Occupational exposure to antineoplastic drugs: what about hospital sanitation personnel?

OBJECTIVE: Occupational exposure to antineoplastic drugs (ANPs) occurs mainly through dermal contact. Our study was set up to assess the potential exposure of hospital sanitation (HS) personnel, for whom almost no data are available, through contamination of surfaces they regularly touch. METHODS: In the oncology departments of two hospitals around Montreal, surface wipe samples of 120-2000 cm2 were taken at 10 sites cleaned by the HS personnel and five other sites frequently touched by nursing and pharmacy personnel. A few hand wipe samples were collected to explore skin contamination. Wipes were analyzed by ultra-performance liquid chromatography tandem-mass spectrometry for 10 ANPs. RESULTS: Overall, 60.9% of 212 surface samples presented at least one ANP above the limits of detection (LOD). Cyclophosphamide and gemcitabine were most often detected (52% and 31% of samples respectively), followed by 5-fluorouracil and irinotecan (15% each). Highest concentrations of five ANPs were found in outpatient clinics on toilet floors (5-fluorouracil, 49 ng/cm2; irinotecan, 3.6 ng/cm2), a perfusion pump (cyclophosphamide, 19.6 ng/cm2) and on a cytotoxic waste bin cover (gemcitabine, 4.97 ng/cm2). Floors in patient rooms had highest levels of cytarabine (0.12 ng/cm2) and methotrexate (6.38 ng/cm2). Hand wipes were positive for two of 12 samples taken on HS personnel, seven of 18 samples on nurses, and two of 14 samples on pharmacy personnel. CONCLUSIONS: A notable proportion of surfaces showed measurable levels of ANPs, with highest concentrations found on surfaces cleaned by HS personnel, who would benefit from appropriate preventive training. As potential sources of worker exposure, several hospital surfaces need to be regularly monitored to evaluate environmental contamination and efficacy of cleaning.

Cross-Sectional Evaluation of Surface Contamination with Antineoplastic Drugs in Canadian Health Care Centres.

BACKGROUND: Surfaces in health care centres are often contaminated with traces of antineoplastic drugs. Such contamination should be limited as much as possible, to reduce workers' exposure. OBJECTIVES: The primary objective was to monitor environmental contamination with 9 antineoplastic drugs in oncology pharmacy and patient care areas of Canadian health care centres. The secondary objective was to explore the use of sodium hypochlorite as a cleaning agent for cyclophosphamide contamination. METHODS: This cross-sectional evaluation was conducted from January to April 2018. Twelve standardized sites were sampled at each participating centre: 6 in the oncology pharmacy and 6 in patient care areas. Six of the antineoplastic drugs (cyclophosphamide, ifosfamide, methotrexate, gemcitabine, 5-fluorouracil, and irinotecan) were quantified by ultra-performance liquid chromatography - tandem mass spectrometry. For the other 3 antineoplastic drugs (docetaxel, paclitaxel, and vinorelbine), samples were screened for contamination but not quantified. The effect of using sodium hypochlorite as a cleaning agent was evaluated with a Kolmogorov-Smirnov test for independent samples. RESULTS: Of 202 Canadian centres invited, 79 participated. A total of 887 surface samples were analyzed, 467 from pharmacy areas and 420 from patient care areas. Cyclophosphamide was the drug most often found as a contaminant (32.2% [286/887] of samples positive, 75th percentile of measured contamination 0.0017 ng/cm2, 90th percentile 0.021 ng/cm2). The front grille inside the hood (80.8% [63/78] of samples positive for at least one antineoplastic drug), treatment chair armrest (78.9% [60/76]), storage shelf in pharmacy (61.5% [48/78]), and floor in front of the hood (60.3% [47/78]) were the most frequently contaminated surfaces. Cleaning with a sodium hypochlorite solution was highly variable. Among centres that reported using sodium hypochlorite to clean armrests on patient chairs, the concentration of cyclophosphamide was lower (0.00866 versus 0.0300 ng/cm2, p = 0.014). CONCLUSIONS: Despite growing awareness and implementation of new safe-handling guidelines, surfaces in health care centres were contaminated with traces of many antineoplastic drugs. Providing centres with attainable goals (e.g., 75th to 90th percentile relative to other similar centres) would help in identifying the sampling sites where improvements are needed and in achieving lower surface contamination.

