👤 Mary Helen Black

Individuals with atrial fibrillation (AF) are at increased risk of stroke, cognitive impairment and dementia. Observational studies suggest that anticoagulation may reduce the risk of cognitive decline in patients with AF and elevated thromboembolic risk, implicating subclinical cerebral emboli as a potential mechanistic link. Whether anticoagulation prevents cognitive deterioration in patients with AF at low risk of stroke remains uncertain. Here we conducted a multicenter, double-blind, placebo-controlled trial in which participants with AF and low thromboembolic risk (CHA Show less

This case describes the resolution of refractory chronic idiopathic urticaria (CIU) with setmelanotide in a patient with Bardet-Biedl Syndrome (BBS). While setmelanotide is approved for syndromic obesity, this report highlights a novel anti-inflammatory effect potentially mediated by melanocortin-4 receptor (MC4R) agonism, expanding current understanding of its immunomodulatory role in mast cell-mediated disease. The patient presented with lifelong hyperphagia and class III obesity beginning in childhood, along with multiple features fulfilling BBS clinical criteria: visual impairment with night vision difficulties, renal abnormalities with proteinuria, irregular menses with polycystic ovaries, and neurodevelopmental delay. She also reported refractory CIU with pruritic hives occurring several times weekly despite dual antihistamine therapy. On examination, BMI was 40.5 kg/m Genetic testing revealed a heterozygous pathogenic Setmelanotide produced durable remission of refractory CIU in a patient with BBS, suggesting MC4R agonism may exert immunomodulatory effects beyond weight regulation. This finding underscores the broader potential of precision therapies for rare obesity syndromes to reveal new mechanisms of inflammation control. Show less

Previous reports of distal deletions in chromosome 10q in patients have described distinct facial features combined with other neurodevelopmental abnormalities, including intellectual disability. However, the association of interstitial deletions in chromosome 10q with global developmental delay, musculoskeletal abnormalities, and dysmorphic features has not been previously reported. Genetic testing using whole exome sequencing (WES) was performed on three patients with neurodevelopmental delay, musculoskeletal abnormalities and dysmorphic features. Sequencing reads were aligned to the human genome build GRCh37/UCSC hg19 and analysed for both sequence and copy number variants. WES identified similar interstitial deletions in the 10q21.1q21.3 locus in all three patients. The deleted region includes online Mendelian inheritance in man (OMIM)-annotated genes with clinical significance, such as This is the first report associating interstitial deletions in the 10q21.1q21.3 locus with neurodevelopmental delay, musculoskeletal abnormalities and dysmorphic features. Our findings highlight the clinical significance of this deleted region and suggest possible mechanisms underlying the observed pathological phenotypes. Show less

Cerebral small vessel disease (SVD) and amyloid beta (Aβ) pathology frequently co-exist. The impact of concurrent pathology on the pattern of hippocampal atrophy, a key substrate of memory impacted early and extensively in dementia, remains poorly understood. In a unique cohort of mixed Alzheimer's disease and moderate-severe SVD, we examined whether total and regional neuroimaging measures of SVD, white matter hyperintensities (WMH), and Aβ, as assessed by Frontal WMH, occipital WMH, and Aβ were independently associated with smaller hippocampal volume. Frontal WMH had a spatially distinct impact on hippocampal shape relative to Aβ. In contrast, hippocampal shape alterations associated with occipital WMH spatially overlapped with Aβ-vulnerable subregions. Hippocampal degeneration is differentially sensitive to SVD and Aβ pathology. The pattern of hippocampal atrophy could serve as a disease-specific biomarker, and thus guide clinical diagnosis and individualized treatment strategies for mixed dementia. Show less

