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Abstract: Low-dose methotrexate has been proposed as therapy for patients with severely disabling psoriasis and psoriatic arthritis. However, it can be associated with severe toxicity, such as pancytopenia, characterized by anemia (hemoglobin level <13 g/dL in men), thrombocytopenia (platelet count <150 × 109/L), and neutropenia or agranulocytosis (neutrophil count <1.5 × 109/L and 0.5 × 109/L, respectively). Here, we report a challenging clinical scenario characterized by pancytopenia and acute renal failure to inform clinicians about potential drug-drug interactions and subclinical renal insufficiency., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Competing Interests: The authors declare no conflict of interest.

Background: The case study discusses a complex scenario involving the use of amoxicillin in a critically ill patient undergoing intermittent renal replacement therapy.Severe infections are complicated by septic shock and organ failure, requiring urgent and effective antibiotic treatment., Methods: The patient's comorbidities, including obesity and acute kidney injury, required careful consideration of the amoxicillin dosing strategies., Results: Therapeutic drug monitoring is critical for dose adjustment during treatment., Conclusions: This case highlights the importance of a collaborative approach between clinicians and therapeutic drug monitoring consultants to optimize antibiotic therapy for critically ill patients with renal impairment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background and Objectives: Narcolepsy type 1 (NT1) is due to the loss of hypothalamic neurons that produce orexin (ORX), by a suspected immune-mediated process. Rare postmortem studies are available and failed to detect any inflammation in the hypothalamic region, but these brains were collected years after the first symptoms. In vivo studies close to disease onset are lacking. We aimed to explore microglia density in the hypothalamus and thalamus in NT1 compared with controls using [ 18 F]DPA-714 PET and to study in NT1 the relationships between microglia density in the hypothalamus and in other regions of interest (ROIs) with disease duration, severity, and ORX levels., Methods: Patients with NT1 and controls underwent a standardized clinical evaluation and [ 18 F]DPA-714 PET imaging using a radiolabeled ligand specific to the 18 kDa translocator protein (TSPO). TSPO genotyping determined receptor affinity. Images were processed on peripheral module interface using standard uptake value (SUV) on ROIs: hypothalamus, thalamus, frontal area, cerebellum, and the whole brain. SUV ratios (SUVr) were calculated by normalizing SUV with cerebellum uptake., Results: A total of 41 patients with NT1 (21 adults, 20 children, 10 with recent disease onset <1 year) and 35 controls were included, with no significant difference between groups for [ 18 F]DPA-714 binding (SUV/SUVr) in the hypothalamus and thalamus. Unexpectedly, significantly lower SUVr in the whole brain was found in NT1 compared with controls (0.97 ± 0.06 vs 1.08 ± 0.22, p = 0.04). The same finding between NT1 and controls in the whole brain was observed in those with high or mixed TSPO affinity ( p = 0.03 and p = 0.04). Similar trend was observed in the frontal area in NT1 (0.96 ± 0.09 vs 1.09 ± 0.25, p = 0.05). In NT1, no association was found between SUVr in different ROIs and age, disease duration, severity, or ORX levels., Discussion: We found no evidence of in vivo increased microglia density in NT1 compared with controls, even close to disease onset, and even unexpectedly a decrease in the whole brain of these patients. These findings do not support the presence of neuroinflammation in the destruction process of ORX neurons., Trial Registration Information: ClinicalTrials.org NCT03754348.

