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Background: Volenrelaxin is a half-life-extended recombinant human relaxin protein developed for improving kidney perfusion and cardiorenal function. This study assessed the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of volenrelaxin following single- and multiple-ascending doses (SAD and MAD) administration., Methods: In this Phase 1, four-part, randomized, double-blinded, placebo-controlled SAD and MAD study in healthy participants, SAD participants (n = 56) received an intravenous or subcutaneous dose of volenrelaxin or placebo in a dose-ascending manner. MAD participants (n = 77) received volenrelaxin or placebo subcutaneously once weekly for 5 weeks. Effective renal plasma flow (ERPF) and measured glomerular filtration rate (mGFR) were determined by para-aminohippurate and iohexol clearance, respectively., Results: Volenrelaxin demonstrated an extended half-life and increased acute and chronic placebo-adjusted ERPF change from baseline by 50% and 44%, respectively (P < .0001). mGFR was unchanged, while filtration fraction and afferent/efferent renal arteriolar resistances were reduced. Systolic and diastolic blood pressures decreased, and pulse rate increased with increasing volenrelaxin exposures, demonstrating maximal model-derived placebo-adjusted changes (90% confidence interval) of -6.16 (-8.04, -4.28) mmHg, -6.10 (-7.61, -4.58) mmHg and +4.39 (+3.38, +5.39) bpm, respectively. Adverse events were mild, with no difference in orthostatic hypotension between volenrelaxin and placebo., Conclusion: Volenrelaxin was well-tolerated, safe and suitable for weekly subcutaneous dosing. Volenrelaxin showed a sustained improvement in kidney perfusion upon repeated dosing, supporting further clinical development in chronic kidney disease and chronic heart failure., Clinical Trial Registration: NCT04768855., (© The Author(s) 2024. Published by Oxford University Press on behalf of the ERA.)

Adverse drug events (ADEs) account for a significant mortality, morbidity, and cost burden. Pharmacogenetic testing has the potential to reduce ADEs and inefficacy. The objective of this INGENIOUS trial (NCT02297126) analysis was to determine whether conducting and reporting pharmacogenetic panel testing impacts ADE frequency. The trial was a pragmatic, randomized controlled clinical trial, adapted as a propensity matched analysis in individuals (N = 2612) receiving a new prescription for one or more of 26 pharmacogenetic-actionable drugs across a community safety-net and academic health system. The intervention was a pharmacogenetic testing panel for 26 drugs with dosage and selection recommendations returned to the health record. The primary outcome was occurrence of ADEs within 1 year, according to modified Common Terminology Criteria for Adverse Events (CTCAE). In the propensity-matched analysis, 16.1% of individuals experienced any ADE within 1-year. Serious ADEs (CTCAE level ≥ 3) occurred in 3.2% of individuals. When combining all 26 drugs, no significant difference was observed between the pharmacogenetic testing and control arms for any ADE (Odds ratio 0.96, 95% CI: 0.78-1.18), serious ADEs (OR: 0.91, 95% CI: 0.58-1.40), or mortality (OR: 0.60, 95% CI: 0.28-1.21). However, sub-group analyses revealed a reduction in serious ADEs and death in individuals who underwent pharmacogenotyping for aripiprazole and serotonin or serotonin-norepinephrine reuptake inhibitors (OR 0.34, 95% CI: 0.12-0.85). In conclusion, no change in overall ADEs was observed after pharmacogenetic testing. However, limitations incurred during INGENIOUS likely affected the results. Future studies may consider preemptive, rather than reactive, pharmacogenetic panel testing., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Prior to the fitting of hearing aids, clinicians and patients must discuss the best treatment options for the physical and audiologic needs of the patients. To be able to confidently make these decisions, the clinician should complete a medical and audiological case history. Additionally, clinicians need accurate results from a comprehensive audiologic evaluation. The evaluation should include the following: pure-tone testing, word recognition testing, speech-in-noise testing, and loudness discomfort level measures. This article will outline the process and procedures for acquiring this information in line with the Audiology Practice Standards Organization (APSO) Guidelines for Adult Hearing Aid Fittings Standards 1 and 4. This article will also discuss how results can affect decision-making during the hearing aid selection and fitting process., Competing Interests: Conflict of Interest None declared., (Thieme. All rights reserved.)