CONTEXTE: Les surfaces dans les centres de santé sont souvent contaminées par des traces de médicaments antinéoplasiques. Une telle contamination devrait être limitée autant que faire se peut afin de réduire l'exposition des employés à ces produits. OBJECTIFS: L'objectif principal consistait à mesurer la contamination environnementale provenant de neuf médicaments antinéoplasiques dans la section de la pharmacie oncologique et celle des soins offerts aux patients dans des centres de soins de santé canadiens. L'objectif secondaire consistait à explorer l'action nettoyante de l'hypochlorite de sodium pour éliminer la contamination par la cyclophosphamide. MÉTHODES: Cette évaluation transversale a été menée de janvier à avril 2018. Des échantillons ont été prélevés dans douze endroits standardisés de chaque centre participant : six dans la section de la pharmacie oncologique et six dans celle des soins donnés aux patients. La présence de six des médicaments antinéoplasiques examinés (cyclophosphamide, ifosfamide, méthotrexate, gemcitabine, 5-fluorouracil et irinotécan) a été quantifiée par chromatographie liquide à haute performance (HPLC) avec spectrométrie de masse en tandem. Quant aux trois autres échantillons de médicaments antinéoplasiques (docetaxel, paclitaxel et vinorelbine), ils ont été analysés pour rechercher la présence d'une contamination qui n'a pas été quantifiée. L'action nettoyante de l'hypochlorite de sodium a été évaluée à l'aide d'un test de Kolmogorov-Smirnov pour les échantillons indépendants. RÉSULTATS: Sur 202 centres canadiens invités à participer à l'étude, 79 ont répondu à l'invitation. L'analyse a porté sur 887 échantillons de surfaces des lieux sélectionnés : 467 dans la section de la pharmacie et 420 dans la section des soins donnés aux patients. La cyclophosphamide était le médicament contaminant le plus souvent décelé (32,2 % d'échantillons positifs [286/887], 75e percentile de contamination mesurée 0,0017 ng/cm2, 90e percentile 0,021 ng/cm2). La grille frontale à l'intérieur de la hotte de laboratoire (80,8 % des échantillons [63/78] étaient positifs pour au moins un médicament antinéoplasique), l'accoudoir de la chaise du patient (78,9 % [60/76]), l'étagère de stockage dans la pharmacie (61,5 % [48/78]) et le sol en face de la hotte (60,3% [47/78]) étaient les surfaces le plus souvent contaminées. L'usage d'une solution d'hypochlorite de sodium pour le nettoyage variait grandement d'un centre à l'autre. Dans les centres qui indiquaient utiliser cet agent pour nettoyer les accoudoirs des chaises du patient, la concentration de cyclophosphamide sur les accoudoirs était moins élevée (0,00866 contre 0,0300 ng/cm2, p = 0,014). CONCLUSIONS: Malgré la prise de conscience et la mise en place croissantes de nouvelles lignes directrices en matière de manipulation sécuritaire, les surfaces de certains endroits des centres de santé sont contaminées par des traces de nombreux médicaments antinéoplasiques. La fixation d'objectifs atteignables pour les centres (p. ex., entre le 75e et le 90e percentile par rapport aux autres centres similaires) aide à déterminer les sites d'échantillonnage où des améliorations sont nécessaires et à diminuer la contamination des surfaces.

Collaborative Method Performance Study of the Measurement of Nicotine, Its Metabolites, and Total Nicotine Equivalents in Human Urine.

Background: Biomarkers of tobacco exposure have a central role in studies of tobacco use and nicotine intake. The most significant exposure markers are nicotine itself and its metabolites in urine. Therefore, it is important to evaluate the performance of laboratories conducting these biomarker measurements.Methods: This report presents the results from a method performance study involving 11 laboratories from 6 countries that are currently active in this area. Each laboratory assayed blind replicates of seven human urine pools at various concentrations on three separate days. The samples included five pools blended from smoker and nonsmoker urine sources, and two additional blank urine samples fortified with pure nicotine, cotinine, and hydroxycotinine standards. All laboratories used their own methods, and all were based on some form of liquid chromatography/tandem mass spectrometry.Results: Overall, good agreement was found among the laboratories in this study. Intralaboratory precision was good, and in the fortified pools, the mean bias observed was < + 3.5% for nicotine, approximately 1.2% for hydroxycotinine, and less than 1% for cotinine (1 outlier excluded in each case). Both indirect and direct methods for analyzing the glucuronides gave comparable results.Conclusions: This evaluation indicates that the experienced laboratories participating in this study can produce reliable and comparable human urinary nicotine metabolic profiles in samples from people with significant recent exposure to nicotine.Impact: This work supports the reliability and agreement of an international group of established laboratories measuring nicotine and its metabolites in urine in support of nicotine exposure studies. Cancer Epidemiol Biomarkers Prev; 27(9); 1083-90. ©2018 AACR.

