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International audience; In this study, drawbacks of the use of 243Am as a tracer in order to quantify curium isotopes during a complex chemical procedure were highlighted. Origins of these drawbacks were investigated through a speciation computation simulation. This simulation was based on the compilation of over 50 constants and allowed to confirm that in order to quantify curium isotopes, more reliable results are obtained using a specific curium isotope as a tracer. A new curium standard was then characterized and used to quantify a wide 244Cm activity range in environmental samples. Obtained results were in excellent agreement with spiked activities and more consistent than those obtained when using a 243Am tracer.

Artículo con numerosos autores, sólo se recogen el primero y los pertenecientes a la UAM, Background and study aim: Advanced therapy medicinal products (ATMP) frequently lack of clinical data on efficacy to substantiate a future clinical use. This study aims to evaluate the efficacy to heal long bone delayed unions and non-unions, as secondary objective of the EudraCT 2011-005441-13 clinical trial, through clinical and radiological bone consolidation at 3, 6 and 12 months of follow-up, with subgroup analysis of affected bone, gender, tobacco use, and time since the original fracture. Patients and methods: Twenty-eight patients were recruited and surgically treated with autologous bone marrow derived mesenchymal stromal cells expanded under Good Manufacturing Practices, combined to bioceramics in the surgical room before implantation. Mean age was 39 ±13 years, 57% were males, and mean Body Mass Index 27 ±7. Thirteen (46%) were active smokers. There were 11 femoral, 4 humeral, and 13 tibial non-unions. Initial fracture occurred at a mean ±SD of 27.9 ±31.2 months before recruit- ment. Efficacy results were expressed by clinical consolidation (no or mild pain if values under 30 in VAS scale), and by radiological consolidation with a REBORNE score over 11/16 points (value of or above 0.6875). Means were statistically compared and mixed models for repeated measurements estimated the mean and confidence intervals (95%) of the REBORNE Bone Healing scale. Clinical and radiological con- solidation were analyzed in the subgroups with Spearman correlation tests (adjusted by Bonferroni). Results: Clinical consolidation was earlier confirmed, while radiological consolidation at 3 months was 25.0% (7/28 cases), at 6 months 67.8% (19/28 cases), and at 12 months, 92.8% (26/28 cases including the drop-out extrapolation of two failures). Bone biopsies confirmed bone formation surrounding the bioce- ramic granules. All locations showed similar consolidation, although this was delayed in tibial non-unions. No significant gender difference was found in 12-month consolidation (95% confidence). Higher consoli- dation scale values were seen in non-smoking patients at 6 ( p = 0.012, t -test) and 12 months ( p = 0.011, t -test). Longer time elapsed after the initial fracture did not preclude the occurrence of consolidation. Conclusion: Bone consolidation was efficaciously obtained with the studied expanded hBM-MSCs com- bined to biomaterials, by clinical and radiological evaluation, and confirmed by bone biopsies, with lower consolidation scores in smokers, This research received funding from the European Union’s Sev- enth Framework Programme ( FP7/FP7-HEALTH-2009 ) with the RE- BORNE Project (under G.A. 241876), and the European Union’s Horizon 2020 Programme ( H2020-SC1 2016-2017 ), with the OR- THOUNION Project (under G.A. 7333288)

La mise sur le marché de biosimilaires requiert une démonstration stricte de la similarité avec l’anticorps de référence, au travers d’études précliniques et cliniques. Cet article synthétise l’ensemble des analyses physicochimiques et fonctionnelles mises en œuvre in vitro, préalables à la réalisation d’études cliniques. Pour chaque caractéristique critique de l’anticorps, nous avons détaillé les techniques analytiques communément employées, leur principe de fonctionnement, ainsi que le type d’informations que ces techniques permettent d’obtenir.

PURPOSE Chemotherapies are considered high-risk drugs for patient and staff safety. Considering the rising burden of cancer and the increasing use of chemotherapy drugs in low- and middle-income countries (LMICs), promoting continuous improvements in the safety and quality of practices in these settings is essential. This paper describes the development and proof of concept of a toolkit to audit chemotherapy handling practices in the health care facilities of LMICs. METHODS A steering committee defined the audit method and the toolkit content. Several checklists were developed to facilitate the audit and data collection. Items included in checklists were derived from key reference works on safe handling. Different tools were validated using Delphi surveys and expert reviews. Audits of pilot sites were performed to test the toolkit's applicability and relevance. RESULTS The toolkit contains a 134-item global assessment tool for the different processes at each step of the medication pathway and three step-specific observation checklists to assess different health workers' practices during the prescription, preparation, and administration of chemotherapies. The toolkit also proposes using a surface-wipe sampling method to measure any cytotoxic contamination of the immediate environment. The toolkit was tested in three teaching hospitals in Africa. CONCLUSION The toolkit developed was successfully implemented in a variety of LMIC settings, providing a comprehensive evaluation of the quality and safety of the chemotherapy drug handling practices in participating health care facilities. This toolkit can help facilities in LMICs to implement a new approach to continuously improving the quality and safety of their practices and ultimately ensure patient and staff safety., A toolkit for implementing safe chemotherapy handling practices to ensure patient and staff safety in LMICs

International audience; Alumina production from bauxite ore gives rise to a large amount of liquid and solid waste since the production of 1 kg of alumina involves the same weight of solid residue, the well-known red muds enriched in iron and heavy metals. Up to now, without any valorization of by-products, the red muds have been stored in artificial ponds which are a potential source of pollutants for the surroundings due to the uplift of red dust under strong wind conditions. The goal of the current work is to perform an in situ study of the transfer of red mud micro particles in the atmosphere and in the terrestrial environment around the red mud disposition basins. To this end, the activity concentration of NORM (Naturally Occurring Radioactive Materials such as U-238, decay products and Th-232) determined in plant leaves (Quercus robur and leaf vegetables) and in grains (wheat) is compared with the activity of the atmospheric micro particles, both taken in the environment of the red mud disposition basins (100-1,500 m). The activity ratios of NORM in vegetation and atmospheric micro particles are also compared to the ratios of potential sources of dust, such as soil and, above all, the red mud and bauxite emitted from the basins and from the piles, respectively.The increase in Th-232 and U-238 activity concentration in a few tree leaves (by a factor of 9 and 4, respectively) and in some aerosol samples is accompanied by a decrease in U-238/Th-232 activity ratio for these matrices. Low U-238/Th-232 is also observed in bauxite and red mud. This suggests that the airborne particles emitted by bauxite piles and red mud basins first contaminate the atmosphere and then the leafs surfaces, after deposition. Locally produced foodstuffs at a distance greater than 1,000 m from the ponds do not show any excess radionuclides, suggesting the low influence of airborne particles from the alumina production. Further variations in the Po-210/Pb-210 ratio in the plants and the atmospheric micro particles are consistent with the variation over time of these radionuclides coming in all likelihood from the atmosphere rather than from the aluminum industry.

