Practical and Safe Method of Long-Term Cryopreservation for Clinical Application of Human Adipose-Derived Mesenchymal Stem Cells Without a Programmable Freezer Or Serum.

BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) have emerged as a promising modality for cellular therapy. However, techniques of ADSC cryopreservation, which can facilitate their clinical application, haven't been established yet. OBJECTIVE: To determine optimal conditions for ADSC cryopreservation. MATERIALS AND METHODS: We used three cryoprotectants [serum containing 10% dimethyl sulfoxide; CP-1TM (5% dimethyl sulfoxide, serum-free); Stem-CellBankerTM (dimethyl sulfoxide and serum-free)], two storage temperatures (-80°C, -150°C) and two cell densities (1 × 106, 7 × 106 cells/mL). Storage was up to 18 months using cryovials. We didn't use a rate-controlled freezer or liquid nitrogen storage. RESULTS: We found that CP-1TM was a suitable cryoprotectant. Storage at -150°C and higher cell density (7×106 cells/mL) kept the best viability of ADSCs, but storage at -80°C and a lower cell density (1×106 cells/mL) is acceptable for up to 9 months. We also confirmed large quantities of ADSCs, stored with CP-1 in a cryobag, were still viable after -150°C cryopreservation for 24 months. CONCLUSION: We have developed a safe, cost-effective way to cryopreserve ADSCs that could be used in the clinical setting.

Emerging investigator series: investigation of mercury emission sources using Hg isotopic compositions of atmospheric mercury at the Cape Hedo Atmosphere and Aerosol Monitoring Station (CHAAMS), Japan.

This study conducted mercury (Hg) isotopic analysis, which has been expected as a new indicator for understanding the behavior of atmospheric Hg. The dominant atmospheric Hg species, namely gaseous elemental mercury (GEM, Hg0), were collected at the Cape Hedo Atmosphere and Aerosol Monitoring Station (CHAAMS) in Okinawa, Japan, for evaluating possible source(s) and transformation process(es) of Hg. The Hg isotopic compositions of GEM samples showed that the mass-dependent fractionation (MDF) of δ202Hg and the mass-independent fractionation (MIF) of Δ199Hg ranged from -2.15‰ to 0.79‰ and from -0.32‰ to 0.00‰, respectively. The results were classified into two groups: (1) negative δ202Hg and near-zero Δ199Hg in summer and (2) near-zero δ202Hg and negative Δ199Hg in the other season. According to the NOAA Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) model, the dominant air masses traveled from East Asia during winter and South and East Asia during summer. However, the air masses also traveled from mainland Japan and rotated around Okinawa before reaching CHAAMS. In contrast, clear positive correlations between δ202Hg values and CO and PM2.5 concentrations were observed during summer. A small peak of Ox concentration was observed at three atmospheric monitoring stations, namely Nago, Naha, and Miyako Island during summer. Since Miyako Island is located ∼370 km southwest of CHAAMS, the main emission source of GEM transported to CHAAMS was not from mainland Okinawa but traveled from the southwest during summer.

Comparable outcomes between autologous and allogeneic transplant for adult acute myeloid leukemia in first CR.

Although allogeneic hematopoietic stem cell transplantation from an HLA-matched sibling donor (MSD) is a potentially curative post-remission treatment for adults with acute myeloid leukemia (AML) in their first CR, transplant-related morbidity and mortality remains a major drawback. We retrospectively compared the outcomes of patients who underwent autologous peripheral blood stem cell transplantation (auto-PBSCT; n=375) with those who underwent allogeneic bone marrow transplantation (allo-BMT; n=521) and allo-PBSCT (n=380) from MSDs for adults with AML/CR1, in which propensity score models were used to adjust selection biases among patients, primary physicians and institutions to overcome ambiguity in the patients' background information. Both the multivariate analysis and propensity score models indicated that the leukemia-free survival rate of auto-PBSCT was not significantly different from that of allo-BMT (hazard ratio (HR), 1.23; 95% confidence interval (CI), 0.92 to 1.66; P=0.16) and allo-PBSCT (HR, 1.13; 95% CI, 0.85-1.51; P=0.40). The current results suggest that auto-PBSCT remains a promising alternative treatment for patients with AML/CR1 in the absence of an available MSD.

Decision analysis for donor selection in stem cell transplantation-HLA-8/8 allele-matched unrelated donor vs HLA-1 AG mismatched related donor.

