CD44 regulates survival and memory development in Th1 cells.

Optimal immunity to microorganisms depends upon the regulated death of clonally expanded effector cells and the survival of a cohort of cells that become memory cells. After activation of naive T cells, CD44, a widely expressed receptor for extracellular matrix components, is upregulated. High expression of CD44 remains on memory cells and despite its wide usage as a "memory marker," its function is unknown. Here we report that CD44 was essential for the generation of memory T helper 1 (Th1) cells by promoting effector cell survival. This dependency was not found in Th2, Th17, or CD8(+) T cells despite similar expression of CD44 and the absence of splice variants in all subsets. CD44 limited Fas-mediated death in Th1 cells and its ligation engaged the phosphoinositide 3-kinase-Akt kinase signaling pathway that regulates cell survival. The difference in CD44-regulated apoptosis resistance in T cell subpopulations has important implications in a broad spectrum of diseases.

Pradigastat disposition in humans: in vivo and in vitro investigations.

1. Pradigastat is a potent and specific diacylglycerol acyltransferase-1 (DGAT1) inhibitor effective in lowering postprandial triglycerides (TG) in healthy human subjects and fasting TG in familial chylomicronemia syndrome (FCS) patients. 2. Here we present the results of human oral absorption, metabolism and excretion (AME), intravenous pharmacokinetic (PK), and in vitro studies which together provide an overall understanding of the disposition of pradigastat in humans. 3. In human in vitro systems, pradigastat is metabolized slowly to a stable acyl glucuronide (M18.4), catalyzed mainly by UDP-glucuronosyltransferases (UGT) 1A1, UGT1A3 and UGT2B7. M18.4 was observed at very low levels in human plasma. 4. In the human AME study, pradigastat was recovered in the feces as parent drug, confounding the assessment of pradigastat absorption and the important routes of elimination. However, considering pradigastat exposure after oral and intravenous dosing, this data suggests that pradigastat was completely bioavailable in the radiolabeled AME study and therefore completely absorbed. 5. Pradigastat is eliminated very slowly into the feces, presumably via the bile. Renal excretion is negligible. Oxidative metabolism is minimal. The extent to which pradigastat is eliminated via metabolism to M18.4 could not be established from these studies due to the inherent instability of glucuronides in the gastrointestinal tract.

Identification of Three Novel Ring Expansion Metabolites of KAE609, a New Spiroindolone Agent for the Treatment of Malaria, in Rats, Dogs, and Humans.

KAE609 [(1'R,3'S)-5,7'-dichloro-6'-fluoro-3'-methyl-2',3',4',9'-tetrahydrospiro[indoline-3,1'-pyridol[3,4-b]indol]-2-one] is a potent, fast-acting, schizonticidal agent being developed for the treatment of malaria. After oral dosing of KAE609 to rats and dogs, the major radioactive component in plasma was KAE609. An oxidative metabolite, M18, was the prominent metabolite in rat and dog plasma. KAE609 was well absorbed and extensively metabolized such that low levels of parent compound (≤11% of the dose) were detected in feces. The elimination of KAE609 and metabolites was primarily mediated via biliary pathways (≥93% of the dose) in the feces of rats and dogs. M37 and M23 were the major metabolites in rat and dog feces, respectively. Among the prominent metabolites of KAE609, the isobaric chemical species, M37, was observed, suggesting the involvement of an isomerization or rearrangement during biotransformation. Subsequent structural elucidation of M37 revealed that KAE609, a spiroindolone, undergoes an unusual C-C bond cleavage, followed by a 1,2-acyl shift to form a ring expansion metabolite M37. The in vitro metabolism of KAE609 in hepatocytes was investigated to understand this novel biotransformation. The metabolism of KAE609 was qualitatively similar across the species studied; thus, further investigation was conducted using human recombinant cytochrome P450 enzymes. The ring expansion reaction was found to be primarily catalyzed by cytochrome P450 (CYP) 3A4 yielding M37. M37 was subsequently oxidized to M18 by CYP3A4 and hydroxylated to M23 primarily by CYP1A2. Interestingly, M37 was colorless, whereas M18 and M23 showed orange yellow color. The source of the color of M18 and M23 was attributed to their extended conjugated system of double bonds in the structures.

