Analysis of spontaneous cytomegalovirus clearance after low level reactivation using a pre-emptive treatment threshold of 4,000 IU/mL in allogeneic hematopoietic cell transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) can be a serious complication after allogeneic hematopoietic cell transplant (HCT). CMV viral load is routinely monitored, and pre-emptive therapy is initiated to prevent CMV viremia from developing into CMV organ disease based on institutional thresholds. There is no established universal threshold for pre-emptive therapy and many centers utilize different strategies. METHODS: Allogeneic HCT recipients at WVU Medicine from 2009 to 2021 were routinely initiated on pre-emptive CMV treatment for a PCR viral threshold above 4000 IU/mL. Adult patients with quantifiable values below this threshold, were analyzed to evaluate the rate of spontaneous clearance without initiation of CMV-directed therapy, during their first episode of CMV reactivation. This study excluded any patients that received letermovir prophylaxis. RESULTS: Sixty patients were included in the analysis. The spontaneous clearance rate was 60 %. The risk factors that were associated with a lower spontaneous clearance rate were reactivation within thirty days after transplant (p = 0.031), presence of graft-versus-host-disease (p = 0.031), and CMV PCR values of 2500-4000 IU/mL (p = 0.02). Although these patients had lower rates of spontaneous clearance, they still spontaneously cleared in 42 %, 42 %, and 43 % of the cases, respectively. CONCLUSION: Delaying pre-emptive treatment until a CMV PCR value of 4000 IU/mL is reached appears appropriate and decreases unnecessary treatment toxicity and resistance.

Evaluation of prolonged magnesium infusion after allogeneic hematopoietic cell transplant.

PURPOSE: Calcineurin inhibitor use after allogeneic hematopoietic cell transplantation (allo-HCT) is associated with significant magnesium wasting. Utilization of a prolonged magnesium infusion is thought to lead to a lower serum peak concentration and therefore, decreased renal wasting of magnesium. In November 2017, our institution implemented a modification to our inpatient electrolyte replacement protocol for allo-HCT recipients that extended the magnesium infusion rate from 4 g/2 h to 4 g/4 h based on this theoretical advantage. The primary objective of this study was to compare the median magnesium requirements per day of admission between patients who received magnesium 4 g/2 h to patients who received magnesium 4 g/4 h. Secondary objectives included a comparison of the per-patient median serum magnesium concentration during admission, as well as the median incremental difference in serum magnesium concentration after intravenous replacement per patient per admission. METHODS: Allo-HCT recipients who received prolonged infusion magnesium infusions were compared to a historical cohort of allo-HCT patients who received shorter IV magnesium infusions. Admissions were included if the patient had received an allo-HCT within 100 days prior, was admitted to the Transplant and Cellular Therapy Unit at WVU Medicine J.W. Ruby Memorial Hospital, and received at least one magnesium infusion and one dose of cyclosporine or tacrolimus. Admissions were excluded if the patient received oral magnesium, total parenteral nutrition, aminoglycosides, amphotericin, carboplatin, cisplatin, or foscarnet. RESULTS: The pre-implementation group consisted of 81 admissions (n = 64 patients), while the post-implementation group consisted of 90 admissions (n = 60 patients). Median magnesium requirements per day of admission were not different between groups at 1.4 g of magnesium in the pre-implementation group and 1.9 g of magnesium in the post-implementation group (P = 0.25). Median serum magnesium concentrations and median incremental difference in serum magnesium concentration after intravenous replacement were also not different between groups: 1.65 mg/dL vs 1.60 mg/dL (P = 0.65) and 0.30 mg/dL vs 0.28 mg/dL (P = 0.67), respectively. CONCLUSIONS: Prolonged infusion of magnesium in allo-HCT recipients receiving CNI therapy does not result in improvement in magnesium retention.

Evaluation of the Pharmacokinetic Interaction Between Letermovir and Tacrolimus in Allogeneic Hematopoietic Cell Transplantation Recipients.

