Safety and tolerability of intrathecal delivery of autologous bone marrow nucleated cells in children with cerebral palsy: an open-label phase I trial

Background aimsCerebral palsy (CP) is related to severe perinatal hypoxia with permanent brain damage in nearly 50% of surviving preterm infants. Cell therapy is a potential therapeutic option for CP by several mechanisms, including immunomodulation through cytokine and growth factor secretion.MethodsIn this phase I open-label clinical trial, 18 pediatric patients with CP were included to assess the safety of autologous bone marrow–derived total nucleated cell (TNC) intrathecal and intravenous injection after stimulation with granulocyte colony-stimulating factor. Motor, cognitive, communication, personal-social and adaptive areas were evaluated at baseline and 1 and 6 months after the procedure through the use of the Battelle Developmental Inventory. Magnetic resonance imaging was performed at baseline and 6 months after therapy. This study was registered in ClinicaTrials.gov (NCT01019733).ResultsA median of 13.12 × 10⁸ TNCs (range, 4.83–53.87) including 10.02 × 10⁶ CD34+ cells (range, 1.02–29.9) in a volume of 7 mL (range, 4–10.5) was infused intrathecally. The remaining cells from the bone marrow aspirate were administered intravenously; 6.01 × 10⁸ TNCs (range, 1.36–17.85), with 3.39 × 10⁶ cells being CD34+. Early adverse effects included headache, vomiting, fever and stiff neck occurred in three patients. No serious complications were documented. An overall 4.7-month increase in developmental age according to the Battelle Developmental Inventory, including all areas of evaluation, was observed (±SD 2.63). No MRI changes at 6 months of follow-up were found.ConclusionsSubarachnoid placement of autologous bone marrow–derived TNC in children with CP is a safe procedure. The results suggest a possible increase in neurological function.     

A French single-center experience on allogeneic stem cell transplant cryopreservation during severe acute respiratory syndrome coronavirus 2 pandemic

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-SCT) is a curative treatment for chemo-resistant hematological malignancies. Because of transport restriction imposed by the coronavirus disease 2019 pandemic, regulatory bodies and societies recommended graft cryopreservation before recipient conditioning. However, the freezing and thawing processes, including washing steps, might impair CD34+ cell recovery and viability, thereby impacting the recipient engraftment. Over 1 year (between March 2020 and May 2021), we aimed to analyze the results of frozen/thawed peripheral blood stem cell allografts in terms of stem cell quality and clinical outcomes.MethodsTransplant quality was evaluated by comparing total nucleated cells (TNCs), CD34+ cells and colony-forming unit–granulocyte/macrophage (CFU-GM)/kg numbers as well as TNC and CD34+ cell viabilities before and after thawing. Intrinsic biological parameters such as granulocyte, platelet and CD34+ cell concentrations were analyzed, as they might be responsible for a quality loss. The impact of the CD34+ cell richness of the graft on TNC and CD34 yields was evaluated by designing three groups of transplants based on their CD34 /kg value at collection: >8 × 10 ⁶/kg, between 6 and 8 × 10⁶/kg and <6 × 10⁶/kg. The consequences of cryopreservation were compared in the fresh and thawed group by evaluating the main transplant outcomes.ResultsOver 1 year, 76 recipients were included in the study; 57 patients received a thawed and 19 patients a fresh allo-SCT. None received allo-SCT from a severe acute respiratory syndrome coronavirus 2–positive donor. The freezing of 57 transplants led to the storage of 309 bags, for a mean storage time (between freezing and thawing) of 14 days. For the fresh transplant group, only 41 bags were stored for potential future donor lymphocyte infusions. Regarding the graft characteristics at collection, median number of cryopreserved TNC and CD34+ cells/kg were greater than those for fresh infusions. After thawing, median yields were 74.0%, 69.0% and 48.0% for TNC, CD34+ cells and CFU-GM, respectively. The median TNC dose/kg obtained after thawing was 5.8 × 10⁸, with a median viability of 76%. The median CD34+ cells/kg was 5 × 10⁶, with a median viability of 87%. In the fresh transplant group, the median TNC/kg was 5.9 × 10⁸/kg, and the median CD34+ cells/kg and CFU-GM/kg were 6 × 10⁶/kg and 276.5 × 10⁴/kg, respectively. Sixty-one percent of the thawed transplants were out of specifications regarding the CD34+ cells/ kg requested cell dose (6 × 10⁶/kg) and 85% of them would have had this dose if their hematopoietic stem cell transplant had been infused fresh. Regarding fresh grafts, 15.8% contained less than 6 × 10⁶ CD34+ cells /kg and came from peripheral blood stem cells that did not reach 6 × 10⁶ CD34+ cells /kg at collection. Regarding the factor that impaired CD34 and TNC yield after thawing, no significant impact of the granulocyte count, the platelet count or the CD34+ cells concentration/µL was observed. However, grafts containing more than 8 × 10 ⁶/kg at collection showed a significantly lower TNC and CD34 yield.ConclusionsTransplant outcomes (engraftment, graft-versus-host disease, infections, relapse or death) were not significantly different between the two groups.     