SAR exploration at the C-3 position of tetrahydro-ß-carboline sstr3 antagonists.

MK-4256, a tetrahydro-ß-carboline sstr3 antagonist, was discontinued due to a cardiovascular (CV) adverse effect observed in dogs. Additional investigations revealed that the CV liability (QTc prolongation) was caused by the hERG off-target activity of MK-4256 and was not due to sstr3 antagonism. In this Letter, we describe our extensive SAR effort at the C3 position of the tetrahydro-ß-carboline structure. This effort resulted in identification of 5-fluoro-pyridin-2-yl as the optimal substituent on the imidazole ring to balance sstr3 activity and the hERG off-target liability.

YAP/TAZ Drive Agrin-Matrix Metalloproteinase 12-Mediated Diabetic Skin Wound Healing.

Macroscopic loss of extracellular matrix can lead to chronic defects in skin wound healing, but supplementation of extracellular matrix holds promise for facilitating wound closure, particularly in diabetic wound healing. We recently showed that the extracellular matrix proteoglycan agrin accelerates cutaneous wound healing by improving mechanoperception of migrating keratinocytes and allowing them to respond to mechanical stresses through matrix metalloproteinase 12 (MMP12). RNA-sequencing analysis revealed that in addition to a disorganized extracellular matrix, agrin-depleted skin cells have impaired YAP/TAZ transcriptional outcomes, leading us to hypothesize that YAP/TAZ, as central mechanosensors, drive the functionality of agrin-MMP12 signaling during cutaneous wound repair. In this study, we demonstrate that agrin activates YAP/TAZ during migration of keratinocytes after wounding in vitro and in vivo. Mechanistically, YAP/TAZ sustain agrin and MMP12 protein expression during migration after wounding through positive feedback. YAP/TAZ silencing abolishes agrin-MMP12-mediated force recognition and geometrical constraints. Importantly, soluble agrin therapy accelerates wound closure in diabetic mouse models by engaging MMP12-YAP. Because patients with diabetic foot ulcers and impaired wound healing have reduced expression of agrin-MMP12 that correlates with YAP/TAZ inactivation, we propose that timely activation of YAP/TAZ by soluble agrin therapy can accentuate mechanobiological microenvironments for efficient wound healing, under normal and diabetic conditions.

Chemical interactions between an active pharmaceutical ingredient and its counterion in a tromethamine salt under forced degradation conditions.

In this study, the tromethamine salt of an active pharmaceutical ingredient containing both a carboxylic acid and ethyl ester functionality was subjected to forced degradation conditions. Based on HPLC-MS analysis, it was found that tromethamine formed both amide and ester type condensation products with the API, with amide formation predominating over ester formation. Addition of tromethamine at the carboxylic acid group of the API was favored over addition at the ethyl ester group. Tromethamine condensation products were observed only under the harshest stress conditions (80 degrees and 75% relative humidity), in which the salt physically changed from a crystalline form to a deliquesced state. Under stress conditions in which the crystalline structure of the salt remained intact, good stability was observed. Thus, the interaction between tromethamine and API occurred only in cases where the crystallinity of the salt was compromised.

Agrin-Matrix Metalloproteinase-12 axis confers a mechanically competent microenvironment in skin wound healing.

An orchestrated wound healing program drives skin repair via collective epidermal cell proliferation and migration. However, the molecular determinants of the tissue microenvironment supporting wound healing remain poorly understood. Herein we discover that proteoglycan Agrin is enriched within the early wound-microenvironment and is indispensable for efficient healing. Agrin enhances the mechanoperception of keratinocytes by augmenting their stiffness, traction stress and fluidic velocity fields in retaliation to bulk substrate rigidity. Importantly, Agrin overhauls cytoskeletal architecture via enhancing actomyosin cables upon sensing geometric stress and force following an injury. Moreover, we identify Matrix Metalloproteinase-12 (MMP12) as a downstream effector of Agrin's mechanoperception. We also reveal a promising potential of a recombinant Agrin fragment as a bio-additive material that assimilates optimal mechanobiological and pro-angiogenic parameters by engaging MMP12 in accelerated wound healing. Together, we propose that Agrin-MMP12 pathway integrates a broad range of mechanical stimuli to coordinate a competent skin wound healing niche.

