Distinct Biomarker Profiles in Ex Vivo T Cell Depletion Graft Manipulation Strategies: CD34+ Selection versus CD3+/19+ Depletion in Matched Sibling Allogeneic Peripheral Blood Stem Cell Transplantation

Various approaches have been developed for ex vivo T cell depletion in allogeneic stem cell transplantation to prevent graft-versus-host disease (GVHD). Direct comparisons of T cell depletion strategies have not been well studied, however. We evaluated cellular and plasma biomarkers in 2 different graft manipulation strategies, CD3⁺CD19⁺ cell depletion (CD3/19D) versus CD34⁺ selection (CD34S), and their associations with clinical outcomes. Identical conditions, including the myeloablative preparative regimen, HLA-identical sibling donor, GVHD prophylaxis, and graft source, were used in the 2 cohorts. Major clinical outcomes were similar in the 2 groups in terms of overall survival, nonrelapse mortality, and cumulative incidence of relapse; however, the cumulative incidence of acute GVHD trended to be higher in the CD3/19D cohort compared with the CD34S cohort. A distinct biomarker profile was noted in the CD3/19D cohort: higher levels of ST2, impaired Helios^? FoxP3⁺Treg reconstitution, and rapid reconstitution of naïve, Th2, and Th17 CD4 cells in the early post-transplantation period. In vitro graft replication studies confirmed that CD3/19D disproportionately depleted Tregs and other CD4 subset repertoires in the graft. This study confirms the utility of biomarker monitoring, which can be directly correlated with biological consequences and possible future therapeutic indications.     

2013 Annual Report of the American Association of Poison Control Centers’ National Poison Data System (NPDS): 31st Annual Report

Background: This is the 31st Annual Report of the American Association of Poison Control Centers’ (AAPCC) National Poison Data System (NPDS). As of January 1, 2013, 57 of the nation's poison centers (PCs) uploaded case data automatically to NPDS. The upload interval was 8.08 [7.10, 11.63] (median [25%, 75%]) minutes, creating a near real-time national exposure and information database and surveillance system.

Methodology: We analyzed the case data tabulating specific indices from NPDS. The methodology was similar to that of previous years. Where changes were introduced, the differences are identified. Poison center (PC) cases with medical outcomes of death were evaluated by a team of 38 medical and clinical toxicologist reviewers using an ordinal scale of 1–6 to assess the Relative Contribution to Fatality (RCF) of the exposure to the death.

Results: In 2013, 3,060,122 closed encounters were logged by NPDS: 2,188,013 human exposures, 59,496 animal exposures, 806,347 information calls, 6,116 human-confirmed nonexposures, and 150 animal-confirmed nonexposures. Total encounters showed a 9.3% decline from 2012, while health care facility human exposure calls were essentially flat, decreasing by 0.1%.All information calls decreased 21.4% and health care facility (HCF) information calls decreased 8.5%, medication identification requests (drug ID) decreased 26.8%, and human exposures reported to US PCs decreased 3.8%. Human exposures with less serious outcomes have decreased 3.7% per year since 2008 while those with more serious outcomes (moderate, major or death) have increased by 4.7% per year since 2000.

The top five substance classes most frequently involved in all human exposures were analgesics (11.5%), cosmetics/personal care products (7.7%), household cleaning substances (7.6%), sedatives/hypnotics/antipsychotics (5.9%), and antidepressants (4.2%). Sedative/hypnotics/antipsychotics exposures as a class increased most rapidly (2,559 calls/year) over the last 13 years for cases showing more serious outcomes. The top five most common exposures in children of 5 years or less were cosmetics/personal care products (13.8%), household cleaning substances (10.4%), analgesics (9.8%), foreign bodies/toys/miscellaneous (6.9%), and topical preparations (6.1%). Drug identification requests comprised 50.7% of all information calls. NPDS documented 2,477 human exposures resulting in death with 2,113 human fatalities judged related (RCF of 1, undoubtedly responsible; 2, probably responsible; or 3, contributory).

Conclusions: These data support the continued value of PC expertise and need for specialized medical toxicology information to manage the more severe exposures, despite a decrease in calls involving less severe exposures. Unintentional and intentional exposures continue to be a significant cause of morbidity and mortality in the United States. The near real-time, always current status of NPDS represents a national public health resource to collect and monitor US exposure cases and information calls. The continuing mission of NPDS is to provide a nationwide infrastructure for public health surveillance for all types of exposures, public health event identification, resilience response and situational awareness tracking. NPDS is a model system for the nation and global public health.     

