Search

Michael R Preusch,1,2 Nicholas Ieronimakis,1 Errol S Wijelath,3 Sara Cabbage,1 Jerry Ricks,1 Florian Bea,2 Morayma Reyes,1 Joanne van Ryn,4 Michael E Rosenfeld1,5 1Department of Pathology, University of Washington, Seattle, WA, USA; 2Department of Internal Medicine, University of Heidelberg, Heidelberg, Germany; 3Department of Surgery, University of Washington, Seattle, WA, USA; 4Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany; 5Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA, USA Objective: Thrombin has multiple proatherogenic effects including platelet activation and the induction of inflammatory processes. Recently, the cytokine oncostatin M has been shown to have proinflammatory effects. This study was designed to investigate the effects of thrombin inhibition on the initiation and progression of atherosclerosis and on the expression of oncostatin M. Methods: Apolipoprotein E-deficient mice at different ages were fed the thrombin inhibitor dabigatran etexilate. The mean lesion area was measured in the aortic sinus and in the innominate artery. CD45-positive cells within the aortic tissue were measured by flow cytometry. Oncostatin M expression was measured in the tissue sections by immunocytochemistry. Results: Treatment with dabigatran etexilate resulted in a significant reduction of the mean area of atherosclerotic lesions in the aortic sinus in both the young mice (11,176±1,500 µm2 (control) versus 3,822±836 µm2 (dabigatran etexilate), P

Previous studies have demonstrated the presence of apoprotein (apo) E protein and message in arterial lesions. To determine the source of the synthesized apoE, we performed simultaneous in situ hybridization and immunocytochemistry on human and rabbit atherosclerotic tissue. Studies of serial sections of aortic atherosclerotic lesions from humans and hypercholesterolemic New Zealand White rabbits and Watanabe heritable hyperlipidemic rabbits revealed a similar pattern of macrophage-specific apoE expression in the rabbit and human lesions. In early lesions of rabbit atherosclerotic tissue, in which many macrophages were present, there was abundant expression of apoE mRNA. Northern blot analyses of total mRNA obtained from arterial macrophage-derived foam cells, freshly isolated from ballooned, cholesterol-fed New Zealand White rabbits, demonstrated positive hybridization with an apoE-specific riboprobe. Western blot analyses of conditioned media from the isolated foam cells placed in culture for up to 24 hours demonstrated the presence of secreted apoE. These studies demonstrated that in atherosclerotic lesions, arterial wall macrophages synthesize and secrete apoE and probably account for most of the apoE synthesized in the atherosclerotic artery.

Pure macrophage-derived foam cells (MFC) were isolated from the aortas of rabbits made atherosclerotic by balloon deendothelialization followed by diet-induced hypercholesterolemia. The MFC were isolated under sterile conditions using an enzymatic digestion procedure and discontinuous density gradient centrifugation. The purity of the MFC preparations was verified immunocytochemically with the macrophage specific monoclonal antibody RAM-11. MFC plated in medium containing 0.5% FCS for 24 h contained approximately 600 micrograms cholesterol per mg cell protein, 80% of which was esterified cholesterol. The MFC specifically degraded low density lipoprotein (LDL), acetyl-LDL, copper oxidized LDL, and beta-very low density lipoprotein (beta-VLDL) at rates comparable to mouse peritoneal macrophages (MPM) in 5-h assays. MFC within sections of the atherosclerotic lesions from the ballooned rabbits as well as the MFC isolated from the same lesions in the presence of antioxidants, exhibited positive immunoreactivity with polyclonal guinea pig antisera and mouse monoclonal antibodies directed against malondialdehyde-LDL, and 4-hydroxynonal-LDL. The MFC also exhibited the capacity to induce the oxidation of LDL at rates comparable to those exhibited by MPM and rabbit aortic endothelial cells. These data provide direct evidence that arterial wall macrophages express modified LDL receptors in vivo, contain epitopes found in oxidized-LDL and are capable of oxidizing LDL even when maximally loaded with cholesterol.

Increasing evidence indicates that low density lipoprotein (LDL) has to be modified to induce foam cell formation. One such modification, oxidation of LDL, generates a number of highly reactive short chain-length aldehydic fragments of oxidized fatty acids capable of conjugating with lysine residues of apoprotein B. By immunizing animals with homologous malondialdehyde-modified LDL (MDA-LDL), 4-hydroxynonenal-LDL (4-HNE-LDL), and Cu+(+)-oxidized LDL, we developed polyvalent and monoclonal antibodies against three epitopes found in oxidatively modified LDL. The present article characterizes an antiserum and monoclonal antibody (MAL-2 and MDA2, respectively) specific for MDA-lysine, and an antiserum and monoclonal antibody (HNE-6 and NA59, respectively) specific for 4-HNE-lysine. In addition, a monoclonal antibody (OLF4-3C10) was developed against an as yet undefined epitope generated during Cu++ oxidation of LDL. With these antibodies, we demonstrated that MDA-lysine and 4-HNE-lysine adducts develop on apo-lipoprotein B during copper-induced oxidation of LDL in vitro. The application of these antibodies for immunocytochemical demonstration of oxidized lipoproteins in atherosclerotic lesions of progressive severity is described in the companion article. These antibodies should prove useful in studying the role of oxidatively modified lipoproteins as well as other oxidatively modified proteins in atherogenesis.

Antisera and monoclonal antibodies generated against autologous malondialdehyde-conjugated low density lipoprotein (MDA-LDL), 4-hydroxynonenal conjugated LDL (4-HNE-LDL), and the protein fragments of apoprotein B resulting from the copper oxidation of LDL, as well as antibodies against apoprotein B, were used to immunostain atherosclerotic lesions of varying severity from Watanabe heritable hyperlipemic rabbits. In macrophage-rich fatty streaks and transitional lesions, all of the antibodies recognizing oxidation specific epitopes exhibited predominantly cell-associated staining in particulate and annular patterns. This is in contrast to the limited, extracellular, diffuse staining obtained with the antibodies recognizing apoprotein B. In more advanced lesions containing areas with reduced numbers of cells, there was increased extracellular, diffuse staining with the antibodies against oxidation specific epitopes and co-localization with apoprotein B. In addition, there were annular staining patterns associated with the necrotic core and increased staining of intimal and medial smooth muscle cells. We interpret these data as suggesting that in areas of lesions rich in macrophages, LDL is oxidized and taken up by the cells. In more advanced lesions that are relatively devoid of macrophages, both native and oxidized LDL, as well as oxidation products released from dead and decaying cells, are trapped in the matrix, out of reach of those cells capable of accumulating oxidized LDL.

Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, properties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was degraded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.