Exposure to tobacco smoke and validation of smoking status during pregnancy in the MIREC study.

Given that prenatal exposure to tobacco smoke can lead to increased risks of adverse health effects, having valid measures of exposure is important. In a Canadian cohort (n = 2000), maternal and infant biospecimens were analysed for cotinine. Sensitivity and specificity of self-reported active smoking status were estimated. Regression modelling was used to identify potential predictors of maternal and infant plasma cotinine in non-smoking women. During the first trimester, 60.6% of the women reported never smoking, 27.3% were former smokers, 6.1% had quit when they found out they were pregnant, 5.8% were smokers and 42% of the non-smokers reported exposure to secondhand smoke (SHS). Low detection of tobacco biomarkers in meconium limited its ability to identify exposure to SHS. The sensitivity and specificity for self-reported smoking during the 1st trimester were 85.37 and 99.45%, respectively. The lowest sensitivity was found in participants with the highest level of education and income, oldest women and those born outside Canada. Non-smoking women living in an apartment had 1.7 times higher odds of detectable plasma cotinine than those living in a single home after adjusting for other variables. Our results suggest that while self-reports are fairly accurate, they may be less so in populations with higher socio-economic status. This investigation underscores the need to consider the participant socio-economic characteristics and dwelling type when using questionnaires to estimate active and passive tobacco exposure.

Multicenter study of environmental contamination with cyclophosphamide, ifosfamide, and methotrexate in 48 Canadian hospitals.

Context Oncology workers are occupationally exposed to antineoplastic drugs. This exposure can induce adverse health effects. In order to reduce their exposure, contamination on surfaces should be kept as low as possible. Objectives To monitor environmental contamination with cyclophosphamide, ifosfamide, and methotrexate in oncology pharmacy and patient care areas in Canadian hospitals. To describe the impact of some factors that may limit contamination. Methods This is a descriptive study. Twelve standardized sites were sampled in each participating center (six in the pharmacy and six in patient care areas). Samples were analyzed for the presence of cyclophosphamide, ifosfamide, and methotrexate by ultra-performance liquid chromatography tandem mass spectrometry technology. Descriptive statistical analyses were done and results were compared with a Kolmogorov-Smirnov test for independent samples. Results In 2015, 48 hospitals participated in this study (48/202, 24%). Overall, 34% (181/525) of the samples were positive for cyclophosphamide, 8% (41/525) for ifosfamide, and 6% (31/525) for methotrexate. The 75th percentile value of cyclophosphamide surface concentration was 6.9 pg/cm2. For ifosfamide and methotrexate, they were lower than the limit of detection. Centers who prepared more antineoplastic drugs per year and centers who used more cyclophosphamide per year showed significantly higher surface contamination ( p < 0.0001). Over the years, we observed a reduction in surface contamination. Conclusion In comparison with other multicenter studies that were conducted in Canada, the concentration of antineoplastic drugs measured on surfaces is decreasing. Regular environmental monitoring is a good practice in order to maintain contamination as low as reasonably achievable.

Environmental Contamination with Cyclophosphamide, Ifosfamide, and Methotrexate: A Study of 51 Canadian Centres.