White matter (WM) injury is frequently observed along with dementia. Positron emission tomography with amyloid-ligands (Aβ-PET) recently gained interest for detecting WM injury. Yet, little is understood about the origin of the altered Aβ-PET signal in WM regions. Here, we investigated the relative contributions of diffusion MRI-based microstructural alterations, including free water and tissue-specific properties, to Aβ-PET in WM and to cognition. We included a unique cohort of 115 participants covering the spectrum of low-to-severe white matter hyperintensity (WMH) burden and cognitively normal to dementia. We applied a bi-tensor diffusion-MRI model that differentiates between (i) the extracellular WM compartment (represented via free water), and (ii) the fiber-specific compartment (via free water-adjusted fractional anisotropy [FA]). We observed that, in regions of WMH, a decrease in Aβ-PET related most closely to higher free water and higher WMH volume. In contrast, in normal-appearing WM, an increase in Aβ-PET related more closely to higher cortical Aβ (together with lower free water-adjusted FA). In relation to cognitive impairment, we observed a closer relationship with higher free water than with either free water-adjusted FA or WM PET. Our findings support free water and Aβ-PET as markers of WM abnormalities in patients with mixed dementia, and contribute to a better understanding of processes giving rise to the WM PET signal. Show less

The discovery of apolipoprotein A5 (APOA5) in 2001 has raised a number of intriguing questions about its role in lipid transport and triglyceride (TG) homeostasis. Genome-wide association studies have consistently identified APOA5 as a regulator of plasma TG levels, which is further supported by studies in transgenic and knockout mouse models. The present review describes recent concepts pertaining to the roles of APOA5 in TG metabolism as related to the vascular compartment, liver, adipose tissue and the gut. Recent evidence indicates that APOA5 may also affect postprandial TG metabolism through influencing chylomicron formation and transport by the intestine into the intestinal lymph. While substantial evidence supports the notion that APOA5 plays both extracellular and intracellular roles in TG homeostasis, mysteries remain on how this low-abundance, liver-derived protein may modulate TG homeostasis, including via the gut. Given the strong correlation between elevated plasma TG and cardiometabolic diseases, there is great scientific and public interest in understanding the intriguing mysteries presented by APOA5. Show less

In a retrospective analysis of dal-Outcomes, the effect of dalcetrapib on cardiovascular events was influenced by an adenylate cyclase type 9 (ADCY9) gene polymorphism. The dal-GenE study was conducted to test this pharmacogenetic hypothesis. dal-GenE was a double-blind trial in patients with an acute coronary syndrome within 1-3 months and the AA genotype at variant rs1967309 in the ADCY9 gene. A total of 6147 patients were randomly assigned to receive dalcetrapib 600 mg or placebo daily. The primary endpoint was the time from randomization to first occurrence of cardiovascular death, resuscitated cardiac arrest, non-fatal myocardial infarction, or non-fatal stroke. After a median follow-up of 39.9 months, the primary endpoint occurred in 292 (9.5%) of 3071 patients in the dalcetrapib group and 327 (10.6%) of 3076 patients in the placebo group [hazard ratio 0.88; 95% confidence interval (CI) 0.75-1.03; P = 0.12]. The hazard ratios for the components of the primary endpoint were 0.79 (95% CI 0.65-0.96) for myocardial infarction, 0.92 (95% CI 0.64-1.33) for stroke, 1.21 (95% CI 0.91-1.60) for death from cardiovascular causes, and 2.33 (95% CI 0.60-9.02) for resuscitated cardiac arrest. In a pre-specified on-treatment sensitivity analysis, the primary endpoint event rate was 7.8% (236/3015) in the dalcetrapib group and 9.3% (282/3031) in the placebo group (hazard ratio 0.83; 95% CI 0.70-0.98). Dalcetrapib did not significantly reduce the risk of occurrence of the primary endpoint of ischaemic cardiovascular events at end of study. A new trial would be needed to test the pharmacogenetic hypothesis that dalcetrapib improves the prognosis of patients with the AA genotype. Trial registration dal-GenE ClinicalTrials.gov Identifier: NCT02525939. Show less

The prevalence of type 2 diabetes in youth has increased substantially, yet the genetic underpinnings remain largely unexplored. To identify genetic variants predisposing to youth-onset type 2 diabetes, we formed ProDiGY, a multiethnic collaboration of three studies (TODAY, SEARCH, and T2D-GENES) with 3,006 youth case subjects with type 2 diabetes (mean age 15.1 ± 2.9 years) and 6,061 diabetes-free adult control subjects (mean age 54.2 ± 12.4 years). After stratifying by principal component-clustered ethnicity, we performed association analyses on ∼10 million imputed variants using a generalized linear mixed model incorporating a genetic relationship matrix to account for population structure and adjusting for sex. We identified seven genome-wide significant loci, including the novel locus rs10992863 in Show less