The validation and uncertainty assessment of the analytical method developed for the simultaneous determination of 25 anticancer drugs commonly handled in hospital pharmacy was reported. Selected compounds were 5-fluorouracil, cytarabine, fludarabine phosphate, ganciclovir, gemcitabine, dacarbazine, methotrexate, pemetrexed, busulfan, raltitrexed, etoposide phosphate, topotecan, ifosfamide, cyclophosphamide, irinotecan, doxorubicin, epirubicin, daunorubicin, idarubicin, vincristine, vinblastine, vinorelbine, docetaxel and paclitaxel. Accuracy and uncertainty profiles were obtained for all compounds. Quantitative performances were satisfactory in term of specificity, sensitivity, precision and accuracy. Repeatability (1.9–25.4%) and intermediate precision (2.7–29%) were determined for all target compounds. Lower limits of quantification between 1 and 25 ng/mL were obtained. Uncertainty associated to measurement of routine samples was evaluated. The multi-targeted method was specific and reliable and was successfully applied to wipe samples from hospital pharmacy chemotherapy compounding unit and to the determination of outside contamination of vials from pharmaceutical manufacturers.

Although considerable efforts have been made over the last 40 years, occupational exposure to antineoplastic drugs is still a daily concern, since eradicating such contamination from workplaces seems unattainable. Considerable data are currently available on the risks associated with their use at work. Hospital facilities are often cleaned with marketed antimicrobials whose chemical decontamination efficacy certainly differs but remains unknown. To keep compounding facilities sterile, alcohol-based solutions are frequently used but with very limited efficiency. It would be particularly useful if a decontamination method could be added to the means already available so that all conventional antineoplastic drug contamination could be removed. Several degradation methods or desorption methods have previously been experimented, with varying success. They have never been compared or discussed in terms either of efficiency or usability. This review aims to analyse and discuss the results of each degradation or decontamination procedure and to compare them. This should facilitate selection of the method to be implemented in daily practice.

In 2019, we celebrated the 40th anniversary of the famous article published by Falck et al , dealing with the mutagenicity of the urine of nurses who were in daily contact with conventional antineoplastic drugs.1 Since the publication of this article, highlighting the hazard of handling such drugs, extensive literature has flowed, in order to: All this knowledge has raised our level of vigilance on this issue and encouraged us to upgrade our practices constantly. Scientific data have been obtained through technological advances and in many different operating conditions, the latter making it difficult to define a fair protection level. Moreover, a lack of relevant clinical references and toxicological values has led to a continuous exploration for protective measures. Forty years after the first report of … Correspondence to Dr Nicolas Simon, GRITA Grp Rech Formes Injectables, LILLE Cedex 59006, France; nicolas.simon{at}univ-lille.fr

Purpose The stability of busulfan solution in 0.9% sodium chloride and stored in polypropylene syringes or infusion bags was evaluated. Methods Busulfan solutions (0.54 mg/mL) were prepared and transferred to 50-mL polypropylene syringes and 100- and 500-mL polypropylene infusion bags and stored at 2-8 and 23-27 °C. Chemical stability was measured using a stability-indicating, ultrahigh performance liquid chromatography coupled to mass spectrometry method. The stability of busulfan was assessed by measuring the percentage of the initial concentration remaining at the end of each time point of analysis. The initial busulfan concentration was defined as 100%. Stability was defined as retention of at least 90% of the initial busulfan concentration. A visual inspection of the samples for particulate matter, clarity, and color without instrumentation of magnification was conducted at each time point of analysis. Results The visual inspection demonstrated no influence of the storage container when busulfan infusions diluted in 0.9% sodium chloride injection were stored at 23-27 °C. No color change or precipitate was observed at this temperature; however, a rapid decrease of the busulfan content in all containers stored at room temperature was observed. Busulfan in syringes was chemically stable for 12 hours, while busulfan in infusion bags (100 and 500 mL) was stable only for 3 hours at 23-27 °C. Conclusion Busulfan 0.54-mg/mL solution in 0.9% sodium chloride injection was physically and chemically stable for 30 hours when stored in 50-mL polypropylene syringes at 2-8 °C and protected from light.

Objectives Over the past 40 years, numerous actions have been undertaken to decrease the contamination of hospital facilities by intravenous conventional antineoplastic drugs (ICADs) such as centralizing compounding in pharmacies, using personal protective equipment, specific compounding, or infusion devices. As recently proposed in the monograph, an additional specific decontamination step must be envisaged. A recent literature review analysed and discussed the different solutions tested in terms of decontamination efficacy. This article aims to discuss the performance of these solutions in the framework of aseptic compounding. Methods The same dataset used in the previous literature review was reanalysed according to other parameters so as to select decontamination solutions: overall decontamination efficiency (EffQ), tested contaminants, and the risks of use in daily practice. Results Using an EffQ threshold of 90% resulted in discarding 26 out of the 59 solutions. Solutions were tested differently: 8 on 1 contaminant, 11 on 2 contaminants, and 14 solutions on between 3 and 11 contaminants. Three risks were identified to help make choices in routine practice: the mutagenicity of degradation products, the safety of operators and facilities, and respect for the aseptic environment. Conclusions From the results, performance is discussed according to specific situations: a one-time incident or the basic chemical contamination due to daily practice. Accordingly, the decontamination solution selected then required a risk analysis and an evaluation before implementing it in the daily practice of a compounding unit.

PurposeChemotherapy preparation units face peaks in activity leading to high workloads and increased stress. The present study evaluated the impact of work overloads on the safety and accuracy of manual preparations.MethodSimulating overwork, operators were asked to produce increasing numbers of syringes (8, 16, and 24), with markers (phenylephrine or lidocaine), within 1 h, in an isolator, under aseptic conditions. Results were analyzed using qualitative and quantitative criteria. Concentration deviations of 30% from the expected concentration were considered as accurate, weakly accurate, inaccurate, and wrong concentrations, respectively.ResultsTwenty-one pharmacy technicians and pharmacists carried out 63 preparation sessions (n = 1007 syringes). A statistically significant decrease in the manufacturing time for one syringe was observed when workload increased (p 30%), 6 mislabeling, 2 wrong diluents, and 1 wrong drug. There was no statistically significant difference in the mean concentration accuracy of final preparations across the three workloads. The overall error rate increased with the number of preparations made in 1 h: 1.8% for 8 preparations, 2.7% for 16 preparations, and 5.4% for 24 preparations (p ConclusionAlthough pharmacy technicians and pharmacists were able to increase production speeds with no effect on mean concentration accuracy under stressful conditions, there were greater probability errors being made. These results should encourage actions to spread workloads out over the day to avoid peaks in activity.

