👤 John T Gallagher

To determine whether long-term residential air pollution [AP; ozone (O₃) and fine particulate matter (PM₂.₅)] is associated with (1) incident mild cognitive impairment (MCI) or Alzheimer’s disease (AD), (2) biomarkers of core and AD-relevant pathology, and (3) whether these relationships are moderated by APOE4+/- (carrier/non-carrier of one or both ε4 alleles) status or mediated by neuroinflammation. Sample included 795 participants (Mage 68.7 ± 7.9; 68% female) from the Wisconsin Alzheimer’s Disease Research Center and Wisconsin Registry for Alzheimer’s Prevention parent studies, both enriched for AD risk at enrollment based on parental AD history. Residential zip code and 2009–2021 EPA-based annual AP reports were used to estimate individual exposure. Cox proportional hazards models assessed MCI/AD risk. Linear regressions examined the relationships between AP exposure and biomarkers of core and AD-relevant pathology, with and without APOE4 + stratification. Causal mediation analysis examined whether markers of inflammation mediated the AP-AD pathology relationships. Neither O₃ nor PM₂.₅ exposure predicted MCI/AD incidence nor core AD pathology (Ps > 0.05). Higher PM₂.₅ was associated with higher CSF GFAP levels ( Show less

The emergence of Alzheimer's disease (AD) pathology has been the focus of multiple hypotheses, with amyloid β (Aβ) playing a central role due to its presence in both familial and sporadic AD. Therefore, a crucial aspect of AD research is understanding the generation of different Aβ species. Aβ peptides result from the proteolytic processing of Amyloid Precursor Protein (APP) by β- and γ-secretases, with BACE1 being the most prominent β-secretase. However, BACE1-overexpressing mouse models exhibit disadvantages, making them limited for AD research. Importantly, N-terminally truncated Aβ species, which constitute up to 70 % of Aβ in AD brains, are not generated by BACE1. In recent years, alternative proteases capable of cleaving APP have been identified, bridging the gap between N-terminally truncated Aβ species and BACE1-derived Aβ. Among these novel players, the metalloprotease meprin β has emerged as a risk factor in AD pathology, generating both N-terminally truncated and full-length Aβ species. Our primary objective was to develop a mouse model that more accurately resembles the pathology of AD beyond BACE1-overexpressing models, while simultaneously confirming APP cleavage of meprin β in the hippocampus and cerebral cortex. Overexpression of meprin β led to a marked increase in soluble Aβ levels, particularly in the hippocampus, indicating a higher vulnerability or elevated meprin β activity in this region compared to the cerebral cortex. Notably, this biochemical change occurred without any observable behavioral deficits, suggesting a region-specific role of meprin β in AD pathology that may extend beyond immediate functional impairment. Show less

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy, affecting 1 in 500 people. With growing access to genetic testing and incorporation of genetics in diagnosis and management of HCM, it is important to identify phenotypic predictors of HCM genotype, to improve genetic targeting and counselling as well as cascade testing for first-degree relatives. This was a retrospective analysis of consecutive probands, aged over 18 years referred to a tertiary centre for HCM gene panel testing. Demographic information was obtained from clinic data. Left ventricular hypertrophy (LVH) pattern was classified based on trans-thoracic echocardiogram (TTE). Pathogenicity of variants was classified per the American College of Medical Genetics (ACMG) criteria. 166 patients were included for analysis. Mean age was 53 years (SD 14.28). 128 (77%) were male. 59 had a history of hypertension and 19 had a family history of sudden cardiac death (SCD). The most frequent HCM pattern at baseline was concentric HCM (31.9% n = 53). 48 patients had a likely pathogenic (LP) or pathogenic (P) variant, giving a genetic testing yield of 28.9%. The most common sarcomeric genes were MYBPC3 and MYH7 accounting for 57% of cases. Younger age, female sex, and reverse curve LVH pattern were predictors of a LP or P gene variant identification. Reverse curve morphology was found to be a significant predictor for a sarcomere variant (p < 0.001). Genetic testing was appropriately offered in this cohort. Younger age, female sex, family history of SCD, normal/well controlled blood pressure and reverse pattern LVH on TTE predicted a higher yield of pathogenic variant identification. Reverse curve morphology was found to be a significant predictor for a sarcomere variant. This study has implications for supporting better phenotype-based genetic counselling and resource usage for HCM patients. Show less