Abstract: In this article, we present a case of apixaban elimination prolonged by 450% in a patient with coronavirus disease 2019 because of multiple conditions, including drug-drug interaction, severe inflammation, and acute kidney injury. Therapeutic drug monitoring was used to explain unusual routine coagulation assays. This grand round highlights the importance of dialog between the clinician and a therapeutic drug monitoring consultant for optimal patient care., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Abstract: Coronavirus disease (COVID-19) patients frequently develop liver biochemical abnormality. However, liver biochemical abnormality in COVID-19 patients with liver cirrhosis is under-recognized.Patients hospitalized during COVID-19 pandemic in China (ie, from February to April 2020) were screened. All of 17 COVID-19 patients with liver cirrhosis consecutively admitted to the Wuhan Huoshenshan Hospital were identified. Meanwhile, 17 age-, sex-, and severity-matched COVID-19 patients without liver cirrhosis admitted to this hospital were selected as a control group; all of 14 cirrhotic patients without COVID-19 consecutively admitted to the Department of Gastroenterology of the General Hospital of Northern Theater Command were selected as another control group. Incidence of liver biochemical abnormality and decompensated events were primarily compared.Among the COVID-19 patients with liver cirrhosis, the incidence of liver biochemical abnormality at admission and during hospitalization were 76.50% and 84.60%, respectively; 7 (41.20%) had decompensated events at admission; 1 was transferred to intensive care unit due to gastrointestinal bleeding. Among the COVID-19 patients without liver cirrhosis, the incidence of liver biochemical abnormality at admission and during hospitalization were 58.80% (P = .271) and 60.00% (P = .150), respectively. Among the cirrhotic patients without COVID-19, the incidence of liver biochemical abnormality at admission and during hospitalization were 69.20% (P = .657) and 81.80% (P = .855), respectively; 11 (78.60%) had decompensated events at admission (P = .036). None died during hospitalization among the three groups.Liver biochemical abnormality is common in COVID-19 patients with liver cirrhosis. Management of decompensated events in cirrhotic patients without COVID-19 should not be neglected during COVID-19 pandemic., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: To assess the safety and efficacy of percutaneous short-segment pedicle instrumentation compared with conventionally open short-segment pedicle instrumentation and provide recommendations for using these procedures to treat thoracolumbar fractures., Methods: The Medline database, Cochrane database of Systematic Reviews, Cochrane Clinical Trial Register, and Embase were searched for articles published. The randomized controlled trials (RCTs) and non-RCTs that compared percutaneous short-segment pedicle instrumentation to open short-segment pedicle instrumentation and provided data on safety and clinical effects were included. Demographic characteristics, clinical outcomes, radiological outcomes, and adverse events were manually extracted from all of the selected studies. Methodological quality of included studies using Methodological Index for Non-Randomized Studies scale and Cochrane collaboration's tool for assessing the risk of bias by 2 reviewers independently., Results: Nine studies encompassing 433 patients met the inclusion criteria. Subgroup meta-analyses were performed according to the study design. The pooled results showed there were significant differences between the 2 techniques in short- and long-term visual analog scale, intraoperative blood loss, operative time, postoperative draining loss, hospital stay, and incision size, although there were no significant differences in postoperative radiological outcomes, Oswestry Disability Index, hospitalization cost, intraoperative fluoroscopy time, and adverse events., Conclusion: Percutaneous short-segment pedicle instrumentation in cases with achieve satisfactory results, could replace in many cases extensive open surgery and not increased related complications. However, further high-quality RCTs are needed to assess the long-term outcome of patients between 2 techniques.

Objective: The aim of this study was to validate the accuracy of the Accutension XYZ-110 blood pressure (BP) kit according to the American National Standards Institute/Association for the Advancement of Medical Instrumentation/International Organization for Standardization (ANSI/AAMI/ISO) 81060-2:2013 standard., Participants and Methods: Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured simultaneously on the same arm in 85 Chinese adults (female : male=48 : 37) with a mean age of 43.2 years using the mercury sphygmomanometer (two observers) and the Accutension XYZ-110 device (one supervisor). The ANSI/AAMI/ISO 81060-2:2013 standard for the validation of BP-measuring devices in adults was followed precisely. A total of 255 comparison pairs were obtained for analysis., Results: The mean device-observer difference in the 255 separate BP data pairs was 2.45±2.24 mmHg for SBP and 0.69±2.09 mmHg for DBP. The data were in accordance with the criterion 1 of the ANSI/AAMI/ISO 81060-2:2013 standard requirements (≤5±8 mmHg). In addition, the mean device-observer difference of the 85 participants was 2.45±1.47 mmHg for SBP and 0.69±1.36 mmHg for DBP. The device accuracy also fulfilled the criterion 2 with the SD of less than or equal to 6.47 for SBP and less than or equal to 6.90 mmHg for DBP., Conclusion: The Accutension XYZ-110 BP kit fulfilled the requirements of the ANSI/AAMI/ISO 81060-2:2013 standard, and hence could be recommended for both clinical and self/home BP measurement in adults.