Background Several decontamination methods are currently available to reduce the occupational exposure of hospital facilities to conventional anti-neoplastic drugs. Alcohol-based microbicides are not sufficiently efficient in removing chemical contamination and data are lacking on many marketed biocides. Recent data confirm that using a specific chemical decontamination solution is helpful in removing traces of contaminants. Purpose To perform a literature review in order to help pharmacists in choosing a chemical decontamination solution to implement in their compounding unit. Material and methods Articles were searched on Pubmed using the following requests: ‘antineoplastic agents AND cleaning’ or ‘antineoplastic agents AND chemical degradation’ or ‘antineoplastic agents AND chemical decontamination’. Criteria used to classify the performance and usability of decontamination solutions were: decontamination efficiency, number and nature of tested contaminants, hazardousness of the decontamination solution, implementation difficulties and respect of the aseptic environment. Results Two-hundred and seventy-four articles were retrieved following the request application. Two-hundred and fifty-seven articles were discarded for different reasons leading to the analysis of 17 articles. Fifty-nine methods were tested as degradation (n=19) or desorption methods (n=40) with various decontamination efficiencies ranging from ≤10% to 100%. Applying the selection criteria, three decontamination solutions were chosen: sodium hypochlorite, admixture of 10-2 M sodium dodecyl sulfate (SDS) and 70% isopropanol (80/20), marketed two steps towelettes kit (1. Quaternary ammonium solution, 2. Isopropanol). Their inertness to facilities’ surfaces is different and sodium hypochlorite solutions oxide metals. Solutions involving tension-active agents such as SDS may form a film on the facilities surface, which may alter the sterility environment. Conclusion The applied selection criteria led to select only three decontamination solutions. Their application modalities are also to be discussed regarding the biological and chemical facilities’ monitoring. As the solutions were assessed with various methodologies, further studies are necessary to compare them in the same conditions. Because each solution has been tested with different contaminants, new studies are required to confirm their ability to decontaminate other conventional anti-neoplastic drugs. References and/or acknowledgements Vasseur, et al. EJHP 2017;24(Suppl 1):A218. doi:10.1136/ejhpharm-2017–000640.485 Vasseur, et al. PLos One 2018. No conflict of interest.

The wastewater contamination of a Swiss university hospital by active pharmaceutical ingredient (API) residues was evaluated with a three months monitoring campaign at the outlet of the main building. Flow-proportional samples were collected with an automatic refrigerated sampler and analyzed for 15 API, including antibiotics, analgesics, antiepileptic and anti-inflammatory drugs, by using a validated LC-MS/MS method. The metals Gd and Pt were also analyzed using ICP-MS. Measured concentrations were compared to the predicted ones calculated after the drug average consumption data obtained from the hospital pharmacy. The hospital contribution to the total urban load was calculated according to the consumption data obtained from city pharmacies. Lastly, the environmental hazard and risk quotients (RQ) related to the hospital fraction and the total urban consumption were calculated. Median concentrations of the 15 selected compounds were ranging from 0.04 to 675 μg/L, with a mean detection frequency of 84%. The ratio between predicted and measured environmental concentrations (PEC/MEC) has shown a good accuracy for 5 out of 15 compounds, revealing over- and under-estimations of the PEC model. Mean daily loads were ranging between 0.01 and 14.2g/d, with the exception of paracetamol (109.7 g/d). The hospital contribution to the total urban loads varied from 2.1 to 100% according to the compound. While taking into account dilution and removal efficiencies in wastewater treatment plant, only the hospital fraction of the antibiotics ciprofloxacin and sulfamethoxazole showed, respectively, a high (RQ>1) and moderate (RQ>0.1) risk for the aquatic ecosystems. Nevertheless, when considering the total urban consumption, 7 compounds showed potential deleterious effects on aquatic organisms (RQ>1): gabapentin, sulfamethoxazole, ciprofloxacin, piperacillin, ibuprofen, diclofenac and mefenamic acid. In order to reduce inputs of API residues originating from hospitals various solutions can be envisioned. With results of the present study, hospital managers can start handling this important issue.

The Channel Islands are located in the Normand-Breton Gulf (NBG), in the mid-part of the English Channel (France, Normandy). In the northern part, off Cap La Hague, controlled amounts of radioactive liquid waste are discharged by the ORANO La Hague nuclear fuel reprocessing plant (RP). Radionuclides were monitored in the NBG to assess the dispersion of radioactive discharges from the RP in the marine environment. The temporal and spatial distribution of the data are consistent with the history of the discharges, with most gamma emitter radionuclide environmental levels being close to or below the current limits of detection. A clear fingerprint of H-3, C-14 and I-129 radionuclides discharged from the RP is measured. The hydrodynamics in the NBG do not yield a simple gradient with linear distance from the outfall of the RP. Modelling tools were used to understand how radioactive discharges spread from the source of input. Dispersion patterns clearly illustrate the different behaviours of soluble and non-soluble radionuclides. The study indicated that the footprint of radioactive liquid discharges by French nuclear facilities was still measurable in species collected from the NBG for the mostly dissolved radionuclides. The less conservative ones, with a high affinity for suspended matter, are potentially influenced by old releases. These pathways could be investigated by dedicated hydrodynamic dispersion models. Overall, in the Channel Islands the levels are low and consistent with the general decrease in liquid radionuclide discharges by the RP since the 1990s.

A liquid chromatography separation with electrospray ionisation and tandem mass spectrometry detection method was developed for the simultaneous quantification of ten commonly handled cytotoxic drugs in a hospital pharmacy. These cytotoxic drugs are cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. The chromatographic separation was carried out by RPLC in less than 21min, applying a gradient elution of water and acetonitrile in the presence of 0.1% formic acid. MS/MS was performed on a triple quadrupole in selected reaction monitoring mode. The analytical method was validated to determine the limit of quantification (LOQ) and quantitative performance: lowest LOQs were between 0.25 and 2ngmL−1 for the ten investigated cytotoxic drugs; trueness values (i.e. recovery) were between 85% and 110%, and relative standard deviations for both repeatability and intermediate precision were always inferior to 15%. The multi-compound method was successfully applied for the quality control of pharmaceutical formulations and for analyses of spiked samples on potentially contaminated surfaces. Figure Preparation of cytotoxic formulations at the Pharmacy of Geneva University Hospitals

Two capillary electrophoresis (CE) methods were developed for the analysis of 16 antineoplastic drugs contained in injectable pharmaceutical formulations. A capillary zone electrophoresis (CZE) method coupled to UV was developed with a background electrolyte (BGE) made of a 100 mM phosphate buffer at pH 2.5 containing 50% (v/v) of acetonitrile and dynamic coating of capillaries with Ceofix®. This method allowed the analysis of doxorubicin, epirubicin, idarubicin, daunorubicin, irinotecan, topotecan, vincristine, vindesine, vinblastine, and vinorelbine in less than 8 min. A micellar electrokinetic chromatography (MEKC) method coupled to UV was also developed for the determination of methotrexate, pemetrexed, etoposide, etoposide phosphate, fludarabine phosphate and 5-fluorouracil. A run time of 16 min was obtained with a BGE made of 50 mM borate buffer at pH 9.2 with 80 mM of sodium dodecyl sulfate (SDS) and 20% (v/v) of acetonitrile. For both methods, the applied voltage was 30 kV and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in fused silica capillaries with an internal diameter of 50 µm and a total length of 64.5 cm. Both methods were validated and trueness values between 99.4% and 101.3% were obtained with repeatability and intermediate precision values of 0.5-1.8% for all drugs. These methods were found appropriate for controlling injectable pharmaceutical formulations containing antineoplastic drugs and successfully applied in quality control.