A cross-sectional study of umbilical cord blood donor profiles and their influence on umbilical cord blood collection in a Brazilian hospital

Background aimsUmbilical cord blood (UCB) cells are a new alternative to bone marrow source for hematopoietic stem cell transplantation and their use has increased in the last decade. Thus efforts are being made to improve the umbilical cord blood unit's quality. Besides compatibility, other factors, such as the total nucleated cell (TNC) count and the percentage of CD34⁺ cells in the product, are very important for a successful transplant outcome. Our aim was to describe our donor population and assess the best cord blood collection technique at Hospital Israelita Albert Einstein's cord blood bank (São Paulo, Brazil).MethodsThis was a retrospective study in which all analyses were performed simultaneously. A Student's t-test was used for qualitative variables for non-matched samples. For qualitative analyses, we used either the chi-square test or the exact Fisher's test.ResultsThe stratification of the population characteristics allowed us to determine which ones had an impact on unit volume, TNC count and percentage of CD34⁺ cells. A significant correlation was observed between donor characteristics and the quality of UCB units as related to maternal and gestational age, type of pregnancy, route of delivery, cord blood collection technique and birth weight.ConclusionsWe found that cord blood collection technique and newborn weight were significantly correlated with the TNC content. The collection technique used at our institution significantly improved the UCB unit volume and consequently the TNC count. Some findings, such as the impact of maternal age and newborn weight, have led us to re-evaluate our protocol in order to achieve better results.     

Long-term effects of cryopreservation on clinically prepared hematopoietic progenitor cell products

Background aimsThe question of how long hematopoietic progenitor cells (HPCs) destined for clinical applications withstand long-term cryopreservation remains unanswered. To increase our basic understanding about the stability of HPC products over time, this study focused on characterizing long-term effects of cryopreservation on clinically prepared HPC products.MethodsCryovials (n = 233) frozen for an average of 6.3 ± 14.2 years (range, 0.003–14.6 years) from HPC products (n = 170) representing 75 individual patients were thawed and evaluated for total nucleated cells (TNCs), cell viability, viable CD34+ (vCD34+) cells and colony-forming cells (CFCs). TNCs were determined by use of an automated cell counter, and cell viability was measured with the use of trypan blue exclusion. Viable CD34 analysis was performed by means of flow cytometry and function by a CFC assay.ResultsSignificant losses in TNCs, cell viability, vCD34+ cells and CFC occurred on cryopreservation. However, once frozen, viable TNCs, vCD34+ cells and CFC recoveries did not significantly change over time. The only parameter demonstrating a change over time was cell viability, which decreased as the length of time that an HPC product was stored frozen increased. A significant negative correlation (correlation coefficient = −0.165) was determined between pre-freeze percent granulocyte content and post-thaw percent viability (n = 170; P = 0.032). However, a significant positive correlation was observed between percent viability at thaw and pre-freeze lymphocyte concentration.ConclusionsOnce frozen, HPC products were stable for up to 14.6 years at <−150°C. Post-thaw viability was found to correlate negatively with pre-freeze granulocyte content and positively with pre-freeze lymphocyte content.     

