Targeting the MR1-MAIT cell axis improves vaccine efficacy and affords protection against viral pathogens.

Mucosa-associated invariant T (MAIT) cells are MR1-restricted, innate-like T lymphocytes with tremendous antibacterial and immunomodulatory functions. Additionally, MAIT cells sense and respond to viral infections in an MR1-independent fashion. However, whether they can be directly targeted in immunization strategies against viral pathogens is unclear. We addressed this question in multiple wild-type and genetically altered but clinically relevant mouse strains using several vaccine platforms against influenza viruses, poxviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), a riboflavin-based MR1 ligand of bacterial origin, can synergize with viral vaccines to expand MAIT cells in multiple tissues, reprogram them towards a pro-inflammatory MAIT1 phenotype, license them to bolster virus-specific CD8+ T cell responses, and potentiate heterosubtypic anti-influenza protection. Repeated 5-OP-RU administration did not render MAIT cells anergic, thus allowing for its inclusion in prime-boost immunization protocols. Mechanistically, tissue MAIT cell accumulation was due to their robust proliferation, as opposed to altered migratory behavior, and required viral vaccine replication competency and Toll-like receptor 3 and type I interferon receptor signaling. The observed phenomenon was reproducible in female and male mice, and in both young and old animals. It could also be recapitulated in a human cell culture system in which peripheral blood mononuclear cells were exposed to replicating virions and 5-OP-RU. In conclusion, although viruses and virus-based vaccines are devoid of the riboflavin biosynthesis machinery that supplies MR1 ligands, targeting MR1 enhances the efficacy of vaccine-elicited antiviral immunity. We propose 5-OP-RU as a non-classic but potent and versatile vaccine adjuvant against respiratory viruses.

Characterization of a Vimentinhigh /Nestinhigh proteome and tissue regenerative secretome generated by human pancreas-derived mesenchymal stromal cells.

Multipotent/mesenchymal stromal cells (MSCs) exist within a variety of postnatal tissues; however, global proteomic analyses comparing tissue-specific MSC are limited. Using human bone marrow (BM)-derived MSCs as a gold standard, we used label-free mass spectrometry and functional assays to characterize the proteome, secretome, and corresponding function of human pancreas-derived MSCs (Panc-MSCs) with a classical phenotype (CD90+/CD73+/CD105+/CD45-/CD31-). Both MSC subtypes expressed mesenchymal markers vimentin, α-SMA, and STRO-1; however, expression of nestin was increased in Panc-MSCs. Accordingly, these Vimentinhigh /Nestinhigh cells were isolated from fresh human pancreatic islet and non-islet tissues. Next, we identified expression of >60 CD markers shared between Panc-MSCs and BM-MSCs, including validated expression of CD14. An additional 19 CD markers were differentially expressed, including reduced pericyte-marker CD146 expression on Panc-MSCs. Panc-MSCs also showed reduced expression of proteins involved in lipid and retinoid metabolism. Accordingly, Panc-MSCs showed restricted responses to adipogenic stimuli in vitro, although both MSC types demonstrated trilineage differentiation. In contrast, Panc-MSCs demonstrated accelerated growth kinetics and competency to pro-neurogenic stimuli in vitro. The secretome of Panc-MSCs was highly enriched for proteins associated with vascular development, wound healing and chemotaxis. Similar to BM-MSCs, Panc-MSCs conditioned media augmented endothelial cell survival, proliferation, and tubule formation in vitro. Importantly, the secretome of both MSC types was capable of stimulating chemotactic infiltration of murine endothelial cells in vivo and reduced hyperglycemia in STZ-treated mice following intrapancreatic injection. Overall, this study provides foundational knowledge to develop Panc-MSCs as a unique MSC subtype with functional properties beneficial in regenerative medicine for diabetes and vascular disease.

Characterization of extracellular vesicles derived from mesenchymal stromal cells by surface-enhanced Raman spectroscopy.

