Blood Transcriptomics and Metabolomics for Mersonalized Medicine

Molecular analysis of blood samples is pivotal to clinical diagnosis and has been intensively investigated since the rise of systems biology. Recent developments have opened new opportunities to utilize transcriptomics and metabolomics for personalized and precision medicine. Efforts from human immunology have infused into this area exquisite characterizations of subpopulations of blood cells. It is now possible to infer from blood transcriptomics, with fine accuracy, the contribution of immune activation and of cell subpopulations. In parallel, high-resolution mass spectrometry has brought revolutionary analytical capability, detecting N10,000 metabolites, together with environmental exposure, dietary intake, microbial activity, and pharmaceutical drugs. Thus, the re-examination of blood chemicals by metabolomics is in order. Transcriptomics and metabolomics can be integrated to provide a more comprehensive understanding of the human biological states. We will review these new data and methods and discuss how they can contribute to personalized medicine

RNA from stabilized whole blood enables more comprehensive immune gene expression profiling compared to RNA from peripheral blood mononuclear cells

Monitoring changes in the immune profile in blood samples can help identifying changes in tumor biology and therapy responsiveness over time. Immune-related gene expression profiles offer a highly reproducible method to monitor changes of the immune system. However, measuring gene expression profiles in whole blood samples can be complicated because of the high protein and enzyme abundancy that affect the stability and quality of the RNA. Peripheral blood mononuclear cells (PBMCs) are one the most commonly used source for immune cell RNA extraction, though, this method does not reflect all components of the peripheral blood. The aim of this study was to determine the differences in immune-related gene expression between RNA isolated from stabilized whole blood and RNA isolated from PBMCs. Whole blood samples from 12 pancreatic cancer patients were collected before and after chemotherapy (n = 24). Blood samples were collected in both EDTA tubes, and Tempus tubes containing an RNA stabilizer (total n = 48). PBMCs were isolated from EDTA samples using Ficoll and were snap frozen. Subsequently, immune-related gene expression was profiled using the PanCancer Immune Profiling Panel of NanoString technology. Gene expression profiles of PBMCs were compared to that of Tempus tubes using the Advanced Analysis module of nSolver software. Both types of samples provided good quality RNA and gene expression measurements. However, RNA isolated from Tempus tubes resulted in significantly higher gene counts than PBMCs; 107/730 genes were exclusively detected in Tempus samples, while under the detection limit in PBMCs. In addition, 192/730 genes showed significantly higher gene counts in Tempus samples, 157/730 genes showed higher gene counts in PBMCs. Thus, RNA isolated from whole blood stabilizing blood tubes, such as Tempus tubes, enable higher gene counts and more comprehensive measurements of gene expression profiles compared to RNA isolated from PBMCs

Metabolic pathways and immunometabolism in rare kidney diseases

Objectives To characterise renal tissue metabolic pathway gene expression in different forms of glomerulonephritis. Methods Patients with nephrotic syndrome (NS), antineutrophil cytoplasmic antibody-associated vasculitis (AAV), systemic lupus erythematosus (SLE) and healthy living donors (LD) were studied. Clinically indicated renal biopsies were obtained at time of diagnosis and microdissected into glomerular and tubulointerstitial compartments. Microarray-derived differential gene expression of 88 genes representing critical enzymes of metabolic pathways and 25 genes related to immune cell markers was compared between disease groups. Correlation analyses measured relationships between metabolic pathways, kidney function and cytokine production. Results Reduced steady state levels of mRNA species were enriched in pathways of oxidative phosphorylation and increased in the pentose phosphate pathway (PPP) with maximal perturbation in AAV and SLE followed by NS, and least in LD. Transcript regulation was isozymes specific with robust regulation in hexokinases, enolases and glucose transporters. Intercorrelation networks were observed between enzymes of the PPP (eg, transketolase) and macrophage markers (eg, CD68) (r=0.49, p<0.01). Increased PPP transcript levels were associated with reduced glomerular filtration rate in the glomerular (r=-0.49, p<0.01) and tubulointerstitial (r=-0.41, p<0.01) compartments. PPP expression and tumour necrosis factor activation were tightly co-expressed (r=0.70, p<0.01). Conclusion This study demonstrated concordant alterations of the renal transcriptome consistent with metabolic reprogramming across different forms of glomerulonephritis. Activation of the PPP was tightly linked with intrarenal macrophage marker expression, reduced kidney function and increased production of cytokines. Modulation of glucose metabolism may offer novel immune-modulatory therapeutic approaches in rare kidney diseases

White Blood Cell Differentials Enrich Whole Blood Expression Data in the Context of Acute Cardiac Allograft Rejection

