Long-term survival after unrelated donor marrow transplantation for aplastic anaemia after optimized conditioning regimen: a retrospective multicentre cohort study.

Background: Almost all acquired severe aplastic anaemia is immune mediated and characterised by hypocellular bone marrow and ≥2 affected haematopoietic lineages. The optimal preparartive regimen for unrelated donor transplantation remains to be established. We aimed to study long-term outcomes after unrelated donor transplantation for severe aplastic anaemia with de-escalation of cyclophosphamide (Cy) dose in steps of 50 mg/kg (150, 100, 50, 0 mg/kg) in combination with total body irradiation (TBI) 2 Gy, anti-thymocyte globulin (ATG) and fludarabine. Methods: Ninety-six patients with severe aplastic anaemia aged ≤65 years with adequate organ function enrolled on a trial of human leukocyte antigen (HLA)-matched or 1 HLA-locus mismatched unrelated donor marrow transplantation conducted between 02/2006 and 12/2013 in the United States (NCT00326417). Exclusion criteria were Karnofsky performance status of less than 60, clonal cytogenetic abnormalities and inherited marrow failure syndormes. The primary outcome was day-100 engraftment (achievement of absolute neutrophil recovery to at least 0.5 × 109/L without subsequent decline) and day-100 survival. The trial determined the lowest effective Cy dose as 50 mg/kg (n = 38) for day-100 engraftment and survival. Cy dose 100 mg/kg (n = 41) was also acceptable. Accrual to Cy doses 150 mg/kg (n = 15) and 0 mg/kg (n = 3) was terminated early for toxicities. The current study is an extended follow up of patients enrolled on the trial (NCT00326477) and includes 76 of 96 patients alive ≥1 year after transplantation. There were 20 deaths in the first year after transplantation (Cy 0 mg/kg [n = 2], Cy 50 mg/kg [n = 1], Cy 100 mg/kg [n = 10], Cy 150 mg/kg [n = 7]). Patients were followed prospectively from transplantation and data reported using standardized data collection forms until death, loss to follow up or last contact through November 2023. The incidence of graft failure was calculated using the cumulative incidence estimator and the probability of survival using the Kaplan-Meier estimator. Findings: The median follow up of the cohort is 8.02 (IQR) 5.16-10.12) years. With Cy 50 mg/kg, there was one graft failure and five deaths ≥1 year after transplantation. With Cy 100 mg/kg there was only one late death and no graft failure. The 8-year probabilities of survival were 85.0% (95% CI 67.3-93.5) and 75.6% (95% CI 59.4-86.1) after Cy 50 mg/kg and 100 mg/kg, respectively, P = 0.31. With Cy 0 mg/kg and 150 mg/kg, there were no graft failures or death ≥1 year after transplantation. Regardless of Cy dose 12 of 15 patients aged ≥50 years died. Interpretation: Cy 50 mg/kg or 100 mg/kg with TBI 2 Gy, ATG and fludarabine are effective conditioning regimens for unrelated donor marrow transplants for aplastic anaemia. Identification of an optimized transplantation approach for patients aged ≥50 years is needed. Funding: US National Institutes of Health.

Sex-dependent effects in the aged melanoma tumor microenvironment influence invasion and resistance to targeted therapy.

There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain unclear. While biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the tumor-surrounding microenvironment, contextually in aging, has been overlooked. Here, we show that skin fibroblasts undergo age-mediated, sex-dependent changes in their proliferation, senescence, ROS levels, and stress response. We find that aged male fibroblasts selectively drive an invasive, therapy-resistant phenotype in melanoma cells and promote metastasis in aged male mice by increasing AXL expression. Intrinsic aging in male fibroblasts mediated by EZH2 decline increases BMP2 secretion, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of invasive phenotypes and sensitizes melanoma cells to BRAF/MEK inhibition.

Bone Accrual Trajectories in Children and Adolescents with Perinatal HIV Infection.