Extracellular vesicles (EVs) are secreted by all cells into bodily fluids and play an important role in intercellular communication through the transfer of proteins and RNA. There is evidence that EVs specifically released from mesenchymal stromal cells (MSCs) are potent cell-free regenerative agents. However, for MSC EVs to be used in therapeutic practices, there must be a standardized and reproducible method for their characterization. The detection and characterization of EVs are a challenge due to their nanoscale size as well as their molecular heterogeneity. To address this challenge, we have fabricated gold nanohole arrays of varying sizes and shapes by electron beam lithography. These platforms have the dual purpose of trapping single EVs and enhancing their vibrational signature in surface-enhanced Raman spectroscopy (SERS). In this paper, we report SERS spectra for MSC EVs derived from pancreatic tissue (Panc-MSC) and bone marrow (BM-MSC). Using principal component analysis (PCA), we determined that the main compositional differences between these two groups are found at 1236, 761, and 1528 cm-1, corresponding to amide III, tryptophan, and an in-plane -C=C- vibration, respectively. We additionally explored several machine learning approaches to distinguish between BM- and Panc-MSC EVs and achieved 89 % accuracy, 89 % sensitivity, and 88 % specificity using logistic regression.

Human Multipotent Stromal Cell Secreted Effectors Accelerate Islet Regeneration.

Human multipotent stromal cells (hMSC) can induce islet regeneration after transplantation via the secretion of proteins that establish an islet regenerative niche. However, the identity of hMSC-secreted signals and the mechanisms by which pancreatic islet regeneration is induced remain unknown. Recently, mammalian pancreatic α-cells have been shown to possess considerable plasticity, and differentiate into ß-like cells after near complete ß-cell loss or overexpression of key transcriptional regulators. These studies have generated new excitement that islet regeneration during diabetes may be possible if we can identify clinically applicable stimuli to modulate these key regulatory pathways. Herein, we demonstrate that intrapancreatic-injection of concentrated hMSC-conditioned media (CM) stimulated islet regeneration without requiring cell transfer. hMSC CM-injection significantly reduced hyperglycemia, increased circulating serum insulin concentration, and improved glucose tolerance in streptozotocin-treated mice. The rate and extent of endogenous ß-cell mass recovery was dependent on total protein dose administered and was further augmented by the activation of Wnt-signaling using GSK3-inhibition during CM generation. Intrapancreatic hMSC CM-injection immediately set in motion a cascade of regenerative events that included the emergence of proliferating insulin+ clusters adjacent to ducts, NKX6.1 expression in glucagon+ cells at days 1-4 suggesting the acquisition of ß-cell phenotype by α-cells, and accelerated ß-cell maturation with increased MAFA-expression for >1 month postinjection. Discovery and validation of islet regenerative hMSC-secreted protein may lead to the development of cell-free regenerative therapies able to tip the balance in favor of ß-cell regeneration versus destruction during diabetes. Stem Cells 2019;37:516-528.

Multi-site investigation of strategies for the clinical implementation of CYP2D6 genotyping to guide drug prescribing.

PURPOSE: A number of institutions have clinically implemented CYP2D6 genotyping to guide drug prescribing. We compared implementation strategies of early adopters of CYP2D6 testing, barriers faced by both early adopters and institutions in the process of implementing CYP2D6 testing, and approaches taken to overcome these barriers. METHODS: We surveyed eight early adopters of CYP2D6 genotyping and eight institutions in the process of adoption. Data were collected on testing approaches, return of results procedures, applications of genotype results, challenges faced, and lessons learned. RESULTS: Among early adopters, CYP2D6 testing was most commonly ordered to assist with opioid and antidepressant prescribing. Key differences among programs included test ordering and genotyping approaches, result reporting, and clinical decision support. However, all sites tested for copy-number variation and nine common variants, and reported results in the medical record. Most sites provided automatic consultation and had designated personnel to assist with genotype-informed therapy recommendations. Primary challenges were related to stakeholder support, CYP2D6 gene complexity, phenotype assignment, and sustainability. CONCLUSION: There are specific challenges unique to CYP2D6 testing given the complexity of the gene and its relevance to multiple medications. Consensus lessons learned may guide those interested in pursuing similar clinical pharmacogenetic programs.