Atherosclerosis has been effectively avoided with many therapies that lower low-density lipoprotein cholesterol. However, significant cardiovascular burden remains. The effect of raising high-density lipoprotein (HDL) has been confounded by other factors (such as lowering triglycerides or LDL) and unsuccessful when attempting to solely increase HDL. Reviewing the available data, the failures of previous strategies may reflect the complexity of HDL in human metabolism and the heterogeneity of human genetics. dal-GenE (NCT02525939) represents the first large cardiovascular outcomes study to use a selective genomic test to identify the target population most likely to receive therapeutic benefit and uses a cholesterol ester transfer protein inhibitor, dalcetrapib. Both the cholesterol ester transfer protein target and the ADCY9 polymorphism identified by the diagnostic test are based on inheritance and an evolving understanding of inborn risk. Selective treatment of subpopulations may be the key to the conundrum of HDL as an actionable risk factor. Show less

Incident type 2 diabetes is common among patients with recent acute coronary syndrome and is associated with an adverse prognosis. Some data suggest that cholesteryl ester transfer protein (CETP) inhibitors reduce incident type 2 diabetes. We compared the effect of treatment with the CETP inhibitor dalcetrapib or placebo on incident diabetes in patients with recent acute coronary syndrome. In the dal-OUTCOMES trial, 15,871 patients were randomly assigned to treatment with dalcetrapib 600 mg daily or placebo, beginning 4-12 weeks after an acute coronary syndrome. Absence of diabetes at baseline was based on medical history, no use of antihyperglycemic medication, and hemoglobin A At baseline, 10,645 patients (67% of the trial cohort) did not have diabetes. During a median follow-up of 30 months, incident diabetes was identified in 403 of 5,326 patients (7.6%) assigned to dalcetrapib and in 516 of 5,319 (9.7%) assigned to placebo, corresponding to absolute risk reduction of 2.1%, hazard ratio of 0.77 (95% CI 0.68-0.88; In patients with a recent acute coronary syndrome, incident diabetes is common and is reduced substantially by treatment with dalcetrapib. Show less

The objectives of precision medicine are to better match patient characteristics with the therapeutic intervention to optimize the chances of beneficial actions while reducing the exposure to unneeded adverse drug experiences. In a retrospective genome-wide association study of the overall neutral placebo-controlled dal-Outcomes trial, the effect of the cholesteryl ester transfer protein (CETP) modulator dalcetrapib on the composite of cardiovascular death, myocardial infarction or stroke was found to be influenced by a polymorphism in the adenylate cyclase type 9 (ADCY9) gene. Whereas patients with the AA genotype at position rs1967309 experienced fewer cardiovascular events with dalcetrapib, those with the GG genotype had an increased rate and the heterozygous AG genotype exhibited no difference from placebo. Measurements of cholesterol efflux and C-reactive protein (CRP) offered directionally supportive genotype-specific findings. In a separate, smaller, placebo-controlled trial, regression of ultrasonography-determined carotid intimal-medial thickness was only observed in dalcetrapib-treated patients with the AA genotype. Collectively, these observations led to the hypothesis that the cardiovascular effects of dalcetrapib may be pharmacogenetically determined, with a favorable benefit-risk ratio only for patients with this specific genotype. We describe below the design of dal-GenE, a precision medicine, placebo-controlled clinical outcome trial of dalcetrapib in patients with a recent acute myocardial infarction with the unique feature of selecting only those with the AA genotype at rs1967309 in the ADCY9 gene. Show less

