% to -11%) and less productive cough (-11% treatment difference; 95% CI, -19% to -3%) compared with placebo participants. CONCLUSION In children and adolescents with CF uninfected with P. aeruginosa, treatment with azithromycin for 24 weeks did not result in improved pulmonary function. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00431964.

Open‐label, follow‐on study of azithromycin in pediatric patients with CF uninfected with Pseudomonas aeruginosa

We previously performed a randomized placebo‐controlled trial to examine the effects of azithromycin in children and adolescents 6–18 years of age with cystic fibrosis uninfected with Pseudomononas aeruginosa and demonstrated that while azithromycin did not acutely improve pulmonary function, azithromycin‐reduced pulmonary exacerbations, decreased the initiation of new oral antibiotics, and improved weight gain. We now report the results of the open‐label, follow‐on study to assess durability of response to azithromycin and continued safety and tolerability.

The natural occurence of circulating idiotype-anti-idiotype complexes during a secondary immune response to phosphorylcholine

Abstract This report deals with the direct demonstration of circulating idiotype-anti-idiotype immune complexes in Balb c mice immunized with R36a, a pneumococcus vaccine which bears phosphorylcholine (PC) as a major antigenic determinant. Balb c mice developing a secondary response to R36a were found to produce anti-PC antibodies which had the same idiotype as the Balb c IgA myeloma protein TEPC-15 (T15 idiotype), as well as antibodies directed against the T15 idiotype. Anti-PC plaque-forming cells (PFC) were detected after 4–6 days while anti-T15 idiotype PFC were found after 6–9 days. Immune complexes reacting with conglutinin-coated tubes were detected in the circulation from Days 5 to 12. Some of the conglutinin-bound complexes were able to bind 125 I-PC-BSA and 125 I-TEPC-15 myeloma protein, but not 125 I-HPC-16, a Balb c IgM anti-PC antibody which does not bear the T15 idiotype. Complexes detected on Days 5–9 selectively bound 125 I-PC-BSA whereas complexes detected on Days 10–12 bound both 125 I-PC-BSA and 125 I-TEPC-15 myeloma protein. These results demonstrate that during the course of a secondary response to PC determinants, immune complexes are formed in two phases. First, immune complexes appear which bind additional PC determinants and, therefore, contain free anti-PC antibody-binding sites, but do not contain free PC antigenic determinants nor free anti-T15 idiotype antibody sites. Later, immune complexes appear which contain both free anti-PC antibody sites and free anti-T15 antibody sites, but still contain no free PC antigenic determinants. This sequence of events suggests that T15 idiotype-anti-T15 idiotype antibody complexes are first formed with a relatively large excess of T15 idiotype, then concomitantly with the increased production of anti-T15 idiotype antibodies, free anti-T15 idiotype antibody-binding sites become available within the complexes. The anti-idiotypic response was first detected at the end of a first peak of anti-PC antibody, but was maximal immediately before a second peak of anti-PC antibody bearing the the same T15 idiotype. This cyclic response may reflect an enhancement of the anti-PC response by anti-idiotype antibodies or by the idiotype-anti-idiotype immune complexes. These results suggest that self-idiotypic recognition may represent a potential source of immune complexes which would not depend directly on the presence in the circulation of the antigen inducing the original immune response.

Development of lymphocyte subsets in pigtailed macaques

The early development of eight lymphocyte subsets was determined for pigtailed macaque infants from 0 to 800 days of age using two-color flow cytometry and fluorescein- and R-phycoerythrin-conjugated monoclonal antibodies specific for human leukocyte antigens. Four major lymphocyte subsets in monkeys (B, CD4+ T, CD8+ T, and NK cells) could be further divided using two-color analysis. In neonates, the frequency of lymphocyte subpopulations having surface phenotypes found principally on dense, resting cells (IgD+ B cells, Lp220+ CD4+ T cells, and CD18dull CD8+ T cells) was much higher than subpopulations having phenotypes present principally on buoyant, activated cells (IgD- B cells, Lp220- CD4+ T cells, CD18bri CD8+ T cells). There was a complete absence of two CD18bri CD8+ subsets (CD8dull and CD8bri) during the first 300 days of life. The relative proportion of lymphocyte subsets with resting phenotype decreased with increasing age, while the subpopulations associated with activation gradually increased with age. These findings suggest that during early development immunocompetent cells gradually differentiate into activated lymphocytes.