CONTEXT: Low bone mineral density (BMD) has been reported in children and adolescents living with perinatally-acquired HIV (PHIV). Little is known about their bone accrual through puberty compared to an uninfected healthy cohort. OBJECTIVE: To compare bone accrual in PHIV and healthy children. DESIGN: PHIV children aged 7-16 years had dual energy X-ray absorptiometry (DXA) at entry, 2 years, and then at least 2 years later. Bone accrual was compared to healthy children from the Bone Mineral Density in Childhood Study (BMDCS). SETTING: United States academic clinical research centers. PATIENTS: 172 PHIV; 1321 BMDCS. ANALYSIS: We calculated height-adjusted whole-body and spine BMD and bone mineral content (BMC) Z-scores in PHIV using BMDCS reference curves. We fit piecewise weighted linear mixed effects models with change points at 11 and 15 years, adjusted for age, sex, race, height Z-score, and Tanner stage, to compare BMD and BMC Z-scores across actual age by cohort.Main Outcome Measure: BMD/BMC Z-scores. RESULTS: Height-adjusted whole-body BMD and BMC Z-scores in PHIV were lower across age compared to BMDCS children. Spine BMD Z-score across age was higher in PHIV after height adjustment. Whole-body and spine bone area tended to be lower in PHIV. PHIV had slower accrual in whole-body and spine bone area before 14 years. After 15 years, bone area accruals were similar, as were height-adjusted spine BMC Z-scores, across age. CONCLUSIONS: PHIV had persistent deficits in all measures except height-adjusted spine BMD and BMC Z-scores. Data are needed on PHIV followed to adulthood.

Aged fibroblast-derived extracellular vesicles promote angiogenesis in melanoma.

Advancing age is a negative prognostic factor for cutaneous melanoma. However, the role of extracellular vesicles (EVs) within the melanoma tumor microenvironment (TME) has remained unexplored in the context of aging. While the size and morphology of the EVs isolated from young vs. aged fibroblasts remained unaltered, the contents of the protein cargo were changed. Aging reduced the expression of the tetraspanin CD9 in both the dermal fibroblasts and released EVs. CD9 is a crucial regulator of EV cargo sorting. Modulating the CD9 expression in fibroblasts was sufficient to alter its levels in EVs. Mass spectrometry analysis of EVs released by CD9 knockdown (KD) vs. control cells revealed a significant increase in angiopoietin-like protein 2 (ANGPTL2), an angiogenesis promoter. Analysis of primary endothelial cells confirmed increased sprouting under CD9 KD conditions. Together, our data indicate that aged EVs play an important role in promoting a tumor-permissive microenvironment.

A novel framework for ketamine-assisted couple therapy.

Intimate relationship distress is prevalent and is associated with poorer health, mental health, and mortality outcomes. Evidence-based couple therapies target cognitive, behavioral, and emotional processes that underlie relationship dysfunction. Increasing research and clinical evidence supports the efficacy of ketamine-assisted psychotherapy (KAP) for addressing clinical mental health concerns, including depression, anxiety disorders, posttraumatic stress disorder, and more. The purported mechanisms of KAP are also likely to improve psychosocial and relational functioning for patients and may be useful for supporting change mechanisms in couple therapy. This paper reviews the current evidence for therapeutic ketamine and KAP and outlines how the mechanisms of ketamine therapy may also augment the cognitive, behavioral, and emotional interventions in the most commonly used evidence-based couple therapies. Key mechanisms include increased neuroplasticity, changes in functional connectivity, adaptive dissociation, decreased inhibition, and reduced avoidance. Given the reciprocal interaction between relationship dysfunction and mental health problems, ketamine may also help alleviate relationship distress by directly treating clinical mental health symptoms. We then outline a proposed framework for ketamine-assisted couple therapy, addressing the application of KAP preparation, dosing, and integration to a dyadic intervention framework in a way that can be applied to different couple therapy modalities. This clinical framework for couples' KAP may be useful for clinicians and researchers working to improve the efficacy of couple therapy, particularly when one or both partners has accompanying mental health concerns.

