The Hand-Held Fan and the Calming Hand for People With Chronic Breathlessness: A Feasibility Trial.

CONTEXT: The battery-operated hand-held fan ("fan") and the Calming Hand (CH), a cognitive strategy, are interventions used in clinical practice to relieve chronic breathlessness. OBJECTIVE: To test the feasibility of a Phase III randomized controlled trial (RCT) evaluating the impact of the fan and/or CH compared with exercise advice alone for the relief of chronic breathlessness due to respiratory conditions. METHODS: A single-site, feasibility "2 × 2" factorial, nonblinded, mixed-methods RCT was performed. Participants randomly allocated to four groups: fan + exercise advice, CH + exercise advice, fan + CH + exercise advice, and exercise advice alone. Measures included recruitment, acceptability, data quality and study outcomes (baseline and day 28), modified Incremental Shuttle Walk Test (mISWT), recovery time from exertion-induced breathlessness, life-space questionnaire, General Self-Efficacy Scale, and breathlessness numerical rating scales. Willing participants and carers were interviewed at study end. RESULTS: Recruitment/acceptability/data completion: 53 people were screened, 40 randomized and completed (mean age 72 years (SD 9.8), 70% male). There were few missing data (mISWT, n = 2). Recovery time (seconds) from exertion-induced breathlessness showed most improvement for the fan; mean reduction from baseline -33.5 vs. CH mean increase from baseline 5.7. This represents a recovery speed at day 28 (-20.4%) faster for the fan vs. 4.1% slower for the CH. Qualitative data indicated participants valued the faster recovery and identified the fan as a useful "medical" device but found the CH unhelpful. CONCLUSION: A Phase III RCT is feasible. Mixed-methods data synthesis supports recovery time as a novel, meaningful outcome measure.

A randomised controlled trial of three or one breathing technique training sessions for breathlessness in people with malignant lung disease.

BACKGROUND: About 90 % of patients with intra-thoracic malignancy experience breathlessness. Breathing training is helpful, but it is unknown whether repeated sessions are needed. The present study aims to test whether three sessions are better than one for breathlessness in this population. METHODS: This is a multi-centre randomised controlled non-blinded parallel arm trial. Participants were allocated to three sessions or single (1:2 ratio) using central computer-generated block randomisation by an independent Trials Unit and stratified for centre. The setting was respiratory, oncology or palliative care clinics at eight UK centres. Inclusion criteria were people with intrathoracic cancer and refractory breathlessness, expected prognosis ≥3 months, and no prior experience of breathing training. The trial intervention was a complex breathlessness intervention (breathing training, anxiety management, relaxation, pacing, and prioritisation) delivered over three hour-long sessions at weekly intervals, or during a single hour-long session. The main primary outcome was worst breathlessness over the previous 24 hours ('worst'), by numerical rating scale (0 = none; 10 = worst imaginable). Our primary analysis was area under the curve (AUC) 'worst' from baseline to 4 weeks. All analyses were by intention to treat. RESULTS: Between April 2011 and October 2013, 156 consenting participants were randomised (52 three; 104 single). Overall, the 'worst' score reduced from 6.81 (SD, 1.89) to 5.84 (2.39). Primary analysis [n = 124 (79 %)], showed no between-arm difference in the AUC: three sessions 22.86 (7.12) vs single session 22.58 (7.10); P value = 0.83); mean difference 0.2, 95 % CIs (-2.31 to 2.97). Complete case analysis showed a non-significant reduction in QALYs with three sessions (mean difference -0.006, 95 % CIs -0.018 to 0.006). Sensitivity analyses found similar results. The probability of the single session being cost-effective (threshold value of £20,000 per QALY) was over 80 %. CONCLUSIONS: There was no evidence that three sessions conferred additional benefits, including cost-effectiveness, over one. A single session of breathing training seems appropriate and minimises patient burden. TRIAL REGISTRATION: Registry: ISRCTN; TRIAL REGISTRATION NUMBER: ISRCTN49387307; http://www.isrctn.com/ISRCTN49387307 ; registration date: 25/01/2011.

Assessment of umbilical cord tissue as a source of mesenchymal stem cell/endothelial cell mixtures for bone regeneration.