It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can give rise to foam cells, the key component of the fatty streak lesion of atherosclerosis. Oxidation of LDL probably generates a broad spectrum of conjugates between fragments of oxidized fatty acids and apolipoprotein B. We now present three mutually supportive lines of evidence for oxidation of LDL in vivo: (i) Antibodies against oxidized LDL, malondialdehyde-lysine, or 4-hydroxynonenal-lysine recognize materials in the atherosclerotic lesions of LDL receptor-deficient rabbits; (ii) LDL gently extracted from lesions of these rabbits is recognized by an antiserum against malondialdehyde-conjugated LDL; (iii) autoantibodies against malondialdehyde-LDL (titers from 512 to greater than 4096) can be demonstrated in rabbit and human sera.

Babesiosis is usually acquired from a tick bite or through a blood transfusion. We report a case of babesiosis in an infant for whom vertical transmission was suggested by evidence of Babesia spp. antibodies in the heel-stick blood sample and confirmed by detection of Babesia spp. DNA in placenta tissue. [ABSTRACT FROM AUTHOR]

Background: Exposure to air pollution is associated with worldwide morbidity and mortality. Diesel exhaust (DE) emissions are important contributors which induce vascular inflammation and metabolic disturbances by unknown mechanisms. We aimed to determine molecular pathways activated by DE in the liver that could be responsible for its cardiometabolic toxicity., Methods: Apolipoprotein E knockout (ApoE KO) mice were exposed to DE or filtered air (FA) for two weeks, or DE for two weeks followed by FA for 1 week. Expression microarrays and global metabolomics assessment were performed in the liver. An integrated transcriptomic and metabolomic analytical strategy was employed to dissect critical pathways and identify candidate genes that could dissect DE-induced pathogenesis. HepG2 cells were treated with an organic extract of DE particles (DEP) vs. vehicle control to test candidate genes., Results: DE exposure for 2 weeks dysregulated 658 liver genes overrepresented in whole cell metabolic pathways, especially including lipid and carbohydrate metabolism, and the respiratory electron transport pathway. DE exposure significantly dysregulated 118 metabolites, resulting in increased levels of triglycerides and fatty acids due to mitochondrial dysfunction as well as increased levels of glucose and oligosaccharides. Consistently, DEP treatment of HepG2 cells led to increased gluconeogenesis and glycogenolysis indicating the ability of the in-vitro approach to model effects induced by DE in vivo. As an example, while gene network analysis of DE livers identified phosphoenolpyruvate carboxykinase 1 (Pck1) as a key driver gene of DE response, DEP treatment of HepG2 cells resulted in increased mRNA expression of Pck1 and glucose production, the latter replicated in mouse primary hepatocytes. Importantly, Pck1 inhibitor mercaptopicolinic acid suppressed DE-induced glucose production in HepG2 cells indicating that DE-induced elevation of hepatic glucose was due in part to upregulation of Pck1 and increased gluconeogenesis., Conclusions: Short-term exposure to DE induced widespread alterations in metabolic pathways in the liver of ApoE KO mice, especially involving carbohydrate and lipid metabolism, together with mitochondrial dysfunction. Pck1 was identified as a key driver gene regulating increased glucose production by activation of the gluconeogenesis pathway., Competing Interests: Declarations. Ethical approval: Animal procedures were approved by the Animal Care and Use Committees of the University of Washington and UCLA. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Mycoplasma hominis is a facultative anaerobic bacterium commonly present in the urogenital tract. In recent years, M. hominis has increasingly been associated with extra-urogenital tract infections, particularly in immunosuppressed patients. Detecting M. hominis in a diagnostic laboratory can be challenging due to its slow growth rate, absence of a cell wall, and the requirements of specialized media and conditions for optimal growth. Consequently, it is necessary to establish guidelines for the detection of this microorganism and to request the appropriate microbiological work-up of immunosuppressed patients., Case Presentation: We hereby present two cases of solid organ transplant patients who developed M. hominis infection. Microscopic examination of the bronchial lavage and pleural fluid showed no microorganisms. However, upon inoculating the specimens onto routine microbiology media, the organism was successfully identified and confirmation was performed using 16S rDNA sequencing. Both patients received appropriate treatment resulting in the resolution of M. hominis infection., Conclusions: The prompt detection of M. hominis in a clinical specimen can have a significant impact on patient care by allowing for early intervention and ultimately resulting in more favorable clinical outcomes, especially in transplant patients., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Limited data support use of pneumococcal urinary antigen testing (PUAT) for patients with community-acquired pneumonia (CAP) as an antimicrobial stewardship tool. At our institution, CAP guidelines and admission order set were standardized to include universal PUAT., Methods: This was a retrospective study of adults hospitalized in 2019 who had PUAT performed. We compared incidence and timing of de-escalation in PUAT- positive vs -negative groups and described patients' outcomes., Results: We evaluated 910 patients, 121 (13.3%) of whom were PUAT positive. No difference in baseline characteristics, including severity of illness, was observed between groups. Initial de-escalation occurred in 82.9% and 81.2% of PUAT-positive and -negative patients, respectively ( P  = .749). Median time to de-escalation was shorter in the PUAT-positive group (1 [interquartile range {IQR}, 0-2] day vs 1 [IQR, 1-2] day, P  = .01). Within 24 hours of PUAT, more patients in the PUAT-positive group had atypical coverage discontinued (61.3% vs 47.2%, P  = .026) without difference in methicillin-resistant Staphylococcus aureus (MRSA) agent discontinuation (or antipseudomonal de-escalation). Among the PUAT-positive group, unadjusted analysis demonstrated shorter median length of stay in patients who were de-escalated compared to those who were not (6 [IQR, 4-10] vs 8 [IQR, 7-12] days, P  = .0005), without difference in the incidence of Clostridioides difficile , in-hospital mortality, or 30-day infection-related readmission., Conclusions: We observed earlier de-escalation in the PUAT-positive group. This seems to be due to discontinuation of atypical rather than anti-MRSA or antipseudomonal coverage. Further antimicrobial stewardship interventions are warranted., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Introduction: Declining renal function results in the accumulation of solutes normally excreted by healthy kidneys. Data suggest that some of the protein-bound solutes mediate accelerated cardiovascular disease. Many of the poorly dialyzable protein-bound uremic retention solutes are products of gut bacterial metabolism., Methods: We performed a blinded-randomized controlled trial comparing the changes in plasma concentrations of a panel of protein-bound solutes and microbiome structure in response to the once-weekly oral administration of 250 mg of vancomycin or placebo over a period of 12 weeks in a cohort of stable patients with end-stage kidney disease. We also examined the pattern of recovery of the solutes and gut microbiome over 12 weeks of placebo administration following vancomycin., Results: We enrolled 15 subjects. Ten subjects provided sufficient plasma and stool samples to permit us to examine the effect of vancomycin on plasma solute levels. We showed that a weekly dose of vancomycin resulted in a reduction in the plasma concentration of 7 colon-derived solutes. We described a significant effect of vancomycin on the microbiome structure with a decrease in alpha diversity and change in beta diversity. Multiple taxa decreased with vancomycin including genera Clostridium and Bacteroides . We demonstrated microbiome recovery after stopping vancomycin. However, recovery in the solutes was highly variable between subjects., Conclusions: We demonstrated that microbiome suppression using vancomycin resulted in changes in multiple gut-derived uremic solutes. Future studies are needed to address whether reduction in those uremic solutes results in improvement of cardiovascular outcomes in ESKD patients., (© 2021 International Society of Nephrology. Published by Elsevier Inc.)