In vivo administration of anti-CD4 monoclonal antibody prolongs survival in longtailed macaques with experimental allergic encephalomyelitis

The in vivo administration of monoclonal antibody (mAb) to the CD4 antigen associated with helper T cells has been successful in prolonging the survival of nonhuman primates with experimental allergic encephalomyelitis (EAE). EAE was induced in 17 outbred longtailed macaques (Macaca fascicularis) by inoculation of homologous myelin basic protein (BP) in complete Freunds adjuvant (CFA). Treatment was begun at the onset of clinical signs. Eleven animals were treated with anti-CD4 mAb Leu3a (eight) or OKT4a (three). Of the six control animals, two received anti-CD8 mAb (Leu2a), and four were treated with saline. Specific T- and B-cell subsets which have been implicated in the development of EAE were monitored throughout the course of the disease by one- and two-color immunofluorescence (IF). The monkey anti-BP antibody and anti-mouse immunoglobulin (IgG) responses were measured by enzyme-linked immunoassay (ELISA) techniques, as were the levels of free-circulating murine IgG. The nature of the infiltrating lymphocytes in the brain was evaluated histologically post mortem. Our results indicate that anti-CD4 mAb can prolong survival and in some cases completely reverse the clinical appearance of the disease; however, relapses did occur. Treatments with Leu3a or OKT4a anti-CD4 mAbs reversed the ongoing depletion of CD4+ and CD8+ cells caused by the development of EAE and appeared to reduce the size and degree of inflammation in brain lesions. These treatments did not induce immunologic tolerance to mouse IgG since all of the anti-CD4-treated animals produced high titers of anti-mouse IgG antibodies. Treatment with Leu2a (anti-CD8) had no effect on the development of EAE. These results suggest that CD4+ cells are important to the pathogenesis of EAE in macaques and that manipulation of this subset with monoclonal antibodies may provide effective treatment of human demyelinating disease.

Fluctuations of T- and B-cell subsets in basic protein-induced experimental allergic encephalomyelitis (EAE) in long-tailed macaques☆

Experimental allergic encephalomyelitis (EAE) was induced in long-tailed macaques (Macaca fascicularis) by inoculation of autologous myelin basic protein (BP) in complete Freunds adjuvant. Natural killer (NK) cell activity and lymphocyte subsets detected by one- and two-color immunofluorescence were monitored longitudinally in these animals. A decrease in NK cell activity was detected at the onset of clinically defined disease. During the preclinical phase of EAE (5-7 days before the onset of clinical signs) the absolute number of T helper (CD4+) and T suppressor (CD8+) cells in the peripheral blood decreased significantly. Analysis of peripheral blood B cells revealed a selective depletion of IgD+ B cells and a corresponding increase in the number of IgD- B cells prior to and during the onset of clinical signs. Total B-cell numbers were not significantly different between EAE and normal groups. The increased proportion of IgD- B cells in BP-sensitized animals corresponded with the appearance of high titers of circulating anti-BP antibodies. Thus two-color analysis of B-cell subsets may be a sensitive indicator of B-cell activation and of abnormal immune status in EAE. Changes in lymphocyte subsets in macaques with EAE are compared with those in humans with multiple sclerosis.

Experimental allergic encephalomyelitis in non-human primates: diffusion imaging of acute and chronic brain lesions

Diffusion imaging and T2-weighted magnetic resonance imaging were performed on 16 monkeys with experimental allergic encephalomyelitis (EAE), a model of the human demyelinating disease MS. The purpose of this study was to determine whether local changes in diffusion image intensity could be correlated with the formation of acute and chronic demyelinating lesions. Diffusion image analysis was restricted to the internal capsule of the brain because of its anatomic orientation offiber pathways. Acute inflammatory EAE lesions were large and monophasic, as visualized by T2-weighted MRI, and were accompanied by a decrease in the diffusion MR image signal with the diffusion-sensitizing gradient in ail three orthogonal directions (n=27 brain regions, P<0.005). Chronic demyelinating lesions were preceded by multiple inflammatory attacks, as visualized by MRI, and by a decrease in diffusion MR image signal with the diffusion-sensitizing gradient in the two orthogonal directions perpendicular to the fibers of the internal capsule (n=18 brain regions, P<0.005). However, for the chronic group, there was no significant change m the diffusion MR image signal with diffusion-sensitizing gradient parallel to the fibers of the internal capsule at the terminal scan, suggesting little change in the water diffusion within the nerve fibers. These results suggest that diffusion imaging holds promise for measuring subtle changes in water diffusion due to different types of brain damage.