Design, Construction, and Test of Compact, Distributed-Charge, X-Band Accelerator Systems that Enable Image-Guided, VHEE FLASH Radiotherapy.

The design and optimization of laser-Compton x-ray systems based on compact distributed charge accelerator structures can enable micron-scale imaging of disease and the concomitant production of beams of Very High Energy Electrons (VHEEs) capable of producing FLASH-relevant dose rates. The physics of laser-Compton x-ray scattering ensures that the scattered x-rays follow exactly the trajectory of the incident electrons, thus providing a route to image-guided, VHEE FLASH radiotherapy. The keys to a compact architecture capable of producing both laser-Compton x-rays and VHEEs are the use of X-band RF accelerator structures which have been demonstrated to operate with over 100 MeV/m acceleration gradients. The operation of these structures in a distributed charge mode in which each radiofrequency (RF) cycle of the drive RF pulse is filled with a low-charge, high-brightness electron bunch is enabled by the illumination of a high-brightness photogun with a train of UV laser pulses synchronized to the frequency of the underlying accelerator system. The UV pulse trains are created by a patented pulse synthesis approach which utilizes the RF clock of the accelerator to phase and amplitude modulate a narrow band continuous wave (CW) seed laser. In this way it is possible to produce up to 10 µA of average beam current from the accelerator. Such high current from a compact accelerator enables production of sufficient x-rays via laser-Compton scattering for clinical imaging and does so from a machine of "clinical" footprint. At the same time, the production of 1000 or greater individual micro-bunches per RF pulse enables > 10 nC of charge to be produced in a macrobunch of < 100 ns. The design, construction, and test of the 100-MeV class prototype system in Irvine, CA is also presented.

Longitudinal changes in mitochondrial-associated measures and insulin resistance in youth with perinatally-acquired HIV in the U.S.

HIV infection and its treatment are associated with mitochondrial dysfunction and metabolic derangement. However, longitudinal changes in oxidative phosphorylation activities [Complex I (C1) and Complex IV (C4)], or venous lactate/pyruvate ratios (LPR), and their relationships with insulin resistance (IR), remain unclear in youth living with perinatally-acquired HIV (YPHIV). We measured venous LPR, C1, and C4 activities in blood cells and homeostatic model assessment for IR (HOMA-IR) over two years. Limited longitudinal differences in mitochondrial-related measures and IR were observed in YPHIV vs youth perinatally HIV-exposed but uninfected. There were no systematic differences in C1, C4, or HOMA-IR between the groups.

Comparing behavioral measures of aggression in the laboratory: Taylor Aggression Paradigm versus Point-Subtraction Aggression Paradigm.

Aggression refers to a wide range of behaviors with lasting individual and societal consequences. Recurrent, unplanned aggressive behavior is the core diagnostic criterion for intermittent explosive disorder (IED). In this study, we compared two behavioral measures of aggression in the laboratory: the Taylor Aggression Paradigm (TAP) and the Point-Subtraction Aggression Paradigm (PSAP). This sample (n = 528) included community participants who met DSM-5 criteria for IED (n = 156), met DSM-5 criteria for a nonaggressive psychiatric disorder (n = 205), or did not meet DSM-5 criteria for any psychiatric disorder (n = 167). All participants completed the TAP, a single-session PSAP, and relevant self-report measures. MANOVA analyses demonstrated differences between IED participants and nonaggressive participants; however, these group differences were no longer significant for the PSAP after including demographic variables. Correlation analyses found that the TAP and PSAP were positively related to one another and the composite variables associated with aggressive behavior (i.e., history of aggression, impulsivity, and propensity to experience anger) and; dependent correlations revealed that past aggression and trait anger were more strongly related to the TAP. Differences in TAP and PSAP outcomes may be partially attributed to operationalizations of aggression and methods of aggression and provocation. Further, as aggressive and nonaggressive participants differed on the PSAP somewhat mirroring the TAP, our results add to growing evidence of the validity of a single-session PSAP; further research is needed to fully establish single-session PSAP as a laboratory aggression task compared to the multi-session PSAP.