High-density lipoprotein cholesterol (HDL-C) concentration is inversely related to risk of major adverse cardiovascular events (MACE) in epidemiologic studies but is a poorer predictor of MACE in patients with established coronary heart disease. HDL particle concentration (HDLP) has been proposed as a better predictor of risk. We investigated whether HDLP is associated with risk of MACE after acute coronary syndrome (ACS). The dal-Outcomes trial compared the CETP inhibitor dalcetrapib with placebo in patients with recent ACS. In a nested case-cohort analysis, total, large, medium, and small HDLPs were measured by nuclear magnetic resonance spectroscopy at baseline (4-12 weeks after ACS) in 476 cases with MACE and 902 controls. Hazard ratios (HRs; case-control) for 1-SD increment of HDLP or HDL-C at baseline were calculated with and without adjustment for demographic, clinical, laboratory, and treatment variables. Similarly, HRs for MACE were calculated for changes in HDLP or HDL-C from baseline to month 3 of assigned treatment. Over median follow-up of 28 months, the risk of MACE was not associated with baseline HDLP (adjusted HR = 0.98, 95% CI = 0.84-1.15, P = .81), any HDLP subclass, or HDL-C. Dalcetrapib increased HDL-C and total, medium, and large HDLP and decreased small HDLP but had no effect on MACE compared with placebo. There were no association of risk of MACE with change in HDLP or HDL-C and no interaction with assigned study treatment. Neither baseline HDLP nor the change in HDLP on treatment with dalcetrapib or placebo was associated with risk of MACE after ACS. Show less

The role of omega-3 fatty acid in multiple sclerosis (MS) susceptibility is unclear. To determine whether fish/seafood intake or genetic factors that regulate omega-3 fatty acids levels are associated with MS risk. We examined the association of fish and shrimp consumption and 13 tag single nucleotide polymorphisms (SNPs) in Consuming fish/seafood at least once a week or at least once a month with regular fish oil use was associated with 44% reduced odds of MS/CIS (adjusted OR = 0.56; 95% CI = 0.41-0.76; These findings suggest that omega-3 fatty acid intake may be an important modifiable risk factor for MS. This is consistent with the other known health benefits of fish consumption and complementary genetic studies supporting a key role for omega-3 regulation. Show less

Lung cancer has the highest mortality rate of all cancers worldwide. Lung cancer is a very heterogeneous disease that is often diagnosed at later stages which have a poor prognosis. Aberrant alternative splicing patterns found in lung cancer contribute to important cell functions. These include changes in splicing for the BCL2L1, MDM2, MDM4, NUMB and MET genes during lung tumourigenesis, to affect pathways involved in apoptosis, cell proliferation and cellular cohesion. Global analyses of RNASeq datasets suggest there may be many more potentially influential aberrant splicing events that need to be investigated in lung cancer. Changes in expression of the splicing factors that regulate alternative splicing events have also been identified in lung cancer. Of these, changes in expression of QKI, RBM4, RBM5, RBM6, RBM10 and SRSF1 proteins regulate many of the most frequently referenced aberrant splicing events in lung cancer. The expanding list of genes known to be aberrantly spliced in lung cancer along with the altered expression of splicing factors that regulate them are providing new clues as to how lung cancer develops, and how these events can be exploited for better treatment. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm. Show less

The cholesterol ester transfer protein (CETP) inhibitor dalcetrapib has been under evaluation for its potential to prevent cardiovascular (CV) events for almost two decades. The current clinical development program, representing new advances in precision medicine and focused on a genetically defined population with acute coronary syndrome (ACS), is supported by a large body of pharmacokinetic and pharmacodynamic data as well as substantial clinical experience in over 13,000 patients and volunteers. Dalcetrapib treatment of 600 mg/day produces significant inhibition of CETP activity, and has been utilized in phase II and III studies, including CV endpoint trials. Numerous studies have investigated the interactions between dalcetrapib and most drugs commonly prescribed to CV patients and have not demonstrated any clinically significant effects. Evaluations in patients with renal and hepatic impairment demonstrate a greater exposure to dalcetrapib than in the non-impaired population, but long-term clinical studies including patients with mild to moderate hepatic and renal dysfunction demonstrate no increase in adverse events. Safety pharmacology and toxicology studies as well as the clinical safety experience support the continuing development of dalcetrapib as an adjunct to 'standard of care' for the ACS population. This article provides a full review of the pharmacokinetics, as well as pharmacodynamics and pharmacology, of dalcetrapib in the context of a large clinical program. Show less