Initial cord blood unit volume affects mononuclear cell and CD34+ cell-processing efficiency in a non-linear fashion

Background aimsUmbilical cord blood (UCB) is a source of hematopoietic stem cells that initially was used exclusively for the hematopoietic reconstitution of pediatric patients. It is now suggested for use for adults as well, a fact that increases the pressure to obtain units with high cellularity. Therefore, the optimization of UCB processing is a priority.MethodsThe present study focused on parameters influencing total nucleated cell (TNC), mononucleated cell (MNC) and CD34 ⁺ cell (CD34C) recovery after routine volume reduction of 1553 UCB units using hydroxyethyl starch-induced sedimentation with an automated device, under routine laboratory conditions.ResultsWe show that the unit volume rather than the TNC count significantly affects TNC, MNC and CD34C processing efficiency (PEf), and this in a non-linear fashion: when units were sampled according to the collection volume, including pre-loaded anticoagulant (gross volume), PEf increased up to a unit volume of 110–150 mL and decreased thereafter. Thus units with initial gross volumes < 90 mL and > 170 mL similarly exhibited a poor PEf.ConclusionsThese data identify unit gross volume as a major parameter influencing PEf and suggest that fractionation of large units should be contemplated only when the resulting volume of split units is > 90 mL.     

影响脐带血采集质量的相关性因素分析

目的回顾性分析影响脐带血采集质量的母婴因素和采集处理因素。 方法记录389份脐带血的采集量、母亲年龄、孕龄、新生儿体重、分娩方式、新生儿性别、胎次及脐带血采集至计数间隔以及采集方式。用KX-21型全自动血液分析仪进行细胞计数并计算有核细胞总数（TNC）。采用Pearson相关和Spearman秩相关进行相关性分析,采用t检验、Mann-Whitney U检验、方差分析及Kruskal-Wallis H检验进行分组比较,分析影响脐带血采集量和TNC的相关因素。? 结果脐带血采集量与TNC显著相关（r = 0.723,P < 0.001）,脐带血采集量大于80 ml时的TNC显著高于低体积者。在母婴因素中,母亲年龄与采集量及TNC差异均无统计学意义;孕龄与采集量负相关（r = -0.119,P = 0.019）,而与TNC正相关（r = 0.138,P = 0.007）,足月儿脐带血的TNC显著高于早产儿（P = 0.038）;婴儿出生体重与采集量及TNC均正相关（r = 0.236,P < 0.001;r = 0.275,P < 0.001）,体重较大婴儿脐带血的采集量和TNC均显著高于体重较小者（P?< 0.001）;剖宫产的脐带血采集量虽高于阴道分娩（P < 0.001）,但其TNC不及阴道分娩;男婴与女婴在脐带血采集量和TNC之间无显著差异。在采集处理因素中,脐带血采集至计数的时间间隔与采集量及TNC均无显著相关。 结论为提高脐带血的保存质量,应侧重选择胎儿体重较大、经阴道分娩的产妇作为脐带血供者,实验室应首先处理采集量较大的脐带血。     

Iodine Status of Preterm Infants Born in an Area of Iodine Sufficiency: Are They at Risk of Iodine Deficiency?