We investigated the acute and chronic effects of efavirenz, a widely used antiretroviral drug, and CYP2B6 genotypes on the disposition of racemic and stereoisomers of bupropion (BUP) and its active metabolites, 4-hydroxyBUP, threohydroBUP and erythrohydroBUP. The primary objective of this study was to test how multiple processes unique to the efavirenz- CYP2B6 genotype interaction influence the extent of efavirenz-mediated drug-drug interaction (DDI) with the CYP2B6 probe substrate BUP. In a three-phase, sequential, open-label study, healthy volunteers (N=53) were administered a single 100 mg oral dose of BUP alone (control phase), with a single 600 mg oral efavirenz dose (inhibition phase), and after 17-days pretreatment with efavirenz (600 mg/day) (induction phase). Compared to the control phase, we show for the first time that efavirenz significantly decreases and chronically increases the exposure of hydroxyBUP and its diastereomers, respectively, and these interactions were CYP2B6 genotype dependent. Chronic efavirenz enhances the elimination of racemic BUP and its enantiomers as well as of threo- and erythro-hydroBUP and their diastereomers, suggesting additional novel mechanisms underlying efavirenz interaction with BUP. The effects of efavirenz and genotypes were nonstereospecific. In conclusion, acute and chronic administration of efavirenz inhibits and induces CYP2B6 activity. Efavirenz-BUP interaction is complex involving time- and CYP2B6 genotype-dependent inhibition and induction of primary and secondary metabolic pathways. Our findings highlight important implications to the safety and efficacy of BUP, study design considerations for future efavirenz interactions, and individualized drug therapy based on CYP2B6 genotypes. Significance Statement The effects of acute and chronic doses of efavirenz on the disposition of racemic and stereoisomers of BUP and its active metabolites were investigated in healthy volunteers. Efavirenz causes an acute inhibition, but chronic induction of CYP2B6 in a genotype dependent manner. Chronic efavirenz induces BUP reduction and the elimination of BUP active metabolites. Efavirenz's effects were non-stereospecific. These data reveal novel mechanisms underlying efavirenz DDI with BUP and provide important insights into time- and CYP2B6 genotype dependent DDIs., (Copyright © 2020 American Society for Pharmacology and Experimental Therapeutics.)

Purpose: We developed an accessible method for labeling small extracellular vesicles (sEVs) without disrupting endogenous ligands. Using labeled sEVs administered to conscious rats, we developed a multiple compartment pharmacokinetic model to identify potential differences in the disposition of sEVs from three different cell types., Methods: Crude sEVs were labeled with a non-homologous oligonucleotide and isolated from cell culture media using a commercial reagent. Jugular vein catheters were used to introduce EVs to conscious rats (n = 30) and to collect blood samples. Digital PCR was leveraged to allow for quantification over a wide dynamic range. Non-linear mixed effects analysis with first order conditional estimation - extended least squares (FOCE ELS) was used to estimate population-level parameters with associated intra-animal variability., Results: 86.5% ± 1.5% (mean ± S.E.) of EV particles were in the 45-195 nm size range and demonstrated protein and lipid markers of endosomal origin. Incorporated oligonucleotide was stable in blood and detectable over five half-lives. Data were best described by a three-compartment model with one elimination from the central compartment. We performed an observation-based simulated posterior predictive evaluation with prediction-corrected visual predictive check. Covariate and bootstrap analyses identified cell type having an influence on peripheral volumes (V2 and V3) and clearance (Cl3)., Conclusions: Our method relies upon established laboratory techniques, can be tailored to a variety of biological questions regarding the pharmacokinetic disposition of extracellular vesicles, and will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics., (© 2021. The Author(s).)

Extracellular vesicles (EVs) convey information used in cell-to-cell interactions. Lipid analysis of EVs remains challenging because of small sample amounts available. Lipid discovery using traditional mass spectrometry platforms based on liquid chromatography and high mass resolution typically employs milligram sample amounts. We report a simple workflow for lipid profiling of EVs based on multiple reaction monitoring (MRM) profiling that uses microgram amounts of sample. After liquid-liquid extraction, individual EV samples were injected directly into the electrospray ionization (ESI) ion source at low flow rates (10 μl/min) and screened for 197 MRM transitions chosen to be a characteristic of several classes of lipids. This choice was based on a discovery experiment, which applied 1,419 MRMs associated with multiple lipid classes to a representative pooled sample. EVs isolated from 12 samples of human lymphocytes and 16 replicates from six different rat cells lines contained an estimated amount of total lipids of 326 to 805 μg. Samples showed profiles that included phosphatidylcholine (PC), sphingomyelin (SM), cholesteryl ester (CE), and ceramide (Cer) lipids, as well as acylcarnitines. The lipid profiles of human lymphocyte EVs were distinguishable using principal component and cluster analysis in terms of prior antibody and drug exposure. Lipid profiles of rat cell lines EV's were distinguishable by their tissue of origin., (© 2020 John Wiley & Sons Ltd.)