Inhibition of Aldehyde Dehydrogenase-Activity Expands Multipotent Myeloid Progenitor Cells with Vascular Regenerative Function.

Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDHhi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDHhi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDHhi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDHhi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDHhi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased >70-fold, the frequency of cells that retained ALDHhi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDHhi /CD34+ cell number by ≥10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB-inhibition of ALDH-activity delayed hematopoietic differentiation and expanded multipotent myeloid cells that accelerated vascular regeneration following i.m. transplantation in vivo. Stem Cells 2018;36:723-736.

Prediction of chemotherapy-induced nausea and vomiting from patient-reported and genetic risk factors.

PURPOSE: Chemotherapy-induced nausea and vomiting (CINV) is common among cancer patients. Early identification of patients at risk for CINV may help to personalize anti-emetic therapies. To date, few studies have examined the combined contributions of patient-reported and genetic risk factors to CINV. The goal of this study was to evaluate these risk factors. METHODS: Prior to their first chemotherapy infusion, participants completed demographic and risk factor questionnaires and provided a blood sample to measure genetic variants in ABCB1 (rs1045642) and HTR3B (rs45460698) as well as CYP2D6 activity score. The M.D. Anderson Symptom Inventory was completed at 24 h and 5-day post-infusion to assess the severity of acute and delayed CINV, respectively. RESULTS: Participants were 88 patients (55% female, M = 60 years). A total of 23% experienced acute nausea and 55% delayed nausea. Younger age, history of pregnancy-related nausea, fewer hours slept the night prior to infusion, and variation in ABCB1 were associated with more severe acute nausea; advanced-stage cancer and receipt of highly emetogenic chemotherapy were associated with more severe delayed nausea (p values < 0.05). In multivariable analyses, ABCB1 added an additional 5% predictive value beyond the 13% variance explained by patient-reported risk factors. CONCLUSIONS: The current study identified patient-reported and genetic factors that may place patients at risk for acute nausea despite receipt of guideline-consistent anti-emetic prophylaxis. Additional studies examining other genetic variants are needed, as well as the development of risk prediction models including both patient-reported and genetic risk factors.

Proteomic characterisation reveals active Wnt-signalling by human multipotent stromal cells as a key regulator of beta cell survival and proliferation.

AIMS/HYPOTHESIS: Novel strategies to stimulate the expansion of beta cell mass in situ are warranted for diabetes therapy. The aim of this study was to elucidate the secretome of human bone marrow (BM)-derived multipotent stromal cells (MSCs) with documented islet regenerative paracrine function. We hypothesised that regenerative MSCs will secrete a unique combination of protein factors that augment islet regeneration. METHODS: Human BM-derived MSCs were examined for glucose-lowering capacity after transplantation into streptozotocin-treated NOD/severe combined immunodeficiency (SCID) mice and segregated into samples with regenerative (MSCR) vs nonregenerative (MSCNR) capacity. Secreted proteins associated with islet regenerative function were identified using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics. To functionally validate the importance of active Wnt signalling, we stimulated the Wnt-signalling pathway in MSCNR samples during ex vivo expansion using glycogen synthase kinase 3 (GSK3) inhibition (CHIR99201), and the conditioned culture media (CM) generated was tested for the capacity to support cultured human islet cell survival and proliferation in vitro. RESULTS: MSCR showed increased secretion of proteins associated with cell growth, matrix remodelling, immunosuppressive and proangiogenic properties. In contrast, MSCNR uniquely secreted proteins known to promote inflammation and negatively regulate angiogenesis. Most notably, MSCR maintained Wnt signalling via Wnt5A/B (~2.5-fold increase) autocrine activity during ex vivo culture, while MSCNR repressed Wnt signalling via Dickkopf-related protein (DKK)1 (~2.5-fold increase) and DKK3 secretion. Inhibition of GSK3 activity in MSCNR samples increased the accumulation of nuclear ß-catenin and generated CM that augmented beta cell survival (13% increases) and proliferation when exposed to cultured human islets. CONCLUSIONS/INTERPRETATION: Maintenance of active Wnt signalling within human MSCs promotes the secretion of matricellular and proangiogenic proteins that formulate a niche for islet regeneration.