Data describing the magnitude of the pharmacokinetic interaction between letermovir and tacrolimus in allogeneic hematopoietic cell transplantation (allo-HCT) recipients are limited, and varying outcomes have been reported. The need for empiric dose adjustment of tacrolimus on initiation of letermovir has not been established; instead, guidelines suggest closely monitoring the tacrolimus trough concentration and adjusting the dose as needed. A better understanding of this interaction is imperative to accurately manage the narrow therapeutic window of tacrolimus post-transplantation. The primary objective of this study was to determine the percent change in tacrolimus concentration-to-dose ratio over the 14-day period after initiation of letermovir. Secondary objectives were to describe the frequency of tacrolimus dose adjustments after initiation of letermovir, the percent change in daily tacrolimus dose over the 14-day period after initiation of letermovir, and the incidence of both subtherapeutic and supratherapeutic tacrolimus trough concentrations. This retrospective chart review included adult allo-HCT recipients at our institution who received tacrolimus in combination with oral letermovir and had been taking tacrolimus for at least 5 days before letermovir initiation. Patients receiving strong CYP3A4 inhibitors or i.v. tacrolimus were excluded. Thirty-five patients were included in the analysis. The median percent increase in tacrolimus concentration-to-dose ratio over the 14-day period after initiation of letermovir was 22% on days 2 to 4, 47% on days 5 to 7, 66% on days 8 to 11, and 81% on days 12 to 14. The mean frequency of tacrolimus dose adjustments was 0.66 on days 2 to 4, 0.69 on days 5 to 7, 1.06 on days 8 to 11, and 0.57 on days 12 to 14. The results of this study show that the pharmacokinetic interaction between tacrolimus and letermovir is substantial and continues to affect tacrolimus concentration over the 14-day period after letermovir initiation. Close monitoring of tacrolimus trough concentration on initiation of letermovir should be considered.

Toxicity assessment of concurrent gabapentin/pregabalin administration with high-dose melphalan in autologous hematopoietic cell transplant recipients.

A theoretical pharmacokinetic interaction mediated through L-amino acid transporter 1 and 2 exists between gabapentin (GP) and pregabalin (PG) with melphalan. Peripheral neuropathy is a common toxicity of various multiple myeloma regimens commonly utilized prior to autologous hematopoietic cell transplant (auto-HCT) with high-dose melphalan (HD-Mel). Therefore, it is likely concurrent administration of either GP or PG will occur in patients receiving HD-Mel conditioning for auto-HCT, which could potentially increase cellular uptake and worsen the mucosal injury. A retrospective chart review of adult patients from January 2012 to July 2016 who received HD-Mel (140-200 mg/m2) at West Virginia University Medicine was performed to assess toxicity and outcomes in these patients. A total of 80 patients were included in the study, with 30 patients receiving GP or PG and 50 control patients. There were no significant differences in grade 2 or higher mucositis, admissions for nausea/vomiting/diarrhea, intravenous opioid requirements, oral topical therapies, antidiarrheal medication use, rescue anti-emetics, days of nausea or vomiting, pain scores, neutrophil or platelet engraftment, treatment-related mortality, progression-free survival, or overall survival. Our data suggest that it is safe to continue GP/PG therapy throughout HD-Mel therapy, with no negative transplant outcomes. Prospective studies or evaluations of larger databases are necessary to better characterize the clinical effect of concomitant therapy.

Prospective assessment of Clostridioides (formerly Clostridium) difficile colonization and acquisition in hematopoietic stem cell transplant patients.

BACKGROUND: Patients undergoing hematopoietic stem cell transplant (HSCT) possess numerous risk factors for Clostridioides (formerly Clostridium) difficile infection (CDI) and experience a high rate of diarrhea. Colonization rates of Clostridium difficile vary greatly among subgroup analyses with recent studies demonstrating colonization rates in the blood and marrow transplant units up to nine times that of the general population. METHODS: The primary objectives of this study were to identify the rate of C difficile colonization and acquisition in HSCT patients admitted to the blood and marrow transplant unit. This was a prospective study that included all adult patients admitted for hematopoietic stem cell transplantation. Stool specimens were routinely collected on admission and weekly thereafter for a maximum of six samples per patient. RESULTS: Forty-two patients met inclusion criteria and had baseline samples available for analysis. The rate of C difficile colonization on admission was 24%, and an additional 9% of patients acquired the organism during admission. Twelve percent of patients developed CDI that was diagnosed clinically. Univariate analysis showed an increased risk of colonization for patients with three or more prior chemotherapy cycles. CONCLUSIONS: Given high colonization rates coupled with high risk of CDI in this population, providers must be judicious when testing for CDI and interpreting test results for HSCT patients.

Comparison of Graft Acquisition and Early Direct Charges of Haploidentical Related Donor Transplantation versus Umbilical Cord Blood Transplantation.