Objective: The prevalence of atherosclerotic cardiovascular disease (ASCVD) in younger adults has increased over the past decade. However, it is less well established whether patient reported outcomes differ between younger and older adults with ASCVD. We sought to evaluate age-specific differences in patient reported outcomes among adults with ASCVD., Methods: This was a retrospective cross-sectional survey study. We used data from the 2006-2015 Medical Expenditure Panel Survey (MEPS), a nationally representative sample of the United States population. Adults ≥18 years with a diagnosis of ASCVD, ascertained by ICD9 codes or self-reported data, were included. Logistic regression was used to compare self-reported patient-clinician communication, patient satisfaction, perception of health, emergency department (ED) visits, and use of preventive medications (aspirin and statins) by age category [Young: 18-44, Middle: 45-64, Older: ≥65 years]. We used two-part econometric modeling to evaluate age-specific annual healthcare expenditure., Results: There were 21,353 participants included. Over 9000 (42.6%-weighted) of the participants were young or middle aged, representing ~9.9 million adults aged <65 years with ASCVD nationwide. Compared with older adults, middle-aged and young adults with ASCVD were more likely to report poor patient-clinician communication [OR 1.73 (95% CI 1.28-2.33) and 2.49 (1.76-3.51), respectively], poor healthcare satisfaction, and poor perception of health status, have increased ED utilization and were also less likely to be using aspirin and statins. The mean annual healthcare expenditure was highest among middle-aged adults [$10,798 (95% CI, $10,012 to $11,583)]., Conclusion: Compared with older adults, younger adults with ASCVD were more likely to report poor patient experience and poor health status and less likely to be using preventive medications. More effort needs to be geared towards understanding the age-specific differences in healthcare quality and delivery to improve outcomes among high-risk young adults with ASCVD., (© 2020 The Authors.)

Importance: Cardiovascular disease (CVD) is the leading cause of death and disability among women. Achievement of recommended physical activity (PA) levels is an essential component of CVD management., Objective: To describe trends, sociodemographic factors, and health care expenditures associated with suboptimal PA among a nationally representative sample of US women with CVD., Design, Setting, and Participants: This cross-sectional study used serial data from the Medical Expenditure Panel Survey from 2006 through 2015. The analyses were conducted in August 2018. Women who had self-reported and/or International Classification of Diseases, Ninth Revision, diagnosis of CVD were included., Main Outcomes and Measures: Recommended PA was defined as 30 minutes or more of moderate- to vigorous-intensity exercise, 5 or more days per week. Weighted logistic regression was used to examine the associations of various sociodemographic factors with suboptimal PA, adjusted for comorbidities. A 2-part econometric model was used to assess health care expenditures., Results: A total of 18 027 women were included in this study. The results were weighted to provide estimates for approximately 19.5 million adult women in the United States with CVD (mean [SD] age, 60.4 [16.9] years). More than half of the women with CVD reported suboptimal PA, a trend that increased during the 10-year period, with 58.2% (95% CI, 55.9%-60.5%) of participants reporting suboptimal PA in 2006-2007 vs 61.9% (95% CI, 59.7%-64.2%) in 2014-2015 (P = .004). The proportion of women with suboptimal PA differed by sociodemographic factors. In adjusted models, compared with non-Hispanic white women, African American women (odds ratio, 1.22; 95% CI, 1.08-1.38) and Hispanic women (odds ratio, 1.33; 95% CI, 1.13-1.58) were more likely to have suboptimal PA. Women from low- or very low-income strata (compared with high-income strata), enrolled in public insurance (compared with private insurance), and with less than high school education (compared with at least some college education) were more likely to have suboptimal PA. Health care costs among women with CVD with suboptimal PA were higher compared with those among women who met the recommended PA, and this increased through time, from a mean total health care expenditure of $12 724 (95% CI, $11 627-$13 821) in 2006-2007 to $14 820 (95% CI, $13 521-$16 119) in 2014-2015., Conclusions and Relevance: The proportion of women with CVD not meeting recommended PA is high and increasing, particularly among certain racial/ethnic and socioeconomic groups, and is associated with significant health care costs. More must be done to improve PA for secondary prevention and reduction of expenditures among women with CVD.

Background: Unhealthy lifestyles and inflammation contribute to cardiovascular disease (CVD). GlycA is a novel biomarker of systemic inflammation representing post-translational glycosylation of acute phase reactants and associated with increased clinical CVD risk., Hypothesis: We hypothesized that ideal cardiovascular health (CVH), as assessed by (higher) Life's Simple 7 (LS7) scores, would be associated with lower GlycA levels among individuals free of CVD in a multiethnic community-based population., Methods: This was a cross-sectional study of 6479 Multi-Ethnic Study of Atherosclerosis participants [53% women; mean age 62 ± 10 years] with GlycA levels measured at baseline by nuclear magnetic resonance spectroscopy. The LS7 metrics (smoking, physical activity, diet, body mass index, blood pressure, cholesterol, and glucose) were each scored as ideal (2), moderate (1), or poor (0). Total scores were summed and categorized as optimal (12-14), average (8-11), and inadequate (0-7). Linear regression assessed percent difference in GlycA by LS7 scores, after adjusting for age, sex, ethnicity, education, income, family history of CVD, and other inflammatory biomarkers., Results: GlycA levels were 403.4 ± 63.1, 374.4 ± 59.2, and 350.3 ± 56.2 micromoles per liter (μmol/L) for inadequate, average, and optimal CVH, respectively (P-trend <0.001). After multivariable adjustment, GlycA remained independently and inversely associated with CVH categories, with a lower mean GlycA level of 5 μmol/L (95% confidence interval 4.5-5.8) for each one unit increment in LS7 score., Conclusions: Among this group of ethnically diverse individuals without CVD, suboptimal CVH is associated with higher GlycA levels, independent of traditional inflammatory biomarkers. Strategies aimed at improving CVH might reduce GlycA, which could be a marker of reduced risk of future CVD events., (© 2018 Wiley Periodicals, Inc.)