Metabolic syndrome (MetS) is linked to later-life cognitive decline and brain aging, but early detection of vulnerability in midlife remains challenging. This study applied two methods to detect subtle changes in midlife adults with MetS: (1) latent profile analysis (LPA) to identify cognitive performance patterns and (2) an MRI-derived brain-predicted age metric to assess structural brain aging. Participants were cognitively unimpaired, community-dwelling adults from prior studies on metabolic and brain health ( MetS prevalence differed across cognitive profiles (χ Cognitive profiles and brain-predicted age metrics identify early vulnerability in midlife MetS, underscoring the importance of early intervention. Show less

Dendritic cells (DCs) are key in the initiation of the adaptive T cell responses to tailor adequate immunity that corresponds to the type of pathogen encountered. Oppositely, DCs control the resolution phase of inflammation and are able to induce tolerance after receiving anti-inflammatory cytokines or upon encounter of self-associated molecular patterns, such as α2-3 linked sialic acid (α2-3sia). We here investigated whether α2-3sia, that bind immune inhibitory Siglec receptors, would alter signaling and reprogramming of LPS-stimulated human monocyte-derived DCs (moDCs). Transcriptomic analysis of moDCs stimulated with α2-3sia-conjugated dendrimers revealed differentially expressed genes related to metabolic pathways, cytokines, and T cell differentiation. An increase in genes involved in ATPase regulator activity, oxidoreductase activity, and glycogen metabolic processes was detected. Metabolic extracellular flux analysis confirmed a more energetic moDC phenotype upon α2-3sia binding as evidenced by an increase in both glycolysis and mitochondrial oxidative phosphorylation. T In conclusion, we demonstrate that α2-3sia binding to moDCs, phosphorylates Siglec-9, alters metabolic pathways, cytokine signaling, and T cell differentiation processes in moDCs and promotes regulatory T cells. The sialic acid-Siglec axis on DCs is therefore, a novel target to induce tolerance and to explore for immunotherapeutic interventions aimed to restore inflammatory processes. Show less

Limited knowledge exists of the extent of epigenetic alterations, such as DNA methylation, in heart failure (HF). We conducted targeted DNA methylation sequencing to identify DNA methylation alterations in coding and noncoding RNA (ncRNA) across different etiological subtypes of HF. A targeted bisulfite sequence capture sequencing platform was applied to DNA extracted from cardiac interventricular septal tissue of 30 male HF patients encompassing causes including hypertrophic obstructive cardiomyopathy, ischemic cardiomyopathy, dilated cardiomyopathy, and 9 control patients with nonfailing hearts. We detected 62 678 differentially methylated regions in the studied HF cohort. By comparing each HF subgroup to the nonfailing control group, we identified 195 unique differentially methylated regions: 5 in hypertrophic obstructive cardiomyopathy, 151 in dilated cardiomyopathy, and 55 in ischemic cardiomyopathy. These translated to 4 genes/1 ncRNA in hypertrophic obstructive cardiomyopathy, 131 genes/17 ncRNA in dilated cardiomyopathy, and 51 genes/5 ncRNA in ischemic cardiomyopathy. Subsequent gene/ncRNA expression analysis was assessed using quantitative reverse transcription polymerase chain reaction and revealed 6 genes: 4 hypermethylated ( HEY2, MSR1, MYOM3, and COX17), 2 hypomethylated ( CTGF and MMP2); and 2 microRNA: 1 hypermethylated (miR-24-1), 1 hypomethylated (miR-155) with significantly upregulated or downregulated expression levels consistent with the direction of methylation in the particular HF subgroup. For the first time DNA methylation alterations and associated gene expression changes were identified in etiologically variant pathological HF tissue. The methylation-sensitive and disease-associated genes/ncRNA identified from this study represent a unique cohort of loci that demonstrate a plausible potential as a novel diagnostic and therapeutic target in HF and warrant further investigation. Show less

