Infusion of mesenchymal stromal cells after deceased liver transplantation: A phase I-II, open-label, clinical study.

BACKGROUND & AIMS: Mesenchymal stromal cell (MSC) infusion could be a means to establish tolerance in solid organ recipients. The aim of this prospective, controlled, phase I study was to evaluate the feasibility, safety and tolerability of a single infusion of MSCs in liver transplant recipients. METHODS: Ten liver transplant recipients under standard immunosuppression received 1.5-3×106/kg third-party unrelated MSCs on postoperative day 3±2, and were prospectively compared to a control group of ten liver transplant recipients. As primary endpoints, MSC infusion toxicity was evaluated, and infectious and cancerous complications were prospectively recorded until month 12 in both groups. As secondary endpoints, rejection rate, month-6 graft biopsies, and peripheral blood lymphocyte phenotyping were compared. Progressive immunosuppression weaning was attempted from month 6 to 12 in MSC recipients. RESULTS: No variation in vital parameters or cytokine release syndrome could be detected during and after MSC infusion. No patient developed impairment of organ functions (including liver graft function) following MSC infusion. No increased rate of opportunistic infection or de novo cancer was detected. As secondary endpoints, there was no difference in overall rates of rejection or graft survival. Month-6 biopsies did not demonstrate a difference between groups in the evaluation of rejection according to the Banff criteria, in the fibrosis score or in immunohistochemistry (including Tregs). No difference in peripheral blood lymphocyte typing could be detected. The immunosuppression weaning in MSC recipients was not successful. CONCLUSIONS: No side effect of MSC infusion at day 3 after liver transplant could be detected, but this infusion did not promote tolerance. This study opens the way for further MSC or Treg-based trials in liver transplant recipients. LAY SUMMARY: Therapy with mesenchymal stromal cells (MSCs) has been proposed as a means to improve results of solid organ transplantation. One of the potential MSC role could be to induce tolerance after liver transplantation, i.e. allowing the cessation of several medications with severe side effects. This study is the first-in-man use of MSC therapy in ten liver transplant recipients. This study did not show toxicity after a single MSC infusion but it was not sufficient to allow withdrawal of immunosuppression. CLINICAL TRIAL REGISTRATION NUMBER: Eudract: # 2011-001822-81, ClinicalTrials.gov: # NCT 01429038.

[« Doctor, I would like to stay at home ¼ About the partnership between a specialized palliative home care team and general practitioners]. / « J'aimerais rester chez moi, Docteur ¼. A propos du partenariat entre l'équipe mobile de soinspalliatifs et les médecins de premier recours.

During home care, general practitioners are faced with complex end-of-life situations. This leads to difficulties in taking care of the patient alone, and may require support from a multi-professional specialized palliative home care team. The palliative care teams follow these situations upon request from the GPs or home nurses, in order to provide help in encountered difficulties. The palliative care teams' activity is based on mentoring, anticipating possible future difficulties and managing crises. In order to have a real working partnership between the home palliative care teams and the GPs it is essential to have a mutual understanding of how each entity works and what kind of help they can provide.

Les médecins de premier recours peuvent être confrontés au domicile à des situations de fin de vie lourdes et complexes, difficiles à prendre en charge seuls, dans lesquelles le soutien d'une équipe multi-professionnelle spécialisée en soins palliatifs est nécessaire. L'équipe mobile de soins palliatifs intervient sur demande des médecins et des équipes soignantes pour les aider à faire face à ces difficultés. Son activité est centrée sur le compagnonnage de ces équipes par une aide à l'anticipation et à la gestion de la crise. Un partenariat efficace entre l'équipe mobile de soins palliatifs et les médecins de premier recours implique une connaissance réciproque, à la fois du fonctionnement et des prestations offertes par chacun des partenaires.

Clinical-scale expansion of mesenchymal stromal cells: a large banking experience.