International audience; ObjectiveTo define whether good manufacturing practice (GMP)-clinical grade adipose stem cell (ASC)-derived conditioned medium (CM) is as effective as GMP-ASC in modulating inflammatory and catabolic factors released by both osteoarthritis (OA) chondrocytes or synoviocytes.MethodsOA chondrocytes and synoviocytes were treated with ASC-CM or co-cultured with ASC. Inflammatory factors (IL6, CXCL1/GROα,CXCL8/IL8, CCL2/MCP-1, CCL3/MIP-1α and CCL5/RANTES) and proteinases, such as metalloproteinase (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS4, ADAMTS5) and their tissue metalloproteinase inhibitors (TIMP1, TIMP3) were evaluated by qRT-PCR or immunoassays. The involvement of prostaglandin E2 (PGE2) was also analyzed.ResultsMost ASC-CM ratios tested did not decrease IL6, CCL2/MCP-1, CCL3/MIP1-α, CCL5/RANTES on basal inflamed chondrocytes or synoviocytes in contrast to what we found using ASC in co-culture. CXCL8/IL8 and CXCL1/GROα were not decreased by ASC-CM on synoviocytes but were only partially reduced on chondrocytes. Moreover, ASC-CM was less efficient both on basal inflamed OA chondrocytes and synoviocytes in reducing proteinases, such as MMP13, ADAMTS4, ADAMTS5 and increasing TIMP1 and TIMP3 compared to ASC in co-culture. The different ratios of ASC-CM contain lower amounts of PGE2 which were not sufficient to reduce inflammatory factors.ConclusionsThese data show that ASC-CM has a limited ability to decrease inflammatory and proteinases factors produced by OA chondrocytes or synoviocytes. ASC-CM is not sufficient to recapitulate the beneficial effect demonstrated using ASC in co-culture with inflamed OA chondrocytes and synoviocytes and shows that their use in clinical trials is fundamental to counteract OA progression.

Adult stem cells have more restricted differentiation potential than embryonic stem cells (ESCs), but upon appropriate stimulation can differentiate into cells of different germ layers. Epigenetic factors, including DNA modifications, take a significant part in regulation of pluripotency and differentiation of ESCs. Less is known about the epigenetic regulation of these processes in adult stem cells. Gene expression profile and location of DNA modifications in adipose-derived stem cells (ADSCs) and their osteogenically differentiated lineages were analyzed using Agilent microarrays. Methylation-specific PCR and restriction-based quantitative PCR were applied for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) detection in selected loci. The level of DNA modifications in the POU5F1 locus was quantified with deep sequencing. Expression levels of selected genes were assayed by real-time PCR. ADSCs differentiation into osteogenic lineages involved marked changes in both 5mC and 5hmC profiles, but 5hmC changes were more abundant. 5mC losses and 5hmC gains were the main events observed during ADSCs differentiation, and were accompanied by increased expression of TET1 (P = 0.009). In ADSCs, POU5F1 was better expressed than NANOG or SOX2 (P ≤ 0.001). Both 5mC and 5hmC marks were present in the POU5F1 locus, but only hydroxymethylation of specific cytosine showed significant effect on the gene expression. In summary, the data of our study suggest significant involvement of changes in 5hmC profile during the differentiation of human adult stem cells.

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5–10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876)., The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/FP7-HEALTH-2009); REBORNE Project (GA: 241876)

Summary Objective To define if adipose mesenchymal stromal cell (ASC) treatment mediated switching of the pro-inflammatory profile of M1-like macrophages as a means to develop a tailored in vitro efficacy/potency test. Design We firstly performed immunohistochemical analysis of CD68, CD80 (M1-like) and CD206 (M2-like) macrophages in osteoarthritic (OA) synovial tissue. ASC were co-cultured in contact and in transwell with activated (GM-CSF + IFNγ)-M1 macrophages. We analyzed IL1β, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9, IL10, CD163 and CD206 by qRT-PCR or immunoassays. Prostaglandin E2 (PGE2) blocking experiments were performed using PGE2 receptor antagonist. Results In moderate grade OA synovium we did not always find a higher percentage of CD80 with respect to CD206. M1-like-activated macrophage factors IL1β, TNFα, IL6, MIP1α/CCL3, S100A8 and S100A9 were down-modulated both in contact and in transwell by ASC. However, in both systems ASC induced the typical M2-like macrophage markers IL10, CD163 and CD206. Activated-M1-like macrophages pre-treated with PGE2 receptor antagonist failed to decrease secretion of TNFα, IL6 and to increase that of IL10, CD163 and CD206 when co-cultured with ASC confirming a PGE2 specific role. Conclusions We demonstrated that ASC are responsible for the switching of activated-M1-like inflammatory macrophages to a M2-like phenotype, mainly through PGE2. This evidenced that activated-M1-like macrophages may represent a relevant cell model to test the efficacy/potency of ASC and suggests a specific role of ASC as important determinants in therapeutic dampening of synovial inflammation in OA.

International audience; BACKGROUND: Despite the use of closed system drug transfer devices (CSTD), residual contamination from antineoplastic drugs is still detected inside isolators. The aim of this study was to compare the decontamination level obtained using a CSTD + standard cleaning procedure with a CSTD + standard cleaning procedure + specific decontamination procedure. METHODS AND FINDINGS: A comparative and prospective study was carried out in a newly opened compounding unit. Compounding was performed with a CSTD (BD-Phaseal, Becton-Dickinson). In the Control isolator (C), the cleaning process was completed daily with a standard biocide solution (AnioxysprayTM, Anios, France). In the Intervention isolator (I), weekly decontamination with a homemade admixture of sodium dodecyl sulfate 10-2 M/70% isopropanol (80/20, v/v) was added. Monitoring was performed via a validated LC-MS/MS method. Eight drugs (cyclophosphamide, cytarabine, dacarbazine, fluorouracile, gemcitabine, ifosfamide, irinotecan and methotrexate) were monitored daily over 14 consecutive weeks on three sites inside the isolators: gloves, workbench and window. Results are presented as the odds-ratio (OR) of contamination and as overall decontamination efficiency (EffQ, %). The proportion of EffQ ≥ 90% was assessed by a Fisher's exact test (p