Macrophage polarization plays a central role in both protective immunity and immunopathology. While the role of cytokines in driving macrophage polarization is well characterized, less is understood about the role of chemokines. The purpose of this study was to determine if CC chemokine 2 (CCL2/MCP1) could influence macrophage polarization in response to subsequent activation with cytokines and microbial products. Treatment of bone marrow-derived macrophages with CCL2 alone did not result in increased expression of either classical or alternatively-activated macrophage genes as compared to standard skewing cytokines or Toll-like receptor agonists. However, subsequent stimulation of CCL2 pre-treated macrophages with classical activation stimuli resulted in enhanced expression of genes associated with classical activation. This enhancement correlated with increased phosphorylation of ERK1/2 kinases, a decrease in expression of the ERK phosphatase Dusp6 and enhanced expression of miR-9. These results indicate that CCL2 supports the classical activation of macrophages, with miR-9 mediated down-regulation of Dusp6 and enhanced ERK-mediated signal transduction possibly mediating this enhanced pro-inflammatory gene expression. Show less

Ventricular noncompaction cardiomyopathy is a rare form of congenital cardiomyopathy with increasing evidence of genetic etiology, especially when presenting in childhood. Fetal presentation is rare. We describe a case of fetal hydrops, presenting at 24 weeks gestation and leading to intrapartum death at 26 weeks gestation. Autopsy examination revealed characteristic features of left ventricular noncompaction. A genetic analysis identified a constellation of variants of unknown significance in MYH6, TNNC1, and MYBPC3, genes known to be important in sarcomeric function. Additionally, the variant in MYBPC3 was homozygous. While this case did not demonstrate a conventional single-gene mutation as the cause of the ventricular noncompaction, a broader genomic investigation revealed several variants in sarcomeric genes which may act synergistically to impact cardiac function. Show less

Low back pain (LBP) is responsible for more than 2.5 million visits to US emergency departments (EDs) annually. These patients are usually treated with nonsteroidal anti-inflammatory drugs, acetaminophen, opioids, or skeletal muscle relaxants, often in combination. To compare functional outcomes and pain at 1 week and 3 months after an ED visit for acute LBP among patients randomized to a 10-day course of (1) naproxen + placebo; (2) naproxen + cyclobenzaprine; or (3) naproxen + oxycodone/acetaminophen. This randomized, double-blind, 3-group study was conducted at one urban ED in the Bronx, New York City. Patients who presented with nontraumatic, nonradicular LBP of 2 weeks' duration or less were eligible for enrollment upon ED discharge if they had a score greater than 5 on the Roland-Morris Disability Questionnaire (RMDQ). The RMDQ is a 24-item questionnaire commonly used to measure LBP and related functional impairment on which 0 indicates no functional impairment and 24 indicates maximum impairment. Beginning in April 2012, a total of 2588 patients were approached for enrollment. Of the 323 deemed eligible for participation, 107 were randomized to receive placebo and 108 each to cyclobenzaprine and to oxycodone/acetaminophen. Follow-up was completed in December 2014. All participants were given 20 tablets of naproxen, 500 mg, to be taken twice a day. They were randomized to receive either 60 tablets of placebo; cyclobenzaprine, 5 mg; or oxycodone, 5 mg/acetaminophen, 325 mg. Participants were instructed to take 1 or 2 of these tablets every 8 hours, as needed for LBP. They also received a standardized 10-minute LBP educational session prior to discharge. The primary outcome was improvement in RMDQ between ED discharge and 1 week later. Demographic characteristics were comparable among the 3 groups. At baseline, median RMDQ score in the placebo group was 20 (interquartile range [IQR],17-21), in the cyclobenzaprine group 19 (IQR,17-21), and in the oxycodone/acetaminophen group 20 (IQR,17-22). At 1-week follow-up, the mean RMDQ improvement was 9.8 in the placebo group, 10.1 in the cyclobenzaprine group, and 11.1 in the oxycodone/acetaminophen group. Between-group difference in mean RMDQ improvement for cyclobenzaprine vs placebo was 0.3 (98.3% CI, -2.6 to 3.2; P = .77), for oxycodone/acetaminophen vs placebo, 1.3 (98.3% CI, -1.5 to 4.1; P = .28), and for oxycodone/acetaminophen vs cyclobenzaprine, 0.9 (98.3% CI, -2.1 to 3.9; P = .45). Among patients with acute, nontraumatic, nonradicular LBP presenting to the ED, adding cyclobenzaprine or oxycodone/acetaminophen to naproxen alone did not improve functional outcomes or pain at 1-week follow-up. These findings do not support use of these additional medications in this setting. clinicaltrials.gov Identifier: NCT01587274. Show less