ObjectiveTo the assess the iodine status of preterm infants born in an area of iodine sufficiency using the urinary iodine concentration (UIC) and thyroid-stimulating hormone (TSH) levels and compare these values across different feeding practices during the first 7 days of life.MethodsIn this cross-sectional study, 88 preterm infants born at 30 to 34 weeks of gestation and admitted to the neonatal intensive care unit of a referral hospital in Tehran (Iran) were included. The infant UIC and TSH levels and breast milk iodine concentration in mothers who were exclusively breastfeeding were measured.ResultsMedian (interquartile range [IQR]) UIC and TSH levels in the study population were 81 (39-189) μg/L and 1.60 (0.80-2.85) mIU/L, respectively. When preterm infants were stratified by the type of feeding, the median (IQR) UICs were 64 (42-126) μg/L in parenteral nutrition, 125 (41-195) μg/L in exclusively breastfeeding, 57 (28-123) μg/L in formula feeding, and 45 (35-132) μg/L in mixed feeding, with no statistically significant difference between the groups (P = .31). The median (IQR) breast milk iodine concentration was 271 (177-521) μg/L in preterm infants exclusively fed their mothers’ own milk. There was no significant difference in the proportion of the TSH levels of >5 mIU/L between preterm infants who received enteral and parenteral nutrition (P = .27).ConclusionPreterm infants are at risk of iodine deficiency even in an area where the general population has adequate iodine. Only the preterm infants who received exclusively their mothers’ own milk had marginally adequate iodine status. Further studies are warranted to determine the necessity of iodine supplementation for this vulnerable group.     

Predictive analytics and cord blood banking: toward utilization-based unit selection

Background aimsTotal nucleated cell (TNC) and CD34+ cell doses are considered among the most important parameters when assessing the suitability of a human leukocyte antigen-matched cord blood unit (CBU) for allogeneic hematopoietic stem cell transplantation (HSCT). Cord blood banks therefore frequently select CBUs for cryopreservation based on pre-process TNC content. However, cell loss during processing can lead to a significant quantity of CBUs that do not meet desired post-process quality criteria, and such grafts are less likely to be selected by transplant centers for HSCT. Here the authors present a multi-parameter linear regression (MLR) model capable of identifying CBUs that would process poorly, despite meeting established pre-process TNC and CD34+ quality thresholds.MethodsHistorically processed CBUs were graded from A+ to D depending on post-process cell content, and the utilization rate of each grade category was examined. Eight pre-process predictors of post-process cell content were used to train the MLR model, including red blood cell (RBC) content; CBU volume; age of CBU when received; and TNC constituent cell subsets. The selection efficacy of this model was then compared to that of methods conventionally used to select CBUs for processing, with receiver operating characteristic (ROC) and mean inventory quality analysis forming the basis of assessment.ResultsWithin the Anthony Nolan Cell Therapy Centre, CBUs graded ‘D’ accounted for 37% of processing expenditures despite providing only 11% of grafts shipped for HSCT. The MLR model significantly improved pre-process identification of 'D' grade CBUs relative to thresholds based primarily on CD34+ cell content (P < 0.0001) and TNC content (P < 0.0001). At a comparable financial investment, this translated to a banked graft inventory of significantly higher quality than that produced by CD34+ (+8.8% mean increase, P = 0.007) and TNC (+9.9% mean increase, P = 0.010) selection methods.ConclusionsA predictive modelling approach to pre-process CBU selection is a simple and effective means to increase graft inventory quality and potentially future graft utilization, at no additional financial investment.     

Non-cryopreserved hematopoietic stem cells in autograft patients with lymphoma: a matched-pair analysis comparing a single center experience with the use of cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry

Background aimsAround 50 000 autologous stem cell transplantations are done each year worldwide using cryopreserved peripheral blood stem cells (PBSCs). Cryopreservation is time-consuming and expensive. Since 2007, several retrospective studies have shown that PBSCs can be stored at 4°C for 2–3 days, allowing autologous stem cell transplantation in patients with multiple myeloma receiving high-dose melphalan. Data with non-cryopreserved PBSCs in patients autografted for lymphoma following longer pre-conditioning regimens are limited. In addition, no controlled comparison has been able to detect unforeseen differences.MethodsThe authors compared outcomes of 94 consecutive adult patients with lymphoma (66 with Hodgkin lymphoma) autografted in our department in Oran (Algeria) using PBSCs stored at 4°C, from 2009 to 2018, with patients receiving cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry. Patients autografted in Oran were matched with patients receiving cryopreserved PBSCs in the registry (four controls per patient in Oran).ResultsNeutrophil engraftment was significantly faster with cryopreserved PBSCs (P = 0.003). By day 10, only 17% of patients receiving non-cryopreserved PBSCs engrafted versus 48% for cryopreserved PBSCs. Likewise, platelet recovery to 20 000/mm³ was significantly faster in patients receiving cryopreserved PBSCs (P = 0.01). However, all patients in both groups had recovered by day 20. There were no significant differences in non-relapse mortality (9% versus 7%, P = 0.4), relapse incidence (22% versus 32%, P = 0.13), progression-free survival (70% versus 61%, P = 0.4) or overall survival (85% versus 75%, P = 0.3).ConclusionsThis analysis suggests that, in patients with lymphoma receiving pre-transplant regimens such as carmustine, etoposide, cytarabine and melphalan, PBSCs stored at 4°C for up to 6 days can be used safely in centers with no cryopreservation facility. However, the kinetics of hematopoietic recovery showed a significant, albeit small, delay in engraftment for both neutrophils and platelets, which favors the use of cryopreservation if available.     

Hematopoietic Stem Cells in Neonates: Any Differences between Very Preterm and Term Neonates?

Background

In the last decades, human full-term cord blood was extensively investigated as a potential source of hematopoietic stem and progenitor cells (HSPCs). Despite the growing interest of regenerative therapies in preterm neonates, only little is known about the biological function of HSPCs from early preterm neonates under different perinatal conditions. Therefore, we investigated the concentration, the clonogenic capacity and the influence of obstetric/perinatal complications and maternal history on HSPC subsets in preterm and term cord blood.

Methods

CD34+ HSPC subsets in UCB of 30 preterm and 30 term infants were evaluated by flow cytometry. Clonogenic assays suitable for detection of the proliferative potential of HSPCs were conducted. Furthermore, we analyzed the clonogenic potential of isolated HSPCs according to the stem cell marker CD133 and aldehyde dehydrogenase (ALDH) activity.

Results

Preterm cord blood contained a significantly higher concentration of circulating CD34+ HSPCs, especially primitive progenitors, than term cord blood. The clonogenic capacity of HSPCs was enhanced in preterm cord blood. Using univariate analysis, the number and clonogenic potential of circulating UCB HSPCs was influenced by gestational age, birth weight and maternal age. Multivariate analysis showed that main factors that significantly influenced the HSPC count were maternal age, gestational age and white blood cell count. Further, only gestational age significantly influenced the clonogenic potential of UCB HSPCs. Finally, isolated CD34+/CD133+, CD34+/CD133– and ALDH^high HSPC obtained from preterm cord blood showed a significantly higher clonogenic potential compared to term cord blood.

Conclusion

We demonstrate that preterm cord blood exhibits a higher HSPC concentration and increased clonogenic capacity compared to term neonates. These data may imply an emerging use of HSPCs in autologous stem cell therapy in preterm neonates.     

The circadecadal rhythm of oscillation of umbilical cord blood parameters correlates with geomagnetic activity – An analysis of long-term measurements (1999–2011)

Recently, we have shown that the contents of total nucleated cells (TNCs) and CD34⁺ hematopoietic stem and progenitor cells (CD34⁺ HSPCs) as well as the cord blood volume (CBV) in umbilical cord blood (UCB) show a circadecadal (~10 years) rhythm of oscillation. This observation was based on an analysis of 17,936 cord blood donations collected during 1999–2011. The aim of the present study was to investigate whether this circadecadal rhythm of oscillation in TNCs, CD34⁺ HSPCs and CBV is related to geomagnetic activity. For the analysis, the yearly averages of TNCs, CD34⁺ HSPCs and CBV in UCB were correlated with geomagnetic activity (Dcx index). Our analysis revealed that (i) all three UCB parameters were statistically significantly correlated with the level of geomagnetic activity, (ii) CBV showed a linear correlation with the Dcx index (r = 0.5290), (iii) the number of TNCs and CD34⁺ HSPCs were quadratic inversely correlated with the Dcx index (r = ?0.5343 and r = ?0.7749, respectively). Furthermore, (iv) CBV and the number of TNCs were not statistically significantly correlated with the number of either modest or intense geomagnetic storms per year, but (v) the number of CD34⁺ HSPCs was statistically significantly correlated with the number of modest (r = 0.9253) as well as intense (r = 0.8683) geomagnetic storms per year. In conclusion, our study suggests that UCB parameters correlate with the state of the geomagnetic field (GMF) modulated by solar activity. Possible biophysical mechanisms underlying this observation, as well as the outcome of these findings, are discussed.     