Background Osteoarthritis (OA) is the most common joint disease and the major cause of pain and disability in the aging population [1]. Adipose stromal cells (ASC) are promising candidate for cell therapy in OA, because they have immunomodulatory, trophic and differentiation capacities [2,3]. Synovial inflammation is accepted as important OA feature for the symptoms and disease progression [4]. Synovial tissue is mainly composed of synovial fibroblasts (SF), macrophages (SM) and a low percentage of other cell types [5]. Objectives Aim of the study was to analyze the effects of adipose stromal cells in co-culture with SF and SM. Methods GMP clinical grade ASC were isolated from subcutaneous adipose tissue. Synoviocytes were isolated from synovial tissue of OA patients undergoing total joint replacement. Synovial cells at passage 1 and 5 were analyzed for: 1. different phenotypical markers by flow cytometric analysis, 2. inflammatory factors by multiplex immunoassay, 3.anabolic and degradative factors by qRT-PCR. Both p.1 (mix of SF and SM) and p.5 (only SF) synovial cells, as different cell models, were co-cultured with adipose stem cells (ASC) to define their effects. Furthermore macrophages type 1 (M1) were isolated and co-cultured with ASC. Results Synoviocytes at passage 1 were positive to typical markers of SM (CD14,CD16,CD68, CD80,CD163) and SF (CD55,CD73,CD90,CD105,CD106), whereas at passage 5 were positive only to SF markers and showed a higher percentage of CD55 and CD106. At p.1 synovial cells released a significantly higher amount of all inflammatory (IL6,CXCL8,CCL2,CCL3,CCL5) and anabolic (IL10) factors than those at p.5. Moreover, p.1 synovial cells expressed also higher amount of some degradative factors (MMP13, S100A8, S100A9) than p.5 synovial cells. Co-culture experiments showed that the amount of SM in p.1 synovial cells specifically orchestrate the up or down-modulation of some inflammatory (IL6,CXCL8,CCL2,CCL3,CCL5) and degradative factors (ADAMTS5,MMP13, S100A8,S100A9) analyzed. Interestingly, p.5 synovial cells induced all factors analyzed, except CCL5. Finally, we demonstrated that ASC effects were strictly dependent by M1, that decreased the release of typical macrophages cytokines (IL1β, IL6, TNFα and CCL3/MIP1α) and that ASC effects are responsible for the switching by M1 like inflammatory macrophages to M2 like phenotype mainly due to PGE2 involvement. Conclusions These data demonstrate that the GMP-ASC effects on OA synovial inflammation are strictly dependent by macrophages, that orchestrate the switching activated-M1 inflammatory macrophages to a M2-like phenotype, mainly through PGE2 References Loeser RF (2012) Arthritis Rheum 64:1697–707. C. Manferdini (2013) Arthritis Rheum 65:1271–81. M. Maumus (2013) Stem Cell Research 11, 834–844. Y. Henrotin (2014) Semin Arthritis Rheum 43(5):579–87. MD. Smith (2011) The Open Rheumatology Journal 5:100–6. Acknowledgements This research was supported by Horizon 2020 Programme (project ADIPOA2, grant agreement no: 643809). The materials presented and views expressed here are the responsibility of the authors only. The EU Commission takes no responsibility for any use made of the information set out. Disclosure of Interest None declared

Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics. ADCs show unprecedented intrinsic heterogeneity and address new quality attributes which have to be assessed. Liquid chromatography is one of the most frequently used analytical method for the characterization of ADCs. This review summarizes recent results in the chromatographic characterization of ADCs and supposed to provide a general overview on the possibilities and limitations of current approaches for the evaluation of drug load distribution, determination of average drug to antibody ratio (DARav), and for the analysis of process/storage related impurities. Hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC) and multidimensional separations are discussed focusing on the analysis of marketed ADCs. Fundamentals and aspects of method development are illustrated with applications for each technique. Future perspectives in hydrophilic interaction chromatography (HILIC), HIC, SEC and ion exchange chromatography (IEX) are also discussed.

Background Busulfan is an alkylating antineoplastic drug extensively used in myeloablative regimens prior to haematopoietic stem cell transplantation. Administration every 6 hours for 4 consecutive days and the low stability of this product induces organisational problems for cytotoxic preparation units in hospital pharmacies. A few studies have already been conducted to investigate the stability of diluted busulfan solutions but published results showed important variability. Purpose The purpose of this study was to evaluate the stability of busulfan solutions diluted to 0.54 mg/mL with 0.9% sodium chloride and stored in polypropylene (PP) syringes or PP infusion bags with a highly sensitive, specific and rapid ( Material and methods Busulfan solutions (0.54 mg/mL) were prepared from three Busilvex batch numbers and transferred into 50 mL PP syringes (n=12), or 100 mL (n=12) or 500 mL (n=4) PP infusion bags (stored at 2–8°C or 23–27°C). The chemical stability of busulfan was assessed by measuring in each container the percentage of the initial concentration remaining at each time point of analysis (max 100 hours) with an ultra high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) method. Busulfan and [ 2 H 8 ]-busulfan (internal standard) were detected in selected ion recording mode as ammonium adducts at m/z: 264.1 and 272.1, respectively. Stability was defined as retention of at least 90% of the initial busulfan concentration. Physical stability was assessed by visual inspection. Results Busulfan in PP syringes proved to be stable for 30 hours at 2–8°C and 12 hours at 23–27°C. A reduced stability of 12 hours and 9 hours were obtained at 2–8°C for 100 mL and 500 mL PP bags, respectively. At 23–27°C, all PP bags presented stability for 3 hours. The main effects observed were hydrolysis at 23–27°C and precipitation at 2–8°C. Conclusion This study demonstrated that the stability of busulfan diluted solutions depends on the container type and storage temperature. Stability for 30 hours in PP syringes stored at 2–8°C is a considerable improvement compared with the manufacturer’s recommendations (12 hours). The extended shelf life of busulfan infusion in syringes allows better organisation of the chemotherapy preparation unit by reducing the number of busulfan production to once a day. No conflict of interest

Adipose tissue represents an abundant source of stem cells. Along with anti-inflammatory effects, ASC secrete various factors that may modulate metabolism of extracellular matrix in osteoarthritic (OA) cartilage, suggesting that the presence of ASC could be advantageous for OA cartilage due to the recovery of homeostasis between matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs). To evaluate these effects, cartilage explants (CE) were cocultured with ASC for 3 and 7 days under stimulation with or without IL-1β.The pattern of gene expression in CE was modified by ASC, including the upregulation ofCOL1A1andCOL3A1and the downregulation ofMMP13andCOL10A1. The production of MMP-1, MMP-3, and MMP-13 by ASC was not significant; moreover, cocultures with ASC reduced MMP-13 production in CE. In conclusion, active production of TIMP-1, TIMP-2, TIMP-3, IL-6, IL-8, and gelatinases MMP-2 and MMP-9 by ASC may be involved in the extracellular matrix remodelling, as indicated by the altered expression of collagens, the downregulated production of MMP-13, and the reduced chondrocyte apoptosis in the cocultured CE. These data suggest that ASC modulated homeostasis of MMPs/TIMPs in degenerated OA cartilage in vitro and might be favourable in case of the intra-articular application of ASC therapy for the treatment of OA.