Although the evidence linking apoA-IV expression and triglyceride (TG)-rich lipoprotein assembly and secretion is compelling, the intracellular mechanisms by which apoA-IV could modulate these processes remain poorly understood. We therefore examined the functional impact of apoA-IV expression on endogenous apoB, TG, and VLDL secretion in stably transfected McA-RH7777 rat hepatoma cells. Expression of apoA-IV modified with the endoplasmic reticulum (ER) retention signal KDEL (apoA-IV-KDEL) dramatically decreased both the rate and efficiency of endogenous apoB secretion, suggesting a presecretory interaction between apoA-IV-KDEL and apoB or apoB-containing lipoproteins. Expression of native apoA-IV using either a constitutive or tetracycline-inducible promoter delayed the initial rate of apoB secretion and reduced the final secretion efficiency by ∼40%. However, whereas apoA-IV-KDEL reduced TG secretion by 75%, expression of native apoA-IV caused a 20-35% increase in TG secretion, accompanied by a ∼55% increase in VLDL-associated apoB, an increase in the TG:phospholipid ratio of secreted d < 1.006 lipoproteins, and a 10.1 nm increase in peak VLDL(1) particle diameter. Native apoA-IV expression had a negligible impact on expression of the MTP gene. These data suggest that by interacting with apoB in the secretory pathway, apoA-IV alters the trafficking kinetics of apoB-containing TG-rich lipoproteins through cellular lipidation compartments, which in turn, enhances particle expansion and increases TG secretion. Show less

Heparan sulfate proteoglycans (HSPG) encompass some of the most abundant macromolecules on the surface of almost every cell type. Heparan sulfate (HS) chains provide a key interaction surface for the binding of numerous proteins such as growth factors and morphogens, helping to define the ability of a cell to respond selectively to environmental cues. The specificity of HS-protein interactions are governed predominantly by the order and positioning of sulfate groups, with distinct cell types expressing unique sets of HS epitopes. Embryos deficient in HS-synthesis (Ext1(-/-)) exhibit pre-gastrulation lethality and lack recognizable organized mesoderm and extraembryonic tissues. Here we demonstrate that embryonic stem cells (ESCs) derived from Ext1(-/-) embryos are unable to differentiate into hematopoietic lineages, instead retaining ESC marker expression throughout embryoid body (EB) culture. However hematopoietic differentiation can be restored by the addition of soluble heparin. Consistent with specific size and composition requirements for HS:growth factor signaling, chains measuring at least 12 saccharides were required for partial rescue of hematopoiesis with longer chains (18 saccharides or more) required for complete rescue. Critically N- and 6-O-sulfate groups were essential for rescue. Heparin addition restored the activity of multiple signaling pathways including bone morphogenic protein (BMP) with activation of phospho-SMADs re-established by the addition of heparin. Heparin addition to wild-type cultures also altered the outcome of differentiation, promoting hematopoiesis at low concentrations, yet inhibiting blood formation at high concentrations. Thus altering the levels of HS and HS sulfation within differentiating ESC cultures provides an attractive and accessible mechanism for influencing cell fate. Show less