Determinants of resistance and response to melanoma therapy.

Metastatic melanoma is among the most enigmatic advanced cancers to clinically manage despite immense progress in the way of available therapeutic options and historic decreases in the melanoma mortality rate. Most patients with metastatic melanoma treated with modern targeted therapies (for example, BRAFV600E/K inhibitors) and/or immune checkpoint blockade (for example, anti-programmed death 1 therapy) will progress, owing to profound tumor cell plasticity fueled by genetic and nongenetic mechanisms and dichotomous host microenvironmental influences. Here we discuss the determinants of tumor heterogeneity, mechanisms of therapy resistance and effective therapy regimens that hold curative promise.

A Radioactivity-mass Spectrometry Calibration Method Coupled with Biosynthesis to Generate a Metabolite Standard for Enzyme Kinetics Studies.

In early stages of drug development, the absence of authentic metabolite standards often results in semi-quantitative measurements of metabolite formation in reaction phenotyping studies using mass spectrometry (MS), leading to inaccuracies in the determination of enzyme kinetic parameters, such as the Michaelis constant (Km). Moreover, it is impossible to ascertain the maximum rate of enzyme-catalyzed reactions (kcat or Vmax). The use of radiolabeled parent compounds can circumvent this problem. However, radiometric detection exhibits significantly lower sensitivity compared to MS. To address these challenges, we have developed a stepwise approach that leverages biosynthesized radiolabeled and non-radiolabeled metabolites as standards, enabling accurate determination of Km, kcat or Vmax without the need for authentic metabolite standards. This approach, using the carbon-14 [14C] labeled metabolite to calibrate the unlabeled metabolite (14C calibration method), combines radiometric with LC-MS/MS detection to generate both [14C]-labeled and unlabeled metabolite standard curves to ensure that the sample concentrations measured are accurately quantitated. Two case studies were presented to demonstrate the utility of this method. We first compared the accuracy of the 14C calibration method to the use of authentic standards for quantitating imipramine metabolites. Next, we biosynthesized and quantitated the metabolites of BI 894416 using 14C calibration method and evaluated the enzyme kinetics of metabolite formation. The Km values of the metabolite formation demonstrated substantially improved accuracy compared to MS semi-quantitation. Moreover, the 14C calibration method offers a streamlined approach to prepare multiple metabolite standards from a single biosynthesis, reducing the time required for structure elucidation and metabolite synthesis.

The effect of rhizosphere pH on removal of naphthenic acid fraction compounds from oil sands process-affected water in a willow hydroponic system.

The extraction and processing of bitumen from the oil sands in northern Alberta, Canada generates large volumes of oil sands process-affected water (OSPW). OSPW contains a complex mixture of inorganic and organic compounds, including naphthenic acid fraction compounds (NAFCs) that are of particular concern due to their toxicity to aquatic organisms. Phytoremediation is a cost-effective, scalable approach that has the potential to remove NAFCs from OSPW and reduce OSPW toxicity. Environmental pH influences the chemical form and bioavailability of NAFCs. However, little is known about the influence of pH on the uptake of NAFCs in plant systems. This study sought to elucidate the impact of rhizosphere pH on the uptake of NAFCs using a sandbar willow (Salix interior) hydroponic system. To mimic and maintain the naturally low pH conditions of the root, OSPW solutions in these systems were adjusted to a low pH level (pH 5.0) and their NAFC uptake from solution was compared to that of OSPW at native pH (pH 8.0). Our findings revealed that the lower pH hydroponic systems demonstrated enhanced NAFC removal from solution as determined by LC-MS analysis, where up to 26% of NAFCs were removed from OSPW over 72 h at pH 5.0 compared to 8% removed at pH 8.0. Similarly, analysis of spike-in 13C-labeled NAs demonstrated that the OSPW hydroponic system rapidly removed a relatively labile NA (13C-cyclohexane carboxylic acid) from solution at both pH levels, whereas near complete removal of a recalcitrant NA (13C-1-adamantane carboxylic acid) was observed in pH 5.0 solutions only. These results provide insight into the importance of rhizosphere pH on efficient NAFC uptake by plant root systems. Further research will determine whether OSPW phytoremediation efficiency can be enhanced using field treatment conditions that promote low rhizosphere pH levels.