Risk of relapse during the unrelated donor coordination period biases comparisons between allogeneic hematopoietic stem cell transplantation from an HLA 8 of 8 allele-matched unrelated donor (8/8 MUD) and that from a related donor with an HLA-1 antigen mismatch in the graft-versus-host (GVH) direction (RD/1AGMM-GVH). To reduce this bias, we performed a decision analysis focusing on acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in first complete remission (CR1). The primary outcome measure was 5-year survival probability with or without quality-of-life (QOL) adjustment. A baseline analysis showed that the decision to perform MUD transplantation was superior to that to perform RD/1AGMM-GVH transplantation for patients with AML or ALL. However, in the ALL cohort, the direction of superiority was reversed when the interval between CR1 and 8/8 MUD transplantation was >5.5 months (without QOL adjustment) or >6 months (after QOL adjustment) or when overall survival of RD/1AGMM-GVH transplantation improved by 1.3% without QOL adjustment and 2.1% after QOL adjustment. In conclusion, 8/8 MUD should be prioritized in transplantation for AML and ALL in CR1. However, the MUD coordination period and improvements in RD/1AGMM-GVH transplantation might change the donor selection priority in transplantation for ALL in CR1.

Successful hyperbaric oxygen therapy for refractory BK virus-associated hemorrhagic cystitis after cord blood transplantation.

BK virus-associated hemorrhagic cystitis (BKV-HC) is a common and major cause of morbidity in recipients of allogeneic hematopoietic stem cell transplantation. A 32-year-old woman developed severe BKV-HC on day 24 after cord blood transplantation (CBT). Despite supportive therapies - such as hyperhydration, forced diuresis, and urinary catheterization - macroscopic hematuria and bladder irritation persisted for over a month. Hyperbaric oxygen (HBO) therapy at 2.1 atmospheres for 90 min per day was started on day 64 after CBT. Macroscopic hematuria resolved within a week, and microscopic hematuria was no longer detectable within 2 weeks. Hematuria did not recur after 11 sessions of HBO therapy, and no significant side effects were observed during or after treatment. HBO therapy could thus be useful in controlling refractory BKV-HC after CBT.

Relative incidences and outcomes of Clostridium difficile infection following transplantation of unrelated cord blood, unrelated bone marrow, and related peripheral blood in adult patients: a single institute study.

BACKGROUND: Clostridium difficile is a major cause of nosocomial diarrhea. The incidence and prognosis of C. difficile-associated diarrhea (CDAD) has not yet been assessed in adult patients after unrelated cord blood transplantation (uCBT). METHODS: The medical records of 135 adult unrelated cord blood transplant recipients were reviewed retrospectively to investigate the clinical features of CDAD after uCBT. These data were compared to medical records of 39 unrelated bone marrow transplant recipients and 27 related peripheral blood stem cell transplant recipients as controls. RESULTS: A total of 17 recipients developed CDAD, with onset occurring at a median of 22 days (range, 0-56 days) after transplantation. Among the unrelated cord blood transplant recipients, 11 (9%) developed CDAD. These results were comparable with those of CDAD after unrelated bone marrow transplantation (uBMT) (2/39, 6%) and related peripheral blood stem cell transplantation (rPBSCT) (4/27, 16%) (P=0.37). Fifteen of the infected recipients were successfully treated with oral metronidazole, vancomycin, or cessation of antibiotics. The remaining 2 recipients who developed CDAD after uCBT died of other causes. The development of CDAD did not negatively affect overall survival after uCBT. CONCLUSIONS: These data indicate that the incidence and prognosis of CDAD after uCBT are comparable with those after uBMT and rPBSCT.

Pharmacodynamics of the glucagon-like peptide-1 receptor agonist lixisenatide in Japanese and Caucasian patients with type 2 diabetes mellitus poorly controlled on sulphonylureas with/without metformin.