BACKGROUND: Mesenchymal stromal cells (MSC) are largely investigated in clinical trials aiming to control inappropriate immune reactions (GVHD, Crohn's disease, solid organ transplantation). As the percentage of MSC precursors in bone marrow is very low, these must be expanded in vitro to obtain therapeutic cell doses. We describe here the constitution of an allogeneic human third-party MSC bank from screened healthy volunteer donors in compliance with quality specifications and ISCT-release criteria and report follow-up of different aspects of this activity since 2007. METHODS: 68 clinical-grade large-scale MSC cultures were completed and analyzed. The whole process was described, including volunteer donor screening, bone marrow collection, mononuclear cell isolation and expansion over 4 weeks, harvesting, cryopreservation, release, administration and quality controls of the cells (including microbiology, phenotype, and potency assays). RESULTS: From 59 validated donors, 68 cultures were completed (mean of final yields: 886 × 10(6) cells/culture) and a total of 464 MSC aliquots have been produced and stored in liquid nitrogen (mean of 132.8 × 10(6) cells/bag). Each MSC batch underwent extensive testing to verify its conformity with EBMT and ISCT release criteria and was individually validated. As of June 1 2015, 314 bags have been released and infused to patients included in 6 different clinical protocols. All thawed MSC units satisfied to release criteria and no infusion-related toxicity was reported. CONCLUSION: In conclusion, despite low passage cultures, we have been able to create an allogeneic "off-the-shelf" MSC bank with a large number of frozen aliquots and report here an efficient clinical-grade MSC banking activity in place for more than 7 years. Our challenge now is to produce MSC in compliance with good manufacturing practices (GMP) as, in the meantime, MSC have become considered as advanced therapy medicinal products (ATMP). Another significant challenge remains the development of relevant potency assay.

Infusion of clinical-grade enriched regulatory T cells delays experimental xenogeneic graft-versus-host disease.

BACKGROUND: We investigated the ability of clinical-grade enriched human regulatory T cells (Treg) to attenuate experimental xenogeneic graft-versus-host disease (GVHD) induced by peripheral blood mononuclear cells (PBMNCs; autologous to Treg) infusion in NSG mice, as well as verified their inability to induce xenogeneic GVHD when infused alone. STUDY DESIGN AND METHODS: Human Treg were isolated from peripheral blood apheresis products with a cell separation system (CliniMACS, Miltenyi Biotec GmbH) using a two-step procedure (simultaneous CD8 and CD19 depletion followed by CD25-positive selection) in six independent experiments with six different healthy volunteer donors. Sublethally (2.5 Gy) irradiated NSG mice were given 2 × 10(6) cytapheresis (PBMNC) product cells intravenously (IV) without (PBMNC group) or with 1 × 10(6) Treg (PBMNC + Treg group), while other NSG mice received 2 × 10(6) enriched Treg alone (also in IV; Treg group). RESULTS: The first five procedures were successful at obtaining a relatively pure Treg population (defined as >50%), while the sixth procedure, due to a technical problem, was not (Treg purity, 42%). Treg cotransfusion significantly delayed death from xenogeneic GVHD in the first five experiments, (p < 0.0001) but not in the sixth experiment. Importantly, none of the mice given enriched Treg alone (Treg group) experienced clinical signs of GVHD, while, interestingly, the CD4+ cells found in these mice 26 days after transplantation were mainly conventional T cells (median CD25+FoxP3+ cells among human CD4+ total cells were only 2.1, 3.1, and 12.2% in spleen, marrow, and blood, respectively). CONCLUSIONS: Infusion of clinical-grade enriched Treg delayed the occurrence of xenogeneic GVHD without inducing toxicity in this murine model.

Effectiveness of a real-life program (DIAfit) to promote physical activity in patients with type 2 diabetes: a pragmatic cluster randomized clinical trial.