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, created an unprecedented need for comprehensive laboratory testing of populations, in order to meet the needs of medical practice and to guide the management and functioning of our society. With the greater New York metropolitan area as an epicenter of this pandemic beginning in March 2020, a consortium of laboratory leaders from the assembled New York academic medical institutions was formed to help identify and solve the challenges of deploying testing. This report brings forward the experience of this consortium, based on the real-world challenges which we encountered in testing patients and in supporting the recovery effort to reestablish the health care workplace. In coordination with the Greater New York Hospital Association and with the public health laboratory of New York State, this consortium communicated with state leadership to help inform public decision-making addressing the crisis. Through the length of the pandemic, the consortium has been a critical mechanism for sharing experience and best practices in dealing with issues including the following: instrument platforms, sample sources, test performance, pre- and post-analytical issues, supply chain, institutional testing capacity, pooled testing, biospecimen science, and research. The consortium also has been a mechanism for staying abreast of state and municipal policies and initiatives, and their impact on institutional and laboratory operations. The experience of this consortium may be of value to current and future laboratory professionals and policy-makers alike, in dealing with major events that impact regional laboratory services., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

The Infectious Diseases Society of America has identified the use of SARS-CoV-2 genomic load for prognostication purposes as a key research question. We designed a retrospective cohort study that included adult patients with COVID-19 pneumonia who had at least 2 positive nasopharyngeal tests at least 24 hours apart to study the correlation between the change in the genomic load of SARS-CoV-2, as reflected by the Cycle threshold (Ct) value of the RT-PCR, with change in clinical status. The Sequential Organ Failure Assessment (SOFA) score was used as a surrogate for patients' clinical status. Among 457 patients with COVID-19 pneumonia between 3/31/2020-4/10/2020, we identified 42 patients who met the inclusion criteria. The median initial SOFA score was 2 (IQR 2-3). 20 out of 42 patients had a lower SOFA score on their subsequent tests. We identified a statistically significant inverse correlation between the change in SOFA score and change in the Ct value with a decrease in SOFA score by 0.05 (SE 0.02; p<0.05) for an increase in Ct values by 1. This correlation was independent of the duration of symptoms. Our findings suggest that an increasing Ct value in sequential tests may be of prognostic value for patients diagnosed with COVID-19 pneumonia., Competing Interests: The authors have declared that no competing interests exist.

There have been multiple descriptions of seizures during the acute infectious period in patients with COVID-19. However, there have been no reports of status epilepticus after recovery from COVID-19 infection. Herein, we discuss a patient with refractory status epilepticus 6 weeks after initial infection with COVID-19. Extensive workup demonstrated elevated inflammatory markers, recurrence of a positive nasopharyngeal SARS-CoV-2 polymerase chain reaction, and hippocampal atrophy. Postinfectious inflammation may have triggered refractory status epilepticus in a manner similar to the multisystemic inflammatory syndrome observed in children after COVID-19., (© 2020 International League Against Epilepsy.)

The recent emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed formidable challenges for clinical laboratories seeking reliable laboratory diagnostic confirmation. The swift advance of the crisis in the United States has led to Emergency Use Authorization (EUA) facilitating the availability of molecular diagnostic assays without the more rigorous examination to which tests are normally subjected prior to FDA approval. Our laboratory currently uses two real-time reverse transcription-PCR (RT-PCR) platforms, the Roche Cobas SARS-CoV2 and the Cepheid Xpert Xpress SARS-CoV-2. The two platforms demonstrate comparable performances; however, the run times for each assay are 3.5 h and 45 min, respectively. In search for a platform with a shorter turnaround time, we sought to evaluate the recently released Abbott ID Now COVID-19 assay, which is capable of producing positive results in as little as 5 min. We present here the results of comparisons between Abbott ID Now COVID-19 and Cepheid Xpert Xpress SARS-CoV-2 using nasopharyngeal swabs transported in viral transport media and comparisons between Abbott ID Now COVID-19 and Cepheid Xpert Xpress SARS-CoV-2 using nasopharyngeal swabs transported in viral transport media for Cepheid and dry nasal swabs for Abbott ID Now. Regardless of method of collection and sample type, Abbott ID Now COVID-19 had negative results in a third of the samples that tested positive by Cepheid Xpert Xpress when using nasopharyngeal swabs in viral transport media and 45% when using dry nasal swabs., (Copyright © 2020 American Society for Microbiology.)

This study was performed as a contribution for a better understanding of Chlamydia pneumoniae frequency in children with respiratory infections. A total of 416 children were recruited from two clinical centers in Sao Luis, Brazil. Of these patients, 165 children had upper respiratory tract infections (URTI), 150 had community-acquired pneumonia (CAP), and 101 were asymptomatic volunteer children. Clinical and epidemiological data from the participants were recorded. Nasopharyngeal swab samples were collected to extract DNA. C. pneumoniae DNA positivity and copy numbers were obtained by an absolute quantitative real-time PCR method. RESULTS: Positivity for C. pneumoniae DNA was higher in samples from URTI children (38.2%) and from CAP children (18.0%) than in those from the control group (7.9%; p < 0.001). Moreover, C. pneumoniae DNA was denser in children with URTI than in asymptomatic children. Considering the cutoff, the highest value of C. pneumoniae DNA found in asymptomatic children of the 3.98 log10 copies/mL, 8.5% (14/165) of the children with URTI, and 3.3% (5/150) with CAP presented high copy numbers of C. pneumoniae DNA. CONCLUSION: Taken together, these results revealed a high frequency of C. pneumoniae in both children with URTI and CAP.

Objective: Air pollution is associated with increased cardiovascular morbidity and mortality, as well as dyslipidemia and metabolic syndrome. Our goal was to dissect the mechanisms involved. Approach and Results: We assessed the effects of exposure to air pollution on lipid metabolism in mice through assessment of plasma lipids and lipoproteins, oxidized fatty acids 9-HODE (9-hydroxyoctadecadienoic) and 13-HODE (13-hydroxyoctadecadienoic), lipid, and carbohydrate metabolism. Findings were corroborated, and mechanisms were further assessed in HepG2 hepatocytes in culture. ApoE knockout mice exposed to inhaled diesel exhaust (DE, 6 h/d, 5 days/wk for 16 weeks) exhibited elevated plasma cholesterol and triglyceride levels, increased hepatic triglyceride content, and higher hepatic levels of 9-HODE and 13-HODE, as compared to control mice exposed to filtered air. A direct effect of DE exposure on hepatocytes was demonstrated by treatment of HepG2 cells with a methanol extract of DE particles followed by loading with oleic acid. As observed in vivo, this led to increased triglyceride content and significant downregulation of ACAD9 mRNA expression. Treatment of HepG2 cells with DE particles and oleic acid did not alter de novo lipogenesis but inhibited total, mitochondrial, and ATP-linked oxygen consumption rate, indicative of mitochondrial dysfunction. Treatment of isolated mitochondria, prepared from mouse liver, with DE particles and oleic acid also inhibited mitochondrial complex activity and β-oxidation., Conclusions: DE exposure leads to dyslipidemia and liver steatosis in ApoE knockout mice, likely due to mitochondrial dysfunction and decreased lipid catabolism.