The widely used oscillometric automated blood pressure (BP) monitor was continuously questioned on its accuracy. A novel BP kit named Accutension which adopted Korotkoff auscultation method was then devised. Accutension worked with a miniature microphone, a pressure sensor, and a smartphone. The BP values were automatically displayed on the smartphone screen through the installed App. Data recorded in the phone could be played back and reconfirmed after measurement. They could also be uploaded and saved to the iCloud. The accuracy and consistency of this novel electronic auscultatory sphygmomanometer was preliminarily verified here. Thirty-two subjects were included and 82 qualified readings were obtained. The mean differences ± SD for systolic and diastolic BP readings between Accutension and mercury sphygmomanometer were 0.87 ± 2.86 and -0.94 ± 2.93 mm Hg. Agreements between Accutension and mercury sphygmomanometer were highly significant for systolic (ICC = 0.993, 95% confidence interval (CI): 0.989-0.995) and diastolic (ICC = 0.987, 95% CI: 0.979-0.991). In conclusion, Accutension worked accurately based on our pilot study data. The difference was acceptable. ICC and Bland-Altman plot charts showed good agreements with manual measurements. Systolic readings of Accutension were slightly higher than those of manual measurement, while diastolic readings were slightly lower. One possible reason was that Accutension captured the first and the last korotkoff sound more sensitively than human ear during manual measurement and avoided sound missing, so that it might be more accurate than traditional mercury sphygmomanometer. By documenting and analyzing of variant tendency of BP values, Accutension helps management of hypertension and therefore contributes to the mobile heath service.

Study Design: A case report of pyogenic intradural abscess is described., Objectives: The rarity of the presentation and its successful management are discussed., Summary of Background Data: Intradural abscesses are exceptionally rare., Method: The abscess was drained by performing a posterior midline lumbar durotomy, and intravenous antibiotics were initiated., Result: At the 1 year follow-up, the patient has made significant neurologic recovery., Conclusion: Intradural pyogenic abscess secondary to chronic pyogenic spondylodiscitis is a rare manifestation. MRI is a vital component in diagnosis, which revealed key pathologic features within the dural sac as well as in the vertebral column. An emergency decompression and appropriate antibiotic regimen is the solution for a favorable outcome.

Background: Abnormal sarcoplasmic reticulum calcium (Ca) cycling is increasingly recognized as an important mechanism for increased ventricular automaticity that leads to lethal ventricular arrhythmias. Previous studies have linked lethal familial arrhythmogenic disorders to mutations in the ryanodine receptor and calsequestrin genes, which interact with junctin and triadin to form a macromolecular Ca-signaling complex. The essential physiological effects of junctin and its potential regulatory roles in sarcoplasmic reticulum Ca cycling and Ca-dependent cardiac functions, such as myocyte contractility and automaticity, are unknown., Methods and Results: The junctin gene was targeted in embryonic stem cells, and a junctin-deficient mouse was generated. Ablation of junctin was associated with enhanced cardiac function in vivo, and junctin-deficient cardiomyocytes exhibited increased contractile and Ca-cycling parameters. Short-term isoproterenol stimulation elicited arrhythmias, including premature ventricular contractions, atrioventricular heart block, and ventricular tachycardia. Long-term isoproterenol infusion also induced premature ventricular contractions and atrioventricular heart block in junctin-null mice. Further examination of the electrical activity revealed a significant increase in the occurrence of delayed afterdepolarizations. Consistently, 25% of the junctin-null mice died by 3 months of age with structurally normal hearts., Conclusions: Junctin is an essential regulator of sarcoplasmic reticulum Ca release and contractility in normal hearts. Ablation of junctin is associated with aberrant Ca homeostasis, which leads to fatal arrhythmias. Thus, normal intracellular Ca cycling relies on maintenance of junctin levels and an intricate balance among the components in the sarcoplasmic reticulum quaternary Ca-signaling complex.