Introduction: The aim of this study was to evaluate the effectiveness of a real-life clinical physical activity program (DIAfit) on improving physical fitness, body composition, and cardiometabolic health in an unselected population with type 2 diabetes mellitus, and to compare the effects of two variants a different exercise frequencies on the same outcomes. Research design and methods: This was a cluster randomized-controlled assessor-blind trial conducted in 11 clinical centres in Switzerland. All participants in the clinical program with type 2 diabetes were eligible and were randomized to either standard (3 sessions/week for 12 weeks) or alternative (1 session/week for the first four weeks, then 2 sessions/week for the rest of 16 weeks) physical activity program each consisting of 36 sessions of combined aerobic and resistance exercise. Allocation was concealed by a central office unrelated to the study. The primary outcome was aerobic fitness. Secondary outcome measures included: body composition, BMI, HbA1c, muscle strength, walking speed, balance, flexibility, blood pressure, lipid profile. Results: All 185 patients with type 2 diabetes (mean age 59.7 +-10.2 years, 48% women) agreed to participate and were randomized in two groups: a standard group (n=88) and an alternative group (n=97)). There was an 11% increase in aerobic fitness after the program (12.5 Watts; 95% CI 6.76 to 18.25; p<0.001). Significant improvements in physical fitness, body composition, and cardiometabolic parameters were observed at the end of the DIAfit program (improvements between 2-29%) except for lean body mass, triglycerides and cholesterol. No differences were observed between both programs, except for a larger weight reduction of -0.97kg (95% CI -0.04 to -1.91; p=0.04) in the standard program. Conclusions: Both frequency variants of the nation-wide DIAfit program had beneficial effects on physical fitness, HbA1c, body composition, and blood pressure in type 2 diabetes patients and differences were negligible. Clinical trial registration: clinicaltrials.gov, identifier NCT01289587.

Methadone Switching for Cancer Pain: A New Classification of Initiation Protocols, Based on a Critical Literature Review.

Background: The initiation of methadone, a known effective analgesic for cancer pain, is complex. The existing protocols are often inadequately described; therefore, a classification of literature is needed. We reviewed and classified the recent literature on methadone initiation protocols in cancer patients experiencing severe pain. Objective: To provide a new classification of initiation protocols, based on a critical literature review. Data Sources: The MEDLINE database was searched for articles published until March 25, 2021, using the terms "cancer pain," "methadone," "methadone introduction," or "methadone initiation." The search was limited to human studies, randomized controlled trials (RCTs), other clinical trials, meta-analyses, and case reports. Selected articles were assessed for initiation details (rapid or progressive), administered dose (fixed rescue dose or ad libitum), and dose calculation (fixed or progressive ratios using morphine equivalent daily dose [MEDD] for daily or unitary dose). Results: Twenty-four publications that met our inclusion criteria were analyzed. No large-scale prospective double-blind RCTs with robust design were identified. Most studies assessed relatively small numbers of patients. Eight initiation types were identified, of which three involved seven "high quality" studies: "rapid switch-fixed doses and rescue dose-progressive daily ratio," "progressive switch-fixed dose and rescue dose-progressive daily ratio," and "rapid switch-ad libitum-fixed ratio for unitary dose" protocols. This classification provides the latest information on methadone initiation protocols. The total daily dose of methadone varied largely across protocols. Conclusion: We recommend a maximal daily methadone dose of 100 mg (3 doses of 30 mg or 5 doses of 20 mg) for MEDD <500 mg, when the two "ad libitum" protocols are used. Further clinical research on this topic is warranted.

Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of alpha4 integrin- and CXCR4-mediated engraftment in NOD/SCID beta2-microglobulin-null mice.