AIMS: The PDY6797 study evaluated efficacy, safety and pharmacodynamics of lixisenatide in Japanese and Caucasian patients with type 2 diabetes mellitus (T2DM) insufficiently controlled with sulphonylureas with/without metformin. METHODS: This randomized, double-blind, placebo-controlled trial comprised a single-dose assessment of lixisenatide 5 and 10 µg, and a 5- to 6-week repeated dose-escalation assessment of lixisenatide 5 to 30 µg once (QD) or twice daily (BID). The primary endpoint was change in postprandial plasma glucose (PPG) area under the curve (AUC)[0:29-4:30 h] after a standardized breakfast at the highest tolerated lixisenatide dose. Change from baseline in glycated haemoglobin (HbA1c), 2-h PPG and fasting plasma glucose (FPG) were assessed, as were adverse events. RESULTS: Change from baseline in PPG AUC[0:29-4:30 h] with lixisenatide QD and BID was significantly greater than placebo (p < 0.0001 for all study populations), with particularly prominent effects in Japanese patients. Greater reductions in PPG AUC[0:29-4:30 h] were seen with lixisenatide QD versus BID, while the totality of evidence suggested that the lixisenatide 20 µg dose was optimal. In the overall population, changes from baseline for 2-h PPG, HbA1c and FPG were significant with lixisenatide QD and BID versus placebo (p < 0.01 for all). Lixisenatide was well tolerated. CONCLUSIONS: Lixisenatide significantly reduced PPG AUC[0:29-4:30 h] versus placebo at the highest well-tolerated dose in patients with T2DM treated with sulphonylureas with/without metformin and had a good safety and tolerability profile. Japanese patients experienced particular benefits with lixisenatide in terms of reductions in PPG excursions.

Randomized, double-blind, placebo-controlled trial of the once-daily GLP-1 receptor agonist lixisenatide in Asian patients with type 2 diabetes insufficiently controlled on basal insulin with or without a sulfonylurea (GetGoal-L-Asia).

AIMS: To assess the efficacy and safety of once-daily lixisenatide versus placebo in Asian patients with type 2 diabetes insufficiently controlled on basal insulin ± sulfonylurea. METHODS: In this 24-week, randomized, double-blind, placebo-controlled, parallel-group, multicentre study, participants (mean baseline HbA(1c) 8.53%) from Japan, Republic of Korea, Taiwan and the Philippines received lixisenatide (n = 154) or placebo (n = 157) in a stepwise dose increase to 20 µg once daily. The primary endpoint was HbA(1c) change from baseline to week 24. RESULTS: Once-daily lixisenatide significantly improved HbA(1c) versus placebo (LS mean difference vs. placebo = -0.88% [95%CI= -1.116, -0.650]; p < 0.0001), and allowed more patients to achieve HbA(1c) <7.0% (35.6 vs. 5.2%) and ≤ 6.5% (17.8 vs. 1.3%). Lixisenatide also significantly improved 2-h postprandial plasma glucose and glucose excursion, average 7-point self-monitored blood glucose and fasting plasma glucose. Lixisenatide was well tolerated; 86% of patients on lixisenatide completed the study versus 92% on placebo. Ten (6.5%) lixisenatide and 9 (5.7%) placebo patients experienced serious adverse events. More lixisenatide patients [14 (9.1%)] discontinued for adverse events versus placebo [5 (3.2%)], mainly with gastrointestinal causes. Nausea and vomiting were reported in 39.6 and 18.2% of patients on lixisenatide versus 4.5 and 1.9% on placebo. Symptomatic hypoglycaemia was more frequent with lixisenatide (42.9%) versus placebo (23.6%), but was similar between groups (32.6 vs. 28.3%, respectively), in those not receiving sulfonylureas. No severe hypoglycaemia was reported. CONCLUSIONS: In an Asian type 2 diabetes population insufficiently controlled by basal insulin ± sulfonylurea, once-daily lixisenatide significantly improved glycaemic control, with a pronounced postprandial effect, and was well tolerated.

Successful treatment of Trichosporon fungemia in a patient with refractory acute myeloid leukemia using voriconazole combined with liposomal amphotericin B.

Trichosporon fungemia is a rare and fatal fungal infection that occurs in patients with prolonged neutropenia associated with hematologic malignancies. A 21-year-old male developed Trichosporon fungemia during remission induction therapy for acute myeloid leukemia (AML). Although two courses of induction therapy failed to induce a remission of AML, combination therapy with voriconazole and liposomal amphotericin B (L-AmB) followed by monocyte colony-stimulating factor ameliorated the Trichosporon fungemia and enabled the patient to receive reduced-intensity bone marrow transplantation (BMT) from his human leukocyte antigen-A one-locus mismatched mother. The patient achieved a durable remission after BMT without exacerbation of Trichosporon fungemia. The combination therapy with voriconazole and L-AmB may therefore be useful in controlling Trichosporon fungemia associated with prolonged neutropenia after remission induction therapy for AML.