Purpose Ganciclovir is increasingly provided by hospital pharmacy production unit in a ready-to-use form, in order to improve the safety of healthcare workers and the efficiency of the organisation. The objective of this study was to develop a stability-indicating method to assay ganciclovir and determine the stability of ganciclovir in syringes (5 mg/mL) and infusion bags (0.25 and 5 mg/mL) at two different temperatures. Method Ganciclovir solutions (0.25 mg/mL and 5 mg/mL) in 0.9% sodium chloride were prepared in 50 mL polypropylene syringes or 100 mL polypropylene infusion bags and stored at 2–8℃ and 23–27℃. The chemical stability was measured using a stability-indicating Ultra High Performance Liquid Chromatography coupled to mass spectrometry method. Physical stability was assessed by visual inspection. Results No significant loss of ganciclovir under any of the tested conditions was observed in this study. All solutions remained clear through the study period. Conclusion All tested formulations remained stable for at least 185 days independently of container type, temperature or concentration studied.

A large amount of residues from active pharmaceutical ingredients (API) that are currently in use are known to reach aquatic ecosystems and have potentially adverse effects on living organisms. Prioritization methods are useful tools for both regulation and surveillance purposes in the environmental policy of APIs. Their use has largely increased over the last decade, and the different existing methodologies can lead to large discrepancies between the highlighted substances. This chapter aims at discussing studies conducted in the context of hospitals. Perhaps more important than the results themselves, the methodologies with the set of selected criteria are discussed, as well as their advantages and associated uncertainties. A case study of API prioritization applied to a Swiss university hospital is presented with two different approaches: a ranking-based OPBT approach (Occurrence, Persistence, Bioaccumulation, and Toxicity) and an environmental risk assessment (ERA), with the calculation of risk quotient (RQ). The ERA results combined with those of other studies dealing with ERA-based API prioritization in hospitals highlighted several compounds presenting high risks for the aquatic ecosystems (RQ > 1): antibiotics (ciprofloxacin, amoxicillin, piperacillin, azithromycin), anti-inflammatory drugs (diclofenac, mesalazine), as well as the hormone estradiol and the antidiabetic metformin. Nevertheless, only the antibiotic ciprofloxacin was commonly determined as problematic. Finally, the most critical issues for API prioritization in hospitals were identified from the literature overview and the results of the presented case study: handling of the consumption data, involvement of expert judgment, uncertainties linked with the predicted environmental concentration (PEC) calculation, and quality of the hazard evaluation. Although prioritization procedures applied to hospitals can be burdensome to apply in practice and many associated uncertainties remain, they represent essential tools to establish lists of priority molecules to follow via monitoring programs and allow their theoretical risk assessment.

The number of patients suffering from cancer is constantly increasing and, consequently, the number of different chemotherapy treatments administered is increasing. Given the high reactivity and toxicity of antineoplastic drugs, analytical methods are required in all pharmaceutical fields, from drug development to their elimination in wastewater; including formulation quality control, environment and human exposure and therapeutic drug monitoring. The aim of this paper is to provide an overview of the analytical methods available for the determination of antineoplastic drugs in different matrices such as pharmaceutical formulations, biological and environmental samples. The applicability and performance of the reported methods will be critically discussed, with focus on the most commonly used antineoplastic drugs. Only conventional compounds and small molecules for targeted therapy will be considered in the present review.

Non-revascularizable critical limb ischemia (CLI) is the most severe stage of peripheral arterial disease, with no therapeutic option. Extensive preclinical studies have demonstrated that adipose-derived stroma cell (ASC) transplantation strongly improves revascularization and tissue perfusion in ischemic limbs. This study, named ACellDREAM, is the first phase I trial to evaluate the feasibility and safety of intramuscular injections of autologous ASC in non-revascularizable CLI patients.Seven patients were consecutively enrolled, on the basis of the following criteria: (i) lower-limb rest pain or ulcer; (ii) ankle systolic oxygen pressure 50 or 70 mm Hg for non-diabetic and diabetic patients, respectively, or first-toe systolic oxygen pressure 30 mm Hg or 50 mm Hg for non-diabetic and diabetic patients, respectively; (iii) not suitable for revascularization. ASCs from abdominal fat were grown for 2 weeks and were then characterized.More than 200 million cells were obtained, with almost total homogeneity and no karyotype abnormality. The expressions of stemness markers Oct4 and Nanog were very low, whereas expression of telomerase was undetectable in human ASCs compared with human embryonic stem cells. ASCs (10(8)) were then intramuscularly injected into the ischemic leg of patients, with no complication, as judged by an independent committee. Trans-cutaneous oxygen pressure tended to increase in most patients. Ulcer evolution and wound healing showed improvement.These data demonstrate the feasibility and safety of autologous ASC transplantation in patients with objectively proven CLI not suitable for revascularization. The improved wound healing also supports a putative functional efficiency.

Objectives To improve the safety of the preparation and its availability in ophthalmology, a ready-to-use cefuroxime syringe prepared under aseptic conditions according to good manufacturing practice was developed by the hospital pharmacy. Methods The chemical stability of cefuroxime solution at 10 mg/mL in 0.9% sodium chloride in syringes (0.5 mL) was achieved at −18°C by a capillary electrophoresis method. Syringes of the last acceptable time were then stored at 4°C and the study was continued for one additional month. The pH was measured throughout the study and the presence of particle matter was controlled. Sterility testing was performed to test the integrity of the syringes. Results An optimal period of 4 months at −18°C was defined for storage of syringes of cefuroxime without significant loss in potency and an acceptable rate (4%) of degradation products. After thawing and storage at 4°C, degradation products progressively increased. As these derivatives can represent up to 10% of cefuroxime in terms of peak area after 1 month, it is recommended that the syringes are used within 1 h after thawing. The pH did not change appreciably during the study and the sterility testing results were negative. Moreover, in all cases the syringes fulfilled all the European Pharmacopoeia criteria with regard to non-visible particles. Conclusions The availability of cefuroxime for use in ophthalmology is improved as ready-to-use syringes can be stored in the ward freezer and administered immediately after thawing.

Introduction The external contamination and cross-contamination by cytotoxic drugs on the surface (outside and septum) of 133 vials from various manufacturers and available on the Swiss market were evaluated. All of the tested vials contained one of the following active ingredients: cyclophosphamide, cytarabine, doxorubicin, epirubicin, etoposide phosphate, gemcitabine, ifosfamide, irinotecan, methotrexate or vincristine. Methods and materials The validated wiping liquid chromatography-mass spectrometry method used in this study allowed for the simultaneous determination of these 10 cytotoxic drugs in less than 30 min. Results External contamination by cytotoxic drugs was detected on 63% of tested vials (outside and septum). The highest contamination level was observed on etoposide phosphate vials with 1896.66 ng of active ingredient on the outside of the vial. Approximately 20% of the contaminated vials had greater than 10 ng of cytotoxic drugs. Chemical contamination on the septum was detected on 38% of the vials. No contamination or very low levels of cytotoxic drugs, less than 1 ng per vial, were detected on the vials protected by plastic shrink-wrap. Traces of cytotoxic drugs different from the active ingredient were detected on 35% of the tested vials. Conclusion Handling cytotoxic vials with gloves and having a procedure for the decontamination of vials are of the utmost importance for reducing exposure to cytotoxic drugs. Moreover, manufacturers must improve their procedures to provide products free from any contamination.