Key Lessons Learned from Moffitt's Molecular Tumor Board: The Clinical Genomics Action Committee Experience.

BACKGROUND: The increasing practicality of genomic sequencing technology has led to its incorporation into routine clinical practice. Successful identification and targeting of driver genomic alterations that provide proliferative and survival advantages to tumor cells have led to approval and ongoing development of several targeted cancer therapies. Within many major cancer centers, molecular tumor boards are constituted to shepherd precision medicine into clinical practice. MATERIALS AND METHODS: In July 2014, the Clinical Genomics Action Committee (CGAC) was established as the molecular tumor board companion to the Personalized Medicine Clinical Service (PMCS) at Moffitt Cancer Center in Tampa, Florida. The processes and outcomes of the program were assessed in order to help others move into the practice of precision medicine. RESULTS: Through the establishment and initial 1,400 patients of the PMCS and its associated molecular tumor board at a major cancer center, five practical lessons of broad applicability have been learned: transdisciplinary engagement, the use of the molecular report as an aid to clinical management, clinical actionability, getting therapeutic options to patients, and financial considerations. Value to patients includes access to cutting-edge practice merged with individualized preferences in treatment and care. CONCLUSIONS: Genomic-driven cancer medicine is increasingly becoming a part of routine clinical practice. For successful implementation of precision cancer medicine, strategically organized molecular tumor boards are critical to provide objective evidence-based translation of observed molecular alterations into patient-centered clinical action. Molecular tumor board implementation models along with clinical and economic outcomes will define future treatment standards. The Oncologist 2017;22:144-151Implications for Practice: It is clear that the increasing practicality of genetic tumor sequencing technology has led to its incorporation as part of routine clinical practice. Subsequently, many cancer centers are seeking to develop a personalized medicine services and/or molecular tumor board to shepherd precision medicine into clinical practice. This article discusses the key lessons learned through the establishment and development of a molecular tumor board and personalized medicine clinical service. This article highlights practical issues and can serve as an important guide to other centers as they conceive and develop their own personalized medicine services and molecular tumor boards.

Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions.

Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDH(hi) ) stimulate islet regeneration after transplantation into mice with streptozotocin-induced ß cell deletion. However, ALDH(hi) cells represent a rare progenitor subset and widespread use of UCB ALDH(hi) cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDH(hi) cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDH(hi) cells diminished as culture time progressed such that total ALDH(hi) cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDH(lo) subset) from less differentiated progeny with high ALDH-activity (ALDH(hi) subset). The ALDH(hi) subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34(+) /CD38(-) cells, 37.0% ± 6.9% CD34(+) /CD133(+) cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDH(lo) subset. Notably, bulk cells or ALDH(lo) cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDH(hi) subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ.

Brief Report: Elastin Microfibril Interface 1 and Integrin-Linked Protein Kinase Are Novel Markers of Islet Regenerative Function in Human Multipotent Mesenchymal Stromal Cells.