Utility of Body Weight, Urine Color, and Thirst Perception (WUT) in Determining Hydration in Young Adults.

OBJECTIVE: The primary aim of this study was to assess the efficacy of the weight, urine, thirst (WUT) framework in predicting dehydration after a body water manipulation protocol, while concurrently determining the individual and interactive contributions of the model components. METHODS: The total study sample was 93 participants (female, n = 47), recruited from two institutions. Phase 1 involved collecting daily hydration measures from free-living participants (Study 1, 58 participants for 3 days; Study 2, 35 participants for 7 days). Phase 2 entailed a two-hour passive heating protocol, where participants from Study 2 were randomly assigned to one of three groups that manipulated total body water over 24-hours using passive heating and fluid restriction. During each Phase, participants provided urine samples, underwent body mass measurements, and completed questionnaires pertaining to thirst perception. Morning and 24-hour urine samples were assessed for color, osmolality, and specific gravity. Differences between intervention groups, based on the probability of hydration status, were examined (ANOVA) and ridge regression analysis assessed the relative importance of variables within the WUT model. RESULTS: The study revealed significant differences among the intervention groups for predicted probability of dehydration, as determined by changes in body mass (p = 0.001), urine color (p = 0.044), and thirst perception (p < 0.001). Binomial ridge regression indicated that change in body mass (58%) and thirst perception (26%) were the most influential predictors of dehydration. CONCLUSIONS: These data support use of an enhanced version of the WUT model, underscoring the significance of changes in body mass and thirst perception in the assessment of hydration status.

Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis.

Aged patients with melanoma (>65 years old) have more aggressive disease relative to young patients (<55 years old) for reasons that are not completely understood. Analysis of the young and aged secretome from human dermal fibroblasts identified >5-fold levels of IGF-binding protein 2 (IGFBP2) in the aged fibroblast secretome. IGFBP2 functionally triggers upregulation of the PI3K-dependent fatty acid biosynthesis program in melanoma cells. Melanoma cells co-cultured with aged dermal fibroblasts have higher levels of lipids relative to those co-cultured with young dermal fibroblasts, which can be lowered by silencing IGFBP2 expression in fibroblasts prior to treating with conditioned media. Conversely, ectopically treating melanoma cells with recombinant IGFBP2 in the presence of conditioned media from young fibroblasts or overexpressing IGFBP2 in melanoma cells promoted lipid synthesis and accumulation in melanoma cells. Treatment of young mice with rIGFBP2 increases tumor growth. Neutralizing IGFBP2 in vitro reduces migration and invasion in melanoma cells, and in vivo studies demonstrate that neutralizing IGFBP2 in syngeneic aged mice reduces tumor growth and metastasis. Our results suggest that aged dermal fibroblasts increase melanoma cell aggressiveness through increased secretion of IGFBP2, stressing the importance of considering age when designing studies and treatment. SIGNIFICANCE: The aged microenvironment drives metastasis in melanoma cells. This study reports that IGFBP2 secretion by aged fibroblasts induces lipid accumulation in melanoma cells, driving an increase in tumor invasiveness. Neutralizing IGFBP2 decreases melanoma tumor growth and metastasis.