Background Setting up an automated compounder device (ACD) takes time and money. Daily production of paediatric parenteral nutrition (PN) solutions in our pharmacy decreased after implementing standard PN for neonatology. To keep the production of a limited number of PN bags cost effective, we wished to set up the ACD weekly rather than daily. Objectives This study aims to validate (operational and performance qualification (PQ)) use of an ACD (Baxa MM12) for once-a-day and once-a-week set up. Methods Accuracy and precision of the ACD were evaluated using water (operational qualification) on a daily basis, and using the products for preparing PN solutions (PQ) on a daily and a weekly basis. Nutrient dosage was measured for specific PN solutions after daily or weekly ACD set ups. Particle contamination of PN solutions produced were controlled throughout the week. To validate the aseptic process, media-fill tests of the once-a-week set ups were carried out over three consecutive weeks. Results Accuracy and precision of the ACD were validated for small (0.5 ml) and high (100 ml) volumes. Precision improved when pumped volumes increased. Nutrient dosage in PN solutions following daily and weekly set ups were always in conformity with specification limits. No particles or microbiological contaminations were detected following the week-long set ups. Conclusions Validation proved good accuracy, precision and aseptic conditions existed throughout each week. PN solutions can be produced safely all week long with one set up. A week-long set up saves technician time and money.

Objective To examine the effect of different sources of Good Manufacturing Practice clinical grade adipose-derived mesenchymal stem cells (AD-MSCs) on inflammatory factors in osteoarthritic (OA) chondrocytes and synoviocytes. Methods AD-MSCs from infrapatellar Hoffa fat, subcutaneous (SC) hip fat, and SC abdominal fat were cocultured in Transwells with chondrocytes or synoviocytes. Inflammatory factors (interleukin-1β [IL-1β], tumor necrosis factor α, IL-6, CXCL1/growth-related oncogene α, CXCL8/IL-8, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α, and CCL5/RANTES) were evaluated by quantitative reverse transcription–polymerase chain reaction or multiplex bead–based immunoassay. The role of different immunomodulators was analyzed. Results All the inflammatory factors analyzed were down-modulated at the messenger RNA or protein level independently by all 3 AD-MSC sources or by allogeneic AD-MSCs used in coculture with chondrocytes or synoviocytes. Inflammatory factor down-modulation was observed only when AD-MSCs were cocultured with chondrocytes or synoviocytes that produced high levels of inflammatory factors, but no effect was observed in cells that produced low levels of those factors, thus highlighting a dependence of the AD-MSC effect on existing inflammation. The immunomodulators IL-10, IL-1 receptor antagonist, fibroblast growth factor 2, indoleamine 2,3-dioxygenase 1, and galectin 1 were not involved in AD-MSC effects, whereas the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway exerted a role in the mechanism of antiinflammatory AD-MSC action. Conclusion The antiinflammatory effects of AD-MSCs are probably not dependent on AD-MSC adipose tissue sources and donors but rather on the inflammatory status of OA chondrocytes and synoviocytes. AD-MSCs seem to be able to sense and respond to the local environment. Even though a combination of different molecules may be involved in AD-MSC effects, the COX-2/PGE2 pathway may play a role, suggesting that AD-MSCs may be useful for therapies in osteoarticular diseases.

Purpose The results of a study of the accuracy of i.v. medication preparation by anesthesiologists are presented. Methods The accuracy of syringe preparation was assessed by analyzing the contents of 500 unused syringes collected after adult and pediatric surgery procedures. The collected syringes contained various i.v. medication formulations representative of different preparation techniques: atracurium 1, 2.5, and 5 μg/mL and fentanyl 10, 20, 25, and 50 μg/mL, which required serial dilution after withdrawal of the drugs from ampuls; thiopental 5, 25, and 50 mg/mL, prepared by diluting reconstituted powdered drug from vials; and lidocaine 10-mg/mL solution, which was withdrawn directly from the ampul into a syringe. Variances between actual and labeled drug concentrations were determined via a validated ultraviolet–visible light spectro-photometry method. Results Overall, 29% of the evaluated syringes were found to contain drug concentrations outside the designated range of acceptability (±10% of the targeted concentration); 18% of preparations deviated from the declared dose by ±20%, 8% deviated by ±50%, and 4% deviated by ±100%. In one instance, the actual drug concentration was at variance with the labeled concentration by >100%. In 4% of cases ( n = 20), discrepancies exceeded 100%, suggesting not just imprecision but errors in the preparation process, such as incorrect dilution calculations and selection of the wrong medication vial by the syringe preparer. Conclusion Analysis of different i.v. formulations of four medications prepared in syringes by anesthesiologists revealed a high rate of discrepancies between ordered and actual drug concentrations, suggesting a need for increased institutional efforts to prevent errors during the preparation process. Am J Health-Syst Pharm. 2013; 70:137–42

Background The aim of the study was to characterize synovial cells from OA synovium with low-grade and moderate-grade synovitis and to define the role of synovial macrophages in cell culture. Methods Synovial tissue explants were analyzed for the expression of typical markers of synovial fibroblasts (SF), synovial macrophages (SM) and endothelial cells. Synovial cells at passage 1 (p.1) and 5 (p.5) were analyzed for different phenotypical markers by flow cytometric analysis, inflammatory factors by multiplex immunoassay, anabolic and degradative factors by qRT-PCR. P.1 and p.5 synovial cells as different cell models were co-cultured with adipose stem cells (ASC) to define SM effects. Results Synovial tissue showed a higher percentage of CD68 marker in moderate compared with low-grade synovitis. Isolated synovial cells at p.1 were positive to typical markers of SM (CD14, CD16, CD68, CD80 and CD163) and SF (CD55, CD73, CD90, CD105, CD106), whereas p.5 synovial cells were positive only to SF markers and showed a higher percentage of CD55 and CD106. At p.1 synovial cells released a significantly higher amount of all inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and some anabolic (IL10) factors than those of p.5. Moreover, p.1 synovial cells also expressed a higher amount of some degradative factors (MMP13, S100A8, S100A9) than p.5 synovial cells. Co-culture experiments showed that the amount of SM in p.1 synovial cells differently induced or down-modulated some of the inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and degradative factors (ADAMTS5, MMP13, S100A8, S100A9). Conclusions We found that p.1 (mix of SM and SF) and p.5 (only SF) synovial cells represent two cell models that effectively reproduce the low- or moderate-grade synovitis environment. The presence of SM in culture specifically induces the modulation of the different factors analyzed, confirming that SM are key effector cells. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0983-4) contains supplementary material, which is available to authorized users.