Apolipoprotein (apo) A-IV overexpression enhances chylomicron (CM) assembly and secretion in newborn swine intestinal epithelial cells by producing larger particles (Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW, Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg RB, Black DD. J Biol Chem 281: 3473-3483, 2006). To determine the impact of apo A-IV on microsomal triglyceride transfer protein (MTTP), IPEC-1 cell lines containing a tetracycline-regulatable expression system were used to overexpress native swine apo A-IV and "piglike" human apo A-IV, a mutant human apo A-IV with deletion of the EQQQ-rich COOH-terminus, previously shown to upregulate basolateral triglyceride (TG) secretion 5-fold and 25-fold, respectively. Cells were incubated 24 h with and without doxycycline and oleic acid (OA, 0.8 mM). Overexpression of the native swine apo A-IV and piglike human apo A-IV increased MTTP lipid transfer activity by 39.7% (P = 0.006) and 53.6% (P = 0.0001), respectively, compared with controls. Changes in mRNA and protein levels generally paralleled changes in activity. Interestingly, native swine apo A-IV overexpression also increased MTTP large subunit mRNA, protein levels, and lipid transfer activity in the absence of OA, suggesting a mechanism not mediated by lipid absorption. Overexpression of piglike human apo A-IV significantly increased partitioning of radiolabeled OA from endoplasmic reticulum (ER) membrane to lumen, suggesting increased net transfer of membrane TG to luminal particles. These results suggest that the increased packaging of TG into nascent CMs in the ER lumen, induced by apo A-IV, is associated with upregulation of MTTP activity at the pretranslational level. Thus MTTP is regulated by apo A-IV in a manner to promote increased packaging of TG into the CM core, which may be important in neonatal fat absorption. Show less

The initial segment of the epididymis is vital for male fertility; therefore, it is important to understand the mechanisms that regulate this important region. Deprival of testicular luminal fluid factors/lumicrine factors from the epididymis results in a wave of apoptosis in the initial segment. In this study, a combination of protein array and microarray analyses was used to examine the early changes in downstream signal transduction pathways following loss of lumicrine factors. We discovered the following cascade of events leading to the loss of protection and eventual apoptosis: in the first 6 h after loss of lumicrine factors, down-regulation of the ERK pathway components was observed at the mRNA expression and protein activity levels. Microarray analysis revealed that mRNA levels of several key components of the ERK pathway, Dusp6, Dusp5, and Etv5, decreased sharply, while the analysis from the protein array revealed a decline in the activities of MAP2K1/2 and MAPK1. Immunostaining of phospho-MAPK3/1 indicated that down-regulation of the ERK pathway was specific to the epithelial cells of the initial segment. Subsequently, after 12 h of loss of lumicrine factors, levels of mRNA expression of STAT and NFKB pathway components increased, mRNA levels of several genes encoding cell cycle inhibitors increased, and levels of protein expression of several proapoptotic phosphatases increased. Finally, after 18 h of loss of protection from lumicrine factors, apoptosis was observed. In conclusion, testicular lumicrine factors protect the cells of the initial segment by activating the ERK pathway, repressing STAT and NFKB pathways, and thereby preventing apoptosis. Show less

Individuals born small for gestational age (SGA) are at increased risk of rapid postnatal weight gain, later obesity and diseases in adulthood such as type 2 diabetes, hypertension and cardiovascular diseases. Environmental risk factors for SGA are well established and include smoking, low pregnancy weight, maternal short stature, maternal diet, ethnic origin of mother and hypertension. However, in a large proportion of SGA, no underlying cause is evident, and these individuals may have a larger genetic contribution. In this study we tested the association between SGA and polymorphisms in genes that have previously been associated with obesity and/or diabetes. We undertook analysis of 54 single nucleotide polymorphisms (SNPs) in 546 samples from the Auckland Birthweight Collaborative (ABC) study. 227 children were born small for gestational age (SGA) and 319 were appropriate for gestational age (AGA). The results demonstrated that genetic variation in KCNJ11, BDNF, PFKP, PTER and SEC16B were associated with SGA and support the concept that genetic factors associated with obesity and/or type 2 diabetes are more prevalent in those born SGA compared to those born AGA. We have previously determined that environmental factors are associated with differences in birthweight in the ABC study and now we have demonstrated a significant genetic contribution, suggesting that the interaction between genetics and the environment are important. Show less