Effect of Different Isolation Methods on Bioaerosol Distribution: An In Vitro Study.

Purpose: The purposes of this in vitro study were to evaluate the effect of three isolation methods to mitigate bioaerosols during stainless steel crown (SSC) preparations and assess the distribution of Streptococcus mutans by aerosolization in closed-room operatories. Methods: Melamine teeth coated in laboratory-grown S. mutans biofilm were prepared for SSCs using three different isolation methods. Agar plates were placed in five locations throughout the operatory and opened during each preparation as well as for 10 minutes immediately following to collect aerosolized S. mutans. Bacterial colonies were counted after incubating plates for 48 hours. Data were analyzed for differences between the isolation method and plate locations. Results: Bacterial colony counts for teeth prepared using high-volume evacuation suction (HVE) with dental dam (DD) isolation were statistically significantly higher than for those prepared using HVE with a DryShield®(DS) and HVE with no isolation at the assistant (A) (P<0.001), operator face shield (FS) (P<0.001), and patient (Pt) (P=0.002) locations. No significant differences were found among isolation methods for parent (Pa) or rear delivery (RD) locations. The location that produced the most bacterial colony counts using HVE with DD isolation was FS (P<0.001), followed by A (P=0.04), Pt (P<0.001), and RD and Pa (P<0.001). Counts produced from teeth prepared with DS isolation were significantly higher at the Pt location than the A (P<0.001), FS (P=0.002), RD (P<0.001), and Pa (P=0.008) locations. Conclusion: The use of dental dam with high-volume evacuation suction during stainless steel crown preparations increased bioaerosols near the procedure, while dental evacuation systems (DryShield®) may effectively limit their spread.

Oral subchronic toxicity study and genetic toxicity evaluation of mitoquinone mesylate.

Mitochondrial dysfunction and excessive reactive oxygen species production contributes to the pathophysiology of aging. Coenzyme Q10 is thought to protect mitochondria from oxidative damage; thus, mitoquinone was developed as mitochondria-targeted analogue with similar antioxidant activity. Mitoquinone is the oxidized form of mitoquinol. Mitoquinone/mitoquinol mesylate has been proposed as a food ingredient. As part of the safety analysis, we performed genotoxicity assays and a 39-week toxicity study to determine overall toxicity potential. Mitoquinone mesylate showed no evidence of genotoxic potential in two in vitro assays, bacterial reverse mutation and human lymphocyte chromosome aberration, nor in the in vivo micronucleus test in rats. In the 39-week study in dogs, there were no findings observed, which were considered to represent adverse systemic toxicity; therefore, the high dose level (40 mg/kg/day) was considered the NOAEL. The principal findings in this study were fecal disturbances and vomiting. These findings were considered to be due to a local, possibly irritant effect of the test substance on the gastrointestinal tract and were not considered adverse as there were no impacts on clinical or histopathology. This highest dose exceeds the expected daily human intake more than 100-fold. Data from well-designed clinical trials actively collecting safety endpoints corroborate that 20 mg/day can be safely consumed and is not likely to result in significant gastrointestinal complaints. These results support the conclusion that the use of mitoquinone/mitoquinol mesylate as a food ingredient is safe.

A Stable Aluminum Tris(dithiolene) Triradical.

A stable aluminum tris(dithiolene) triradical (3) was experimentally realized through a low-temperature reaction of the sterically demanding lithium dithiolene radical (2) with aluminum iodide. Compound 3 was characterized by single-crystal X-ray diffraction, UV-vis and EPR spectroscopy, SQUID magnetometry, and theoretical computations. The quartet ground state of triradical 3 has been unambiguously confirmed by variable-temperature continuous wave EPR experiments and SQUID magnetometry. Both SQUID magnetometry and broken-symmetry DFT computations reveal a small doublet-quartet energy gap [ΔEDQ = 0.18 kcal mol-1 (SQUID); ΔEDQ = 0.14 kcal mol-1 (DFT)]. The pulsed EPR experiment (electron spin echo envelop modulation) provides further evidence for the interaction of these dithiolene-based radicals with the central aluminum nucleus of 3.