Cell surface heparan sulfate (HS) proteoglycans are required in development and postnatal repair. Important classes of ligands for HS include growth factors and extracellular matrix macromolecules. For example, the focal adhesion component syndecan-4 interacts with the III(12-14) region of fibronectin (HepII domain) through its HS chains. The fine structure of HS is critical to growth factor responses, and whether this extends to matrix ligands is unknown but is suggested from in vitro experiments. Cell attachment to HepII showed that heparin oligosaccharides of >or=14 sugar residues were required for optimal inhibition. The presence of N-sulfated glucosamine in the HS was essential, whereas 2-O-sulfation of uronic acid or 6-O-sulfation of glucosamine had marginal effects. In the more complex response of focal adhesion formation through syndecan-4, N-sulfates were again required and also glucosamine 6-O-sulfate. The significance of polymer N-sulfation and sulfated domains in HS was confirmed by studies with mutant Chinese hamster ovary cells where heparan sulfation was compromised. Finally, focal adhesion formation was absent in fibroblasts synthesizing short HS chains resulting from a gene trap mutation in one of the two major glucosaminoglycan polymerases (EXT1). Several separate, specific properties of cell surface HS are therefore required in cell adhesion responses to the fibronectin HepII domain. 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

To examine the role of apolipoprotein A-IV (apoA-IV) in the intracellular trafficking and secretion of apoB, COS cells were cotransfected with microsomal triglyceride transfer protein (MTP), apoB-41 (amino terminal 41% of apoB), and either native apoA-IV or apoA-IV modified with the carboxy-terminal endoplasmic reticulum (ER) retention signal, KDEL (apoA-IV-KDEL). As expected, apoA-IV-KDEL was inefficiently secreted relative to native apoA-IV. Coexpression of apoB-41 with apoA-IV-KDEL reduced the secretion of apoB-41 by approximately 80%. The apoA-IV-KDEL effect was specific, as neither KDEL-modified forms of human serum albumin or apoA-I affected apoB-41 secretion. Similar results were observed in McA-RH7777 rat hepatoma cells, which express endogenous MTP. The full inhibitory effect of apoA-IV-KDEL on apoB secretion was observed only for forms of apoB containing a minimum of the amino-terminal 25% of the protein (apoB-25). However, apoA-IV-KDEL inhibited the secretion of both lipid-associated and lipid-poor forms of apoB-25. Dual-label immunofluorescence microscopy of cells transfected with native apoA-IV and apoB-25 revealed that both apolipoproteins were localized to the ER and Golgi, as expected. However, when apoA-IV-KDEL was cotransfected with apoB-25, both proteins localized primarily to the ER. These data suggest that apoA-IV may physically interact with apoB in the secretory pathway, perhaps reflecting a role in modulating the process of triglyceride-rich lipoprotein assembly and secretion. Show less

Apolipoprotein A-V (apoA-V), the newest member of the plasma apolipoprotein family, was recently discovered by comparison of the mouse and human genomes. Studies in rodents and population surveys of human apoA-V polymorphisms have noted a strong effect of apoA-V on plasma triglyceride levels. Toward the elucidation of the biologic function of apoA-V, we used spectroscopic and surface chemistry techniques to probe its structure and interfacial activity. Computer-assisted sequence analysis of apoA-V predicts that it is very hydrophobic, contains a significant amount of alpha-helical secondary structure, and probably is composed of discrete structural regions with varying degrees of lipid affinity. Fluorescence spectroscopy of recombinant human apoA-V provided evidence of tertiary folding, and light scattering studies indicated that apoA-V transforms dimyristoylphosphatidylcholine vesicles into discoidal complexes with an efficiency similar to that of apoA-I. Surface chemistry techniques revealed that apoA-V displays high affinity, low elasticity, and slow binding kinetics at hydrophobic interfaces, properties we propose may retard triglyceride-rich particle assembly. Metabolic labeling and immunofluorescence studies of COS-1 cells transfected with human apoA-V demonstrated that apoA-V is poorly secreted, remains associated with the endoplasmic reticulum, and does not traffic to the Golgi. Given that overexpression of the apoA-V gene lowers plasma triglycerides in mice, these data together suggest that apoA-V may function intracellularly to modulate hepatic VLDL synthesis and/or secretion. Show less