In the United States, laboratories frequently offer multiple different assays for testing of cerebrospinal fluid (CSF) samples to provide laboratory support for the diagnosis of central nervous system Lyme disease (CNSLD). Often included among these diagnostic tests are the same enzyme immunoassays and immunoblots that are routinely used to detect the presence of antibodies to Borrelia burgdorferi in serum. However, performing these assays on CSF alone may yield positive results simply from passive diffusion of serum antibodies into the CSF. In addition, such tests are only U.S. Food and Drug Administration cleared and well validated for testing serum, not CSF. When performed using CSF, positive results from these assays do not establish the presence of intrathecal antibody production to B. burgdorferi and therefore should not be offered. The preferred test to detect intrathecal production of antibodies to B. burgdorferi is the antibody index assay, which corrects for passive diffusion of serum antibodies into CSF and requires testing of paired serum and CSF collected at approximately the same time. However, this assay also has limitations and should only be used to establish a diagnosis of CNSLD in conjunction with patient exposure history, clinical presentation, and other laboratory findings., (Copyright © 2019 American Society for Microbiology.)

Macrophages are prominent cells in acute and chronic inflammatory diseases. Recent studies highlight a role for macrophage proliferation post-monocyte recruitment under inflammatory conditions. Using an acute peritonitis model, we identify a significant defect in macrophage proliferation in mice lacking the leukocyte transmembrane protease ADAM17. The defect is associated with decreased levels of macrophage colony-stimulating factor 1 (CSF-1) in the peritoneum and is rescued by intraperitoneal injection of CSF-1. Cell surface CSF-1 (csCSF-1) is one of the substrates of ADAM17. We demonstrate that both infiltrated neutrophils and macrophages are major sources of csCSF-1. Furthermore, acute shedding of csCSF-1 following neutrophil extravasation is associated with elevated expression of iRhom2, a member of the rhomboid-like superfamily, which promotes ADAM17 maturation and trafficking to the neutrophil surface. Accordingly, deletion of hematopoietic iRhom2 is sufficient to prevent csCSF-1 release from neutrophils and macrophages and to prevent macrophage proliferation. In acute inflammation, csCSF-1 release and macrophage proliferation are self-limiting due to transient leukocyte recruitment and temporally restricted csCSF-1 expression. In chronic inflammation, such as atherosclerosis, the ADAM17-mediated lesional macrophage proliferative response is prolonged. Our results demonstrate a novel mechanism whereby ADAM17 promotes macrophage proliferation in states of acute and chronic inflammation., (Copyright © 2018 American Society for Microbiology.)

Animal studies are a foundation for defining mechanisms of atherosclerosis and potential targets of drugs to prevent lesion development or reverse the disease. In the current literature, it is common to see contradictions of outcomes in animal studies from different research groups, leading to the paucity of extrapolations of experimental findings into understanding the human disease. The purpose of this statement is to provide guidelines for development and execution of experimental design and interpretation in animal studies. Recommendations include the following: (1) animal model selection, with commentary on the fidelity of mimicking facets of the human disease; (2) experimental design and its impact on the interpretation of data; and (3) standard methods to enhance accuracy of measurements and characterization of atherosclerotic lesions., Competing Interests: The American Heart Association makes every effort to avoid any actual or potential conflicts of interest that may arise as a result of an outside relationship or a personal, professional, or business interest of a member of the writing panel. Specifically, all members of the writing group are required to complete and submit a Disclosure Questionnaire showing all such relationships that might be perceived as real or potential conflicts of interest., (© 2017 American Heart Association, Inc.)

The extended-spectrum-β-lactamase (ESBL)- and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae represent serious and urgent threats to public health. In a retrospective study of multidrug-resistant K. pneumoniae , we identified three clinical isolates, CN1, CR14, and NY9, carrying both bla CTX-M and bla KPC genes. The complete genomes of these three K. pneumoniae isolates were de novo assembled by using both short- and long-read whole-genome sequencing. In CR14 and NY9, bla CTX-M and bla KPC were carried on two different plasmids. In contrast, CN1 had one copy of bla KPC-2 and three copies of bla CTX-M-15 integrated in the chromosome, for which the bla CTX-M-15 genes were linked to an insertion sequence, IS Ecp1 , whereas the bla KPC-2 gene was in the context of a Tn 4401a transposition unit conjugated with a PsP3-like prophage. Intriguingly, downstream of the Tn 4401a-bla KPC-2 -prophage genomic island, CN1 also carried a clustered regularly interspaced short palindromic repeat (CRISPR)- cas array with four spacers targeting a variety of K. pneumoniae plasmids harboring antimicrobial resistance genes. Comparative genomic analysis revealed that there were two subtypes of type I-E CRISPR- cas in K. pneumoniae strains and suggested that the evolving CRISPR- cas , with its acquired novel spacer, induced the mobilization of antimicrobial resistance genes from plasmids into the chromosome. The integration and dissemination of multiple copies of bla CTX-M and bla KPC from plasmids to chromosome depicts the complex pandemic scenario of multidrug-resistant K. pneumoniae Additionally, the implications from this study also raise concerns for the application of a CRISPR- cas strategy against antimicrobial resistance., (Copyright © 2017 American Society for Microbiology.)

Glutathione (GSH) is depleted early in the course of Parkinson's disease (PD), and deficiency has been shown to perpetuate oxidative stress, mitochondrial dysfunction, impaired autophagy, and cell death. GSH repletion has been proposed as a therapeutic intervention. The objective of this study was to evaluate whether intranasally administered reduced GSH, (in)GSH, is capable of augmenting central nervous system GSH concentrations, as determined by magnetic resonance spectroscopy in 15 participants with mid-stage PD. After baseline GSH measurement, 200 mg (in)GSH was self-administered inside the scanner without repositioning, then serial GSH levels were obtained over ~1 h. Statistical significance was determined by one-way repeated measures analysis of variance. Overall, (in)GSH increased brain GSH relative to baseline ( P <0.001). There was no increase in GSH 8 min after administration, although it was significantly higher than baseline at all of the remaining time points ( P <0.01). This study is the first to demonstrate that intranasal administration of GSH elevates brain GSH levels. This increase persists at least 1 h in subjects with PD. Further dose-response and steady-state administration studies will be required to optimize the dosing schedule for future trials to evaluate therapeutic efficacy., Competing Interests: The authors declare no conflict of interest.