The primary goal of amplification is to restore audibility without causing discomfort; for someone with severe-to-profound hearing loss, the reduced dynamic range poses unique challenges in hearing-assistive device fitting. These challenges, including physiological limitation, processing difficulties, technology constraints, and other confounding factors, must be considered when selecting, fitting, and counseling for appropriate amplification. Many of the advanced features in hearing aids do not adequately address the unique characteristics of patients with severe-to-profound hearing loss. This review article will attempt to unravel some of the challenges and associated considerations when fitting adults and children with severe-to-profound hearing loss.

Preclinical studies in animals often require frequent blood sampling over prolonged periods. A preferred method in rats is the implantation of a polyurethane catheter into the jugular vein, with heparinized glycerol as a lock solution. However, analysis of various biologic compounds (for example, microRNA) precludes the use of heparin. We used sodium citrate as an alternative to heparin but observed more frequent loss of catheter patency. We hypothesized that this effect was due to evaporation of lock solution at the exteriorized portion of the catheter, subsequent blood infiltration into the catheter, and ultimately clot formation within the catheter. We therefore tested evaporation and its variables in vitro by using 5 common catheter materials. We used the migration of dye into vertically anchored catheters as a measure of lock displacement due to evaporation. Exposure to dry room-temperature air was sufficient to cause dye migration against gravity, whereas a humid environment and adding glycerol to the lock solution mitigated this effect, thus confirming loss of the lock solution from the catheter by evaporation. We tested 4 catheter treatments for the ability to reduce lock evaporation. Results were validated in vivo by using male Sprague-Dawley rats (n = 12) implanted with polyurethane jugular vein catheters and randomized to receive a nitrocellulose-based coating on the exteriorized portion of the catheter. Coating the catheters significantly improved patency, as indicated by a Kaplan-Meier log-rank hazard ratio greater than 5 in untreated catheters. We here demonstrate that a simple nitrocellulose coating reduces evaporation from and thus prolongs the patency of polyurethane catheters in rats.

Colistin sulfate (polymixin E) is an antibiotic prescribed with increasing frequency for severe Gram-negative bacterial infections. As nephrotoxicity is a common side effect, the discovery of pharmacogenomic markers associated with toxicity would benefit the utility of this drug. Our objective was to identify genetic markers of colistin cytotoxicity that were also associated with expression of key proteins using an unbiased, whole genome approach and further evaluate the functional significance in renal cell lines. To this end, we employed International HapMap lymphoblastoid cell lines (LCLs) of Yoruban ancestry with known genetic information to perform a genome-wide association study (GWAS) with cellular sensitivity to colistin. Further association studies revealed that single nucleotide polymorphisms (SNPs) associated with gene expression and protein expression were significantly enriched in SNPs associated with cytotoxicity ( p ≤ 0.001 for gene and p = 0.015 for protein expression). The most highly associated SNP, chr18:3417240 ( p = 6.49 × 10 -8 ), was nominally a cis -expression quantitative trait locus (eQTL) of the gene TGIF1 (transforming growth factor β (TGFβ)-induced factor-1; p = 0.021) and was associated with expression of the protein HOXD10 (homeobox protein D10; p = 7.17 × 10 -5 ). To demonstrate functional relevance in a murine colistin nephrotoxicity model, HOXD10 immunohistochemistry revealed upregulated protein expression independent of mRNA expression in response to colistin administration. Knockdown of TGIF1 resulted in decreased protein expression of HOXD10 and increased resistance to colistin cytotoxicity. Furthermore, knockdown of HOXD10 in renal cells also resulted in increased resistance to colistin cytotoxicity, supporting the physiological relevance of the initial genomic associations.

siRNA stabilized for in vivo applications is filtered and reabsorbed in the renal proximal tubule (PT), reducing mRNA expression transiently. Prior siRNA efforts have successfully prevented upregulation of mRNA in response to injury. We proposed reducing constitutive gene and protein expression of LRP2 (megalin) in order to understand its molecular regulation in mice. Using siRNA targeting mouse LRP2 (si LRP2 ), reduction of LRP2 mRNA expression was compared to scrambled siRNA (siSCR) in mouse PT cells. Mice received si LRP2 administration optimized for dose, administration site, carrier solution, administration frequency, and administration duration. Kidney cortex was collected upon sacrifice. Renal gene and protein expression were compared by qRT-PCR, immunoblot, and immunohistochemistry (IHC). Compared to siSCR, si LRP2 reduced mRNA expression in PT cells to 16.6% ± 0.6%. In mouse kidney cortex, si LRP2 reduced mRNA expression to 74.8 ± 6.3% 3 h and 70.1 ± 6.3% 6 h after administration. mRNA expression rebounded at 12 h (160.6 ± 11.2%). No megalin renal protein expression reduction was observed by immunoblot or IHC, even after serial twice daily dosing for 3.5 days. Megalin is a constitutively expressed protein. Although LRP2 renal mRNA expression reduction was achieved, siRNA remains a costly and inefficient intervention to reduce in vivo megalin protein expression.