Polyclonal adaptive regulatory CD4 cells that can reverse type I diabetes become oligoclonal long-term protective memory cells.

Type 1 diabetes is a CD4 cell-dependent disease that results from destruction of insulin-producing beta cells in pancreatic islets. An ideal therapy would reverse diabetes shortly after onset when islet function in not yet fully ablated, and also prevent re-emergence of disease through the generation of memory cells that control the autoimmune response. In this study, we show that adaptive/induced polyclonal regulatory (TR) cells, which contain islet-reactive cells, fulfill these criteria in the NOD mouse model. CD4 cells induced to express FoxP3, IL-10, and TGF-beta1 in response to TCR signaling and TGF-beta1 can reverse diabetes with clinical restoration of prediabetic serum levels of IL-10. Unlike naturally occurring TR cells, these adaptive TR cells persist indefinitely (>1 year) as FoxP3(+), CD25(-) memory cells that self-renew. Establishment of memory is accompanied by narrowing of the T cell repertoire to usage of a single TCR beta-chain, Vbeta11, implying selection by Ag. With islet-specific adaptive TR cells, we show that memory is functionally stable and transferable. Therefore, adaptive TR cells, which can be readily generated from normal CD4 populations and become focused by Ag with induction of memory, may provide a treatment and a vaccine for the long-term cure of diabetes making them attractive as immunotherapeutic agents.

Direct oxa-Pictet-Spengler cyclization to the natural (3a,5)-trans-stereochemistry in the syntheses of (+)-7-deoxyfrenolicin B and (+)-7-deoxykalafungin.

The pyranonaphthoquinones (+)-7-deoxyfrenolicin B and (+)-7-deoxykalafungin were synthesized in four steps using an oxa-Pictet-Spengler cyclization that directly provided the natural (3a,5)-trans-substituted dihydronaphthopyrans with high diastereoselectivity. This outcome is in contrast to the unnatural (3a,5)-cis-substituted dihydronaphthopyrans reported under similar conditions for the syntheses of (+)-frenolicin B and (+)-kalafungin. Computational modeling is presented that provides insight into this unusual stereoselectivity.

C-5 substituted heteroaryl-3-pyridinecarbonitriles as PKCtheta inhibitors: part II.

We previously reported that a 3-pyridinecarbonitrile analog with a furan substituent at C-5 and a 4-methylindol-5-ylamino substituent at C-4, 1, was a potent inhibitor of PKCtheta (IC50=4.5 nM). Replacement of the C-5 furan ring of 1 with bicyclic heteroaryl rings, led to compounds with significantly improved potency against PKCtheta. Analog 6b with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-1-benzofuran-2-yl group at C-5 had an IC50 value of 0.28 nM for the inhibition of PKCtheta.

Droplet CAR-Wash: continuous picoliter-scale immunocapture and washing.

To address current limitations in adapting solid phase sample capture and washing techniques to continuously flowing droplet microfluidics, we have developed the "Coalesce-Attract-Resegment Wash" (CAR-Wash) approach. This module provides efficient, high-throughput magnetic washing by electrocoalescing magnetic bead-laden input droplets with a washing buffer flow and magnetophoretically transporting beads through the buffer into a secondary droplet formation streamline. In this work, we first characterized the technology in terms of throughput, sample retention, and flow-based exclusion of waste volume, demonstrating >500 Hz droplet processing with >98% bead retention and >100-fold dilution in final droplets. Next, we showed that the technique can be adapted to alternative commercially available magnetic beads with lower magnetite content per particle. Then, we demonstrated the CAR-Wash module's effectiveness in washing away a small molecule competitive inhibitor to restore the activity of magnetic bead-immobilized ß-galactosidase. Finally, we applied the system to immunomagnetically enrich a green fluorescent protein-histone H2B fusion protein from cell lysate while washing away mCherry and other lysate components. We believe this approach will bridge the gap between powerful biochemical and bioanalytical techniques and current droplet microfluidic capabilities, and we envision future application in droplet-based immunoassays, solid phase extraction, and other complex, multi-step operations.