BACKGROUND: Occupational exposure to hazardous drugs may lead to adverse reproductive effects. There is no safe exposure limit for health care professionals. OBJECTIVES: To monitor levels of cyclophosphamide, ifosfamide, and methotrexate contamination in oncology pharmacy and patient care areas in Canadian health care institutions. METHODS: The study was conducted in 2014. Hospitals with at least 50 acute care beds were invited to participate. At each participating centre, 12 standardized sites (6 in pharmacy areas and 6 in patient care areas) were sampled. The samples were analyzed for the presence of cyclophosphamide, ifosfamide, and methotrexate by ultra-performance liquid chromatography tandem mass spectrometry technology. The limits of detection were 0.36 pg/cm(2) for cyclophosphamide, 0.95 pg/cm(2) for ifosfamide, and 0.97 pg/cm(2) for methotrexate. Descriptive statistical analyses were performed to determine the median, 75th percentile, and maximum levels. RESULTS: Fifty-one hospitals participated in this descriptive study, and a total of 584 samples were quantified. Overall, 294 (50%) of the samples were positive for cyclophosphamide, 125 (21%) for ifosfamide, and 54 (9%) for methotrexate. The most frequently contaminated sampling sites in pharmacy areas were the front grille inside the hood and the floor in front of the hood and, in patient care areas, the armrest and outpatient clinic counter. The 75th percentiles for surface concentration were 10.8 pg/cm(2) for cyclophosphamide, 1.59 pg/cm(2) for ifosfamide, and below the limit of detection for methotrexate. CONCLUSIONS: Relative to 3 other multicentre studies conducted in Quebec over the past few years, the proportion of positive samples remained constant. Nonetheless, the 75th percentile surface concentration of antineoplastic drugs has been decreasing and seems to have reached a plateau. Local (country-specific or region-specific) and attainable goals for surface contamination with hazardous drugs should be set annually, so long as no health-based limit is known.

CONTEXTE: L'exposition professionnelle à des médicaments dangereux peut causer des effets indésirables sur la reproduction. Aucune limite d'exposition sécuritaire n'est établie pour les professionnels de la santé. OBJECTIFS: Évaluer les taux de cyclophosphamide, d'ifosfamide et de méthotrexate dans la pharmacie d'oncologie et dans les unités de soins des établissements de santé canadiens. MÉTHODES: L'étude s'est déroulée en 2014. Les hôpitaux disposant d'au moins 50 lits de soins de courte durée ont été invités à participer. Dans chacun des établissements participants, des échantillons ont été prélevés dans 12 zones prédéterminées : 6 dans les pharmacies et 6 dans les unités de soins. On a ensuite analysé les échantillons par chromatographie liquide à très haute performance couplée à la spectrométrie de masse en tandem afin de détecter la présence de cyclophosphamide, d'ifosfamide et de méthotrexate. Le seuil de détection était de 0,36 pg/cm2 pour la cyclophosphamide, de 0,95 pg/cm2 pour l'ifosfamide et de 0,97 pg/cm2 pour le méthotrexate. Des analyses statistiques descriptives ont été effectuées afin de déterminer la médiane, le 75e percentile et les taux maximums. RÉSULTATS: Au total, 51 hôpitaux ont participé à cette étude descriptive et 584 échantillons ont été quantifiés. Dans l'ensemble, 294 (50 %) échantillons étaient positifs pour la cyclophosphamide, 125 (21 %) pour l'ifosfamide et 54 (9 %) pour le méthotrexate. Les zones les plus fréquemment contaminées étaient : en pharmacie, la grille avant dans la hotte et le sol devant la hotte; dans les unités de soins, les accoudoirs et le comptoir des cliniques de consultation externe. Le 75e percentile de la concentration de surface était de 10,8 pg/cm2 pour la cyclophosphamide, 1,59 pg/cm2 pour l'ifosfamide et sous le seuil de détection pour le méthotrexate. CONCLUSIONS: Comparativement à trois autres études multicentriques menées au Québec au cours des dernières années, la proportion de prélèvements positifs demeure la même. Toutefois, le 75e percentile de la concentration de surface d'antinéoplasiques a diminué et semble avoir atteint un plateau. Des objectifs locaux (pour le pays ou selon les régions) et réalisables de contamination de surface par des médicaments dangereux devraient être établis chaque année, et ce, tant qu'aucune limite fondée sur les critères liés à la santé ne sera pas déterminée.

Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures.