Use of ibuprofen to assess inflammatory biomarkers in induced sputum: Implications for clinical trials in cystic fibrosis

BACKGROUND High-dose ibuprofen (HDI) is a clinically beneficial anti-inflammatory regimen that may be a useful reagent to study induced sputum inflammatory marker changes over short study periods appropriate for early-phase CF clinical trials. METHODS We conducted a 28-day, open-label, randomized, controlled trial among 72 clinically stable CF subjects (FEV1≥40% predicted) randomized to HDI or routine care that assessed IL-6, IL-8, TNF-α, IL-1-β, free neutrophil elastase, and white cell counts with differentials change from baseline in induced sputum. RESULTS IL-6 was the only biomarker with significant within-group change: 0.13 log10 pg/mL mean reduction among ibuprofen-treated subjects (p=0.04); and no change in the control group. IL-6 change between groups was statistically significant (p=0.024). No other inflammatory biomarker differences were observed between groups after 28 days. CONCLUSION Although we studied only one agent, HDI, these results suggest that one month may be inadequate to assess anti-inflammatory candidates using markers from induced sputum.

Development of the First Inhaled Antibiotic for the Treatment of Cystic Fibrosis

A session about the challenges that occur during drug development was held at a 2010 NIH forum on promoting collaborations among academia, government, and industry. Development of a New Therapy A meeting was held in Bethesda, Maryland, in February 2010 to discuss ways to promote effective collaborations among academia, government, and industry in the field of translational medicine. One session, summarized in this meeting report, was organized to explore the issues and barriers associated with drug development. Specifically, presenters representing the academic, industry, and foundation partners who discovered, developed, and commercialized the first inhaled antibiotic to treat cystic fibrosis–associated lung infections described the challenges they faced during this process. Tobramycin Inhalation Solution USP (TOBI), a therapy developed to treat lung infections associated with cystic fibrosis (CF), was presented as a demonstration case for collaborative pharmaceutical development at a Clinical and Translational Science Awards Industry Forum on “Promoting Efficient and Effective Collaborations Among Academia, Government, and Industry” held in February 2010. TOBI was developed by PathoGenesis Corporation (Seattle, WA) in collaboration with the academic inventors, the National Institutes of Health, the U.S. Food and Drug Administration, and the CF Foundation. The presenters, representing the academic, industry, and foundation partners, each reviewed the program from their perspectives and identified challenges that existed during the discovery, development, and commercialization of TOBI. The attendees were asked to consider other collaborative opportunities that might have further improved TOBI development, including the optimal roles of the academic researchers, foundations, and other agencies when industry drives development and commercialization decisions.

Resolution of CNS lesions following treatment of experimental allergic encephalomyelitis in macaques with monoclonal antibody to the CD 18 leukocyte integrin

Experimental allergic encephalomyelitis (EAE) in macaques is an acute inflammatory and demyelinating disease of the central nervous system (CNS) which has been studied extensively as a model of the human demyelinating disease multiple sclerosis (MS). The in vivo administration of monoclonal antibodies against CD 18, the common β-chain of a leukocyte integrin, at the onset of clinical disease, significantly prolonged the survival of nine of 11 macaques (82%) and in some cases completely reversed the clinical appearance of disease. Treatment with anti-CD18 mAbs dramatically reduced the extent of inflammation in brain lesions as determined by magnetic resonance imaging (MRI). These improvements confirm that anti-CD18 mAbs are powerful anti-inflammatory agents in vivo and suggest that such mAbs may provide effective treatment of both demyelinating and inflammatory CNS diseases in man.