Toward dynamic lumbar puncture guidance using needle-based single-element ultrasound imaging.

Lumbar punctures (LPs) are interventional procedures that are used to collect cerebrospinal fluid. Since the target window is small, physicians have limited success conducting the procedure. The procedure is especially difficult for obese patients due to the increased distance between bone and skin surface. We propose a simple and direct needle insertion platform, enabling image formation by sweeping a needle with a single ultrasound element at the tip. The needle-shaped ultrasound transducer can not only sense the distance between the tip and a potential obstacle, such as bone, but also visually locate the structures by combining transducer location tracking and synthetic aperture focusing. The concept of the system was validated through a simulation that revealed robust image reconstruction under expected errors in tip localization. The initial prototype was built into a 14 G needle and was mounted on a holster equipped with a rotation shaft allowing one degree-of-freedom rotational sweeping and a rotation tracking encoder. We experimentally evaluated the system using a metal-wire phantom mimicking high reflection bone structures and human spinal bone phantom. Images of the phantoms were reconstructed, and the synthetic aperture reconstruction improved the image quality. These results demonstrate the potential of the system to be used as a real-time guidance tool for improving LPs.

Sequential Payload Release from Acoustically-Responsive Scaffolds Using Focused Ultrasound.

Regenerative processes, such as angiogenesis and osteogenesis, often require multiple growth factors with distinct spatiotemporal patterns and expression sequences. Within tissue engineering, hydrogel scaffolds are commonly used for exogenous growth factor delivery. However, direct incorporation of growth factors within conventional hydrogels does not afford spatiotemporally controlled delivery because release is governed by passive mechanisms that cannot be actively controlled after the scaffold is implanted. We have developed acoustically-responsive scaffolds (ARSs), which are fibrin scaffolds doped with payload-containing, sonosensitive emulsions. Payload release from ARSs can be controlled non-invasively and on demand using focused, megahertz-range ultrasound. In the in vitro study described here, we developed and characterized ARSs that enable sequential release of two surrogate, fluorescent payloads using consecutive ultrasound exposures at different acoustic pressures. ARSs were generated with various combinations and volume fractions of perfluoropentane, perfluorohexane, and perfluoroheptane emulsions. Acoustic droplet vaporization and inertial cavitation thresholds correlated with the boiling point/molecular weight of the perfluorocarbon while payload release correlated inversely. Payload release was longitudinally measured and observed to follow a sigmoidal trend versus acoustic pressure. Perfluoropentane and perfluorohexane emulsions were stabilized when incorporated into ARSs with perfluoroheptane emulsion. These results highlight the potential of using ARSs for sequential, dual-payload release for tissue regeneration.

Protecting group free radical C-H trifluoromethylation of peptides.

Two radical-based approaches have been developed to effect the trifluoromethylation of aryl C-H bonds in native peptides either using stoichiometric oxidant or visible light photoredox catalysis. The reported methods are able to derivatize tyrosine and tryptophan sidechains under biocompatible conditions, and a number of examples are reported involving fully unprotected peptides with up to 51 amino acids. The development of this chemistry adds to the growing array of chemical methods for selectively modifying amino acid residues in the context of complex peptides. The direct incorporation of trifluoromethyl groups into biopolymers enables the study of a range of biological and biochemical systems, and preliminary results indicate this method can be extended to the incorporation of other fluoroalkyl groups for bioconjugation applications.

Controlled release of basic fibroblast growth factor for angiogenesis using acoustically-responsive scaffolds.