Background: Phospholamban (PLN) is an inhibitor of the Ca2+ affinity of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2). The amino acid sequence of PLN is highly conserved, and although all species contain asparagine (Asn), human PLN is unique in containing lysine (Lys) at amino acid 27., Methods and Results: Human PLN was introduced in the null background. Expression of human PLN, at similar levels to mouse wild-type PLN, resulted in significant decreases in the affinity of SERCA2 for Ca2+, attributed to unique spatial conformation of this PLN form and increases in its monomeric active unit compared with mouse PLN. The increased inhibition by human PLN was associated with attenuated cardiac contractility in the intact-animal, organ, and cardiomyocyte levels and with depressed calcium kinetics. These inhibitory effects could not be fully reversed even on maximal isoproterenol stimulation. There were no alterations in the expression levels of SERCA2, calsequestrin, ryanodine receptor, and FKBP12, although the sodium/calcium exchanger and the L-type Ca2+ channel expression levels were upregulated. The depressed function resulted in increased heart/body weight ratios and phosphorylation levels of Akt, p38, and Erk1/2., Conclusions: Human PLN may play a more inhibitory role than that of other species in Ca2+ cycling. Expression of human PLN in the mouse is compensated by alterations in Ca2+-handling proteins and cardiac remodeling in an effort to normalize cardiac contractility. Thus, the unique amino acid sequence of human PLN may be critical in maintaining a high cardiac reserve, which is of paramount importance in the regulation of human cardiac function.

Background: Heat-shock proteins (Hsps) have been shown to render cardioprotection from stress-induced injury; however, little is known about the role of another small heat-shock protein, Hsp20, which regulates activities of vasodilation and platelet aggregation, in cardioprotection against ischemia injury. We recently reported that increased expression of Hsp20 in cardiomyocytes was associated with improved contraction and protection against beta-agonist-induced apoptosis., Methods and Results: To investigate whether overexpression of Hsp20 exerts protective effects in both ex vivo and in vivo ischemia/reperfusion (I/R) injury, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20 (10-fold). TG and wild-type (WT) hearts were then subjected to global no-flow I/R (45 minutes/120 minutes) using the Langendorff preparation. TG hearts exhibited improved recovery of contractile performance over the whole reperfusion period. This improvement was accompanied by a 2-fold decrease in lactate dehydrogenase released from the TG hearts. The extent of infarction and apoptotic cell death was also significantly decreased, which was associated with increased protein ratio of Bcl-2/Bax and reduced caspase-3 activity in TG hearts. Furthermore, in vivo experiments of 30-minute myocardial ischemia, via coronary artery occlusion, followed by 24-hour reperfusion, showed that the infarct region-to-risk region ratio was 8.1+/-1.1% in TG hearts (n=7), compared with 19.5+/-2.1% in WT hearts (n=11, P<0.001)., Conclusions: Our data demonstrate that increased Hsp20 expression in the heart protects against I/R injury, resulting in improved recovery of cardiac function and reduced infarction. Thus, Hsp20 may constitute a new therapeutic target for ischemic heart diseases.

Activation of the sympathetic nervous system is a common compensatory feature in heart failure, but sustained beta-adrenergic activation induces cardiomyocyte death, leading to cardiac remodeling and dysfunction. In mouse cardiomyocytes, we recently reported that prolonged exposure to beta-agonists is associated with transient increases in expression and phosphorylation of a small heat-shock protein, Hsp20. To determine the functional significance of Hsp20, we overexpressed this protein and its constitutively phosphorylated (S16D) or nonphosphorylated (S16A) mutant in adult rat cardiomyocytes. Hsp20 protected cardiomyocytes from apoptosis triggered by activation of the cAMP-PKA pathway, as indicated by decreases in the number of pyknotic nuclei, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling, and DNA laddering, which were associated with inhibition of caspase-3 activity. These protective effects were further increased by the constitutively phosphorylated Hsp20 mutant (S16D), which conferred full protection from apoptosis. In contrast, the nonphosphorylatable mutant (S16A) exhibited no antiapoptotic properties. Immunostaining studies and immunoprecipitations with Hsp20 or actin antibodies demonstrated that Hsp20 translocated to cytoskeleton and associated with actin on isoproterenol stimulation. These findings suggest that Hsp20 and its phosphorylation at Ser16 may provide cardioprotection against beta-agonist-induced apoptosis. Thus, Hsp20 may represent a novel therapeutic target in the treatment of heart failure.