Unlabelled: Membrane drug transporters contribute to the disposition of many drugs. In human liver, drug transport is controlled by two main superfamilies of transporters, the solute carrier transporters (SLC) and the ATP Binding Cassette transporters (ABC). Altered expression of these transporters due to drug-drug interactions can contribute to differences in drug exposure and possibly effect. In this study, we determined the effect of rifampin on gene expression of hundreds of membrane transporters along with all clinically relevant drug transporters., Methods: In this study, primary human hepatocytes (n = 7 donors) were cultured and treated for 24 h with rifampin and vehicle control. RNA was isolated from the hepatocytes, mRNA expression was measured by RNA-seq, and miRNA expression was analyzed by Taqman OpenArray. The effect of rifampin on the expression of selected transporters was also tested in kidney cell lines. The impact of rifampin on the expression of 410 transporter genes from 19 different transporter gene families was compared with vehicle control., Results: Expression patterns of 12 clinically relevant drug transporter genes were changed by rifampin (FDR < 0.05). For example, the expressions of ABCC2, ABCB1, and ABCC3 were increased 1.9-, 1.7-, and 1.2-fold, respectively. The effects of rifampin on four uptake drug transporters (SLCO1B3, SLC47A1, SLC29A1, SLC22A9) were negatively correlated with the rifampin effects on specific microRNA expression (SLCO1B3/miR-92a, SLC47A1/miR-95, SLC29A1/miR-30d#, and SLC22A9/miR-20; r < -0.79; p < 0.05). Seven hepatic drug transporter genes (SLC22A1, SLC22A5, SLC15A1, SLC29A1, SLCO4C1, ABCC2, and ABCC4), whose expression was altered by rifampin in hepatocytes, were also present in a renal proximal tubular cell line, but in renal cells rifampin did not alter their gene expression. PXR expression was very low in the kidney cells; this may explain why rifampin induces gene expression in a tissue-specific manner., Conclusion: Rifampin alters the expression of many of the clinically relevant hepatic drug transporters, which may provide a rational basis for understanding rifampin-induced drug-drug interactions reported in vivo. The relevance of its effect on many other transporters remains to be studied.

Objective: Plasma miRNAs represent potential minimally invasive biomarkers to monitor and predict outcomes from chemotherapy. The primary goal of the current study-consisting of patients with recurrent, platinum-resistant ovarian cancer-was to identify the changes in circulating miRNA concentrations associated with decitabine followed by carboplatin chemotherapy treatment. A secondary goal was to associate clinical response with changes in circulating miRNA concentration., Methods: We measured miRNA concentrations in plasma samples from 14 patients with platinum-resistant, recurrent ovarian cancer enrolled in a phase II clinical trial that were treated with a low dose of the hypomethylating agent (HMA) decitabine for 5 days followed by carboplatin on day 8. The primary endpoint was to determine chemotherapy-associated changes in plasma miRNA concentrations. The secondary endpoint was to correlate miRNA changes with clinical response as measured by progression free survival (PFS)., Results: Seventy-eight miRNA plasma concentrations were measured at baseline (before treatment) and at the end of the first cycle of treatment (day 29). Of these, 10 miRNAs (miR-193a-5p, miR-375, miR-339-3p, miR-340-5p, miR-532-3p, miR-133a-3p, miR-25-3p, miR-10a-5p, miR-616-5p, and miR-148b-5p) displayed fold changes in concentration ranging from -2.9 to 4 (p<0.05), in recurrent platinum resistant ovarian cancer patients, that were associated with response to decitabine followed by carboplatin chemotherapy. Furthermore, lower concentrations of miR-148b-5p after this chemotherapy regimen were associated (P<0.05) with the PFS., Conclusions: This is the first report demonstrating altered circulating miRNA concentrations following a combination platinum plus HMA chemotherapy regiment. In addition, circulating miR-148b-5p concentrations were associated with PFS and may represent a novel biomarker of therapeutic response, with this chemotherapy regimen, in women with recurrent, drug-resistant ovarian cancer.

Plasma-derived microRNAs (miRNAs) are being used as biomarkers, and have been associated with human liver disease and function including fibrosis, inflammation, and drug-induced liver injury. They may also be biomarkers of the drug metabolism function of the liver. In order for plasma miRNA to function as a clinical biomarker, predictable variability is necessary during processing from whole blood to plasma. The current study evaluated the variability of miRNA in whole blood stored for 0.5, 1, 2, 4, 8, and 12 hours following the blood draw under clinical conditions (room temperature) prior to the separation of the plasma. Four healthy volunteers were recruited. Blood from all subjects was collected twice. MicroRNA-16 (miR-16) and miR-223 were evaluated because many studies have shown them to be reliably present in plasma and useful for normalization. miRNA concentrations were measured by real-time polymerase chain reaction. The coefficient of variability of the cycle threshold values for subjects for miR-223 and miR-16 ranged from ∼3.6 to 6.8% and ∼1.48 to 4.1%, respectively, over the 12-hour incubation. A second blood collection was performed to determine interday variability. The coefficient of variance from the initial blood draw compared with the final blood draw for each subject ranged from 0.42 to 7.9% for miR-16 and 1.7 to 8.3% for miR-223, indicating that these miRNAs have limited interday variability. We conclude that plasma miR-16 or miR-223 concentrations are stable in whole blood at room temperature for up to 12 hours.