Isolated rat hepatocytes as a model system for screening chelators for use in cadmium intoxication

The utility of isolated rat hepatocytes as a model system for screening potential chelators in treatment of Cd intoxication was studied. The ability of the chelators diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 2,3-dimercaptosuccinic acid (DMSA), diethyldithiocarbamic acid (DDC), d,1-penicillamine (PEN), nitrilotriacetic acid (NTA), and 2,3-dimercaptopropanol (BAL) to decrease the cellular concentration of Cd was correlated with the previously reported effectiveness of these agents in the treatment of Cd intoxication in vivo. The results of cellular studies with either control or metallothionein-induced hepatocytes were compared to the in vivo effect of the chelators administered before or after the induction of metallothionein, respectively. The effects of DTPA, EDTA, DDC, and BAL in the hepatocyte model screening system correlated well with their reported in vivo effects. The results with NTA, PEN, and DMSA were not correlated as well but were explained by the relative differences between in vivo doses versus in vitro concentrations of the respective chelators. Therefore, the proposed model for screening potential chelators for use in cadmium intoxication appears to be a system which may prove to be an economical and rapid method to facilitate the search for efficacious chelators.     

Decreased effectiveness of chelation therapy with time after acute cadmium poisoning

The effect of increasing the time interval between cadmium (Cd) exposure and chelation therapy was studied in male Swiss Webster mice. The following chelating agents were administered ip at 0, 2, 12, 36, and 72 hr after giving radioactive Cd (1 mg Cd/kg, iv): diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), and 2,3-dimercaptosuccinic acid (DMSA). Daily elimination of Cd into urine and feces was determined for 5 days after which time the mice were killed, and the concentration of Cd was determined in various organs. In most of the tissues examined, including kidney and liver, only administration of chelators immediately after Cd significantly reduced tissue Cd concentrations. The mice treated immediately after Cd administration excreted between 50 and 75% of the Cd into urine in the first 24 hr compared to 0.1% excreted by controls. Treatment at later times (2 to 72 hr) significantly increased Cd excretion but the magnitude of the effect was far less than that seen in mice treated immediately after Cd. In a separate group of mice, the time course for the induction of metallothionein synthesis was determined by administering 1 mg Cd/kg iv at various times before death. Metallothionein concentration increased within 2 hr after Cd administration and reached a maximum within 8 hr. The results show that the length of time before initiating chelation therapy for Cd poisoning greatly influences the effectiveness of the therapy. Hepatic metallothionein may be responsible for this phenomenon.     

Drug-induced torsades de pointes and implications for drug development

Torsades de pointes is a potentially lethal arrhythmia that occasionally appears as an adverse effect of pharmacotherapy. Recently developed understanding of the underlying electrophysiology allows better estimation of the drug-induced risks and explains the failures of older approaches through the surface ECG. This article expresses a consensus reached by an independent academic task force on the physiologic understanding of drug-induced repolarization changes, their preclinical and clinical evaluation, and the risk-to-benefit interpretation of drug-induced torsades de pointes. The consensus of the task force includes suggestions on how to evaluate the risk of torsades within drug development programs. Individual sections of the text discuss the techniques and limitations of methods directed at drug-related ion channel phenomena, investigations aimed at action potentials changes, preclinical studies of phenomena seen only in the whole (or nearly whole) heart, and interpretation of human ECGs obtained in clinical studies. The final section of the text discusses drug-induced torsades within the larger evaluation of drug-related risks and benefits.     

Results of the First Clinical Study in Humans That Combines Hyperbaric Oxygen Pretreatment with Autologous Peripheral Blood Stem Cell Transplantation

Patients undergoing high-dose chemotherapy and autologous hematopoietic cell transplantation (auto-HCT) are at risk for multiple morbidities, including mucosal inflammation and neutropenic fever, both related to neutropenia. Evidence from our preclinical work in an umbilical cord blood (UCB) transplantation murine model suggests that treatment with hyperbaric oxygen (HBO) before UCB infusion improves UCB CD34⁺ cell engraftment by reducing erythropoietin levels. A pilot clinical trial using HBO in patients undergoing UCB transplantation showed improvement in kinetics of blood count recovery. In this study, we evaluated HBO in combination with auto-HCT. Our primary aim was to determine the safety of HBO in this setting and secondarily to determine its efficacy in reducing time to neutrophil and platelet engraftment compared with matched historic controls. Patients with multiple myeloma, non-Hodgkin lymphoma, and Hodgkin disease eligible for auto-HCT were included. On day 0, patients received HBO treatment consisting of exposure to 2.5 atmosphere absolutes for a total of 90 minutes, in a monoplace hyperbaric chamber, breathing 100% oxygen. Six hours after the start of HBO, peripherally mobilized stem/progenitor cells were infused and patients were followed daily for toxicity and blood count recovery. All patients received daily granulocyte colony-stimulating factor starting on day +5 and until absolute neutrophil count (ANC) of ≥1500 or ANC of 500 for 3 consecutive days. A matched historic cohort of 225 patients who received auto-HCT between January 2008 and December 2012 was chosen for comparison and matched on sex, age, conditioning regimen, and disease type. We screened 26 patients for this study; 20 were treated and included in the primary analysis, and 19 completed the HBO therapy and were included in the secondary analysis. Although the median time to neutrophil count recovery was 11 days in both the HBO and control cohorts, the Kaplan-Meier estimates of the full distributions indicate that the time to neutrophil recovery was generally about 1 day sooner for HBO versus historical controls (log-rank P = .005; range, 9 to 13 for HBO patients and 7 to 18 for controls). The median time to platelet count recovery was 16 days (range, 14 to 21) for HBO versus 18 days (range, 11 to 86) for controls (log-rank P < .0001). In the secondary analysis comparing the HBO cohort who completed HBO therapy (n = 19) with our historical cohort, we evaluated neutropenic fever, growth factor use, mucositis, day +100 disease responses, and blood product use. HBO was associated with less growth factor use (median 6 days in HBO cohort versus median 8 days in controls, P < .0001). Packed RBC and platelet transfusion requirements were not statistically different between the 2 cohorts. Mucositis incidence was significantly lower in the HBO cohort (26.3% in HBO cohort versus 64.2% in controls, P = .002). HBO therapy appears to be well tolerated in the setting of high-dose therapy and auto-HCT. Prospective studies are needed to confirm potential benefits of HBO with respect to earlier blood count recovery, reduced mucositis, and growth factor use, and a cost-benefit analysis is warranted.© 2019 American Society for Blood and Marrow Transplantation.     