Pharmacoeconomic impact of up-front use of plerixafor for autologous stem cell mobilization in patients with multiple myeloma

Background aimsStem cell collection can be a major component of overall cost of autologous stem cell transplantation (ASCT). Plerixafor is an effective agent for mobilization; however, it is often reserved for salvage therapy because of its high cost. We present data on the pharmacoeconomic impact of the use of plerixafor as an up-front mobilization in patients with multiple myeloma (MM).MethodsPatients with MM who underwent ASCT between January 2008 and April 2011 at the Mount Sinai Medical Center were reviewed retrospectively. In April 2010, practice changes were instituted for patients with MM to delay initiation of granulocyte-colony-stimulating factor (G-CSF) support from day 0 to day +5 and to add plerixafor to G-CSF as an up-front autologous mobilization. Targets of collection were 5–10 × 10⁶ CD34⁺ cells/kg.ResultsOf 50 adults with MM who underwent ASCT, 25 received plerixafor/filgrastim and 25 received G-CSF alone as an up-front mobilization. Compared with the control, plerixafor mobilization yielded higher CD34⁺ cell content (16.1 versus 8.4 × 10⁶ CD34⁺ cells/kg; P = 0.0007) and required fewer sessions of apheresis (1.9 versus 3.1; P = 0.0001). In the plerixafor group, the mean number of plerixafor doses required per patient was 1.8. Although the overall cost of medications was higher in the plerixafor group, the cost for blood products and overall cost of hospitalization were similar between the two groups.ConclusionsUp-front use of plerixafor is an effective mobilization strategy in patients with MM and does not have a substantial pharmacoeconomic impact in overall cost of hospitalization combined with the apheresis procedure.     

Kinetics of the use of cryopreserved autologous stem cell grafts: a GITMO-SIDEM survey

Background aimsHematopoietic stem cell cryopreservation significantly contributed to autologous stem cell transplantation (ASCT). Cryopreserved stem cell units (SCU) are expected to be used soon after harvesting for most purposes, but, in a number of cases, they remain stored for some time, creating an increasing load for SCU depositories. Disposal policies vary widely in each center, and the existing guidelines are insufficient.MethodsWe conducted a survey of seven Gruppo Italiano Trapianto di Midollo Osseo centers to investigate the outcome of SCU harvested from January 2005 to December 2009 for ASCT. The data from 1603 collections were gathered, for a total of 5822 SCU.ResultsIn our cohort, 79% of patients collected >5 × 10⁶ CD34+ cells/kg, and 3.4% collected <2 × 10⁶ CD34+ cells/kg. Up to 21% of all the patients and 42% of those with acute leukemia did not undergo reinfusion, and 37% of the cryopreserved SCU were excess, resulting from patients not reinfusing or partially reinfusing. Less than one-third of the excess SCU was disposed, and the major causes of disposal were death and, in a minority of cases, withdrawal of the indication for ASCT. In our analysis, very few first reinfusions occurred after 2 years, and those after 5 years were exceptional. Through the use of a multivariate analysis, we sought to identify the risk factors for collection non-use, independent of the centers' policies. Non-use of SCU was significantly associated with patients with acute leukemia, collections of <2 × 10⁶ CD34/kg and lower age groups.ConclusionsThese data serve as a valid basis to support rational recommendations for cost-effective storage and disposal of SCU.     