Chlamydia pneumoniae (Cpn), a common respiratory pathogen of humans, is associated with human cardiovascular disease and the acceleration of atherosclerosis in hyperlipidemic animal models. Our laboratory has demonstrated that murine norovirus (MNV), a prevalent infection of laboratory mice, can unpredictably alter atherosclerosis in hyperlipidemic Ldlr(-/-) and ApoE(-/-) mice. Given that MNV has a tropism for macrophages and may exacerbate atherogenesis, we investigated whether coinfection with MNV and Cpn might alter macrophage phenotypes in vitro and atherosclerosis in ApoE(-/-) mice. In the presence of oxidized low-density lipoprotein, coinfection of ApoE(-/-) bone marrow-derived macrophages (BMDM) with MNV and Cpn resulted in significant increases in gene expression of IL6, MCP1, iNOS, and TNFα compared with Cpn-monoinfected BMDM. On the basis of these findings, we hypothesized that concurrent MNV-Cpn infection might increase plaque lesion size in vivo. As expected, Cpn monoinfection of ApoE(-/-) mice increased mean plaque size by 62% compared with that in uninfected mice. However, MNV did not significantly alter plaque lesion size in MNV-Cpn-coinfected mice compared with Cpn-monoinfected mice. There were no differences in aortic cytokines locally at the site of plaque development or in peritoneal macrophages at 1 wk after infection in MNV-Cpn-coinfected mice compared with Cpn-monoinfected mice. MNV was not detected in the aortic tissue of MNV-infected mice at 1 or 8 wk after infection regardless of Cpn status. These data suggest that MNV infection does not appreciably alter plaque development in Cpn-accelerated atherosclerosis in ApoE(-/-) mice.

Objectives . Oxidative stress contributes to Parkinson's disease (PD) pathophysiology and progression. The objective was to describe central and peripheral metabolites of redox metabolism and to describe correlations between glutathione (Glu) status, age, and disease severity. Methods . 58 otherwise healthy individuals with PD were examined during a single study visit. Descriptive statistics and scatterplots were used to evaluate normality and distribution of this cross-sectional sample. Blood tests and magnetic resonance spectroscopy (MRS) were used to collect biologic data. Spearman's rank-order correlation coefficients were used to evaluate the strength and direction of the association. The Unified PD Rating Scale (UPDRS) and the Patient-Reported Outcomes in PD (PRO-PD) were used to rate disease severity using regression analysis. Results . Blood measures of Glu decreased with age, although there was no age-related decline in MRS Glu. The lower the blood Glu concentration, the more severe the UPDRS ( P = 0.02, 95% CI: -13.96, -1.14) and the PRO-PD ( P = 0.01, 95% CI: -0.83, -0.11) scores. Discussion . These data suggest whole blood Glu may have utility as a biomarker in PD. Future studies should evaluate whether it is a modifiable risk factor for PD progression and whether Glu fortification improves PD outcomes.

Two hallmarks of advanced atherosclerosis are calcification and fibrosis. We hypothesized that Chlamydia pneumoniae infection may contribute to atherosclerosis by inducing the conversion of vascular smooth muscle cells to calcifying cells or by converting mesenchymal stem cells to osteochondrocytic or fibroblastic phenotypes. In this study, direct infection of bovine aortic smooth muscle cells (BSMCs) did not induce the expression of alkaline phosphatase or the deposition of extracellular calcium phosphate. However, conditioned media from C. pneumoniae-infected macrophages accelerated conversion of BSMCs to a calcifying phenotype. Treatment of the conditioned media with an anti-TNF-alpha blocking antibody abrogated this stimulatory effect. Treatment of perivascular Sca-1+, CD31-, CD45- cells from apoE-/- mouse aortas with the conditioned media from infected macrophages induced the Sca-1+ cells to produce collagen II, an additional marker of an osteochondrocytic phenotype. Treatment of mouse coronary perivascular Sca-1+, CD31-, CD45- cells with the supernatant from homogenates of C. pneumoniae-infected mouse lungs as compared to noninfected lungs induced expression of the Collagen 1α1 gene and deposition of collagen. Therefore, an increase in plasma cytokines or other factors in response to respiratory infection with C. pneumoniae or infection of macrophages within the blood vessel could contribute to both calcification and fibrosis of advanced atherosclerotic lesions., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Aims: The goal of this study was to determine whether the A1 adenosine receptor (AR) plays a role in atherosclerosis development and to explore its potential mechanisms., Methods and Results: Double knockout (DKO) mice, deficient in the genes encoding A1 AR and apolipoprotein E (apoE), demonstrated reduced atherosclerotic lesions in aortic arch (en face), aortic root, and innominate arteries when compared with apoE-deficient mice (APOE-KO) of the same age. Treating APOE-KO with an A1 AR antagonist (DPCPX) also led to a concentration-dependent reduction in lesions. The total plasma cholesterol and triglyceride levels were not different between DKO and APOE-KO; however, higher triglyceride was observed in DKO fed a high-fat diet. DKO also had higher body weights than APOE-KO. Plasma cytokine concentrations (IL-5, IL-6, and IL-13) were significantly lower in DKO. Proliferating cell nuclear antigen expression was also significantly reduced in the aorta from DKO. Despite smaller lesions in DKO, the composition of the innominate artery lesion and cholesterol loading and efflux from bone marrow-derived macrophages of DKO were not different from APOE-KO., Conclusion: The A1 AR may play a role in the development of atherosclerosis, possibly due to its pro-inflammatory and mitogenic properties.

Objective: Osteoprotegerin (OPG) is a decoy receptor for the osteoclast differentiation factor receptor activator of NF-κB ligand. OPG regulates bone homeostasis, and its inactivation in mice results in severe osteoporosis. OPG deficiency in apolipoprotein E (ApoE)(-/-) mice results in increased atherosclerotic lesion size and calcification. Furthermore, receptor activator of NF-κB ligand enhances macrophage-dependent smooth muscle cell calcification in vitro. Here, we hypothesized that reconstitution of ApoE(-/-)OPG(-/-) mice with ApoE(-/-)OPG(+/+) bone marrow (BM) would be sufficient to rescue lesion progression and vascular calcification. Conversely, reconstitution of ApoE(-/-)OPG(+/+) mice with ApoE(-/-)OPG(-/-) BM may accelerate lesion progression and vascular calcification., Approach and Results: ApoE(-/-)OPG(-/-) mice transplanted with ApoE(-/-)OPG(+/+) BM developed smaller atherosclerotic lesions and deposited less calcium in the innominate artery than that of ApoE(-/-)OPG(-/-) mice transplanted with ApoE(-/-)OPG(-/-) BM. There were no differences in lesion size and calcification in ApoE(-/-)OPG(+/+) mice transplanted with BM from ApoE(-/-)OPG(-/-) or ApoE(-/-)OPG(+/+) mice. The large lesions observed in the ApoE(-/-)OPG(-/-) mice transplanted with OPG(-/-) BM were rich in chondrocyte-like cells, collagen, and proteoglycans. Importantly, the ApoE(-/-)OPG(-/-) mice transplanted with OPG(+/+) BM remained osteoporotic, and the ApoE(-/-)OPG(+/+) mice did not show signs of bone loss regardless of the type of BM received. In coculture experiments, macrophages and mesenchymal stem cells derived from ApoE(-/-)OPG(-/-) BM induced more vascular smooth muscle cell calcification than cells derived from ApoE(-/-)OPG(+/+) mice., Conclusions: These results indicate that OPG derived either from the BM or from the vessel wall is sufficient to slow down lesion progression and vascular calcification independent of bone turnover.