The clinical translation of pro-angiogenic growth factors for treatment of vascular disease has remained a challenge due to safety and efficacy concerns. Various approaches have been used to design spatiotemporally-controlled delivery systems for growth factors in order to recapitulate aspects of endogenous signaling and thus assist in translation. We have developed acoustically-responsive scaffolds (ARSs), which are fibrin scaffolds doped with a payload-containing, sonosensitive emulsion. Payload release can be controlled non-invasively and in an on-demand manner using focused, megahertz-range ultrasound (US). In this study, we investigate the in vitro and in vivo release from ARSs containing basic fibroblast growth factor (bFGF) encapsulated in monodispersed emulsions. Emulsions were generated in a two-step process utilizing a microfluidic device with a flow focusing geometry. At 2.5 MHz, controlled release of bFGF was observed for US pressures above 2.2 ± 0.2 MPa peak rarefactional pressure. Superthreshold US yielded a 12.6-fold increase in bFGF release in vitro. The bioactivity of the released bFGF was also characterized. When implanted subcutaneously in mice, ARSs exposed to superthreshold US displayed up to 3.3-fold and 1.7-fold greater perfusion and blood vessel density, respectively, than ARSs without US exposure. Scaffold degradation was not impacted by US. These results highlight the utility of ARSs in both basic and applied studies of therapeutic angiogenesis.

The Safety of Prochlorperazine in Children: A Systematic Review and Meta-Analysis.

INTRODUCTION: Prochlorperazine is recommended for adults with breakthrough or refractory chemotherapy-induced nausea and vomiting (CINV). The objective of this review was to describe its safety in children when given for any indication to help define its role for CINV control in children. METHODS: Electronic searches of MEDLINE, EMBASE, PsycINFO, and the Cochrane Central Register of Controlled Trials were performed as of 9 March 2015. All studies in English reporting adverse effects (AEs) associated with prochlorperazine in children (≤18 years) were included. AEs were synthesized for prospective studies. RESULTS: Forty-nine (15 prospective) studies evaluating the use of prochlorperazine in 758 children were included. The most commonly reported AEs in prospective studies of prochlorperazine in children were sedation (multiple-dose studies: 10 %, 95 % CI 5-21) and extrapyramidal symptoms (EPS) (single-dose studies: 9 %, 95 % CI 3-29; multiple-dose studies: 4 %, 95 % CI 1-11). Serious AEs (seizure, neuroleptic malignant syndrome, autonomic collapse, tardive dyskinesia) were rarely associated with prochlorperazine use in children. Five fatalities were reported in children receiving prochlorperazine. LIMITATIONS: The limitations of this systematic review and meta-analysis were that the AEs reported in the included studies were heterogeneous, the prospective use of systematic clinical tools to identify AEs was rare, and the risk of bias in most prospective studies was moderate. CONCLUSIONS: The most common AEs reported with the pediatric use of prochlorperazine are EPS and sedation. Fatalities, life-threatening, and persistent AEs have also been reported.

The Safety of Metoclopramide in Children: A Systematic Review and Meta-Analysis.

INTRODUCTION: Metoclopramide is recommended for adults with breakthrough or refractory chemotherapy-induced nausea and vomiting (CINV) and for CINV prophylaxis in children. The drug regulatory agencies of Canada and the EU have revised the labelling of metoclopramide to contraindicate its use in children aged <1 year and to caution against its use in children aged <5 years and its duration of use beyond 5 days. OBJECTIVE: This review describes the safety of metoclopramide in children when given for any indication. METHODS: We conducted electronic searches in MEDLINE and Embase as of 9 March 2015. All studies in English reporting adverse effects associated with the use of metoclopramide in children (aged ≤18 years) were included. Adverse effects that had a cumulative incidence of at least 1 % and were reported in prospective studies were synthesized. RESULTS: A total of 108 (57 prospective) studies involving 2699 patients (2745 metoclopramide courses) were included. The most common adverse effects reported in prospective studies of metoclopramide in children were extrapyramidal symptoms (EPS; 9 %, 95 % confidence interval [CI] 5-17), diarrhea (6 %, 95 % CI 4-9), and sedation (multiple-dose studies: 6 %, 95 % CI 3-12). Dysrhythmia, respiratory distress/arrest, neuroleptic malignant syndrome, and tardive dyskinesia were rarely associated with metoclopramide use. LIMITATIONS: The definitions of adverse effects reported in the included studies were heterogeneous, and the risk of bias in most studies was moderate. CONCLUSIONS: The most commonly reported adverse effects associated with the use of metoclopramide in children-EPS, diarrhea, and sedation-were reversible and of no long-term significance. Adverse effects that were life threatening or slow to resolve were rarely associated with its use in children.