Hepatic nuclear factor 4α (HNF4A) is a nuclear transcription factor that regulates the expression of many genes involved in drug disposition. To identify additional molecular mechanisms that regulate HNF4A, we identified microRNAs (miRNAs) that target HNF4A expression. In silico analyses suggested that HNF4A is targeted by many miRNAs. We conducted in vitro studies to validate several of these predictions. With use of an HNF4A 3'-untranslated region (UTR) luciferase reporter assay, five of six miRNAs tested significantly down-regulated (∼20-40%) the luciferase activity. In HepG2 cells, miR-34a and miR-449a also down-regulated the expression of both the HNF4A protein and an HNF4A target gene, PXR (∼30-40%). This regulation appeared without reduction in HNF4A mRNA expression, suggesting that they must be blocking HNF4A translation. Using additional bioinformatic algorithms, we identified polymorphisms that are predicted to alter the miRNA targeting of HNF4A. Luciferase assays indicated that miR-34a and miR-449a were less effective in regulating a variant (rs11574744) than the wild-type HNF4A 3'-UTR. In vivo, subjects with the variant HNF4A had lower CYP2D6 enzyme activity, although this result was not statistically significant (p = 0.16). In conclusion, our findings demonstrate strong evidence for a role of miRNAs in the regulation of HNF4A.

Objective: Emerging work has revealed an integral role of the tumor necrosis factor-alpha (TNF-alpha) nuclear factor (NF)-kappaB pathway in the regulation of hematopoiesis. TNF-alpha inhibition of hematopoietic stem/progenitor cell growth involves type I TNF-alpha receptor (TNF-RI) and type II TNF-alpha receptor (TNF-RII). However, the role of TNF-RI vs TNF-RII in mediating this response is less clear. Full induction of NF-kappaB-dependent gene expression through TNF-RI requires the transcriptional coactivator SIMPL (substrate that interacts with mouse pelle-like kinase). To address the role of SIMPL in TNF-alpha-dependent signaling in hematopoiesis, endothelial cells and hematopoietic progenitors expressing SIMPL short hairpin RNA were characterized., Material and Methods: In vitro gene expression and progenitor assays employing SIMPL short hairpin RNA were used to examine the requirement for SIMPL in TNF-alpha-dependent effects upon cytokine gene expression and hematopoietic progenitor cell growth. Competitive repopulation studies were used to extend these studies in vivo., Results: SIMPL is required for full TNF-RI-dependent expression of NF-kappaB-controlled cytokines in endothelial cells. Hematopoietic progenitor cell expansion is not affected if progenitors lacked SIMPL or if progenitors are treated with human TNF-alpha, which signals through TNF-RI. In the absence of SIMPL, human TNF-alpha leads to a dramatic decrease in progenitor cell expansion that is not due to apoptosis. Loss of SIMPL does not affect the activity of transforming growth factor-beta1 and interferon-gamma, other known suppressors of hematopoiesis., Conclusions: Suppression of myeloid progenitor cell expansion requires signaling through TNF-RI and TNF-RII. Signals transduced through the TNF-alpha-TNF-RI-SIMPL pathway support hematopoietic progenitor cell survival, growth and differentiation., (Copyright 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Objective: The intracellular redox state plays an important role in controlling inflammation. Clinical and laboratory data suggest that inflammation can lead to tumor progression. We hypothesized that restoring intracellular redox control would inhibit inflammation and subsequently tumor progression. Our studies were designed to investigate the effect of alpha-lipoic acid (ALA), a naturally occurring antioxidant, on a key inflammatory signaling pathway and cell proliferation in normal and tumorigenic ovarian surface epithelial cells., Methods: Normal and tumorigenic ovarian surface epithelial cells were isolated as described by Roby and coworkers [Roby KF, Taylor CC, Sweetwood JP, Cheng Y, Pace JL, Tawpik O, Persons DL, Smith PG, Terranova PF, Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogen 2000;21 (4):585. [1]]. The effect of ALA on cellular function was measured in cell proliferation and apoptosis assays. p27(kip1) protein levels were measured by Western analysis. Activation of NF-kappaB dependent transcription was assessed in cell cultures transiently transfected with NF-kappaB controlled reporter constructs., Results: Our results reveal that ALA selectively inhibits the growth of tumorigenic as compared to non-tumorigenic ovarian surface epithelial cells. The growth inhibitory effect of ALA is not due to induction of apoptosis but instead is associated with an increase in the half-life of the cyclin-dependent kinase inhibitor, p27(kip1). In parallel to the growth inhibitory effect, ALA also affects a key inflammatory signaling pathway by inhibiting TNFalpha-induced NF-kappaB signaling activity., Conclusions: Our studies are the first to show that ALA treatment has a growth inhibitory effect on malignant surface epithelial cells of ovarian origin. We have also confirmed the reproducibility of the immunocompetent mouse ovarian cancer model originally described by Roby and coworkers [Roby KF, Taylor CC, Sweetwood JP, Cheng Y, Pace JL, Tawpik O, Persons DL, Smith PG, Terranova PF, Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogen 2000;21 (4):585].