Safety of pre-engraftment prophylactic foscarnet administration after allogeneic stem cell transplantation.

Human herpesvirus-6 (HHV-6) is a major cause of limbic encephalitis with a dismal prognosis after allogeneic hematopoietic stem cell transplantation (SCT). Because our previous trial of preemptive therapy with foscarnet sodium (phosphonoformic acid; PFA) failed to prevent HHV-6 encephalitis, we conducted a prospective study to examine the safety of prophylactic PFA administration and elucidate the changes in the plasma HHV-6 DNA levels in the early post-SCT period. Plasma HHV-6 DNA was measured thrice weekly from day 6. PFA, 90 mg/kg/day, was administered from days 7 to 21 after bone marrow or peripheral blood SCT and to day 25 after umbilical cord blood transplantation. Of the 10 patients enrolled, 2 dropped out of the study, 1 because of early death, and 1 with a low glomerular filtration rate. Grade 3 or greater adverse events occurred in 9 of the 10 prophylactic PFA patients and in 7 of the 10 control patients who had clinical backgrounds similar to the study subjects and underwent SCT during the same period. Neurological disorders developed in none of the study subjects but in 4 of the 10 control patients, including 2 with HHV-6 encephalitis. HHV-6 reactivation occurred in 3 of the 10 study subjects. The prophylactic PFA regimen was thus safe and it may reduce the risk of limbic encephalitis, but is not considered to be potent enough to prevent HHV-6 reactivation.

Allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia with t(6;9)(p23;q34) dramatically improves the patient prognosis: a matched-pair analysis.

Acute myeloid leukemia (AML) with t(6;9)(p23;q34) is well known to have a poor prognosis treated with chemotherapy and autotransplantation. The presence of this karyotype is an indicator for allogeneic hematopoietic stem cell transplantation (HSCT); however, the impact of t(6;9)(p23;q34) on the HSCT outcome remains unclear. We conducted a matched-pair analysis of de novo AML patients with and without t(6;9)(p23;q34) using data obtained from the Japanese HSCT data registry. A total of 57 patients with t(6;9)(p23;q34) received transplants between 1996 and 2007, and 171 of 2056 normal karyotype patients matched for age, disease status at HSCT and graft source were selected. The overall survival, disease-free survival, cumulative incidence of relapse and the non-relapse mortality in t(6;9)(p23;q34) patients were comparable to those for normal karyotype patients. A univariate analysis showed that t(6;9)(p23;q34) had no significant impact on the overall survival. These findings suggest that allogeneic HSCT may overcome the unfavorable impact of t(6;9)(p23;q34) as an independent prognostic factor.

A single nucleotide polymorphism of IL-17 gene in the recipient is associated with acute GVHD after HLA-matched unrelated BMT.

IL-17 has an important role in the host defense against extracellular pathogens and the pathophysiology of autoimmune diseases. This study retrospectively examined the impact of a single-nucleotide polymorphism (rs2275913, G197A) in the IL-17 gene of a total 510 recipients with hematologic malignancies and their unrelated donors on the clinical outcomes in HLA-matched myeloablative (discovery study) and nonmyeloablative (validation study) BMT through the Japan Marrow Donor Program (JMDP). In the discovery study, the presence of a 197A genotype in the recipient resulted in a higher incidence of grades II-IV acute GVHD (hazard ratio (HR), 1.87; 95% confidence interval (CI), 1.23-2.85; P=0.004). The donor IL-17A genotype did not significantly influence the transplant outcomes. The validation study showed a trend toward an association of the recipient 197A genotype with an increased risk of grades III-IV acute GVHD (HR, 5.84; 95% CI, 0.75-45.72; P=0.09), as well as a significantly increased risk for chronic GVHD (HR, 3.86; 95% CI, 1.29-11.59; P=0.02). These results suggest an association of the 197A genotype in the recipient side with the development of acute GVHD.

A single-nucleotide polymorphism of the Fcγ receptor type IIIA gene in the recipient predicts transplant outcomes after HLA fully matched unrelated BMT for myeloid malignancies.