In vitro and in vivo assessment of controlled release and degradation of acoustically responsive scaffolds.

Spatiotemporally controlled release of growth factors (GFs) is critical for regenerative processes such as angiogenesis. A common strategy is to encapsulate the GF within hydrogels, with release being controlled via diffusion and/or gel degradation (i.e., hydrolysis and/or proteolysis). However, simple encapsulation strategies do not provide spatial or temporal control of GF delivery, especially non-invasive, on-demand controlled release post implantation. We previously demonstrated that fibrin hydrogels, which are widely used in tissue engineering and GF delivery applications, can be doped with perfluorocarbon emulsion, thus yielding an acoustically responsive scaffold (ARS) that can be modulated with focused ultrasound, specifically via a mechanism termed acoustic droplet vaporization. This study investigates the impact of ARS and ultrasound properties on controlled release of a surrogate payload (i.e., fluorescently-labeled dextran) and fibrin degradation in vitro and in vivo. Ultrasound exposure (2.5MHz, peak rarefactional pressure: 8MPa, spatial peak time average intensity: 86.4mW/cm2), generated up to 7.7 and 21.7-fold increases in dextran release from the ARSs in vitro and in vivo, respectively. Ultrasound also induced morphological changes in the ARS. Surprisingly, up to 2.9-fold greater blood vessel density was observed in ARSs compared to fibrin when implanted subcutaneously, even without delivery of pro-angiogenic GFs. The results demonstrate the potential utility of ARSs in generating controlled release for tissue regeneration. STATEMENT OF SIGNIFICANCE: Simple encapsulation of a molecular payload within a conventional hydrogel scaffold does not provide spatial or temporal control of payload release. Yet, spatiotemporally controlled release of bioactive payloads is critical for tissue regeneration, which often utilizes hydrogel scaffolds to facilitate processes such as angiogenesis. This work investigates the design and performance (both in vitro and in vivo) of hydrogel scaffolds where release of a fluorescent payload is non-invasively and spatiotemporally-controlled using focused ultrasound. We also quantitatively characterize the degradation and vascularization of the scaffolds. Our results may be of interest to groups working on controlled release strategies for implants, especially within the field of tissue engineering.

Evaluation of an electronic medical record system at an opioid agonist treatment program.

OBJECTIVES: The Addiction Research and Treatment Corporation evaluated the impact of an electronic medical record system. METHODS: A prospective pre- and postimplementation design was utilized that examined the domains of quality, productivity, satisfaction, risk management, and financial performance. RESULTS: There were highly statistically significant improvements in timely completion of Annual Medical and 30-Day, 90-Day, and Annual Multidiscipline assessments. There was no statistically significant change in obtaining hepatitis C viral load for hepatitis C antibody-positive patients. The prevalence of risk management events was too low to detect statistically meaningful changes. Patient satisfaction was unchanged pre- and postimplementation, although staff satisfaction trended upward postimplementation. Productivity significantly declined for counseling staff; there was a nonsignificant productivity decline for medical services staff and a nonsignificant productivity increase for case manager staff. Revenue per capita staff increased by 0.6%, while cost per patient visit increased by 5.7%. CONCLUSIONS: Despite less robust results than expected, had we not implemented the electronic system, recent changes in documentation and reimbursement for services would have paralyzed our agency.