Hepatocyte nuclear factor-4alpha (HNF-4alpha) regulates transcription of several genes involved in lipid metabolism, including that of apolipoprotein (apo) A-IV, which is tightly regulated by lipid absorption and enhances enterocyte chylomicron secretion. Studies were performed to define the role of HNF-4alpha in the regulation of apo A-IV gene transcription by dietary fatty acid in neonatal swine small intestine. HNF-4alpha mRNA was expressed in liver > intestine > kidney in suckling, weanling, and weaned pigs. Jejunal HNF-4alpha mRNA and protein and apo A-IV and swine microsomal triglyceride transfer protein (MTP) large subunit mRNA expression were induced in parallel in 2-day-old swine by a 24-h high-fat intraduodenal infusion. In IPEC-1 cells, incubation with oleic acid (OA) resulted in coordinate induction of both HNF-4alpha, apo A-IV, and MTP mRNA, similar to that observed in vivo. When HNF-4alpha expression was driven by doxycycline by using the TET-On system in the absence of OA to observe the effect of HNF-4alpha directly on apo A-IV and MTP mRNA levels in the absence of other factors that might be concomitantly induced by fatty acid absorption, apo A-IV and MTP expression were increased. In luciferase reporter gene assays in IPEC-1 cells using apo A-IV/C-III intergenic region constructs, TET-On-regulated HNF-4alpha expression without OA increased luciferase activity, and incubation with OA did not further increase activity. These data suggest that acute induction of the apo A-IV and MTP genes by dietary lipid in newborn intestine occurs, at least in part, via ligand-independent transactivation by HNF-4alpha that is itself induced by a lipid-mediated mechanism. Show less

The newborn mammal must efficiently absorb dietary fat, predominantly as triacylglycerol, and produce chylomicrons to deliver this lipid to peripheral tissues. The cellular mechanisms involved in enterocyte chylomicron assembly have recently been elucidated, and data on their regulation in the immature gut are beginning to emerge. This review focuses on key proteins involved in chylomicron assembly: apolipoprotein B-48, microsomal triglyceride transfer protein, and apolipoprotein A-IV. Recent studies support a role for apolipoprotein A-IV in enhancing chylomicron secretion by promoting production of larger particles. These proteins are regulated in a manner to maximize the lipid absorptive capacity of the newborn intestine. Show less

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans. Show less

Continuous modes of renal replacement therapy (CRRT) are increasingly being utilized in the intensive care unit. The removal of cytokines and other inflammatory proteins during ultrafiltration may be responsible for some of the beneficial effects of CRRT. We used proteomic tools to identify proteins found in the ultrafiltrate from a patient with acute renal failure. Identification of these proteins could help elucidate the mechanism(s) of improved outcome with continuous renal replacement therapy. Protein was loaded on a reversed-phase C4 column and eluted with stepwise isocratic flows starting with 0%, 5%, 10%, 25%, and 50% of acetonitrile. Effluent was collected, pooled, desalted, and separated by two-dimensional gel electrophoresis (2DE). Reversed-phase separation improved the resolution and the number of spots seen on the gels. Protein spots were digested with trypsin and spotted onto MALDI plates. Proteins were identified by either peptide mass fingerprinting using a MALDI-TOF mass spectrometer or by peptide sequencing using a MALDI-TOF/TOF tandem mass spectrometer. From 196 spots cut, 47 were identified, representing multiple charge forms of 10 different proteins. Proteins identified were albumin, apolipoprotein A-IV, beta-2-microglobulin, lithostathine, mannose-binding lectin associated serine protease 2 associated protein, plasma retinol-binding protein, transferrin, transthyretin, vitamin D-binding protein and Zn alpha-2 glycoprotein. Continuous renal replacement therapy is frequently used in acutely ill patients with renal failure. Removal of proteins occurs during this process. The physiological significance of this protein removal is unclear. Identification of these proteins will lead to better understanding of the role of protein removal in continuous renal replacement therapy. Show less

