Circulating Bacterial-Derived DNA Fragment Level Is a Strong Predictor of Cardiovascular Disease in Peritoneal Dialysis Patients

BackgroundCirculating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients.MethodsWe measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival.ResultsThe average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 – 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005).ConclusionsCirculating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients.     

Elevated Circulating Interleukin 33 Levels in Stable Renal Transplant Recipients at High Risk for Cardiovascular Events

Background

The Major Adverse Cardiovascular Events calculator (CRCRTR-MACE) estimates the burden of cardiovascular risk in renal transplant recipients (RTR). Our recent study of 95 RTR reported the 7-year median risk of cardiovascular events (CVE) to be 9.97%, ranging from 1.93 to 84.27%. Nearly a third (28.4%) of the cohort was above 20% risk for a CVE. Since interleukins (ILs) as part of the inflammatory response may play a role in the pathogenesis of cardiovascular disease (CVD), we extended this study to identify which ILs are associated with high cardiovascular risk in this population.

Methods

Twenty-two ILs were measured by multiplexed fluorescent bead-based immunoassay in 95 RTR and 56 normal controls. Stepwise analysis after multivariate determination of significant demographic and inflammatory variables was performed between the high and low-CVD risk groups (which were arbitrarily set at scores <10% and ≥20%, respectively). Normalized data was presented as mean ± SD and non-normalized data as median (minimum–maximum). Significance was measured at <0.05.

Results

27.5% of the low-risk and 31.3% of the high-risk groups had mean IL levels above the 95 percentile of the normal control levels. In the non-parametric analysis IL-6, 9, 16, 17 and 33 were significantly higher in the high-risk group compared to the control. Univariate analysis (UVA) of the high-risk group identified IL-33 as the only IL that remained significantly higher than the control and low-risk groups (p = 0.000). The percentage of patients with IL-33 levels above the 90 percentile of control value in the low and high-risk groups were 15.6% and 52.0%, respectively (p<0.002). UVA of factors significant to high IL-33 levels included estimated glomerular filtration rate (eGFR), while diabetes mellitus, serum phosphorus, microalbuminuria and age also remained significant in the multivariate analysis.

Conclusion

Circulating IL-33 level is positively associated with high CRCRTR-MACE score. Diminished eGFR, age, diabetes, serum phosphorus and microalbuminurea demonstrate significant relationship with elevated IL-33 levels, supporting the possible pathognomonic role of IL-33 in the cardiovascular burden in RTR.     

Elevated Nerve Growth Factor Levels in the Synovial Fluid of Patients with Inflammatory Joint Disease

A novel pH shock extraction procedure was used to measure nerve growth factor (NGF) levels in both normal and inflamed synovial fluids using a sensitive and specific two-site enzyme linked immunosorbant assay. To date no data is available on NGF levels in normal synovial fluids. Synovial fluids were taken from 5 normal volunteers, 12 patients with rheumatoid arthritis and 10 patients with other inflammatory arthropathies. The mean ± SEM NGF concentration in normal synovial fluids was 95 ± 33.2 pg/ml (range 39.1–143.1 pg/ml), whereas the mean NGF concentration in the synovial fluids taken from patients with rheumatoid arthritis was 532.5 ± 123.8 pg/ml (range 152–1686 pg/ml). The mean NGF concentration in patients with other inflammatory arthropathies was also raised (430.6 ± 90 pg/ml; range 89–1071 pg/ml). The NGF concentrations were significantly higher in the synovial fluids from both inflamed groups (ANOVA p < 0.05) compared to normals. Raised levels of NGF in synovial fluid may contribute directly to joint inflammation via activation of inflammatory cells.     

Circulating Heat Shock Protein 60 Levels Are Elevated in HIV Patients and Are Reduced by Anti-Retroviral Therapy

Circulating heat shock protein 60 (Hsp60) and heat shock protein 10 (Hsp10) have been associated with pro- and anti-inflammatory activity, respectively. To determine whether these heat shock proteins might be associated with the immune activation seen in HIV-infected patients, the plasma levels of Hsp60 and Hsp10 were determined in a cohort of 20 HIV-infected patients before and after effective combination anti-retroviral therapy (cART). We show for the first time that circulating Hsp60 levels are elevated in HIV-infected patients, with levels significantly reduced after cART, but still higher than those in HIV-negative individuals. Hsp60 levels correlated significantly with viral load, CD4 counts, and circulating soluble CD14 and lipopolysaccharide levels. No differences or correlations were seen for Hsp10 levels. Elevated circulating Hsp60 may contribute to the immune dysfunction and non-AIDS clinical events seen in HIV-infected patients.     