Fcγ receptor type IIIA (FCGR3A) has a functional single-nucleotide polymorphism (rs396991), at which a G-to T-point mutation results in an amino acid substitution at position 158 (valine to phenylalanine; V158F). This study examined the effect of the FCGR3A polymorphism in donors and recipients on the clinical outcomes in unrelated HLA fully matched myeloablative BMT. The FCGR3A-V158F genotype was retrospectively analyzed in a total of 99 recipients with myeloid malignancies, and their unrelated donors. The presence of the 158V genotype in recipients showed a statistically better OS (adjusted hazard ratio (HR) 0.49; 95% confidence interval (CI) 0.26-0.93; P=0.03) and TRM (HR 0.30; 95% CI 0.14-0.67; P=0.003) without significant influence on the relapse rate. The recipient 158V genotype was also associated with a significantly reduced risk of chronic GVHD (HR 0.45; 95% CI 0.20-0.99; P=0.049) and a trend toward a reduced risk of grade II-IV acute GVHD (HR 0.55; 95% CI 0.27-1.10; P=0.09), leading to a significantly reduced GVHD-related mortality (HR 0.22; 95% CI 0.06-0.77; P=0.02). The donor FCGR3A polymorphism did not have any effect on the transplant outcomes. These results suggest an association between the recipient FCGR3A genotype and the clinical outcomes after BMT.

Measurement system for particulate nitrate based on the scrubber difference NO-O3 chemiluminescence method in remote areas.

A particulate nitrate analyzer based on a scrubber difference/NO-O3 chemiluminescence method (SD-CL method) has been developed for measuring nitrate concentrations in remote areas. Particulate nitrate concentrations (NO3(-)(p)) were analyzed by the difference between the concentrations of NOy - gaseous nitric acid (HNO3) and NOy - HNO3 - NO3(-)(p). Annular denuders coated with NaCl and PTFE filter were used as the scrubbers for HNO3 and NO3(-)(p), respectively. The transmission efficiency of coarse particles in the denuder was found to be 93.4 ± 5.8%, so the loss of NO3(-)(p) to the denuder was within the uncertainty of the particulate nitrate analyzer (±20%). The measurements of NOy, HNO3, and NO3(-)(p) were conducted from March 15 to April 31, 2008, at Cape Hedo, Okinawa, Japan. Over 99.5% of the observed concentrations of NO3(-)(p) for 10 min integration times were higher than the detection limit of the SD-CL method (0.18 µg m(-3)). The least-squares fit of the R&P nitrate monitor against the SD-CL method yielded a slope of 0.67 ± 0.02 and a correlation coefficient of R = 0.73. This result indicates that this method could also measure NO3(-)(p) when the diameter of aerosols was larger than 10 µm. The SD-CL method was found to be useful as a measurement system for NO3(-)(p) in remote areas where coarse NO3(-)(p) dominates.

Evolution of organic aerosols in the atmosphere.

Organic aerosol (OA) particles affect climate forcing and human health, but their sources and evolution remain poorly characterized. We present a unifying model framework describing the atmospheric evolution of OA that is constrained by high-time-resolution measurements of its composition, volatility, and oxidation state. OA and OA precursor gases evolve by becoming increasingly oxidized, less volatile, and more hygroscopic, leading to the formation of oxygenated organic aerosol (OOA), with concentrations comparable to those of sulfate aerosol throughout the Northern Hemisphere. Our model framework captures the dynamic aging behavior observed in both the atmosphere and laboratory: It can serve as a basis for improving parameterizations in regional and global models.

Immature platelet fraction for prediction of platelet engraftment after allogeneic stem cell transplantation.

Platelet regeneration represents an important and separate element in the engraftment process for allogeneic stem cell transplantation. Fully automated flow cytometry using blood cell counters now allows reliable quantification of reticulated platelets, expressed as the immature platelet fraction (IPF). We studied the kinetics of IPF in six patients grafted with allogeneic peripheral blood stem cell transplantation (PBSCT), 12 patients with bone marrow transplantation (BMT) and seven patients with cord blood transplantation (CBT). Preconditioning therapy caused an immediate and rapid fall in tri-lineage hematopoiesis. IPF rose transiently above 3% after a mean duration of 11 days post-PBSCT, 18 days post-BMT and 19 days post-CBT. This was 1, 4 and 13 days earlier than platelet engraftment, respectively. A linear correlation model showed a close association between the rise of IPF and tri-lineage engraftment after transplantation. IPF counting may thus provide an accessible measure of thrombopoietic activity, leading to early evaluation of marrow function and allowing monitoring of platelet regeneration.