Posttranslational modification of target substrates underlies biological processes through activation/inactivation of signaling cascades. To concurrently identify the phosphoprotein substrates associated with cardiac beta-adrenergic signaling, the mouse myocyte phosphoproteome was analyzed using 2-D gel electrophoresis in combination with 32P autoradiography. Phosphoprotein spots, detected by silver staining, were identified using MALDI-TOF mass spectrometry in conjunction with computer-assisted protein spot matching. Stimulation with isoproterenol (1 micromol/L for 5 minutes) was associated with maximal increases in myocyte contractile parameters, and significant stimulation of the phosphorylation of troponin I (190+/-23%) and succinyl CoA synthetase (160+/-16%), whereas the phosphorylation of pyruvate dehydrogenase (48+/-10%), NADH-ubiquinone oxidoreductase (46+/-6%), heat shock protein 27 (18+/-3%), alphaB-crystallin (20+/-3%), and an unidentified 26-kDa protein (29+/-7%) was significantly decreased, compared with unstimulated cells (100%). After sustained (30 minutes) stimulation with isoproterenol, only the alterations in the phosphorylation levels of troponin I and NADH-ubiquinone oxidoreductase were maintained and de novo phosphorylation of a phosphoprotein (approximately 20 kDa and pI 5.5) was observed. The tryptic peptide fragments of this phosphoprotein were sequenced using postsource decay mass spectrometry, and the protein was subsequently cloned and designated as p20, based on its high sequence homology with rat and human skeletal p20. The mouse cardiac p20 contains the conserved domain sequences for heat shock proteins, and the RRAS consensus sequence for cAMP-PKA substrates. LC-MS/MS phosphorylation mapping confirmed phosphorylation of Ser16 in p20 on beta-agonist stimulation. Adenoviral gene transfer of p20 was associated with significant increases in contractility and Ca transient peak in adult rat cardiomyocytes, suggesting an important role of p20 in cardiac function. These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins to dynamically fine-tune cardiac responses to beta-adrenergic signaling.

Phospholamban, the critical regulator of the cardiac SERCA2a Ca2+ affinity, is phosphorylated at Ser16 and Thr17 during beta-adrenergic stimulation (eg, isoproterenol). To assess the impact of nonphosphorylatable phospholamban, a S16A, T17A double-mutant (DM) was introduced into phospholamban knockout mouse hearts. Transgenic lines expressing DM phospholamban at levels similar to wild types (WT) were identified. In vitro phosphorylation confirmed that DM phospholamban could not be phosphorylated, but produced the same shift in EC50 of SERCA2a for Ca2+ as unphosphorylated WT phospholamban. Rates of basal twitch [Ca2+]i decline were not different in DM versus WT cardiomyocytes. Isoproterenol increased the rates of twitch [Ca2+]i decline in WT, but not DM myocytes, confirming the prominent role of phospholamban phosphorylation in this response. Increased L-type Ca2+ current (ICa) density, with unaltered characteristics, was the major compensation in DM myocytes. Consequently, the normal beta-adrenergic-induced increase in ICa caused larger dynamic changes in absolute ICa density. Isoproterenol increased Ca2+ transients to a comparable amplitude in DM and WT. There were no changes in myofilament Ca2+ sensitivity, or the expression levels and Ca2+ removal activities of other Ca2+-handling proteins. Nor was there evidence of cardiac remodeling up to 10 months of age. Thus, chronic inhibition of SERCA2a by ablation of phospholamban phosphorylation (abolishing its adrenergic regulation) results in a unique cellular adaptation involving greater dynamic ICa modulation. This ICa modulation may partly compensate for the loss in SERCA2a responsiveness and thereby partially normalize beta-adrenergic inotropy in DM phospholamban mice.