Heterogeneity in the clonal outputs of individual human embryonic stem cells (hESCs) confounds analysis of their properties in studies of bulk populations and how to manipulate them for clinical applications. To circumvent this problem we developed a microfluidic device that supports the robust generation of colonies derived from single ESCs. This microfluidic system contains 160 individually addressable chambers equipped for perfusion culture of individual hESCs that could be shown to match the growth rates, marker expression and colony morphologies obtained in conventional cultures. Use of this microfluidic device to analyze the clonal growth kinetics of multiple individual hESCs induced to differentiation revealed variable shifts in the growth rate, area per cell and expression of OCT4 in the progeny of individual hESCs. Interestingly, low OCT4 expression, a slower growth rate and low nuclear to cytoplasmic ratios were found to be correlated responses. This study demonstrates how microfluidic systems can be used to enable large scale live-cell imaging of isolated hESCs exposed to changing culture conditions, to examine how different aspects of their variable responses are correlated.

A human embryonic stem cell line adapted for high throughput screening.

Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However, optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs, including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20, they remain highly useful. In particular, CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus, 10(8) CA1S cells can be generated within one week from 10(6) cells to seed 10(6) wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm, a finding that was confirmed with H1 hESCs, although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines, these cells should ease the development of optimized hESC growth and differentiation protocols. In particular, they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses.

Assessing differentiation status of human embryonic stem cells noninvasively using Raman microspectroscopy.

Raman microspectroscopy is an attractive approach for chemical imaging of biological specimens, including live cells, without the need for chemi-selective stains. Using a microspectrometer, near-infrared Raman spectra throughout the range 663 cm(-1) to 1220 cm(-1) were obtained from colonies of CA1 human embryonic stem cells (hESCs) and CA1 cells that had been stimulated to differentiate for 3 weeks by 10% fetal bovine serum on gelatin. Distributions and intensities of spectral bands attributed to proteins varied significantly between undifferentiated and differentiated cells. Importantly, compared to proteins and lipids, the band intensities of nucleic acids were dominant in undifferentiated cells with a dominance-reversal in differentiated cells. Thus, we could identify intensity ratios of particular protein-related bands (e.g., 757 cm(-1) tryptophan) to nucleic acid bands (784 cm(-1) DNA/RNA composite) that were effective in discriminating between spectra of undifferentiated and differentiated cells. We observed no discernible negative effects due to the laser exposure in terms of morphology, proliferation, or pluripotency of the stem cells. We conclude that Raman microscopy and complementary data processing procedures provide a rapid, noninvasive approach that can distinguish hESCs from differentiated cells. This is the first report to identify specific Raman markers for the differentiation status of hESCs.

Central tolerance to myogenic cell transplants does not include muscle neoantigens.

BACKGROUND: Duchenne muscular dystrophy is a fatal genetic disease caused by lack of dystrophin. Myogenic cell transplantation (MT), a potential therapy for Duchenne muscular dystrophy, can restore dystrophin expression in muscles. Because allogeneic MT is highly resistant to peripheral tolerance, we proposed to induce central tolerance. However, given its immunogenicity, we asked whether central tolerance to donor major histocompatibility complex would allow long-term expression of dystrophin, a tissue-specific neoantigen in dystrophic recipients. METHODS: Central tolerance was induced in C57BL/10J mdx (dystrophic) mice by allogeneic bone marrow transplantation (BMT) after conditioning with either lethal total body irradiation (TBI) or an established nonmyeloablative protocol (anti-CD154, anti-CD8 mAbs, and low-dose TBI). Recipients subsequently received donor-strain MT or skin grafts. RESULTS: Long-term hematopoietic chimeras generated using either lethal TBI or the nonmyeloablative regimen were tolerant to donor skin grafts and both primary and secondary donor MT (>90 days). Myogenic cell transplantation survival was decreased when chimerism was transient, which was most common with nonmyeloablative conditioning and fully rather than haplo-mismatched donors. Interestingly, regardless of conditioning, MT was associated with localized muscle infiltration with Foxp3CD4, CD25CD4, and PerforinCD8 cells, whereas skin grafts lacked infiltration. CONCLUSIONS: Central tolerance achieved using regimens that eliminate nearly all endogenous peripheral lymphocytes (i.e., lethal irradiation) or a nonmyeloablative protocol that depleted peripheral CD8 cells, results in lymphocytic infiltration in muscles that received MT but not in skin allografts. This suggests that muscle-specific infiltration may result from lack of negative selection for peripheral neoantigens in the thymus after BMT and that tolerance after MT may rely on peripheral regulatory mechanisms.

Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts.

Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the phiC31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the phiC31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the phiC31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.

Endosome disruption enhances the functional nuclear delivery of Tat-fusion proteins.

Tat protein from human immunodeficiency virus can deliver biologically active proteins in vivo and is of considerable interest for protein therapeutics. The mechanism responsible for Tat-fusion protein internalization is still poorly understood and controversial. The punctuate distribution, timing, and temperature sensitivity observed in our experiments with Tat-fusion proteins are consistent with endocytosis. After a few hours, Tat-fusion proteins accumulated around the nucleus without any significant visible nuclear targeting. Using a Cre/Lox based functional assay, lysosomotropic agents known to disrupt endosome integrity, increased by up to 23-fold the nuclear delivery of functional Tat-Cre recombinase without increasing cell uptake in a similar fashion. This shows that endosome disruption can significantly increase Tat-fusion protein access to the cytosol and nucleus. In addition, we found that internalized Tat-fusion proteins persisted several hours and that inhibitors of lysosome acidification did not increase functional nuclear delivery of Tat-Cre. This suggests that Tat-fusion proteins enter via the endosomal pathway, circumvent lysosomal degradation, and are then sequestered in the periphery of the nucleus. Most importantly, our work indicates that an inadequate intracellular trafficking is the main factor limiting the efficiency of protein cargo delivery using Tat.

Resetting the problem of cell death following muscle-derived cell transplantation: detection, dynamics and mechanisms.

We conducted a study in mice to reevaluate and clarify many aspects of the early survival of muscle cells following transplantation. Male mouse muscle cells (primary-cultures and T-antigen-immortalized clones) labeled with [14C]thymidine and beta-galactosidase were injected into female muscles. Each label was detected in the muscles after different time periods. TUNEL, alizarin red, and immunodetection of active caspase-3 were done in muscle sections. The donor cell labels disappeared from the muscles following donor cell death, but this was not instantaneous and even if the donor cells were killed before transplantation, the first 6 hours were not enough to clear [14C]thymidine and Y chromosome. Using the cell pellet before injection as the 100% baseline for cells injected to evaluate cell death can lead to misinterpretations: the Y-chromosome band was 5-fold stronger than that of a muscle injected with cells, irrespective of whether the cells were previously killed or not. There was no evidence of an immediate massive donor cell death. Necrosis (detected by alizarin red) and apoptosis (detected by active caspase-3) were present among the donor myoblasts following transplantation. Necrosis seemed to be the most important mechanism during the first hours. T-antigen immortalized cells died earlier and more massively than primary-cultured cells, but the surviving cells proliferated more. Indeed, they seemed to exhibit more apoptosis and they triggered a more rapid CD8+ cell infiltration. As a result of our findings, many concepts concerning the early donor cell death following myoblast transplantation must be reconsidered.

Identification of a putative pathway for the muscle homing of stem cells in a muscular dystrophy model.

Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS(R) multisort method. Clones of MDSCs, which were Sca-1+/CD34-/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34- MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.

Treatment with anti-CD154 antibody and donor-specific transfusion prevents acute rejection of myoblast transplantation.

BACKGROUND: Achieving immunological tolerance to transplanted myoblasts would reduce the adverse effects associated with the sustained immunosuppression required for this experimental therapeutic approach in Duchenne muscular dystrophic patients. METHODS: Mdx mice were transplanted with fully allogeneic BALB/c myoblasts in the tibialis anterior muscles. Seven days before transplantation (-7), host mice received 107 total donor spleen cells i.v. (donor-specific transfusion, DST) with 500 microg of anti-CD154 mAb i.p. on days -7, -4, 0, +4. RESULTS: Results showed a high level of dystrophin expression in 83, 60, and 20% of the mice 1, 3, and 6 months, respectively, after transplantation of myoblasts. No antibodies against the donor cells were produced up to 3 months after transplantation. However, abundant activated cytotoxic cells were present in muscles still expressing high percentage of dystrophin positive fibers. CONCLUSIONS: In conclusion, the DST + anti-CD154 mAb treatments effectively prolonged myoblast survival, but this treatment could not develop tolerance to complete allogeneic myoblast transplantation.