DNA Methylation Profiling for the Diagnosis and Prognosis of Patients with Nontuberculous Mycobacterium Lung Disease

The incidence of nontuberculous Mycobacterium (NTM) lung disease is rapidly increasing; however, its diagnosis and prognosis remain unclear while selecting patients who will respond to appropriate treatment. Differences in DNA methylation patterns between NTM patients with good or poor prognosis could provide important therapeutic targets. We used the Illumina MethylationEPIC (850k) DNA methylation microarray to determine the pattern between differentially methylated regions (DMRs) in NTM patients with good or poor prognosis (n = 4/group). Moreover, we merged and compared 20 healthy controls from previous Illumina Methylation450k DNA methylation microarray data. We selected and visualized the DMRs in the form of heatmaps, and enriched terms associated with these DMRs were identified by functional annotation with the “pathfinder” package. In total, 461 and 293 DMRs (|Log2 fold change| > 0.1 and P < 0.03) were more methylated in patients with four poor and four good prognoses, respectively. Furthermore, 337 and 771 DMRs (|Log2 fold change| > 0.08 and P < 0.001) were more methylated in eight NTM patients and 20 healthy controls, respectively. TGFBr1 was significantly less methylated, whereas HLA-DR1 and HLA-DR5 were more methylated in patients with poor prognosis (compared to those with good prognosis). LRP5, E2F1, and ADCY3 were the top three less-methylated genes in NTM patients (compared with the controls). The mTOR and Wnt signaling pathway-related genes were less methylated in patients with NTM. Collectively, genes related to Th1- cell differentiation, such as TGFBr1 and HLA-DR, may be used as biomarkers for predicting the treatment response in patients with NTM lung disease.     

Elevated Levels of Procoagulant Plasma Microvesicles in Dialysis Patients

Cardiovascular (CV) death remains the largest cause of mortality in dialysis patients, unexplained by traditional risk factors. Endothelial microvesicles (EMVs) are elevated in patients with traditional CV risk factors and acute coronary syndromes while platelet MVs (PMVs) are associated with atherosclerotic disease states. This study compared relative concentrations of circulating MVs from endothelial cells and platelets in two groups of dialysis patients and matched controls and investigated their relative thromboembolic risk. MVs were isolated from the blood of 20 haemodialysis (HD), 17 peritoneal dialysis (PD) patients and 20 matched controls. Relative concentrations of EMVs (CD144^{+ ve}) and PMVs (CD42b^{+ ve}) were measured by Western blotting and total MV concentrations were measured using nanoparticle-tracking analysis. The ability to support thrombin generation was measured by reconstituting the MVs in normal plasma, using the Continuous Automated Thrombogram assay triggered with 1µM tissue factor. The total concentration of MVs as well as the measured sub-types was higher in both patient groups compared to controls (p<0.05). MVs from HD and PD patients were able to generate more thrombin than the controls, with higher peak thrombin, and endogenous thrombin potential levels (p<0.02). However there were no differences in either the relative quantity or activity of MVs between the two patient groups (p>0.3). Dialysis patients have higher levels of circulating procoagulant MVs than healthy controls. This may represent a novel and potentially modifiable mediator or predictor of occlusive cardiovascular events in these patients.     