The common C-480T transition in the hepatic lipase (HL) promoter has been shown to be associated with lower HL activity and increased high density lipoprotein (HDL) cholesterol. We examined the frequency and lipid associations of this HL polymorphism in 385 healthy, young (18- to 28-year-old) men whose fathers had had a premature myocardial infarction (designated cases) and 405 age-matched controls. These individuals were participants in the European Atherosclerosis Research Study II postprandial trial, who had been recruited from 11 European countries in 4 regions (the Baltic; United Kingdom; and central and southern Europe). Overall, the frequency of the T allele was 0.207 in controls and 0.244 in cases (P=0.08). The T allele was associated with higher fasting plasma total cholesterol (P<0.01), triglycerides (P<0.01), and HDL cholesterol (P<0.01). The strongest association was found with apolipoprotein (apo) A-I concentration, which was 10% higher in individuals homozygous for the T allele compared with those homozygous for the C allele (P<0.001). This polymorphism had no effect on the rise in plasma triglyceride levels after a fatty meal. However, before and after the fat load was ingested, levels of particles containing both apoC-III and apoB (LpC-III:B) were higher in carriers of the T allele, with homozygotes having 23% and 27% higher levels preprandially and postprandially, respectively, than those homozygous for the C allele (P<0.05). Thus, our results demonstrate that the C-480T polymorphism in the HL promoter is associated with alterations in plasma lipids and lipoproteins and the accumulation of atherogenic LpC-III:B particles.

Phospholamban, a prominent modulator of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and basal contractility in the mammalian heart, has been proposed to form pentamers in native SR membranes. However, the monomeric form of phospholamban, which is associated with mutating Cys41 to Phe41, was shown to be as effective as pentameric phospholamban in inhibiting Ca2+ transport in expression systems. To determine whether this monomeric form of phospholamban is also functional in vivo, we generated transgenic mice with cardiac-specific overexpression of the mutant (Cys41-->Phe41) phospholamban. Quantitative immunoblotting indicated a 2-fold increase in the cardiac phospholamban protein levels compared with wild-type controls, with approximately equal to 50% of phospholamban migrating as monomers and approximately 50% as pentamers upon SDS-PAGE. The mutant-phospholamban transgenic hearts were analyzed in parallel with transgenic hearts overexpressing (2-fold) wild-type phospholamban, which migrated as pentamers upon SDS-PAGE. SR Ca(2+)-uptake assays revealed that the EC50 values for Ca2+ were as follows: 0.32 +/- 0.01 mumol/L in hearts overexpressing monomeric phospholamban, 0.49 +/- 0.05 mumol/L in hearts overexpressing wild-type phospholamban, and 0.26 +/- 0.01 mumol/L in wild-type control mouse hearts. Analysis of cardiomyocyte mechanics and Ca2+ kinetics indicated that the inhibitory effects of mutant-phospholamban overexpression (mt) were less pronounced than those of wild-type phospholamban overexpression (ov) as assessed by depression of the following: (1) shortening fraction (25% mt versus 45% ov), (2) rates of shortening (27% mt versus 48% ov), (3) rates of relengthening (25% mt versus 50% ov) (4) amplitude of the Ca2+ signal (21% mt versus 40% ov), and (5) time for decay of the Ca2+ signal (25% mt versus 106% ov) compared with control (100%) myocytes. The differences in basal cardiac, myocyte mechanics and Ca2+ transients among the animal groups overexpressing monomeric or wild-type phospholamban and wild-type control mice were abolished upon isoproterenol stimulation. These findings suggest that pentameric assembly of phospholamban is important for mediating its optimal regulatory effects on myocardial contractility in vivo.

Single-strand conformational polymorphism analysis of the lipoprotein lipase promoter identified a T-->G transition at position -93. The frequency in healthy white men was 3.4% (n = 1575). There was an 83% allelic association between -93T-->G and Asp9-->Asn (D9N); all N9 mutations occurred on a -93G allele, but not all -93G mutations occurred on an N9 allele. It was thus possible to assess the effect on plasma triglyceride (Tg) levels of the rare -93G mutation in the presence of the wild-type D9. Carriers of the -93G, with genotype TG/DD, had significantly lower Tg levels than TT/DD individuals (1.36 versus 1.78 mmol/L, P = .01); carriers of both mutations (TG/DN) had the highest Tg levels (1.93 mmol/L). When the group was stratified above and below the sample mean for body mass index (BMI), carriers of the -93G on a D9 allele (TG/DD) were "protected" against the Tg-raising effect of obesity, as assessed by BMI. In Afro-Caribbeans (n = 91), the carrier frequency of -93G was 18-fold higher (63%), with weaker (17%) allelic association between -93G and N9. In vitro, the -93G promoter had 24% higher activity than the -93T in a rat smooth muscle cell line and 18% higher activity in a human adrenal cell line. A protein identified by band-shift assays bound to the -93G but not to the -93T allele, which may explain the lower Tg levels in -93G carriers.