BACKGROUND: Several studies indicate that ex vivo cytokine-supported expansion induces defective hematopoietic stem cell engraftment. We investigated the role of alpha4 integrin, alpha5 integrin and CXCR4 in engraftment of unmanipulated and cytokine-treated human cord blood CD34(+) cells. DESIGN AND METHODS: Uncultured or expanded CD34(+) cells were infused in NOD/SCID-beta(2)microglobulin-null mice. The function of alpha4, and alpha5 integrins and CXCR4 was assessed by incubating cells with specific neutralizing antibodies, prior to transplant. The activation state of alpha4 integrin was further tested by adhesion and migration assays. RESULTS: Neutralization of either alpha4 integrin or CXCR4 abolished engraftment of uncultured CD34(+) cells at 6 week spost-transplant, while alpha5 integrin neutralization had no significant effect. However, after short-term ex vivo culture, blocking alpha4 integrin or CXCR4 did not affect repopulating activity whereas neutralization of alpha5 integrin inhibited engraftment. Using soluble vascular cell adhesion molecule-1 binding assays, we observed that alpha4 integrin affinity in fresh CD34(+) cells was low and susceptible to stimulation while in cultured CD34(+) cells, it was high and insensitive to further activation. In addition, stromal cell-derived factor-1 stimulated migration across vascular cell adhesion molecule-1 in fresh CD34(+) cells but not in cultured CD34(+) cells. CONCLUSIONS: Our data show that ex vivo culture of hematopoietic progenitor cells is associated with downregulation of both alpha4 integrin- and CXCR4-mediated engraftment. Further investigations suggest that this is caused by supraphysiological increase of alpha4 integrin affinity, which impairs directional migration across vascular cell adhesion molecule-1 in response to stromal cell-derived factor-1. Such changes may underlie the engraftment defect of cytokine-stimulated CD34(+) cells.

Educational Level Is Related to Physical Fitness in Patients with Type 2 Diabetes - A Cross-Sectional Study.

INTRODUCTION: Low educational level (EL) and low physical fitness are both predictors of increased morbidity and mortality in patients with type 2 diabetes. It is unknown if EL is related to physical fitness. This would have important implication for the treatment approach of patients of low EL. MATERIALS AND METHODS: In 2011/12, we invited participants of a new nationwide Swiss physical activity program for patients with type 2 diabetes to participate in this study. EL was defined by self-report and categorized as low (mandatory education), middle (professional education) or high (high school/university). Physical fitness was determined using 5 validated measures that assessed aerobic fitness, functional lower limb muscle strength, walking speed, balance and flexibility. Potential confounder variables such as other socio-cultural factors, physical activity level, body composition, diabetes-related parameters and complications/co-morbidities as well as well-being were assessed. RESULTS: All invited 185 participants (mean age 59.6 ±9.8 yrs, 76 women) agreed to be included. Of all patients, 23.1% had a low, 32.7% a middle and 44.2% a high EL; 41.8% were professionally active. The study population had a mean BMI of 32.4±5.2 kg/m2 and an HbA1c of 7.3±1.3%. The mean diabetes duration was 8.8±7.4 years. In the baseline assessment, higher EL was associated with increased aerobic fitness, increased functional lower limb muscle strength, and increased walking speed using linear regression analysis (values for low, middle and high EL, respectively: 91.8 ± 27.9, 116.4 ± 49.7 and 134.9 ± 60.4 watts for aerobic fitness (p = 0.002), 15 ± 4.7, 13.9 ± 2.7, 12.6 ± 2.9 seconds for strength (p = 0.001) and 8.8 ± 1.6, 8.3 ± 1.4, 7.8 ± 1.4 seconds for walking speed (p = 0.004)). These associations were independent of potential confounders. Overall, aerobic fitness was 46%, functional limb muscle strength 16%, and walking speed 11% higher in patients of high compared to those of low EL. EL was not related to balance or flexibility. DISCUSSION: A main strength of the present study is that it addresses a population of importance and a factor (EL) whose understanding can influence future interventions. A second strength is its relatively large sample size of a high-risk population. Third, unlike studies that have shown an association between self-reported fitness and educational level we assessed physical fitness measures by a quantitative and validated test battery using assessors blinded to other data. Another novelty is the extensive evaluation of the role of many relevant confounder variables. CONCLUSIONS: In conclusion, we show that in patients with type 2 diabetes EL correlates favorably and independently with important health-related physical fitness measures such as aerobic fitness, walking speed, and lower limb strength. Our findings underline that diabetic patients with low EL should be specifically encouraged to participate in physical activity intervention programs to further reduce social disparities in healthcare. Such programs should be structured and integrate the norms, needs and capacities (financial, time, physical capacities and self-efficacy) of this population, and their effectiveness should be tested in future studies. TRIAL REGISTRATION: University of Lausanne clinicaltrials.gov NCT01289587.

Binding and migration across fibronectin and VCAM-1 of cycling hematopoietic progenitor cells.