Multipotent mesenchymal stromal cell (MSC) transplantation is proposed as a novel therapy for treating diabetes by promoting the regeneration of damaged islets. The clinical promise of such treatments may be hampered by a high degree of donor-related variability in MSC function and a lack of standards for comparing potency. Here, we set out to identify markers of cultured human MSCs directly associated with islet regenerative function. Stromal cultures from nine separate bone marrow donors were demonstrated to have differing capacities to reduce hyperglycemia in the NOD/SCID streptozotocin-induced diabetic model. Regenerative (R) and non-regenerative (NR) MSC cultures were directly compared using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics. A total of 1,410 proteins were quantified resulting in the identification of 612 upregulated proteins and 275 downregulated proteins by ± 1.2-fold in R-MSC cultures. Elastin microfibril interface 1 (EMILIN-1), integrin-linked protein kinase (ILK), and hepatoma-derived growth factor (HDGF) were differentially expressed in R-MSCs, and Ingenuity Pathway Analyses revealed each candidate as known regulators of integrin signaling. Western blot validation of EMILIN-1, ILK, and HDGF not only showed significantly higher abundance levels in R-MSCs, as compared with NR-MSCs, but also correlated with passage-induced loss of islet-regenerative potential. Generalized estimating equation modeling was applied to examine the association between each marker and blood glucose reduction. Both EMILIN-1 and ILK were significantly associated with blood glucose lowering function in vivo. Our study is the first to identify EMILIN-1 and ILK as prospective markers of islet regenerative function in human MSCs. Stem Cells 2016;34:2249-2255.

PU.1 opposes IL-7-dependent proliferation of developing B cells with involvement of the direct target gene bruton tyrosine kinase.

Deletion of genes encoding the E26 transformation-specific transcription factors PU.1 and Spi-B in B cells (CD19-CreΔPB mice) leads to impaired B cell development, followed by B cell acute lymphoblastic leukemia at 100% incidence and with a median survival of 21 wk. However, little is known about the target genes that explain leukemogenesis in these mice. In this study we found that immature B cells were altered in frequency in the bone marrow of preleukemic CD19-CreΔPB mice. Enriched pro-B cells from CD19-CreΔPB mice induced disease upon transplantation, suggesting that these were leukemia-initiating cells. Bone marrow cells from preleukemic CD19-CreΔPB mice had increased responsiveness to IL-7 and could proliferate indefinitely in response to this cytokine. Bruton tyrosine kinase (BTK), a negative regulator of IL-7 signaling, was reduced in preleukemic and leukemic CD19-CreΔPB cells compared with controls. Induction of PU.1 expression in cultured CD19-CreΔPB pro-B cell lines induced Btk expression, followed by reduced STAT5 phosphorylation and early apoptosis. PU.1 and Spi-B regulated Btk directly as shown by chromatin immunoprecipitation analysis. Ectopic expression of BTK was sufficient to induce apoptosis in cultured pro-B cells. In summary, these results suggest that PU.1 and Spi-B activate Btk to oppose IL-7 responsiveness in developing B cells.

Budget impact analysis of CYP2C19-guided voriconazole prophylaxis in AML.

OBJECTIVES: The objective of this study was to determine the economic impact of proactive, CYP2C19 genotype-guided voriconazole prophylaxis in AML. METHODS: An Excel-based model was created to project the cost of treating a simulated cohort of severely neutropenic AML patients undergoing antifungal prophylaxis. The model compares (i) standard prophylactic dosing with voriconazole and (ii) CYP2C19 genotyping of all AML patients to guide voriconazole dosing and prescribing. RESULTS: Based on the model, genotype-guided dosing of voriconazole conservatively spares 2.3 patients per year from invasive fungal infections. Implementing proactive genotyping of all AML patients in a simulated 100 patient cohort is expected to save a total of $41467 or $415 per patient. CONCLUSIONS: The model, based on the robust literature of clinical and economic data, predicts that proactive genotype-guided voriconazole prophylaxis is likely to yield modest cost savings while improving patient outcomes. The primary driver of savings is the avoidance of expensive antifungal treatment and extended hospital stays, costing $30 952 per patient, in patients succumbing to fungal infection.

Clinical Implications of Opioid Pharmacogenomics in Patients With Cancer.