AIM: To enumerate and characterize mesenchymal stem cells (MSCs) and endothelial cells (ECs) in umbilical cord (UC) tissue digests. MATERIALS & METHODS: Cultured UC cells were characterized phenotypically, and functionally by using 48-gene arrays. Native MSCs and ECs were enumerated using flow cytometry. RESULTS: Compared with bone marrow (BM) MSCs, UC MSCs displayed significantly lower (range 4-240-fold) basal levels of bone-related transcripts, but their phenotypes were similar (CD73⁺, CD105⁺, CD90⁺, CD45⁻ and CD31⁻). UC MSCs responded well to osteogenic induction, but day 21 postinduction levels remained below those achieved by BM MSCs. The total yield of native UC MSCs (CD90⁺, CD45⁻ and CD235α⁻) and ECs (CD31⁺, CD45⁻ and CD235α⁻) exceeded 150 and 15 million cells/donation, respectively. Both UC MSCs and ECs expressed CD146. CONCLUSION: While BM MSCs are more predisposed to osteogenesis, UC tissue harbors large numbers of MSCs and ECs; such minimally manipulated 'off-the-shelf' cellular mixtures can be used for regenerating bone in patients with compromised vascular supply.

Assay validation for the assessment of adipogenesis of multipotential stromal cells--a direct comparison of four different methods.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed. METHODS: Four previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-γ and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4',6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls. RESULTS: Oil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ~5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ~13-fold, with significant correlations with oil red scoring observed for MSC from other sources. CONCLUSIONS: Flow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources.

NSAIDS inhibit in vitro MSC chondrogenesis but not osteogenesis: implications for mechanism of bone formation inhibition in man.

The non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for analgesia but may inhibit bone formation. We investigated whether the reported NSAID effect on bone is related to inhibition of bone marrow mesenchymal stem cell (MSC) proliferation and osteogenic and chondrogenic differentiation and evaluated both cyclooxygenase (COX)-1 and COX-2 specific drugs. The effects of seven COX-1 and COX-2 inhibitors on MSC proliferation and osteogenic and chondrogenic differentiation were tested using Vybrant, sodium 3'-[1-(phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT), functional and quantitative assays of MSC differentiation. The MSC expression of COX-1 and COX-2 and prostaglandin E2 (PGE-2) levels were evaluated serially during lineage differentiation by quantitative PCR and ELISA. None of the NSAIDs at broad range of concentration (range 10(-3) to 100 µg/ml) significantly affected MSC proliferation. Surprisingly, MSC osteogenic differentiation inhibition was not evident. However, NSAIDs affected chondrogenic potential with a reduction in sulphated glycosaminoglycans (sGAG) content by 45% and 55% with diclofenac and ketorolac, respectively (P < 0.05 compared to controls). Parecoxib and meloxicam, more COX-2 specific reagents inhibited sGAG to a lesser degree, 22% and 27% respectively (P < 0.05 compared to controls). Cartilage pellet immunohistochemistry confirmed the above results. Pellet chondrogenesis was associated with increased COX-1 expression levels but not COX-2, and COX-1 specific drugs suppressed MSC PGE-2 more than COX-2 specific inhibitors. These findings suggest that NSAIDs may inhibit bone formation via blockage of MSC chondrogenic differentiation which is an important intermediate phase in normal endochondral bone formation.

A randomised trial of high vs low intensity training in breathing techniques for breathless patients with malignant lung disease: a feasibility study.