Expanded umbilical cord blood T cells used as donor lymphocyte infusions after umbilical cord blood transplantation

BackgroundUmbilical cord blood (UCB) is an alternative graft source for hematopoietic stem cell transplantation and has been shown to give results comparable to transplantation with other stem cell sources. Donor lymphocyte infusion (DLI) is an effective treatment for relapsed malignancies after hematopoietic stem cell transplantation. However, DLI is not available after UCB transplantation.MethodsIn this study, in vitro–cultured T cells from the UCB graft were explored as an alternative to conventional DLI. The main aim was to study the safety of the cultured UCB T cells used as DLI because such cell preparations have not been used in this context previously. We also assessed potential benefits of the treatment.ResultsThe cultured UCB T cells (UCB DLI) were given to 4 patients with mixed chimerism (n = 2), minimal residual disease (n = 1) and graft failure (n = 1). No adverse reactions were seen at transfusion. Three of the patients did not show any signs of graft-versus-host disease (GVHD) after UCB DLI, but GVHD could not be excluded in the last patient. In the patient with minimal residual disease treated with UCB DLI, the malignant cell clone was detectable shortly before infusion but undetectable at treatment and for 3 months after infusion. In 1 patient with mixed chimerism, the percentage of recipient cells decreased in temporal association with UCB DLI treatment.ConclusionsWe saw no certain adverse effects of treatment with UCB DLI. Events that could indicate possible benefits were seen but with no certain causal association with the treatment.     

PET/CT helps to determine treatment duration in patients with resected as well as inoperable alveolar echinococcosis

PurposeThe aim of the study was to determine the role of ¹⁸F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) at the end of benzimidazole therapy in alveolar echinococcosis.MethodsA total of 22 patients undergoing PET/CT at the end of benzimidazole therapy were retrospectively registered. Maximum standardized uptake values (SUV_max) were measured in remaining echinococcus manifestations and compared to normal liver tissue. Long-term clinical follow-up was performed, and recorded data included laboratory parameters, clinical information and imaging.ResultsAll patients had no detectable levels of Em-18 antibodies and all echinococcus manifestations were negative on PET/CT, i.e. without focally increased FDG uptake or uptake higher than normal/non-infected liver tissue. All manifestations displayed significantly less FDG-uptake than normal liver tissue, i.e. SUVmax 1.8 (interquartile range (IQR) 1.5–3.5) vs. 3.0 (IQR 2.6–5.7), (p < 0.001). Patients were clinically followed for a median of 9.5 years (IQR 6.5–32.0 years) after their initial diagnosis and for 4.5 years (IQR 3.0–14.0 years) after discontinuation of benzimidazole therapy. No patient showed signs of recurrent infection at the last clinical visit. The 10-year and 20-year freedom from all-cause mortality was 95.0% (95% confidence interval 69.5% - 99.3%), for both. Two events occurred in 292 patient years of follow-up; i.e. two patients (9%) died, one because of pancreatic cancer, the other one because of unknown reasons with no detectable antibody levels.ConclusionsNegative FDG-PET/CT results combined with no detectable levels of Em-18 antibodies may allow for the safe discontinuation of benzimidazole therapy in patients with alveolar echinococcosis.     