Background: Strong associations have been observed between exposure to fine ambient particulate matter (PM2.5) and adverse cardiovascular outcomes. In particular, exposure to traffic related PM2.5 has been associated with increases in left ventricular hypertrophy, a strong risk factor for cardiovascular mortality. As much of traffic related PM2.5 is derived from diesel exhaust (DE), we investigated the effects of chronic DE exposure on cardiac hypertrophy and heart failure in the adult mouse by exposing mice to DE combined with either of two mouse models of cardiac hypertrophy: angiotensin II infusion or pressure overload induced by transverse aortic banding., Methods: Wild type male C57BL/6 J mice were either infused with angiotensin II (800 ng/kg/min) via osmotic minipump implanted subcutaneously for 1 month, or underwent transverse aortic banding (27 gauge needle 1 week for observing acute reactions, 26 gauge needle 3 months or 6 months for observing chronic reactions). Vehicle (saline) infusion or sham surgery was used as a control. Shortly after surgery, mice were transferred to our exposure facility and randomly assigned to either diesel exhaust (300 or 400 μg/m(3)) or filtered air exposures. After reaching the end of designated time points, echocardiography was performed to measure heart structure and function. Gravimetric analysis was used to measure the ventricular weight to body weight ratio. We also measured heart rate by telemetry using implanted ambulatory ECG monitors., Results: Both angiotensin II and transverse aortic banding promoted cardiac hypertrophy compared to vehicle or sham controls. Transverse aortic banding for six months also promoted heart failure in addition to cardiac hypertrophy. In all cases, DE failed to exacerbate the development of hypertrophy or heart failure when compared to filtered air controls. Prolonged DE exposure also led to a decrease in average heart rate., Conclusions: Up to 6-months of DE exposure had no effect on cardiac hypertrophy and heart function induced by angiotensin II stimulation or pressure overload in adult C57BL/6 J mice. This study highlights the potential importance of particle constituents of ambient PM2.5 to elicit cardiotoxic effects. Further investigations on particle constituents and cardiotoxicity are warranted.

Several lines of evidence have associated Chlamydia pneumoniae with cardiovascular disease including acceleration of atherosclerotic lesion progression in hyperlipidemic animal models by infection. Many of the pro-atherogenic effects of oxidized low-density lipoprotein (ox-LDL) occur through the activation of the lectin-like ox-LDL receptor-1 (LOX-1). Chlamydia pneumoniae upregulates the expression of the LOX-1 mRNA, promotes the uptake of ox-LDL, and utilizes the LOX-1 receptor for infectivity. The overall goal of this study was to determine whether C. pneumoniae organisms upregulated LOX-1 protein expression in vascular cells and whether upregulation of pro-atherogenic factors by C. pneumoniae occurred through LOX-1. Chlamydia pneumoniae induced LOX-1 protein expression in both endothelial cells and RAW macrophages. Upregulation was prevented by preincubation of cells with LOX-1 antibody prior to infection. Similarly, C. pneumoniae upregulated protein expression of adhesion molecules, MMP-1, and MMP-3, which was mitigated by anti-LOX-1 antibody. Prior treatment of organisms with PNGase, which removes the chlamydial glycan that is N-linked to the major outer membrane, abolished C. pneumoniae upregulation of LOX-1. These studies suggest that activation of LOX-1 expression occurs through binding of the chlamydial glycan and provides one mechanism by which C. pneumoniae infection could play a role in the pathogenesis of atherosclerosis., Competing Interests: The authors have no conflicts of interest to declare., (© 2013 Federation of European Microbiological Societies.)

Objective: To evaluate whether exposure to air pollutants induces oxidative modifications of plasma lipoproteins, resulting in alteration of the protective capacities of high-density lipoproteins (HDLs)., Approach and Results: We exposed apolipoprotein E-deficient mice to diesel exhaust (DE) at ≈ 250 µg/m(3) for 2 weeks, filtered air (FA) for 2 weeks, or DE for 2 weeks, followed by FA for 1 week (DE+FA). DE led to enhanced lipid peroxidation in the brochoalveolar lavage fluid that was accompanied by effects on HDL functionality. HDL antioxidant capacity was assessed by an assay that evaluated the ability of HDL to inhibit low-density lipoprotein oxidation estimated by 2',7'-dichlorofluorescein fluorescence. HDL from DE-exposed mice exhibited 23,053 ± 2844 relative fluorescence units, higher than FA-exposed mice (10,282 ± 1135 relative fluorescence units, P<0.001) but similar to the HDL from DE+FA-exposed mice (22,448 ± 3115 relative fluorescence units). DE effects on HDL antioxidant capacity were negatively correlated with paraoxonase enzymatic activity, but positively correlated with levels of plasma 8-isoprostanes, 12-hydroxyeicosatetraenoic acid, 13-hydroxyoctadecadienoic acid, liver malondialdehyde, and accompanied by perturbed HDL anti-inflammatory capacity and activation of the 5-lipoxygenase pathway in the liver., Conclusions: DE emissions induced systemic pro-oxidant effects that led to the development of dysfunctional HDL. This may be one of the mechanisms by which air pollution contributes to enhanced atherosclerosis.

Lyme disease is transmitted by the bite of certain Ixodes ticks, which can also transmit Anaplasma phagocytophilum, the cause of human granulocytic anaplasmosis (HGA). Although culture can be used to identify patients infected with A. phagocytophilum and is the microbiologic gold standard, few studies have evaluated culture-confirmed patients with HGA. We conducted a prospective study in which blood culture was used to detect HGA infection in patients with a compatible clinical illness. Early Lyme disease was defined by the presence of erythema migrans. The epidemiologic, clinical, and laboratory features of 44 patients with culture-confirmed HGA were compared with those of a convenience sample of 62 patients with early Lyme disease. Coinfected patients were excluded. Patients with HGA had more symptoms (P = 0.003) and had a higher body temperature on presentation (P < 0.001) than patients with early Lyme disease. HGA patients were also more likely to have a headache, dizziness, myalgias, abdominal pain, anorexia, leukopenia, lymphopenia, thrombocytopenia, or elevated liver enzymes. A direct correlation between the number of symptoms and the duration of illness at time of presentation (rho = 0.389, P = 0.009) was observed for HGA patients but not for patients with Lyme disease. In conclusion, although there are overlapping features, culture-confirmed HGA is a more severe illness than early Lyme disease.