Using different experimental approaches, it has been established that transplantability of hematopoietic/stem progenitor cells is ineffective during transit through the cell cycle. Although primitive stem cells are responsive to mitogenic stimulation in optimized ex vivo conditions, defective engraftment of generated cells may limit their detection in standard transplantation models as well as their use in clinical cell therapy. The activation level of adhesion receptors is modulated by stimulation of cytokine receptors via "inside-out" signaling. This prompted us to study the interactions of progenitor cells with fibronectin (Fn) in different phases of the cell cycle. We first demonstrated that adhesion to Fn was stimulated in S/G2 + M as compared to G0/G1, in ex vivo cultured CD34+ cells, with a predominant usage of very late antigen (VLA)-5 over that of VLA-4. We next determined that maximal Fn binding in active phases of the cell cycle limited cell motility toward stromal cell-conditioned medium. It was also observed that VLA-4 and VLA-5 ability to mediate adhesion or migration varied independently during cell cycle transit. Finally, in synchronized progenitor cells executing a first cell cycle ex vivo, a reversible increase in Fn binding was associated with a reversible decrease in adhesion to vascular cell-adhesion molecule (VCAM)-1. Overall, these observations suggest that defective engraftment of cycling stem/progenitor cells may result, at least in part, from abnormal trafficking related to changes in the activation level of adhesion receptors.

Impact of cotransplantation of mesenchymal stem cells on lung function after unrelated allogeneic hematopoietic stem cell transplantation following non-myeloablative conditioning.

BACKGROUND: In the context of hematopoietic stem cell transplantation (HSCT), mesenchymal stem cells (MSC) have been used to promote engraftment and prevent graft-versus-host disease. However, in animal models, MSC were shown to cause pulmonary alterations after systemic administration. The impact of MSC infusion on lung function has not been studied in humans. The objective of the study was to investigate the impact of MSC co-infusion on lung function and airway inflammation as well as on the incidence of pulmonary infections and cytomegalovirus (CMV) reactivation after HSCT. METHODS: We have prospectively followed 30 patients who underwent unrelated HSCT with MSC co-infusion after non-myeloablative conditioning (NMA). Each patient underwent detailed lung function testing (FEV1, FVC, FEV1/FVC, RV, TLC, DLCO, and KCO) and measurement of exhaled nitric oxide before HSCT and 3, 6, and 12 months posttransplant. The incidence of pulmonary infections and CMV reactivation were also monitored. This group was compared with another group of 28 patients who underwent the same type of transplantation but without MSC co-infusion. RESULTS: Lung function tests did not show important modifications over time and did not differ between the MSC and control groups. There was a higher 1-year incidence of infection, particularly of fungal infections, in patients having received a MSC co-infusion. There was no difference between groups regarding the 1-year incidence of CMV reactivation. CONCLUSIONS: MSC co-infusion does not induce pulmonary deterioration 1 year after HSCT with NMA conditioning. MSC appear to be safe for the lung, but close monitoring of pulmonary infections remains essential.

Thinking out of the box--new approaches to controlling GVHD.

Graft-versus-host disease (GVHD) remains a major limitation of allogeneic hematopoietic cell transplantation (allo-HCT). Despite major advances in the understanding of GVHD pathogenesis, standard GVHD prophylaxis regimens continue to be based on the combination of a calcineurin inhibitor with an antimetabolite, while first line treatments still rely on high-dose corticosteroids. Further, no second line treatment has emerged thus far in acute or chronic GVHD patients who failed to respond with corticosteroid treatment. After briefly reviewing current standards of GVHD prevention and treatment, this article will discuss recent approaches that might change GVHD prophylaxis/treatment for decades to come, with a special focus on recently developed immunoregulatory strategies based on infusion of mesenchymal stromal or regulatory T-cells, or injection of low-dose interleukin-2.

Despite inhibition of hematopoietic progenitor cell growth in vitro, the tyrosine kinase inhibitor imatinib does not impair engraftment of human CD133+ cells into NOD/SCIDbeta2mNull mice.