One-hundred and two women had axillary lymphadenectomy for breast cancer and were randomised to early discharge with axillary drain in situ on the third postoperative day or standard duration 7 day hospital stay. The two groups did not differ with respect to seroma formation, wound infection or psychological profile as measured by the Hospital Anxiety and Depression Scale and Spielberger State Trait and Anxiety Inventory. Patient satisfaction levels were high in the early discharge group. The results confirm that early discharge after axillary lymphadenectomy is safe, practicable and satisfactory for patients. Such a policy offers considerable resource savings.

Peritonitis is the most frequent cause for emergency hospital admission in continuous ambulatory peritoneal dialysis (CAPD) patients. Patients may present with 'surgical' peritonitis from other intra-abdominal pathology, but are treated initially as CAPD-related peritonitis. We present nine such cases, each failing to respond to standard conservative treatment, and ultimately coming to laparotomy. Of the nine patients, six survived, five transferring to long-term haemodialysis and one patient returning to CAPD. Failure to respond to standard measures should alert the physician to the possibility of an intra-abdominal emergency. The presence of enteric organisms, particularly E. coli, is an additional suspicious feature. The diagnosis may be difficult and we recommend early surgical referral and appropriate surgical measures (laparotomy rather than simple catheter removal) in order to decrease morbidity and mortality.

One hundred and twenty-one cases of ductal carcinoma in situ, including 26 cases with T1a invasive carcinoma, were reviewed. Seventy-nine patients (65%) were treated by mastectomy and 42 (35%) had conservative surgery. Ductal carcinoma in situ was classified as well differentiated (11%), intermediately differentiated (22%) or poorly differentiated (67%) according to nuclear morphology and the presence or absence of cell polarization. Poorly differentiated lesions were significantly larger than intermediately and well differentiated lesions (P = 0.03 and P = 0.01, respectively) and were significantly associated with the presence of extensive necrosis, marked periductal inflammation and periductal fibrosis (P < 0.0001). Invasive carcinoma was more common in the poorly differentiated group (25% compared with 18% in the intermediate group and 8% in the well differentiated group) but this was not statistically significant. The spectrum of differentiation was similar in symptomatic and mammographically detected ductal carcinoma in situ. Clinical follow-up was available in 90 patients (median period 45 months in patients who had undergone mastectomy and 23 months in those who had conservative surgery). Two incidences of recurrent local disease were recorded in the mastectomy group: one patient had well differentiated and the other poorly differentiated ductal carcinoma in situ. No local recurrences were observed in the conservative surgery group, possibly reflecting the shorter follow up period. All histological grades of ductal carcinoma in situ have the potential to progress to invasive carcinoma and mastectomy does not guarantee a cure.

Gallstone ileus remains a rare but important cause of small bowel obstruction. We report a case of recurrent gallstone ileus, presumably caused by an unidentified second stone resident within the gallbladder at the time of the initial laparotomy. This raises important questions about the traditional surgical management of this interesting condition.

The insulin autoimmune syndrome (IAS), or Hirata's disease, is characterized by the combination of fasting hypoglycemia, high concentration of total serum immunoreactive insulin, and presence of autoantibodies to native human insulin in serum. Autoantibody production is classified as monoclonal or polyclonal, with the majority of IAS cases classified as polyclonal. Previously, we observed a striking association between the human leukocyte antigen (HLA) class II alleles DRB1*0406/DQA1* 0301/DQB1*0302 and Japanese IAS patients with polyclonal insulin autoantibodies (IAAs) and T-cell recognition of human insulin in the context of DRB1*0406 molecules. Because of such a strong HLA association in IAS, we performed intra- and interethnic studies on IAS-associated DRB1 alleles and searched for the critical amino acid residue(s) for IAS pathogenesis. Glutamate at position 74 in the HLA-DR4 beta 1-chain was presumed to be essential to the production of polyclonal IAA in IAS, whereas alanine at the same position of the HLA-DR beta 1-chain might be important in the production of monoclonal IAA.

Surgery, trauma and anaesthesia induce a state of transient immunosuppression. Laparoscopic cholecystectomy has several well documented clinical advantages over traditional cholecystectomy and provokes a lower acute phase response, thought to be a result of the smaller wound size. The influence of laparoscopic cholecystectomy (21 patients) and conventional open cholecystectomy (13 patients) upon components of the cell-mediated immune system was investigated. Cell-mediated immunity was studied by in vitro assays of T lymphocyte proliferation to different mitogens, and by natural killer cell cytotoxicity using a standard 51Cr release assay. Blood samples were taken before and 24 h after the start of the operation. In the sample taken after operation there was significant depression of T lymphocyte proliferation to phytohaemagglutinin (stimulation index 149.4 versus 33.3, P < 0.002), staphylococcal enterotoxin B (85.2 versus 52.6, P = 0.01) and toxic shock syndrome toxin (48.4 versus 14.8, P = 0.08) in the group of patients who underwent open surgery, but not in the group treated by laparoscopic surgery. There was a small but statistically insignificant decrease of natural killer cell cytotoxicity in both groups of patients. These findings suggest that laparoscopic cholecystectomy causes less depression of cell-mediated immunity than open cholecystectomy.