The association of Chlamydia pneumoniae and atherosclerosis has been well documented. Recently, it has been demonstrated that C. pneumoniae up-regulates expression of the lectin-like ox-LDL receptor (LOX-1) in endothelial cells. Many of the pro-atherogenic effects of ox-LDL occur through its activation and uptake by LOX-1. This class E scavenger receptor contains a carbohydrate-recognition domain common to the C type lectin family. Previously, we have demonstrated that the major outer membrane protein of the chlamydiae is glycosylated and glycan removal abrogates infectivity of C. pneumoniae for endothelial cells. In this study, we investigated whether C. pneumoniae binds to LOX-1. The results show that 1) infection of endothelial cells by C. pneumoniae is inhibited by ligands that bind to the LOX-1 receptor, but not by ligands binding to other scavenger receptors; 2) anti-LOX-1 antibody inhibits C. pneumoniae infectivity, while antibodies against other scavenger receptors do not; 3) anti-LOX-1 antibody inhibits attachment of C. pneumoniae to endothelial cells; and 4) C. pneumoniae co-localizes with LOX-1. These effects were not observed for Chlamydia trachomatis. In conclusion, C. pneumoniae binds to the LOX-1 receptor, which is known to promote atherosclerosis., (Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Objective: Glutamate-cysteine ligase (GCL) is the rate-limiting step in glutathione synthesis. The enzyme is a heterodimer composed of a catalytic subunit, GCLC, and a modifier subunit, GCLM. We generated apolipoprotein E (apoE)-/- mice deficient in GCLM (apoE-/-/Gclm-/-) and transgenic mice that overexpress GCLC specifically in macrophages (apoE-/-/Gclc-Tg) to test the hypothesis that significantly altering the availability of glutathione has a measurable impact on both the initiation and progression of atherosclerosis., Methods and Results: Atherosclerotic plaque size and composition were measured in the innominate artery in chow-fed male and female mice at 20, 30, 40, and 50 weeks of age and in the aortic sinus at 40 and 50 weeks of age. The apoE-/-/Gclm-/- mice more rapidly developed complex lesions, whereas the apoE-/-/Gclc-Tg mice had reduced lesion development compared with the littermate apoE-/- control mice. Transplantation of bone marrow from the apoE-/-/Gclm-/- and apoE-/-/Gclc-Tg mice into apoE-/- mice with established lesions also stimulated or inhibited further lesion development at 30 weeks posttransplant., Conclusion: Gain and loss of function in the capacity to synthesize glutathione especially in macrophages has reciprocal effects on the initiation and progression of atherosclerosis at multiple sites in apoE-/- mice.

Rationale: The bone morphogenetic proteins (BMPs), a family of morphogens, have been implicated as mediators of calcification and inflammation in the vascular wall., Objective: To investigate the effect of altered expression of matrix Gla protein (MGP), an inhibitor of BMP, on vascular disease., Methods and Results: We used MGP transgenic or MGP-deficient mice bred to apolipoprotein E mice, a model of atherosclerosis. MGP overexpression reduced vascular BMP activity, atherosclerotic lesion size, intimal and medial calcification, and inflammation. It also reduced expression of the activin-like kinase receptor 1 and the vascular endothelial growth factor, part of a BMP-activated pathway that regulates angiogenesis and may enhance lesion formation and calcification. Conversely, MGP deficiency increased BMP activity, which may explain the diffuse calcification of vascular medial cells in MGP deficient aortas and the increase in expression of activin-like kinase receptor 1 and vascular endothelial growth factor. Unexpectedly, atherosclerotic lesion formation was decreased in MGP-deficient mice, which may be explained by a dramatic reduction in expression of endothelial adhesion molecules limiting monocyte infiltration of the artery wall., Conclusions: Our results indicate that BMP signaling is a key regulator of vascular disease, requiring careful control to maintain normal vascular homeostasis.

The acute phase response to Chlamydia pneumoniae infection was analyzed over a 72 h period post-infection in C57BL/6J mice. A single intra-nasal inoculation stimulated statistically significant increases in the plasma levels of IL-2, IL-5, IL-6, IL-10, IL-12, GM-CSF, IFN-gamma, and serum amyloid A but not TNF-alpha, IL-1beta, IL-4 and serum amyloid P. There was also a decrease in the activity of the HDL protective enzyme paraoxonase as well as a reduced ability of HDL to prevent oxidation of palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine by hydroperoxyoctadecadienoic acid at 48 and 72 h post-infection. To determine whether the C. pneumoniae induced acute phase response had any effect on atherosclerotic plaque stability, we measured the frequency of intra-plaque hemorrhage as a marker of plaque disruption in the innominate arteries of apolipoprotein E deficient mice at 29-30 weeks and 1.5-2.0 years of age. There was an increased frequency of intra-plaque hemorrhage only in the older mice infected with the live organism (8/14) as compared to mice treated with killed C. pneumoniae (2/11) or sham inoculated with PBS (2/12). These results suggest that acute phase reactant proteins produced in response to pulmonary infection with C. pneumoniae may contribute to the progression and destabilization of atherosclerotic lesions., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

Aims: Early growth response gene-1 (Egr-1) regulates the expression of genes important to cardiovascular disease. Within atherosclerotic lesions, Egr-1 is expressed in smooth muscle cells, endothelial cells, and macrophages. Since macrophages play a pivotal role in atherosclerotic lesion initiation and progression, this study investigated the effects of Egr-1 deficiency within bone marrow-derived cells on the development of atherosclerosis in a hyperlipidaemic mouse model., Methods and Results: Bone marrow from Egr-1-deficient mice and wild-type controls was transplanted into lethally irradiated LDL receptor null mice. After 26 weeks on a high fat diet, atherosclerotic lesion size within the aortic sinus of recipients was evaluated. Mice receiving Egr-1-deficient bone marrow had significantly decreased lesion size compared with controls. Lesions of these mice contained fewer macrophages and had reduced expression of vascular cell adhesion molecule-1 (VCAM-1), tissue factor, as well as transforming growth factor receptor type II, which are target genes of Egr-1. These results were validated by in vitro analysis of Egr-1-deficient peritoneal macrophages which, after lipopolysaccharide stimulation, had decreased VCAM-1 and tissue factor mRNA expression compared with wild-type controls., Conclusion: This study demonstrates that bone marrow-derived Egr-1 promotes macrophage accumulation, atherosclerotic lesion development, and lesion complexity.