There is potential interest for combining allogeneic hematopoietic cell transplantation (HCT), and particularly allogeneic HCT with a nonmyeloablative regimen, to the tyrosine kinase inhibitor imatinib (Glivec; Novartis, Basel, Switzerland, http://www.novartis.com) in order to maximize anti-leukemic activity against Philadelphia chromosome-positive leukemias. However, because imatinib inhibits c-kit, the stem cell factor receptor, it could interfere with bone marrow engraftment. In this study, we examined the impact of imatinib on normal progenitor cell function. Imatinib decreased the colony-forming capacity of mobilized peripheral blood human CD133(+) cells but not that of long-term culture-initiating cells. Imatinib also decreased the proliferation of cytokine-stimulated CD133(+) cells but did not induce apoptosis of these cells. Expression of very late antigen (VLA)-4, VLA-5, and CXCR4 of CD133(+) cells was not modified by imatinib, but imatinib decreased the ability of CD133(+) cells to migrate. Finally, imatinib did not decrease engraftment of CD133(+) cells into irradiated nonobese diabetic/severe combined immunodeficient/beta2m(null) mice conditioned with 3 or 1 Gy total body irradiation. In summary, our results suggest that, despite inhibition of hematopoietic progenitor cell growth in vitro, imatinib does not interfere with hematopoietic stem cell engraftment.

Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture.

Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34(+) progenitor cells were synchronized and assayed for adhesion and migration onto fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1) at different stages of a first cell cycle executed ex vivo. During S phase transit, adhesion to Fn was transiently increased while binding to VCAM-1 was reversibly decreased, after which adhesion to both ligands returned to baseline levels with cell cycle completion. Transmigration across Fn and VCAM-1 decreased irreversibly during S phase progression. The function of alpha4 and alpha5 integrins was assessed with specific neutralizing antibodies. In uncultured CD34(+) cells and long-term culture-initiating cells (LTC-ICs), both adhesion and migration on Fn were inhibited by anti-alpha4 but not by anti-alpha5 antibodies. In mitotically activated CD34(+) cells and LTC-ICs, adhesion and migration on Fn were mainly dependent on alpha5 integrin and to a lesser extent on alpha4 integrin. Changes in integrin function were not dependent on parallel modulation of integrin expression. In conclusion, Fn and VCAM-1 binding of progenitor cells fluctuates reversibly during cell cycle transit ex vivo. In addition, our data show that mitogenic activation induces a shift from a dominant alpha4 to a preferential alpha5 integrin-dependent interaction with Fn.

Increased binding and defective migration across fibronectin of cycling hematopoietic progenitor cells.

Engraftment of hematopoietic progenitor cells has been shown to decrease during cell cycle transit. We studied cell cycle-associated changes in adhesion and migration of mitotically activated cord blood CD34+ cells. Migration toward medium conditioned by the stromal-derived factor-1-producing cell line MS-5 was studied in bovine serum albumin- and fibronectin (Fn)-coated transwells. Migration was reduced in cycling CD34+ cells and long-term culture-initiating cells (LTC-ICs) compared with their noncycling counterparts across Fn but not across bovine serum albumin. Conversely, Fn binding was higher in cycling CD34+ cells and LTC-ICs compared with noncycling progenitor cells, while adhesion of both subsets to bovine serum albumin was undetectable. The contribution of alpha4 and alpha5 integrins in mediating adhesion and migration of activated CD34+ cells onto Fn was analyzed by neutralization experiments. While alpha4-mediated Fn binding decreased during G(2)/M, alpha5 integrin-mediated adhesion increased during transit from G(0)/G(1) to S and G(2)/M phases. As for migration, the contribution of alpha4 integrin was similar in all phases, whereas alpha5-directed migration was lower in G(2)/M compared with G(0)/G(1) and S phases. Defective migration of cycling CD34+ cells was not due to differences in alpha5 integrin expression. In conclusion, chemotaxis across Fn is less efficient in cycling progenitor cells in correlation with an increased Fn binding capacity. In addition, alpha4 and alpha5 integrin functions are independently modulated during cell cycle transit.