Allogeneic cord blood regulatory T cells can resolve lung inflammation

Background aimsCD4⁺CD25⁺CD127^lo regulatory T cells (Tregs) are responsible for maintaining immune homeostasis. Tregs can be rendered defective and deficient as a result of the immune imbalance seen in lung injury, and such dysfunction can play a major role in continued tissue inflammation. The authors hypothesized that adoptive therapy with healthy allogeneic umbilical cord blood (UCB)-derived Tregs may be able to resolve inflammation.ResultsEx vivo-expanded UCB Tregs exhibited a unique phenotype with co-expression of CD45RA⁺CD45RO⁺ >80% and lung homing markers, including CD49d. UCB Tregs did not turn pathogenic when exposed to IL-6. Co-culture with increasing doses of dexamethasone led to a synergistic increase in UCB Treg-induced apoptosis of conventional T cells (Tcons), which translated into significantly higher suppression of proliferating Tcons, especially at a lower Treg:Tcon ratio. Multiple injections of UCB Tregs led to their preferential accumulation in lung tissue in an immune injury xenogenic model. A significant decrease in lung resident cytotoxic CD8⁺ T cells (P = 0.0218) correlated with a sustained decrease in their systemic distribution compared with controls (P < 0.0001) (n = 7 per arm) as well as a decrease in circulating human soluble CD40 ligand level (P = 0.031). Tissue architecture was preserved in the treatment arm, and a significant decrease in CD3⁺ and CD8⁺ burden was evident in immunohistochemistry analysis.ConclusionsUCB Treg adoptive therapy is a promising therapeutic strategy for treatment of lung injury.     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

IntroductionHuman fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.MethodsHuman fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.ResultsThe addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.ConclusionThe inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Hypothermia in Preterm Infants in the First Hours after Birth: Occurrence,Course and Risk Factors

BackgroundHypothermia is associated with increased morbidity and mortality rates. Preterm infants frequently have hypothermia when they are admitted to the NICU, but there is no data on the occurrence of hypothermia during the first hours after admission.ObjectiveTo investigate the occurrence of hypothermia in preterm infants in the first three hours of admission and to identify risk factors.MethodsInfants < 32 weeks of gestation included in a randomized trial with admission temperature as primary outcome were retrospectively analyzed for the occurrence of hypothermia (< 36.5°C) in the first three hours after admission. Risk factors were identified using linear regression analysis and logistic regression.ResultsIn total 80 infants were included with a median (IQR) gestational age at birth of 29 (27–30) weeks. In 93% of the infants hypothermia occurred in the first three hours after admission. The median (IQR) duration of hypothermia was 101 (34–162) minutes, of which 24 (7–52) minutes the hypothermia was mild, 45 (4–111) minutes moderate, severe hypothermia hardly occurred. Gestational age and the occurrence of hypothermia at birth were independent risk factors for the occurrence of moderate and severe hypothermia and significantly correlated with duration of hypothermia.ConclusionsHypothermia occurred often and for a long period in preterm infants in the first three hours of life, low gestational age and admission temperature were independent risk factors     

Effects of long-term cryopreservation on peripheral blood progenitor cells

Background aimsThe long-term stability of cryopreserved peripheral blood progenitor cells is an important issue for patients experiencing disease relapse. However, there is no consensus on how to evaluate the long-term effects of cryopreservation. We describe the effect of cryopreservation on viability and progenitor colony activity from 87 individual samples processed at the Scripps Green Hospital Stem Cell Processing Center (La Jolla, CA, USA).MethodsWe randomly selected 87 peripheral blood hematopoietic stem cell (PBHSC) samples from 60 patients and evaluated the effect of cryopreservation on sample viability and red and white cell colony activity after < 24 h and 7, 10 and 15 years of cryopreservation. Viability was assayed via trypan blue dye exclusion and activity was measured following 14 days of culture.ResultsAn age at collection older than 50 years may result in suboptimal activity and viability following long-term cryopreservation, while gender and disease status had no effect. Cryopreservation did not significantly affect white or red cell activity following 10 years of cryopreservation. However, for samples stored longer than 10 years, viability and activity significantly decreased. We noted a positive association between higher pre-cryopreservation %CD34 count and colony activity.ConclusionsCryopreservation of peripheral blood progenitor cells for up to 10 years results in no loss of clonogenic capacity, as determined by culture activity, although longer durations of storage may affect activity. Until validated methods are developed, cryopreserved grafts should be evaluated based on pre-freeze CD34⁺ cell counts as assayed by flow cytometry, and post-thaw sample evaluation should be reserved for patients identified as poor mobilizers.