Since the year 2000, linezolid has been used in the United States to treat infections caused by antimicrobial-resistant Gram-positive cocci. At present, linezolid-resistant (Linr) Staphylococcus aureus and Staphylococcus epidermidis strains are rare and the diversity of their genetic backgrounds is unknown. We performed sequence-based strain typing and resistance gene characterization of 46 Linr isolates that were collected from local and national sources between the years 2004 and 2007. Resistance was found to occur in at least three clonal complexes (CCs; lineages) of S. aureus and in at least four subclusters of a predominant, phylogenetically unstable CC of S. epidermidis. New candidate resistance mutations in 23S rRNA and the L4 riboprotein were identified among the S. epidermidis isolates. These findings suggest that linezolid resistance has emerged independently in multiple clones of S. aureus and with a variety of ribosomal mutations in multiple clones of S. epidermidis.

Background: Standard 2-tiered immunoglobulin G (IgG) testing has performed well in late Lyme disease (LD), but IgM testing early in the illness has been problematic. IgG VlsE antibody testing, by itself, improves early sensitivity, but may lower specificity. We studied whether elements of the 2 approaches could be combined to produce a second-tier IgG blot that performs well throughout the infection., Methods: Separate serum sets from LD patients and control subjects were tested independently at 2 medical centers using whole-cell enzyme immunoassays and IgM and IgG immunoblots, with recombinant VlsE added to the IgG blots. The results from both centers were combined, and a new second-tier IgG algorithm was developed., Results: With standard 2-tiered IgM and IgG testing, 31% of patients with active erythema migrans (stage 1), 63% of those with acute neuroborreliosis or carditis (stage 2), and 100% of those with arthritis or late neurologic involvement (stage 3) had positive results. Using new IgG criteria, in which only the VlsE band was scored as a second-tier test among patients with early LD (stage 1 or 2) and 5 of 11 IgG bands were required in those with stage 3 LD, 34% of patients with stage 1, 96% of those with stage 2, and 100% of those with stage 3 infection had positive responses. Both new and standard testing achieved 100% specificity., Conclusions: Compared with standard IgM and IgG testing, the new IgG algorithm (with VlsE band) eliminates the need for IgM testing; it provides comparable or better sensitivity, and it maintains high specificity.

It is documented that hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis, but whether elevated plasma homocysteine contributes to the progression of atherosclerosis in aged animals with hypercholesterolemia is still unknown. HHcy was induced in apolipoprotein E (ApoE) knockout mice (male, 32 weeks old) by feeding 2% methionine/low folate (1 mg/kg) diet for 20 weeks. HHcy induced by methionine feeding significantly increased oxidative stress, as measured by thiobarbituric-reactive substances in livers (P < .05) and genetic expression of Cu,Zn-superoxide dismutase, in methionine-fed animals compared with controls (P < .05). Furthermore, lipoprotein profiles were changed, in that low-density lipoprotein-cholesterol was shifted to very low-density lipoprotein in the methionine-supplemented group. However, nuclear factor kappaB activity, atherosclerotic lesions, hepatic glutathione level, lipid profiles, and activities of aspartate aminotransferase and alanine aminotransferase were not significantly different. These findings suggest that HHcy induced by methionine may promote disturbances in lipid peroxidation and modify lipoprotein metabolism but not contribute to the progression of atherosclerotic lesion in aged ApoE knockout mice.

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.

Objective: Macrophage-derived products are known to play a crucial role during atherogenesis and vascular calcification. Glucocorticoids (GC) are important modulators of immune cell functions, but their specific effects on macrophages behavior during plaque formation are not defined. The present study was therefore designed to investigate the effects of macrophage-specific deletion of the glucocorticoid receptor (GR(LysMCre)) on atherogenesis and vascular calcification in a hyperlipidemic mouse-model., Methods and Results: Bone marrow was isolated from GR(LysMCre) mice and wild-type controls (GR(flox)) and subsequently transplanted into lethally irradiated LDL-receptor-deficient mice. Animals were fed a Western-type diet for 15 or 24 weeks, and atherosclerotic lesions within the aortic sinus were evaluated. At both time points, no significant difference in serum lipid and corticosterone concentrations, atherosclerotic lesion size and macrophage-content within the lesions could be observed. However, GR(LysMCre) mice showed less calcification as well as a significant reduction of RANKL, BMP2, and Msx2 expression within the vasculature. In vitro studies using conditioned media from macrophages which had been stimulated with dexamethasone demonstrated a dose-dependent increase in calcium deposition by vascular smooth muscle cells., Conclusions: This study demonstrates that macrophage-specific glucocorticoid receptor inactivation reduces vascular calcification without affecting atherosclerotic lesion size in LDL receptor-deficient mice.

Chlamydia pneumoniae, a common respiratory pathogen, has been associated with cardiovascular disease. C. pneumoniae infection accelerates atherosclerotic lesion development in hyperlipidemic animals. Retinoic acid, an anti-oxidant, inhibits infection of endothelial cells by C. pneumoniae. The present study demonstrated that retinoic acid suppresses the acceleration of foam cell lesion development induced by C. pneumoniae in hyperlipidemic C57BL/6J mice. Retinoic acid treatment had no effect on foam cell lesion development in uninfected animals. Lung infection and duration was decreased in treated mice, suggesting one mechanism by which retinoic acid reduces C. pneumoniae-accelerated foam cell lesion formation in hyperlipidemic mice.

Erythema migrans, the most common manifestation of Lyme disease, has been associated with highly variable rates of seropositivity for antibodies to Borrelia burgdorferi. Differences in the sensitivities of serologic assays for the detection of these antibodies, however, may not be the only or even the primary explanation for this observation. We investigated the impacts of four clinical variables on seropositivity--the duration of erythema migrans, the presence of single versus multiple skin lesions, and the gender and age of the patient. In this analysis, three different serologic tests were performed on acute-phase sera from 175 untreated patients with culture-confirmed erythema migrans: the C6 single-peptide enzyme-linked immunosorbent assay (ELISA), a commercially available ELISA in which a whole-cell sonicate of B. burgdorferi was the antigen, and a two-tier procedure. Irrespective of the serologic test performed, the results showed that seropositivity rates increased with the duration of the erythema migrans for patients with single lesions (P < 0.001) but not for those with multiple skin lesions. The variability in seropositivity rates was greatest for the two-tier testing strategy, with a >6-fold-higher rate of seropositivity among patients with a single lesion of 22- to 30-day duration than among those whose skin lesion was of 1- to 7-day duration (85.7 versus 14.1%; P < 0.001). Rates of seropositivity by each of the testing methods were also significantly higher for patients with multiple skin lesions than for those with single lesions (P < 0.001). In contrast, seropositivity rates were not affected by either the gender or the age of the patient. Thus, in patients with erythema migrans, certain clinical variables such as the duration and number of skin lesions had a profound impact on seropositivity rates, irrespective of the serologic assay performed.