Background aims The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL). Methods Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied. Results PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC. Conclusions PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

Background and objectives Centralized chemotherapy preparation units have established systematic strategies to avoid errors. Our work aimed to evaluate the accuracy of manual preparations associated with different control methods. Method A simulation study in an operational setting used phenylephrine and lidocaine as markers. Each operator prepared syringes that were controlled using a different method during each of three sessions (no control, visual double-checking, and gravimetric control). Eight reconstitutions and dilutions were prepared in each session, with variable doses and volumes, using different concentrations of stock solutions. Results were analyzed according to qualitative (choice of stock solution) and quantitative criteria (accurate, 30% deviation). Results Eleven operators carried out 19 sessions. No final preparation (n = 438) contained a wrong drug. The protocol involving no control failed to detect 1 of 3 dose errors made and double-checking failed to detect 3 of 7 dose errors. The gravimetric control method detected all 5 out of 5 dose errors. The accuracy of the doses measured was equivalent across the control methods ( p = 0.63 Kruskal–Wallis). The final preparations ranged from 58% to 60% accurate, 25% to 27% weakly accurate, 14% to 17% inaccurate and 0.9% wrong. A high variability was observed between operators. Discussion Gravimetric control was the only method able to detect all dose errors, but it did not improve dose accuracy. A dose accuracy with

In the last decades, the number of patients receiving chemotherapy has considerably increased. Given the toxicity of cytotoxic agents to humans (not only for patients but also for healthcare professionals), the development of reliable analytical methods to analyse these compounds became necessary. From the discovery of new substances to patient administration, all pharmaceutical fields are concerned with the analysis of cytotoxic drugs. In this review, the use of methods to analyse cytotoxic agents in various matrices, such as pharmaceutical formulations and biological and environmental samples, is discussed. Thus, an overview of reported analytical methods for the determination of the most commonly used anticancer drugs is given.

A simple method based on capillary electrophoresis with a capacitively coupled contactless conductivity detector (CE-C(4)D) was developed for the determination of potassium, sodium, calcium and magnesium in parenteral nutrition formulations. A hydro-organic mixture, consisting of 100 mM Tris-acetate buffer at pH 4.5 and acetonitrile (80:20, v/v), was selected as the background electrolyte. The applied voltage was 30 kV, and sample injection was performed in hydrodynamic mode. All analyses were carried out in a fused silica capillary with an internal diameter of 50 microm and a total length of 64.5 cm. Under these conditions, complete separation between all cations was achieved in less than 4 min. The CE-C(4)D method was validated, and trueness values between 98.6% and 101.8% were obtained with repeatability and intermediate precision values of 0.4-1.3% and 0.8-1.8%, respectively. Therefore, this method was found to be appropriate for controlling potassium, sodium, calcium and magnesium in parenteral nutrition formulations and successfully applied in daily quality control at the Geneva University Hospitals.

The physiological functions of human TCRVgamma9Vdelta2(+) gammadelta lymphocytes reactive to non-peptide phosphoantigens contribute to cancer immunosurveillance and immunotherapy. However, their regulation by mesenchymal stem cells (MSC), multipotent and immunomodulatory progenitor cells able to infiltrate tumors, has not been investigated so far. By analyzing freshly isolated TCRVgamma9Vdelta2(+) lymphocytes and primary cell lines stimulated with synthetic phosphoantigen or B-cell lymphoma cell lines in the presence of MSC, we demonstrated that MSC were potent suppressors of gammadelta-cell proliferation, cytokine production and cytolytic responses in vitro. This inhibition was mediated by the COX-2-dependent production of prostaglandin E2 (PGE(2)) and by MSC through EP2 and EP4 inhibitory receptors expressed by Vgamma9Vdelta2 T lymphocytes. COX-2 expression and PGE(2) production by MSC were not constitutive, but were induced by IFN-gamma and TNF-alpha secreted by activated Vgamma9Vdelta2 T cells. This regulatory cross-talk between MSC and Vgamma9Vdelta2 T lymphocytes involving PGE(2) could be of importance for the antitumor and antimicrobial activities of gammadelta T cells.

SUMMARY: Since the pioneering work of Alexander Friedenstein on multipotent mesenchymal stromal cells (MSCs), a tremendous amount of work has been done to isolate, characterize and culture such cells. Assay of colony forming unit-fibroblasts (CFU-Fs), the hallmark of MSCs, is used to estimate their frequency in tissue. MSCs are adherent cells, so they are easy to isolate, and they show contact inhibition. Thus, several parameters must be taken into account for culture: cell density, number of passages, culture medium, and growth factors used. The purity of the initial material is not a limiting parameter. Similar but not identical cell populations are found in almost all mammal or human tissues. MSCs seem to be very abundant in adipose tissue but at low frequency in blood from umbilical cord or in adult tissue. The culture conditions are very similar, whatever the source of cells. Because of their favorable properties, MSCs are very promising tools for regenerative medicine.

International audience; Recent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal gamopathy of undefined significance (MGUS) and healthy age-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in an MM group. MGUS BMMSCs were interspersed between these two groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs, 46% may account for a tumor-microenvironment cross-talk. Known soluble factors implicated in MM pathophysiologic features (i.e. IL (interleukin)-6, DKK1) were revealed and new ones were found which are involved in angiogenesis, osteogenic differentiation or tumor growth. In particular, GDF15 was found to induce dose-dependent growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an overgrowth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, MM BMMSCs are abnormal and could create a very efficient niche to support the survival and proliferation of the myeloma cells.

Osteoarthritis (OA) is the most widespread musculoskeletal disorder in adults. It leads to cartilage damage associated with subchondral bone changes and synovial inflammation, causing pain and disability. The present study aimed at evaluating the safety of a dose-escalation protocol of intra-articular injected adipose-derived stromal cells (ASCs) in patients with knee OA, as well as clinical efficacy as secondary endpoint. A bicentric, uncontrolled, open phase I clinical trial was conducted in France and Germany with regulatory agency approval for ASC expansion procedure in both countries. From April 2012 to December 2013, 18 consecutive patients with symptomatic and severe knee OA were treated with a single intra-articular injection of autologous ASCs. The study design consisted of three consecutive cohorts (six patients each) with dose escalation: low dose (2 × 106 cells), medium dose (10 × 106), and high dose (50 × 106). The primary outcome parameter was safety evaluated by recording adverse events throughout the trial, and secondary parameters were pain and function subscales of the Western Ontario and McMaster Universities Arthritis Index. After 6 months of follow-up, the procedure was found to be safe, and no serious adverse events were reported. Four patients experienced transient knee joint pain and swelling after local injection. Interestingly, patients treated with low-dose ASCs experienced significant improvements in pain levels and function compared with baseline. Our data suggest that the intra-articular injection of ASCs is a safe therapeutic alternative to treat severe knee OA patients. A placebo-controlled double-blind phase IIb study is being initiated to assess clinical and structural efficacy. Significance Although this phase I study included a limited number of patients without a placebo arm, it showed that local injection of autologous adipose-derived stem cells was safe and well tolerated in patients with knee osteoarthritis. This study also provides encouraging preliminary evidence of efficacy. Larger and controlled long-term studies are now mandatory to confirm whether this new strategy of cell therapy can improve pain and induce structural benefit in osteoarthritis.