A total of 334 patients have been entered into a prospective protocol of breast conserving treatment, which consisted of clinically complete excision, axillary dissection, and radical postoperative radiotherapy given in 20 fractions over 4 weeks. After 7 years' follow-up, 22 patients (6.6%) have had an isolated local recurrence and 24 (7.2%) a local recurrence associated with metastic disease. Cosmetic assessment shows that patients are more satisfied with the result than their treating consultants, and that 81% have scored themselves as having an excellent or very good result more than 5 years after treatment.

The introduction of a national breast cancer screening programme and wider public awareness of breast disease have contributed to an increasing workload for breast surgeons. One method of dealing with this problem efficiently is to encourage day case surgery for breast biopsy patients. We studied our own day case breast biopsy workload, asked other surgeons what proportion of breast biopsies they performed in this way and assessed its acceptability to patients. Of surgeons working in units with facilities available, 40.3% perform 25% or less of breast biopsies as a day case. Of 235 patients undergoing breast biopsy in our unit during an 11-month period, 195 (83%) were performed as day cases. Of these, 192 were under general anesthesia. In all, 97.2% of patients were satisfied with their treatment as a day case, and only 4.5% would have preferred admission postoperatively. Carcinoma was present in 22 (11%) lesions biopsied, 11 (50%) of which occurred in patients under 50 years of age. Surgical complications were encountered in 32 (16.4%) cases; significant bruising 26 (13.3%), wound infections 4 (2.05%), dehiscence 1 (0.51%), and retained suture 1 (0.51%). Breast biopsy is an appropriate and acceptable procedure to undertake as a day case.

Oestrogen-binding sites are present in tissue samples of adenocarcinoma of the pancreas. Uncontrolled studies have suggested that survival of patients with this tumour may be extended by using the antioestrogen drug tamoxifen. Forty-four patients with biopsy-proven irresectable adenocarcinoma of the pancreas were recruited into a randomized placebo-controlled clinical trial of tamoxifen 20 mg twice daily. All patients were assessed at the time of diagnosis and at monthly intervals using the Karnofsky and the Hospital Anxiety and Depression scores for quality of life. Analysis of survival by life-tables and the log rank test revealed no significant difference in the duration of survival of patients treated with tamoxifen or placebo. Quality-of-life assessment revealed no significant difference between the groups. Tamoxifen does not confer significant benefit to patients with irresectable pancreatic cancer.

The serum levels of CA 195 were determined in 52 patients with histologically proven pancreatic carcinoma and compared with carcino-embryonic antigen (CEA), serum bilirubin and albumin. CA 195 levels were raised above the upper limit of 20 U/ml in 42 cases, giving a sensitivity of 80% for the detection of pancreatic carcinoma, whereas CEA was raised in only 55%. The levels of CA 195 and CEA were significantly higher in patients with metastatic disease, but potentially curable cases were not discriminated. Bilirubin and albumin levels were not significantly related to either the presence of metastases or the levels of the tumour markers. At the time of initial presentation, levels of both tumour markers correlated with the eventual duration of survival, but bilirubin and albumin did not. Significant increases in CEA and CA 195 were found in sequential blood samples, as the disease progressed. Neither CA 195 nor CEA was of sufficient sensitivity to be of value for screening, but both give an indication of the presence of metastases and of the subsequent duration of survival. CA 195 appeared to be more sensitive and might help to assess progress of the disease.

From 1985 to 1987 148 patients underwent mastectomy for breast cancer, of whom 91 underwent modified radical mastectomy. Of these patients (median age 60 years (range 31-86 years)), 89 have been assessed for early (< 30 days) and late (> 30 days) non-tumour morbidity and mortality. A total of 41 patients had nodal metastases. Adjunctive therapy used was tamoxifen in 70 patients and radiotherapy in 20. Overall, 47 patients (53%) developed a total of 75 complications, and there was one 30-day mortality. Of the patients, 26 developed one complication, 14 had two complications and 7 three complications. Early complications were lymphocoele/seroma (n = 22), wound infection (n = 9) and cardiopulmonary problems (five deep vein thrombosis, two pulmonary embolus (1 death), one myocardial infarct). Late complications were lymphoedema (n = 10), pectoralis major wasting (n = 6), frozen shoulder (n = 7), intercostobrachial neuralgia (n = 4), and a small number of self-limiting wound problems (n = 9). There were two late deaths (myocardial infarcts). Early complications were not related to nodal status, and late complications were related to neither nodal status nor radiotherapy. Significant morbidity is attached to radical surgery for breast cancer. Most complications are minor and self-limiting, but there are a small number of late complications which may affect quality of life.