A multicentre survey investigating the knowledge, behaviour, and attitudes of surgical healthcare professionals to frailty assessment in emergency surgery: DEFINE(surgery).

PURPOSE: Screening for frailty in people admitted with emergency surgical pathology can initiate timely referrals to enhanced perioperative services such as intensive care and geriatric medicine. However, there has been little research exploring surgical healthcare professionals' opinions to frailty assessment, or accuracy in identification. This study aimed to assess the knowledge, behaviour, and attitudes of healthcare professionals to frailty assessment in emergency surgical admissions. METHODS: We designed a cross-sectional multicentre study developed by a multiprofessional team of surgeons, geriatricians, and supported by patients. A semi-structured survey examined attitudes and behaviours. Knowledge was assessed by comparing respondents' accuracy in scoring twenty-two surgical case vignettes using the Clinical Frailty Scale. RESULTS: Eleven hospitals across England, Wales, and Scotland participated. Two hundred and eleven clinicians responded-20.4% junior doctors, 43.6% middle grade doctors, 24.2% senior doctors, 11.4% nurses and physician associates. Respondents strongly supported perioperative frailty assessment. Most were already assessing for frailty, although frequently not using a standardised tool. There was a strong call for more frailty education. Participants scored 2175 vignettes with 55.4% accurately meeting the gold standard; accuracy improved to 87.3% when categorised into "not frail/mildly frail/severely frail" and 94% when dichotomised to "not frail/frail". CONCLUSION: Frailty assessment is well supported by healthcare professionals working in surgery. However, standardised tools are not routinely being used, and only half of respondents could accurately identify frailty. Better education around frailty assessment is needed for healthcare professionals working in surgery to improve perioperative pathway for people living with frailty.

A novel system to determine activity of individual uridine 5'-diphospho-glucuronosyltransferase (UGT) isoforms: Recombinant UGT-beads.

Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.

Long-Acting Gonadotropin-Releasing Hormone Analogues for Central Precocious Puberty, Including 45-Mg 6-Month Subcutaneous Leuprolide Acetate: Use for Treatment and Treatment Monitoring.

INTRODUCTION: Studies of gonadotropin-releasing hormone analogues (intramuscular [IM] leuprolide acetate [LA] and triptorelin) for treatment monitoring of central precocious puberty (CPP) demonstrate this approach is effective for confirming pubertal hormone suppression. Herein, we provide new data using subcutaneous LA (SC LA), suggesting similar efficacy for treatment monitoring. METHODS: PubMed, Embase, and CINAHL were searched for studies of GnRHa used to monitor treatment of CPP. The titles and the abstracts were reviewed; 5 studies were selected. Additionally, new unpublished data for SC LA from the original phase 3 trial (primary data published by Klein et al.) were evaluated. Serum luteinizing hormone (LH) and leuprolide levels at screening, 1, 4, and 6 h after the first dose SC LA were analyzed and plotted. RESULTS: Data from 162 children (155 girls) were evaluated. SC and IM LA produced overlapping median LH concentration curves and peak LH concentrations after the first dose. For IM LA, subsequent doses yielded suppressed peak LH levels (2.7 IU/L [mean]). For SC LA, subsequent doses also resulted in significant suppressed peak LH levels (0.2 ± 0.02 IU/L) and achieved sex-steroid hormone suppression of >98%. CONCLUSIONS: Compared to IM LA and triptorelin, long-acting SC LA shows similar burst kinetics and rapid LH rise after the first dose, followed by similar suppression of LH and sex steroids after subsequent doses. Since IM LA and triptorelin have demonstrated usefulness that is comparable to that of traditional GnRH stimulation testing for monitoring CPP, we presume that SC LA may be similarly employed.