Migratory properties of ex vivo expanded regulatory T cells: Influence of all-trans retinoic acid and rapamycin.

Adoptively transferred regulatory T-cells represent a promising therapeutic approach for tolerance induction in autoimmunity and transplantation medicine. However, a major hurdle for clinical application is the manufacturing of sufficient Treg cell numbers with respect to the low frequency of naturally occurring Tregs in the peripheral blood. Therefore, ex vivo large-scale expansion is mandatory for most of the clinical conditions. Besides the Treg cell number other parameters of the cell product are of high relevance for safe and efficient clinical Treg cell application like Treg cell purity, suppressive capacity and genetic stability of the Treg cell phenotype. Moreover, migratory properties of ex vivo expanded Tregs should be defined very clearly in order to predict their migration to secondary lymphoid organs as sites of antigen-specific activation, in vivo proliferation and subsequent trafficking to affected target organs. Therefore, we studied different cell culture conditions for Treg large-cell expansion using all-trans retinoic acid (ATRA) and/or rapamycin (Rapa) with focus on their migratory properties. The tested culture conditions revealed comparable chemokine receptor expression profiles (CXCR3, CCR4, CCR6, CCR7) and functional migration capabilities (IP10 and CCL19) with respect to Th1 and Th2 inflammatory conditions. However, the most striking difference was detected for the expansion capacity, suppressive potency and genetic stability likely predisposing large-scale expansion with ATRA and/or Rapa for therapeutic intervention in acute GvHD and without supplementation for chronic GvHD.

Impact of optical coherence tomography scanning density on quantitative analyses in neovascular age-related macular degeneration.

PurposeTo assess the influence of varying B-scan frame-sampling densities on retinal thickness and volume measurements from spectral domain optical coherence tomography (OCT) in eyes with neovascular age-related macular degeneration (AMD).MethodsVolume OCT data (512 × 128 macular cube over 6 × 6 mm) were collected from 39 eyes with neovascular AMD. All 128 B-scans in each image set were manually segmented, allowing quantification of the neurosensory retina, subretinal fluid (SRF), subretinal hyperreflective material (SRHM), and pigment epithelium detachment (PED). Thickness maps were generated for less dense subsets of scans, ranging from every other (64 B-scans) to every 64th (2 B-scans). For each less dense subset, foveal central subfield thickness and total macular volume (TMV) were compared with values obtained using all 128 scans (considered the reference).ResultsFor each parameter, the mean absolute difference compared with the reference increased with reducing B-scan density. However, these differences did not reach statistical significance until frame-sampling density was reduced to every eighth scan (ie, 16 B-scans spaced 375 µm apart) for neurosensory retina, and every fourth scan (ie, 32 B-scans spaced 188 µm apart) for SRF, SRHM, and PED. For neurosensory retina, the mean (% error) and maximum (% error) absolute differences in TMV were 0.02 mm3 (0.24%) and 0.06 mm3 (0.79%), respectively. Similarly, at a density of 32 B-scans, mean and maximum differences for SRF were 0.004 mm3 (3.47%) and 0.02 mm3 (22.22%), respectively. The mean differences for SRHM and PED were 0.01 mm3 (8.03%) and 0.01 mm3 (4.04%), respectively.ConclusionsA minimum of 16 equally spaced B-scans, covering a 6 × 6 mm area, appears necessary to generate retinal thickness measurements similar to those obtained using all 128 B-scans in eyes with choroidal neovascularization (CNV). When considering other CNV lesion features, a minimum of 16 B-scans for SRF and PED, and 32 B-scans for SRHM are required to generate volume maps similar to ground-truth values. These findings may have implications for the design of acquisition and grading protocols for clinical trials using OCT in neovascular AMD.

Characterisation of salmon calcitonin in spray-dried powder for inhalation. Effect of chitosan.

Salmon calcitonin (sCT) powders suitable for inhalation, containing chitosan and mannitol as absorption enhancer and protection agent, respectively, were prepared using a spray-drying process. The effect of chitosan on physicochemical stability of sCT in the dry powder was investigated by different analytical techniques. High-performance liquid chromatography (HPLC) analysis indicated that sCT was chemically stable upon spray-drying. With the proportion of chitosan in spray-drying formulation being increased, dissolution of sCT from the dry powders was decreased both in phosphate buffer and acetate buffer. The thioflavine T fluorescence assay showed that no fibrils were present in the spray-dried powder. However, sCT partly fibrillated in the phosphate buffer, but not in acetate buffer. Fourier transform infrared (FTIR) spectra showed that the secondary structure of sCT was slightly changed in the dry powder, yet no aggregate signal was observed. Circular dichroism analysis indicated that the structure of sCT in an aqueous formulation was slightly altered by addition of chitosan. Nevertheless, recovery of sCT was not influenced by chitosan in the aqueous formulation as indicated by HPLC analysis. This study suggested that sCT, in absence of any additives, was stable during the spray-drying process under certain conditions. Addition of chitosan affects recovery of sCT from spray-dried powders, which may be due to formation of a partially irreversible complex between the protein and chitosan during the spray-drying process.