Reconstructing the Genomic Content of Microbiome Taxa through Shotgun Metagenomic Deconvolution

Metagenomics has transformed our understanding of the microbial world, allowing researchers to bypass the need to isolate and culture individual taxa and to directly characterize both the taxonomic and gene compositions of environmental samples. However, associating the genes found in a metagenomic sample with the specific taxa of origin remains a critical challenge. Existing binning methods, based on nucleotide composition or alignment to reference genomes allow only a coarse-grained classification and rely heavily on the availability of sequenced genomes from closely related taxa. Here, we introduce a novel computational framework, integrating variation in gene abundances across multiple samples with taxonomic abundance data to deconvolve metagenomic samples into taxa-specific gene profiles and to reconstruct the genomic content of community members. This assembly-free method is not bounded by various factors limiting previously described methods of metagenomic binning or metagenomic assembly and represents a fundamentally different approach to metagenomic-based genome reconstruction. An implementation of this framework is available at http://elbo.gs.washington.edu/software.html. We first describe the mathematical foundations of our framework and discuss considerations for implementing its various components. We demonstrate the ability of this framework to accurately deconvolve a set of metagenomic samples and to recover the gene content of individual taxa using synthetic metagenomic samples. We specifically characterize determinants of prediction accuracy and examine the impact of annotation errors on the reconstructed genomes. We finally apply metagenomic deconvolution to samples from the Human Microbiome Project, successfully reconstructing genus-level genomic content of various microbial genera, based solely on variation in gene count. These reconstructed genera are shown to correctly capture genus-specific properties. With the accumulation of metagenomic data, this deconvolution framework provides an essential tool for characterizing microbial taxa never before seen, laying the foundation for addressing fundamental questions concerning the taxa comprising diverse microbial communities.     

Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing ~1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.     

Effect of High-Flux Dialysis on Circulating FGF-23 Levels in End-Stage Renal Disease Patients: Results from a Randomized Trial

Background

In patients undergoing maintenance hemodialysis (HD), increased levels of circulating fibroblast growth factor-23 (FGF-23) are independently associated with cardiovascular events and mortality. Interventional strategies aiming to reduce levels of FGF-23 in HD patients are of particular interest. The purpose of the current study was to compare the impact of high-flux versus low-flux HD on circulating FGF-23 levels.

Methods

We conducted a post-hoc analysis of the MINOXIS study, including 127 dialysis patients randomized to low-flux (n = 62) and high-flux (n = 65) HD for 52 weeks. Patients with valid measures for FGF-23 investigated baseline and after 52 weeks were included.

Results

Compared to baseline, a significant increase in FGF-23 levels after one year of low-flux HD was observed (Delta plasma FGF-23: +4026 RU/ml; p < 0.001). In contrast, FGF-23 levels remained stable in the high flux group (Delta plasma FGF-23: +373 RU/ml, p = 0.70). The adjusted difference of the absolute change in FGF-23 levels between the two treatment groups was statistically significant (p < 0.01).

Conclusions

Over a period of 12 months, high-flux HD was associated with stable FGF-23 levels, whereas the low-flux HD group showed an increase of FGF-23. However, the implications of the different FGF 23 time-trends in patients on high flux dialysis, as compared to the control group, remain to be explored in specifically designed clinical trials.

Trial Registration

German Clinical Trials Register (DRKS) DRKS00007612.     

Sexual Therapy of Patients with Cardiovascular Disease

Physical illness or disability inevitably has a damaging effect on sexual relationships. Physicians are usually unaware of the sexual consequences of illness on their patients, and lack experience in treating sexual dysfunctions.The report of treatment of a couple with serious cardiovascular disease illustrates the potential efficacy of brief sex therapy for improving the quality of a patient''s life. If a primary physician lacks the skills to conduct sex therapy, he may collaborate with nonphysician therapists. The physician''s knowledge of the physiological and psychological effects of a specific illness on his patient is essential to successful therapy. Often, simple education, encouragement or reassurance by the physician is enough to overcome the damaging effects of illness on a patient''s sex life.     

Elevated Levels of the Endosomal-Lysosomal Proteinase Cathepsin D in Cerebrospinal Fluid in Alzheimer Disease

Abstract: Lysosomal hydrolases are normally intracellular enzymes but are abundant extracellularly within senile plaques in Alzheimer disease and in other conditions where β-amyloid accumulates. To examine whether acid hydrolases released from abnormal hydrolase-laden neurons are detectable in CSF, we measured levels of the major aspartic proteinase of lysosomes, cathepsin D (Cat D), in ventricular CSF collected after death from 30 patients with Alzheimer disease, 14 patients with Huntington disease, and seven patients with other neurodegenerative diseases. The levels of Cat D-immunoreactive protein, expressed as micrograms per milliliter of protein, determined by western blot immunoassay using a polyclonal antiserum against human brain Cat D, were more than fourfold higher in the Alzheimer patients than in the other patient groups ( p < 0.0005). Cat D activity, assayed separately against [¹⁴C]methemoglobin at pH 3.2, was also significantly elevated but less than Cat D content. The lower specific activity of Cat D in Alzheimer CSF therefore indicated that the abnormally accumulated Cat D included a high proportion of inactive enzyme. These results indicate that abnormal Cat D release from affected neurons into the extracellular space is an active, ongoing process in Alzheimer brain. In addition, the levels of this enzyme and possibly other lysosomal hydrolases in CSF may prove to be useful biological markers of Alzheimer disease.     