Alternative donor allogeneic hematopoietic cell transplants (HCTs), such as double umbilical cord blood transplants (dUCBT) and haploidentical related donor transplants (haplo-HCT), have been shown to be safe and effective in adult patients who do not have an HLA-identical sibling or unrelated donor available. Most transplant centers have committed to 1 of the 2 alternative donor sources, even with a lack of published randomized data directly comparing outcomes and comparative data on the cost-effectiveness of dUCBT versus haplo-HCT. We conducted a retrospective study to evaluate and compare the early costs and charges of haplo-HCT and dUCBT in the first 100 days at 2 US transplant centers. Forty-nine recipients of haplo-HCT (at 1 center) and 37 with dUCBT (at another center) were included in the analysis. We compared graft acquisition, inpatient/outpatient, and total charges in the first 100 days. The results of the analysis showed a significantly lower cost of graft acquisition and lower total charges (for 100-day HCT survivors) in favor of haplo-HCT. Importantly, to control for the obvious shortcomings of comparing costs at 2 different transplant centers, adjustments were made based on the current (2018) local wage index and inflation rate. In the absence of further guidance from a prospective study, the cost analysis in this study suggests that haplo-HCT may result in early cost savings over dUCBT and may be preferred by transplant centers and for patients with more limited resources.

Low-Dose Acyclovir Prophylaxis for Varicella zoster Reactivation in Autologous Hematopoietic Cell Transplantation Recipients.

Varicella zoster virus (VZV) reactivation after autologous hematopoietic cell transplantation (auto-HCT) may be observed in a quarter of patients. Currently, prophylactic use of acyclovir 800 mg twice daily or valacyclovir 500 mg twice daily is recommended for prophylaxis against VZV reactivation for at least one-year post-HCT, with continued use recommended in immunosuppressed recipients. Acyclovir dosing regimens vary between institutions despite the noted recommendations. In this single-center, retrospective study, recipients of auto-HCT who received at least one year of low-dose antiviral prophylaxis defined as the equivalent of acyclovir 400 mg orally twice daily or valacyclovir 500 mg daily were included. The primary objective of this study was to assess the incidence of VZV reactivation with low-dose acyclovir/valacyclovir prophylaxis in autograft recipients. One hundred and eighty patients undergoing auto-HCT between April 2008 and March 2015 were included. Patients received low-dose acyclovir, for a median duration of 55.5 months (range 12-100). There were no occurrences of VZV reactivation while patients were on these drugs. However, 2 patients (1.1%) had VZV reactivation after discontinuation of therapy, occurring 18.8 and 14 months from transplant and 6.7 and 2 months after stopping prophylaxis, respectively. Our retrospective analysis found low-dose antiviral prophylaxis with oral acyclovir 400 mg twice daily or valacyclovir 500 mg daily to be effective in preventing VZV reactivation in auto-HCT recipients.

SoxB1 Activity Regulates Sensory Neuron Regeneration, Maintenance, and Function in Planarians.

SoxB1 genes play fundamental roles in neurodevelopmental processes and maintaining stem cell multipotency, but little is known about their function in regeneration. We addressed this question by analyzing the activity of the SoxB1 homolog soxB1-2 in the planarian Schmidtea mediterranea. Expression and functional analysis revealed that soxB1-2 marks ectodermal-lineage progenitors, and its activity is required for differentiation of subsets of ciliated epidermal and neuronal cells. Moreover, we show that inhibiting soxB1-2 or its candidate target genes leads to abnormal sensory neuron regeneration that causes planarians to display seizure-like movements or phenotypes associated with the loss of sensory modalities. Our analyses highlight soxB1-2-regulated genes that are expressed in sensory neurons and are homologous to factors implicated in epileptic disorders in humans and animal models of epilepsy, indicating that planarians can serve as a complementary model to investigate genetic causes of epilepsy.

Fixation, Processing, and Immunofluorescent Labeling of Whole Mount Planarians.

Efforts to elucidate mechanisms of regeneration in the planarian Schmidtea mediterranea have included the application of immunocytochemical methods to detect specific molecules and label cells and tissues in situ. Here we describe methods for immunofluorescent labeling of whole mount planarians. We outline protocols for fixation and steps for processing animals prior to immunolabeling, incorporating commonly utilized reagents for mucus removal, pigment bleaching, tissue permeabilization, and antigen retrieval. Because processing steps can mask or degrade antigens, we also recommend protocol parameters that can be tested simultaneously to optimize sample preparation for novel antibodies.

Nervous system development and regeneration in freshwater planarians.