BACKGROUND: Breathlessness remains a refractory symptom in malignant lung disease. Breathing training is an effective, non-pharmacological intervention but it is unclear how this should be delivered. This feasibility study aimed to assess recruitment and retention, best end point and variability of breathlessness scores in order to calculate sample size for a future study. METHOD: This was a single centre, randomised controlled non-blinded parallel group feasibility study. Eligible participants (breathless patients with intrathoracic malignancy) received three breathlessness management training sessions or a single session only. Follow-up was for eight weeks and endpoints were: numerical rating scales (NRS) of breathlessness severity; breathlessness distress; HADS questionnaire; coping (BriefCOPE and our NRS coping question); EQ-5D and EQ-VAS. RESULTS: 22 patients were randomised over 12 months; 55% of expected recruitment from pilot data. Screening logs indicated this resulted, in part, from excluding patients who were receiving or who had recently received chemotherapy or radiotherapy. There was 40% drop-out by week four. The most useful NRS scores for breathlessness severity were for "worst" and "average" over past 24h. From the variability data for "worst breathlessness", a sample size of 270 should allow detection of a 30% improvement in area under the curve in the three-session group compared with single-session, (90% power; p=0.05, two-tailed; 2:1 randomisation single:three sessions) allowing 50% drop out at four weeks. CONCLUSIONS: The follow-on study will test the hypothesis that three sessions of training improve breathlessness better than a single session. It will include patients undergoing palliative anti-cancer therapy. Stratification by centre will allow for differences in rates of chemotherapy or radiotherapy and variations in breathlessness service configuration.

Synovial fluid mesenchymal stem cells in health and early osteoarthritis: detection and functional evaluation at the single-cell level.

OBJECTIVE: Arthritic synovial fluid (SF) contains mesenchymal stem cells (MSCs), which could simply reflect their shedding from diseased joint structures. This study used the bovine model to explore SF MSCs in health and enumerated them at the earliest stages of human osteoarthritis (OA) in radiographically normal joints. METHODS: Clonogenicity and multipotentiality of normal bovine SF MSCs were compared with donor-matched bone marrow (BM) MSCs at the single-cell level. The colony-forming unit-fibroblastic assay was used for MSC enumeration. The XTT assay was employed to assess cell proliferation, and flow cytometry was used to investigate the marker phenotype of bovine and human SF MSCs. RESULTS: Single MSCs were present in normal bovine SF, and 96% of them were able to expand at least 1 million-fold. These cells were CD271-, multipotential, considerably more clonogenic, and less adipogenic than matched BM MSCs. In both pellet assays and on polyglycolic acid scaffolds, SF clones displayed consistent chondrogenic differentiation, while BM clones were variable. MSCs were present in arthroscopically normal human joints and were increased 7-fold in early OA (P = 0.034). Their numbers correlated with numbers of free microscopic synovial tissue fragments (r = 0.826, P < 0.0001). OA SF had a growth-promoting effect on synovial MSCs. CONCLUSION: This study confirms the presence of MSCs in normal SF and shows their numerical increase in early human OA. SF MSCs are likely to originate from synovium. These findings provide a platform for the exploration of the potential role of SF MSCs in joint homeostasis and for investigation of their utility in novel joint regeneration strategies.

Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow.

BACKGROUND: To study the biology of rare bone marrow (BM) multipotent mesenchymal stromal cells (MSCs), recognized protocols are needed. Colony-forming unit-fibroblast (CFU-F) assays have historically been used for the enumeration of MSCs. However, the need to isolate and further analyze MSCs requires new strategies based on cell surface markers. The purpose of this work was to verify the phenotype of BM MSCs in vivo and to develop flow cytometry-based methods for their evaluation. METHODS: Pre-enrichment with D7-FIB-conjugated microbeads, cell sorting for CD45low D7-FIB+ LNGFR+ cells, and CFU-F assay were used to confirm the phenotype of BM MSCs in vivo. Further phenotypic characterization of MSCs was performed using three-color flow cytometry following pre-enrichment or by direct four-color flow cytometry. The sensitivity of direct flow cytometry/rare event analysis for the accurate enumeration of MSCs was validated using 85 samples from patients with neoplastic BM diseases. RESULTS: In normal BM, a significant correlation was found between the frequencies of CFU-Fs and CD45low D7-FIB+ LNGFR+ cells (n = 19, R = 0.719, P = 0.001). Following cell sorting, 15% of these cells were clonogenic. The same cells were enriched using LNGFR-based positive selection, CD45/Glycophorin A-based depletion, or plastic adherence. CD45low D7-FIB+ LNGFR+ cells expressed classic makers of cultured MSCs CD73/SH3 and CD105/SH2 and markers of stromal reticular cells CD106/VCAM and alkaline phosphatase. Novel markers were identified including leukemia inhibitory factor receptor and gp130. CD45low D7-FIB+ LNGFR+ cells were increased fourfold in the floating fat fraction of normal BM aspirates. Their frequency was decreased in chronic lymphocytic leukemia (threefold, n = 13, P = 0.049) and chronic myelogenous leukemia (ninefold, n = 11, P = 0.001) compared with that in age-matched controls (n = 26 and n = 31, respectively). CONCLUSIONS: This study demonstrates the usefulness of flow cytometry-based methods for the detection, enumeration and further phenotypic analysis of BM MSCs. These findings have broad applications for the future evaluation of BM MSCs in health and disease.

Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis.

OBJECTIVE: To evaluate synovial fluid (SF) for the presence of mesenchymal progenitor cells (MPCs), to compare SF MPCs with bone marrow (BM) MPCs, and to enumerate these cells in both inflammatory arthritis and osteoarthritis (OA). METHODS: SF from 100 patients with arthritis (53 rheumatoid arthritis [RA], 20 OA, and 27 other arthropathies) was evaluated. To establish multipotentiality, polyclonal and single cell-derived cultures of SF fibroblasts were examined by standard and quantitative differentiation assays. Their phenotype before and after expansion was determined by multiparameter flow cytometry. A colony-forming unit-fibroblast assay was used for SF MPC enumeration. RESULTS: Regardless of the nature of the arthritis, both polyclonal and single cell-derived cultures of SF fibroblasts possessed trilineage mesenchymal differentiation potentials. The number of MPCs in a milliliter of SF was higher in OA (median 37) than in RA (median 2) (P < 0.00001). No significant differences in MPC numbers were found between early and established RA (median 3 and 2 cells/ml, respectively). Culture-expanded SF and BM MPCs had the same phenotype (negative for CD45 and positive for D7-FIB, CD13, CD105, CD55, and CD10). Rare, uncultured SF fibroblasts were CD45(low) and expressed low-affinity nerve growth factor receptor, similar to in vivo BM MPCs. CONCLUSION: Our findings prove the presence of rare tripotential MPCs, at the single-cell level, in the SF of patients with arthritis. SF MPCs are clonogenic and multipotential fibroblasts that, despite the pathologic environment within a diseased joint, have a phenotype similar to that of uncultured BM MPCs. The higher prevalence of MPCs in OA SF suggests their likely origin from disrupted joint structures. These findings could determine the role of MPCs in the pathogenesis of inflammatory arthritis, together with their role in attempted joint regeneration in degenerative arthritis, which has yet to be established.

Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells.

OBJECTIVE: There is an increased interest in rheumatology in mesenchymal progenitor/stem cells (MPCs) and their roles in rheumatic diseases, but little is known about the phenotype of these cells in vivo. The aim of this study was to isolate and characterize human bone marrow (BM) MPCs. METHODS: Fluorescence microscopy was used to identify putative MPCs among adherent BM cells. To purify them, a positive selection with antifibroblast microbeads was used, combined with fluorescence-activated cell sorting (FACS) for microbead+,CD45(low) cells. A more detailed phenotype of these cells was determined using 4-color flow cytometry, and standard chondrogenic, osteogenic, and adipogenic assays were used to investigate their differentiation potentials. RESULTS: Putative MPCs microscopically identified as large, fibroblast-like, D7-FIB+ cells were purified using positive selection with D7-FIB-conjugated (antifibroblast) microbeads followed by FACS for specifically bound microbead+,CD45(low) cells. These cells represented 0.01% of mononuclear cells in the BM. They were uniformly positive for CD105, LNGFR, HLA-DR, CD10, CD13, CD90, STRO-1, and bone morphogenetic protein receptor type IA (BMPRIA) and were negative for CD14, CD34, CD117, and CD133. Only cells with this phenotype could proliferate and produce adherent cell monolayers capable of chondrogenic, osteogenic, and adipogenic differentiation. D7-FIB- cells in the BM lacked any MPC activity. Uncultured skin fibroblasts had a phenotype similar to that of BM MPCs, but were negative for LNGFR, STRO-1, HLA-DR, and BMPRIA. CONCLUSION: This study shows the distinct phenotype, morphology, and method of isolation of BM MPCs. The findings may have implications for defining the physiologic roles of MPCs in arthritis, bone diseases, and joint regeneration.