A flow cytometry procedure for simultaneous characterization of cell DNA content and expression of intracellular protein kinase C-zeta

A selective involvement of protein kinase C-zeta (PKC-zeta) in the events regulating cell proliferation has been recently proposed. Here we report a flow cytometric method allowing the simultaneous association of intracellular PKC-zeta expression or phosphorylation with each cell cycle phase. Current methods for flow cytometry analysis were applied to several cell lines and compared to the method developed in our laboratory. The latter includes 2% paraformaldehyde (PFA), as fixing agent, a permeabilization/saturation step by means of a solution containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.4, 0.05% NP-40, 0.25% lambda-carrageenan and 0.02% NaN3, followed by labelling with a primary antibody (PKC-zeta or P-PKC-zeta) and with the appropriate FITC-conjugated secondary antibody. Cells processed by such a method disclosed no substantial modification of light scattering features with respect to live cells. In addition, stainability with anti-PKC-zeta or anti-P-PKC-zeta antibodies was well preserved while stoichiometric staining of DNA with PI enabled accurate cell cycle analysis. Results show that a distinct up-regulation of P-PKC-zeta in G2/M phase occurs. The method here described, therefore, represents a simple, reproducible and conservative assay for a simultaneous assessment of intracellular PKC or P-PKC modulations within each cell cycle phase.     

Pharmacokinetic Model of Ascorbic Acid in Healthy Male Volunteers During Depletion and Repletion

Purpose. To develop a new pharmacokinetic model for ascorbic acid (vitamin C) since no previously published model describes ascorbic acid absorption and disposition over a broad physiologic range of doses and plasma concentrations. Methods. A new model was developed through exploratory simulations. The model was fitted to pharmacokinetic data obtained from seven healthy volunteers who underwent ascorbic acid depletion then gradual repletion. Concentrations of ascorbic acid were measured in plasma and urine. Final pharmacokinetic model parameter estimates were obtained using nonlinear regression analysis. Results. The new model included saturable absorption, distribution and renal tubular reabsorption parameters. The model described ascorbic acid concentrations in plasma, cells, and urine during depletion and gradual repletion phases with a residual error less than 15%. Conclusions. The model was useful for obtaining a new understanding of the likely causes for the complex concentration-time profile observed during gradual repletion. At doses of 200 to 2500 mg per day, the plateau in pre-dose concentrations is largely due to apparent saturation of tissue uptake and less a function of oral bioavailability and renal excretion than previously thought.     

Frizzled receptor 6 marks rare, highly tumourigenic stem-like cells in mouse and human neuroblastomas

Wnt signalling is an important component of vertebrate development, required for specification of the neural crest. Ten Wnt receptors [Frizzled receptor 1-10 (Fzd1-10)] have been identified so far, some of which are expressed in the developing nervous system and the neural crest. Here we show that expression of one such receptors, Fzd6, predicts poor survival in neuroblastoma patients and marks rare, HIF1/2 α-positive cells in tumour hypoxic areas. Fzd6 positive neuroblastoma cells form neurospheres with high efficiency, are resistant to doxorubicin killing and express high levels of mesenchymal markers such as Twist1 and Notch1. Expression of Fzd6 is required for the expression of genes of the non-canonical Wnt pathway and the spheres forming activity. When transplanted into immunodeficient mice, neuroblastoma cells expressing the Fzd6 marker grow more aggressively than their Fzd6 negative counterparts. We conclude that Fzd6 is a new surface marker of aggressive neuroblastoma cells with stem cell-like features.