BACKGROUND: Pain can be a significant burden for patients with cancer and may have negative effects on their quality of life. Opioids are potent analgesics and serve as a foundation for pain management. The variation in response to opioid analgesics is well characterized and is partly due to genetic variability. METHODS: We reviewed the results of clinical studies to evaluate the relationships between genetic variants and select genes involved in the pharmacokinetics and pharmacodynamics of opioids, with an emphasis on patients with cancer. RESULTS: In patients with cancer-related pain, genetic variation in OPRM1, COMT, and ABCB1 is associated with response to morphine, which is the most well-studied opioid. Although it has not been studied in patients with cancer-related pain, the effect of CYP2D6 variation is well characterized with codeine and tramadol. Evidence is limited for associating the genetic variation and pain response of oxycodone, hydrocodone, and fentanyl in patients with cancer. CONCLUSION: The clinical availability of pharmacogenomic testing and research findings related to these polymorphic genes suggest that genotyping patients for these genetic variants may allow health care professionals to better predict patient response to pain and, thus, personalize pain treatment.

Umbilical cord blood-derived aldehyde dehydrogenase-expressing progenitor cells promote recovery from acute ischemic injury.

Umbilical cord blood (UCB) represents a readily available source of hematopoietic and endothelial precursors at early ontogeny. Understanding the proangiogenic functions of these somatic progenitor subtypes after transplantation is integral to the development of improved cell-based therapies to treat ischemic diseases. We used fluorescence-activated cell sorting to purify a rare (<0.5%) population of UCB cells with high aldehyde dehydrogenase (ALDH(hi) ) activity, a conserved stem/progenitor cell function. ALDH(hi) cells were depleted of mature monocytes and T- and B-lymphocytes and were enriched for early myeloid (CD33) and stem cell-associated (CD34, CD133, and CD117) phenotypes. Although these cells were primarily hematopoietic in origin, UCB ALDH(hi) cells demonstrated a proangiogenic transcription profile and were highly enriched for both multipotent myeloid and endothelial colony-forming cells in vitro. Coculture of ALDH(hi) cells in hanging transwells promoted the survival of human umbilical vein endothelial cells (HUVEC) under growth factor-free and serum-free conditions. On growth factor depleted matrigel, ALDH(hi) cells significantly increased tube-like cord formation by HUVEC. After induction of acute unilateral hind limb ischemia by femoral artery ligation, transplantation of ALDH(hi) cells significantly enhanced the recovery of perfusion in ischemic limbs. Despite transient engraftment in the ischemic hind limb, early recruitment of ALDH(hi) cells into ischemic muscle tissue correlated with increased murine von Willebrand factor blood vessel and CD31+ capillary densities. Thus, UCB ALDH(hi) cells represent a readily available population of proangiogenic progenitors that promote vascular regeneration. This work provides preclinical justification for the development of therapeutic strategies to treat ischemic diseases using UCB-derived ALDH(hi) mixed progenitor cells.

Annexin A5 Inhibits Endothelial Inflammation Induced by Lipopolysaccharide-Activated Platelets and Microvesicles via Phosphatidylserine Binding.

Sepsis is caused by a dysregulated immune response to infection and is a leading cause of mortality globally. To date, no specific therapeutics are available to treat the underlying septic response. We and others have shown that recombinant human annexin A5 (Anx5) treatment inhibits pro-inflammatory cytokine production and improves survival in rodent sepsis models. During sepsis, activated platelets release microvesicles (MVs) with externalization of phosphatidylserine to which Anx5 binds with high affinity. We hypothesized that recombinant human Anx5 blocks the pro-inflammatory response induced by activated platelets and MVs in vascular endothelial cells under septic conditions via phosphatidylserine binding. Our data show that treatment with wildtype Anx5 reduced the expression of inflammatory cytokines and adhesion molecules induced by lipopolysaccharide (LPS)-activated platelets or MVs in endothelial cells (p < 0.01), which was not observed with Anx5 mutant deficient in phosphatidylserine binding. In addition, wildtype Anx5 treatment, but not Anx5 mutant, improved trans-endothelial electrical resistance (p < 0.05) and reduced monocyte (p < 0.001) and platelet (p < 0.001) adhesion to vascular endothelial cells in septic conditions. In conclusion, recombinant human Anx5 inhibits endothelial inflammation induced by activated platelets and MVs in septic conditions via phosphatidylserine binding, which may contribute to its anti-inflammatory effects in the treatment of sepsis.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for CYP2D6, CYP2C19, CYP2B6, SLC6A4, and HTR2A Genotypes and Serotonin Reuptake Inhibitor Antidepressants.