Planarians have a long history in the fields of developmental and regenerative biology. These animals have also sparked interest in neuroscience due to their neuroanatomy, spectrum of simple behaviors, and especially, their almost unparalleled ability to generate new neurons after any type of injury. Research in adult planarians has revealed that neuronal subtypes homologous to those found in vertebrates are generated from stem cells throughout their lives. This feat is recapitulated after head amputation, wherein animals are capable of regenerating whole brains and regaining complete neural function. In this review, we summarize early studies on the anatomy and function of the planarian nervous system and discuss our present knowledge of the molecular mechanisms governing neurogenesis in planarians. Modern studies demonstrate that the transcriptional programs underlying neuronal specification are conserved in these remarkable organisms. Thus, planarians are outstanding models to investigate questions about how stem cells can replace neurons in vivo. WIREs Dev Biol 2017, 6:e266. doi: 10.1002/wdev.266 For further resources related to this article, please visit the WIREs website.

A functional genomics screen identifies an Importin-α homolog as a regulator of stem cell function and tissue patterning during planarian regeneration.

BACKGROUND: Planarians are renowned for their regenerative capacity and are an attractive model for the study of adult stem cells and tissue regeneration. In an effort to better understand the molecular mechanisms underlying planarian regeneration, we performed a functional genomics screen aimed at identifying genes involved in this process in Schmidtea mediterranea. METHODS: We used microarrays to detect changes in gene expression in regenerating and non-regenerating tissues in planarians regenerating one side of the head and followed this with high-throughput screening by in situ hybridization and RNAi to characterize the expression patterns and function of the differentially expressed genes. RESULTS: Along with five previously characterized genes (Smed-cycD, Smed-morf41/mrg-1, Smed-pdss2/dlp1, Smed-slbp, and Smed-tph), we identified 20 additional genes necessary for stem cell maintenance (Smed-sart3, Smed-smarcc-1, Smed-espl1, Smed-rrm2b-1, Smed-rrm2b-2, Smed-dkc1, Smed-emg1, Smed-lig1, Smed-prim2, Smed-mcm7, and a novel sequence) or general regenerative capability (Smed-rbap46/48-2, Smed-mcm2, Smed-ptbp1, and Smed-fen-1) or that caused tissue-specific defects upon knockdown (Smed-ddc, Smed-gas8, Smed-pgbd4, and Smed-b9d2). We also found that a homolog of the nuclear transport factor Importin-α plays a role in stem cell function and tissue patterning, suggesting that controlled nuclear import of proteins is important for regeneration. CONCLUSIONS: Through this work, we described the roles of several previously uncharacterized genes in planarian regeneration and implicated nuclear import in this process. We have additionally created an online database to house our in situ and RNAi data to make it accessible to the planarian research community.

Novel monoclonal antibodies to study tissue regeneration in planarians.

BACKGROUND: Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians. RESULTS: We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments. CONCLUSIONS: The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand planarian biology and to characterize phenotypes following RNAi experiments. In addition, we present alterations to fixation protocols and demonstrate how these changes can increase the labeling efficiencies of antibodies used to stain whole planarians.

Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases.

Chromatin regulation is a fundamental mechanism underlying stem cell pluripotency, differentiation, and the establishment of cell type-specific gene expression profiles. To examine the role of chromatin regulation in stem cells in vivo, we study regeneration in the freshwater planarian Schmidtea mediterranea. These animals possess a high concentration of pluripotent stem cells, which are capable of restoring any damaged or lost tissues after injury or amputation. Here, we identify the S. mediterranea homologs of the SET1/MLL family of histone methyltransferases and COMPASS and COMPASS-like complex proteins and investigate their role in stem cell function during regeneration. We identified six S. mediterranea homologs of the SET1/MLL family (set1, mll1/2, trr-1, trr-2, mll5-1 and mll5-2), characterized their patterns of expression in the animal, and examined their function by RNAi. All members of this family are expressed in the stem cell population and differentiated tissues. We show that set1, mll1/2, trr-1, and mll5-2 are required for regeneration and that set1, trr-1 and mll5-2 play roles in the regulation of mitosis. Most notably, knockdown of the planarian set1 homolog leads to stem cell depletion. A subset of planarian homologs of COMPASS and COMPASS-like complex proteins are also expressed in stem cells and implicated in regeneration, but the knockdown phenotypes suggest that some complex members also function in other aspects of planarian biology. This work characterizes the function of the SET1/MLL family in the context of planarian regeneration and provides insight into the role of these enzymes in adult stem cell regulation in vivo.

A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells.

Using a flow cytometry-based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.