Dietary lipid acutely upregulates apolipoprotein (apo) A-IV expression by sevenfold at the pretranslational level in neonatal swine jejunum. To determine the mechanism of this regulation, two-day-old female swine received intraduodenal infusions of low- and high-triacylglycerol (TG) isocaloric diets for 24 h. Nuclear runoff assay confirmed apo A-IV gene transcriptional regulation by the high-TG diet. Footprinting analysis using the swine apo A-IV proximal promoter sequence (+14 to -246 bp) demonstrated three regions protected by the low-TG extracts. Of these three motifs, only ACCTTC showed 100% homology to the human sequence and was further studied. EMSA was performed using probes containing wild-type (WT) and mutant (M) motifs. A shift was noted with the low-TG nuclear extracts with the WT probe but not with the M probe. Excess unlabeled free WT probe competed out the shift, whereas the M probe did not. No significant shift occurred with either probe using high-TG extracts. These results suggest that a repressor protein binds to the ACCTTC motif and becomes unbound during lipid absorption, allowing transcriptional activation of the apo A-IV gene in newborn swine small intestine. Show less

Apolipoprotein A-IV (apoA-IV) has myriad functions, including roles as a post-prandial satiety factor and lipid antioxidant. ApoA-IV is expressed in mammalian small intestine and is up-regulated in response to lipid absorption. In newborn swine jejunum, a high fat diet acutely induces a 7-fold increase in apoA-IV expression. To determine whether apoA-IV plays a role in the transport of absorbed lipid, swine apoA-IV was overexpressed in a newborn swine enterocyte cell line, IPEC-1, followed by analysis of the expression of genes related to lipoprotein assembly and lipid transport, as well as quantitation of lipid synthesis and secretion. A full-length swine apoA-IV cDNA was cloned, sequenced, and inserted into a Vp and Rep gene-deficient adeno-associated viral vector, containing the cytomegalovirus immediate early promoter/enhancer and neomycin resistance gene, and was used to transfect IPEC-1 cells. Control cells were transfected with the same vector minus the apoA-IV insert. Using neomycin selection, apoA-IV-overexpressing (+AIV) and control (-AIV) clones were isolated for further study. Both undifferentiated (-D) and differentiated (+D) +AIV cells expressed 40- to 50-fold higher levels of apoA-IV mRNA and both intracellular and secreted apoA-IV protein compared with -AIV cells. Expression of other genes was not affected by apoA-IV overexpression in a manner that would contribute to enhanced lipid secretion. +D +AIV cells secreted 4.9-fold more labeled triacylglycerol (TG), 4.6-fold more labeled cholesteryl ester (CE), and 2-fold more labeled phospholipid (PL) as lipoproteins, mostly in the chylomicron/very low density lipoprotein (VLDL) density range. ApoA-IV overexpression in IPEC-1 cells enhances basolateral TG, CE, and PL secretion in chylomicron/VLDL particles. This enhancement is not associated with up-regulation of other genes involved in lipid transport. ApoA-IV may play a role in facilitating enterocyte lipid transport, particularly in the neonate receiving a diet of high fat breast milk. Show less

Phospholipid (PL) from both dietary sources and biliary secretions may be important in the regulation of intestinal apolipoprotein (apo) synthesis. We previously demonstrated the up-regulation of apo A-I secretion by phosphatidylcholine (PC) in a newborn piglet intestinal epithelial cell line. We hypothesized that dietary PC increases small intestinal apo A-I synthesis in vivo in the newborn piglet. Two-day-old female swine were fed by gavage for 48 h. Diets consisted of a formula containing 51% of calories as triacylglycerol providing 180 kcal/kg/24 h. The experimental group (+PC, n = 7) received 1 g/L added soybean PC, and the control group (-PC, n = 7) received no added PC. At the end of the study period, jejunal apo A-I, B, and A-IV synthesis was measured, and apo A-I mRNA levels were quantitated. Jejunal mucosal PL content and serum lipids and apo B and A-I levels were measured. Jejunal apo A-I synthesis was almost twice as high in the +PC group as compared to the -PC group with no difference in apo A-I mRNA levels. Jejunal content of PL was higher in the +PC group than in the -PC group. There were no differences in jejunal apo B and A-IV synthesis or serum levels of lipids and apo-lipoproteins between the two groups. Dietary PC supplementation in newborn swine up-regulated jejunal apo A-I synthesis. Apo A-IV synthesis, which is sensitive to fatty acid flux, was not significantly increased, which suggests a specific effect of PC on apo A-I synthesis. Lumenal PC may be important in the regulation of intestinal apo A-I synthesis in the neonate. Show less