Human and Extracellular DNA Depletion for Metagenomic Analysis of Complex Clinical Infection Samples Yields Optimized Viable Microbiome Profiles

1. Download high-res image (230KB)
2. Download full-size image

     

Elevated Serum Levels of Macrophage Migration Inhibitory Factor Are Associated with Progressive Chronic Cardiomyopathy in Patients with Chagas Disease

Clinical symptoms of chronic Chagas disease occur in around 30% of the individuals infected with Trypanosoma cruzi and are characterized by heart inflammation and dysfunction. The pathogenesis of chronic chagasic cardiomyopathy (CCC) is not completely understood yet, partially because disease evolution depends on complex host-parasite interactions. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that promotes numerous pathophysiological processes. In the current study, we investigated the link between MIF and CCC progression.Immunohistochemical analysis demonstrated MIF overexpression in the hearts from chronically T. cruzi-infected mice, particularly those showing intense inflammatory infiltration. We also found that MIF exogenously added to parasite-infected murine macrophage cultures is capable of enhancing the production of TNF-α and reactive oxygen species, both with pathogenic roles in CCC. Thus, the integrated action of MIF and other cytokines and chemokines may account for leukocyte influx to the infected myocardium, accompanied by enhanced local production of multiple inflammatory mediators. We further examined by ELISA the level of MIF in the sera from chronic indeterminate and cardiomyopathic chagasic patients, and healthy subjects. CCC patients displayed significantly higher MIF concentrations than those recorded in asymptomatic T. cruzi-infected and uninfected individuals. Interestingly, increased MIF levels were associated with severe progressive Chagas heart disease, in correlation with elevated serum concentration of high sensitivity C-reactive protein and also with several echocardiographic indicators of left ventricular dysfunction, one of the hallmarks of CCC. Our present findings represent the first evidence that enhanced MIF production is associated with progressive cardiac impairment in chronic human infection with T. cruzi, strengthening the relationship between inflammatory response and parasite-driven pathology. These observations contribute to unravel the elements involved in the pathogenesis of CCC and may also be helpful for the design of novel therapies aimed to control long-term morbidity in chagasic patients.     

Global Profiling of Carbohydrate Active Enzymes in Human Gut Microbiome

Motivation

Carbohydrate Active enzyme (CAZyme) families, encoded by human gut microflora, play a crucial role in breakdown of complex dietary carbohydrates into components that can be absorbed by our intestinal epithelium. Since nutritional wellbeing of an individual is dependent on the nutrient harvesting capability of the gut microbiome, it is important to understand how CAZyme repertoire in the gut is influenced by factors like age, geography and food habits.

Results

This study reports a comprehensive in-silico analysis of CAZyme profiles in the gut microbiomes of 448 individuals belonging to different geographies, using similarity searches of the corresponding gut metagenomic contigs against the carbohydrate active enzymes database. The study identifies a core group of 89 CAZyme families that are present across 85% of the gut microbiomes. The study detects several geography/age-specific trends in gut CAZyme repertoires of the individuals. Notably, a group of CAZymes having a positive correlation with BMI has been identified. Further this group of BMI-associated CAZymes is observed to be specifically abundant in the Firmicutes phyla. One of the major findings from this study is identification of three distinct groups of individuals, referred to as ''CAZotypes'', having similar CAZyme profiles. Distinct taxonomic drivers for these CAZotypes as well as the probable dietary basis for such trends have also been elucidated. The results of this study provide a global view of CAZyme profiles across individuals of various geographies and age-groups. These results re-iterate the need of a more precise understanding of the role of carbohydrate active enzymes in human nutrition.