Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines.

Manufacturing procedures for cellular therapies are continuously improved with particular emphasis on product safety. We previously developed a dendritic cell (DC) cancer vaccine technology platform that uses clinical grade lipopolysaccharide (LPS) and interferon (IFN)-y for the maturation of monocyte derived DCs. DCs are frozen after 6 hrs exposure at a semi-mature stage (smDCs) retaining the capacity to secret interleukin (IL)-12 and thus support cytolytic T-cell responses, which is lost at full maturation. We compared closed systems for monocyte enrichment from leucocyte apheresis products from healthy individuals using plastic adherence, CD14 selection, or CD2/19 depletion with magnetic beads, or counter flow centrifugation (elutriation) using a clinical grade in comparison to a research grade culture medium for the following DC generation. We found that elutriation was superior compared to the other methods showing 36 +/- 4% recovery, which was approximately 5-fold higher as the most frequently used adherence protocol (8 +/- 1%), and a very good purity (92 +/- 5%) of smDCs. Immune phenotype and IL-12 secretion (adherence: 1.4 +/- 0.4; selection: 20 +/- 0.6; depletion: 1 +/-0.5; elutriation: 3.6 +/- 1.5 ng/ml) as well as the potency of all DCs to stimulate T cells in an allogeneic mixed leucocyte reaction did not show statistically significant differences. Research grade and clinical grade DC culture media were equally potent and freezing did not impair the functions of smDCs. Finally, we assessed the functional capacity of DC cancer vaccines manufactured for three patients using this optimized procedure thereby demonstrating the feasibility of manufacturing DC cancer vaccines that secret IL-12 (9.4 +/- 6.4 ng/ml). We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing.

Negative expiratory pressure: a new tool for evaluating lung function in children?

The negative expiratory pressure technique (NEP) has been applied in adults with chronic obstructive pulmonary disease (COPD), demonstrating flow limitation in many of these patients. Because this technique does not require patient cooperation, it is of potential interest for application in the pediatric population. This study was performed to test the feasibility of NEP in children, and to further investigate it in children with asthma and cystic fibrosis (CF). We performed NEP (0.3-0.7 kPa) measurements in 14 healthy children (13.3 years, +/- 2.4), in 12 children with asthma (11.7 years, +/- 3.0), and in 17 children with CF (13.3 years, +/- 2.7). NEP-derived flow-volume loops were visually analyzed for flow limitation at tidal breathing. In addition, expiratory flow at 50% of tidal volume (TEF(50)) was measured. In healthy children, the intraclass coefficient of correlation was 77%, and intraindividual short- and long-term variability was 5.8% and 10.8%, respectively. In asthmatics, TEF(50) was lower compared with controls, and increased after inhalation of salbutamol. However, appropriate size-correction has still to be established. Measurement of TEF(50) using NEP is feasible in children. Despite good reproducibility in individual patients, the high intersubject variability may limit its usefulness as a clinical tool. In addition, the lack of flow limitation using NEP even in severely obstructed patients with CF warrants further investigation.