Adenoviral gene transfer has potential use to attenuate the immunogenicity of hepatic allografts. However, the clinical application of adenoviral gene therapy is currently impeded by the potent host immune response to the virus that limits the duration of its effects. In these studies, we identify the cellular and humoral immune responses to recombinant adenovirus in the liver of mice and define the immunologic barriers to the successful application of this technology to transplantation. The immunobiology of recombinant adenovirus was studied in mouse liver using vectors containing the lacZ and alkaline phosphatase marker genes. The duration of transgene expression was studied in various immunodeficient mice to determine the mechanism of viral clearance. Adoptive transfer of serum to B lymphocyte deficient mice and neutralizing antibody assays were used to define the antiviral humoral response. Hepatic adenoviral transgene expression was prolonged in animals deficient in CD4+ or CD8+ T cells indicating their importance in viral clearance. Unexpectedly, mice lacking B lymphocytes also had delayed elimination of virus suggesting that B cells play a role in the primary immune response. Effective repeat gene transfer was blocked by adenoviral-specific neutralizing antibody. Therefore, a T lymphocyte response results in viral elimination after a primary intravenous inoculation of recombinant adenovirus and a potent humoral response inhibits effective repeat adenoviral gene transfer. The immunogenicity of the vector must be overcome for adenoviral gene therapy to have therapeutic application for hepatic transplantation.

Phospholamban ablation is associated with significant increases in the sarcoplasmic reticulum Ca(2+)-ATPase activity and the basal cardiac contractile parameters. To determine whether the observed phenotype is due to loss of phospholamban alone or to accompanying compensatory mechanisms, hearts from phospholamban-deficient and age-matched wild-type mice were characterized in parallel. There were no morphological alterations detected at the light microscope level. Assessment of the protein levels of the cardiac sarcoplasmic reticulum Ca(2+)-ATPase, calsequestrin, myosin, actin, troponin I, and troponin T revealed no significant differences between phospholamban-deficient and wild-type hearts. However, the ryanodine receptor protein levels were significantly decreased (25%) upon ablation of phospholamban, probably in an attempt to regulate the release of Ca2+ from the sarcoplasmic reticulum, which had a significantly higher diastolic Ca2+ content in phospholamban-deficient compared with wild-type hearts (16.0 +/- 2.2 versus 8.6 +/- 1.0 mmol Ca2+/kg dry wt, respectively). The increases in Ca2+ content were specific to junctional sarcoplasmic reticulum stores, as there were no alterations in the Ca2+ content of the mitochondria or A band. Assessment of ATP levels revealed no alterations, although oxygen consumption increased (1.6-fold) to meet the increased ATP utilization in the hyperdynamic phospholamban-deficient hearts. The increases in oxygen consumption were associated with increases (2.2-fold) in the active fraction of the mitochondrial pyruvate dehydrogenase, suggesting increased tricarboxylic acid cycle turnover and ATP synthesis. 31P nuclear magnetic resonance studies demonstrated decreases in phosphocreatine levels and increases in ADP and AMP levels in phospholamban-deficient compared with wild-type hearts. However, the creatine kinase activity and the creatine kinase reaction velocity were not different between phospholamban-deficient and wild-type hearts. These findings indicate that ablation of phospholamban is associated with downregulation of the ryanodine receptor to compensate for the increased Ca2+ content in the sarcoplasmic reticulum store and metabolic adaptations to establish a new energetic steady state to meet the increased ATP demand in the hyperdynamic phospholamban-deficient hearts.