Serotonin reuptake inhibitor antidepressants, including selective serotonin reuptake inhibitors (SSRIs; i.e., citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline), serotonin and norepinephrine reuptake inhibitors (i.e., desvenlafaxine, duloxetine, levomilnacipran, milnacipran, and venlafaxine), and serotonin modulators with SSRI-like properties (i.e., vilazodone and vortioxetine) are primary pharmacologic treatments for major depressive and anxiety disorders. Genetic variation in CYP2D6, CYP2C19, and CYP2B6 influences the metabolism of many of these antidepressants, which may potentially affect dosing, efficacy, and tolerability. In addition, the pharmacodynamic genes SLC6A4 (serotonin transporter) and HTR2A (serotonin-2A receptor) have been examined in relation to efficacy and side effect profiles of these drugs. This guideline updates and expands the 2015 Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 and CYP2C19 genotypes and SSRI dosing and summarizes the impact of CYP2D6, CYP2C19, CYP2B6, SLC6A4, and HTR2A genotypes on antidepressant dosing, efficacy, and tolerability. We provide recommendations for using CYP2D6, CYP2C19, and CYP2B6 genotype results to help inform prescribing these antidepressants and describe the existing data for SLC6A4 and HTR2A, which do not support their clinical use in antidepressant prescribing.

A clinician's guide for counseling patients on results of a multigene pharmacogenomic panel.

PURPOSE: This article explores approaches to pharmacogenomic counseling for patients who have undergone multigene panel testing by describing the collective experience of 5 institutions. SUMMARY: Multigene panel pharmacogenomic testing has the potential to unlock a myriad of information about a patient's past, present, and future drug response. The multifaceted nature of drug response coupled with the complexity of genetic results necessitates some form of patient education through pharmacogenomic counseling. Published literature regarding disclosure of pharmacogenomic test results is limited. This article compares the counseling practices of pharmacists from 5 different institutions with pharmacogenomics clinics whose experience represents perspectives ranging from academia to community clinical environments. Overarching counseling themes discussed during result disclosure center around (1) pharmacogenomic results, (2) gene-drug interactions, (3) gene-drug-drug interactions, (4) drug changes (5) future, familial, or disease-risk implications, (6) updates in the interpretation and application of pharmacogenomic results, (7) gauging patient comprehension, and (8) sharing results and supplemental information. CONCLUSION: Dedicating time to counseling patients on the results of a multigene pharmacogenomic panel is important given the lifelong applications of a test that is generally performed only once. The content and methods of disclosing test results shared by the experiences of pharmacists at 5 different institutions serve as guide to be further refined as research addresses effective communication strategies that enhance patient comprehension of pharmacogenomic results.

Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity.

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity, a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells, mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus, human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.

Synthesis of major pharmacogenomics pretest counseling themes: a multisite comparison.

The accessibility of pharmacogenomic (PGx) testing has grown substantially over the last decade and with it has arisen a demand for patients to be counseled on the use of these tests. While guidelines exist for the use of PGx results; objective determinants for who should receive PGx testing remain incomplete. PGx clinical services have been created to meet these screening and education needs and significant variability exists between these programs. This article describes the practices of four PGx clinics during pretest counseling sessions. A description of the major tenets of the benefits, limitations and risks of testing are compiled. Additional tools are provided to serve as a foundation for those wishing to begin or expand their own counseling service.