Acute cardiac allograft rejection is a serious complication of heart transplantation. Investigating molecular processes in whole blood via microarrays is a promising avenue of research in transplantation, particularly due to the non-invasive nature of blood sampling. However, whole blood is a complex tissue and the consequent heterogeneity in composition amongst samples is ignored in traditional microarray analysis. This complicates the biological interpretation of microarray data. Here we have applied a statistical deconvolution approach, cell-specific significance analysis of microarrays (csSAM), to whole blood samples from subjects either undergoing acute heart allograft rejection (AR) or not (NR). We identified eight differentially expressed probe-sets significantly correlated to monocytes (mapping to 6 genes, all down-regulated in ARs versus NRs) at a false discovery rate (FDR) ≤ 15%. None of the genes identified are present in a biomarker panel of acute heart rejection previously published by our group and discovered in the same data***

Circulating microbial RNA and health

© 2015, Nature Publishing Group. All rights reserved. Measurement of health indicators in the blood is a commonly performed diagnostic procedure. Two blood studies one involving extended observations on the health of an individual by integrative Personal Omics Profiling (iPOP), and the other tracking the impact of Left Ventricular Assist Device (LVAD) placement on nine heart failure patients were examined for the association of change in health status with change in microbial RNA species. Decrease in RNA expression ratios of human to bacteria and viruses accompanying deteriorated conditions was evident in both studies. Despite large between-subject variations in bacterial composition before LVAD implantation among all the patients, on day 180 after the implantation they manifested apparent between-subject bacterial similarity. In the iPOP study three periods, namely, pre-respiratory syncytial virus (RSV) infection with normal blood glucose level, RSV infection with normal blood glucose level, and post-RSV infection with high blood glucose level could be defined. The upsurge of Enterobacteria phage PhiX 174 sensu lato and Escherichia coli gene expression, in which membrane transporters, membrane receptors for environment signalling, carbohydrate catabolic genes and carbohydrate-active enzymes were enriched only throughout the second period, which suggests a potentially overlooked microbial response to or modulation of the host blood glucose level.Link_to_subscribed_fulltex

Exercise-induced molecular mechanisms in untrained and life-long highly trained individuals

Regular physical activity results in extensive systemic and functional adaptation effects in the human body that contribute to physical performance, such as muscular strength and endurance, and can have beneficial health effects on the cardiorespiratory, vascular and immune systems as well as on bone density and metabolic control. Adaptation to regular exercise requires translation of exercise-related signals into molecular responses, including epigenetic modification and other molecular processes. Over time, such processes result in accumulating cellular and sub-cellular biochemical and structural changes of tissues and organ systems. Exercise-related signals that challenge the body as a functional system include hypoxia, flux of energy rich substrates, changes in body temperature, lactate-induced pH changes, changed abundance of metabolites and mechanical shear stress. To overcome such challenges and improve future preparedness, tissues adapt for example by increased mitochondrial content in skeletal muscle, optimized temperature management and circulation, increased plasma volume and altered cell content in circulating blood, increased vascularization or by structural reinforcement and increased ability to develop force in skeletal muscle. In life-long trained athletes, the adaptations can result in outstanding, sports-specific performance. However, not all contributing mechanisms and sports-specific differences are well understood, especially in the context of elite athletes. The results presented in this thesis are based on five papers in which skeletal muscle biopsies (paper I-V) and blood samples (paper V) were collected at different timepoints around acute (papers I, IV, V) and long-term exercise (papers I, II, III). The subjects in papers I-II were young, healthy, normally active men and women, in papers III-V, the subjects were healthy middle-aged men and women, either with a life-long history of sedentary lifestyle or high-level physical activity in enduranceor resistancebased sports. Five experimental models were used: acute bipedal cycling for 60 minutes (paper I), a 12-week unilateral leg extension endurance training protocol consisting of 4x45min of exercise per week (paper I), a 10-week protocol with unilateral leg press and leg extension resistance training at 70-85% 1RM (paper II), acute bipedal cycling for 30 minutes and acute leg extension at 80% 1RM in a cross-over design (papers IV-V). Paper III consisted of a cross-sectional study design without any acute intervention. Collected samples were analyzed by qPCR, western blot (papers I-II), bisulfite-transformation, pyrosequencing and phosphorylation analysis (paper II), immunohistochemistry, citrate synthase assay, RNA sequencing (papers III-V) and FACS sorting (paper V). The overall aim of this thesis was to investigate molecular mechanisms that support and maintain life-long high-level adaptations to exercise training. In paper I, the translation of the biomechanical impulse from contracting skeletal muscle into downstream molecular signaling was investigated. In brief, it was shown that the previously described STARS signaling pathway, which links biomechanical and molecular effects is upregulated immediately following acute cycling exercise and that longterm training neither blunts nor amplifies such an acute response pattern. Furthermore, for the first time it was shown that there is no difference between men and women in STARS response. In paper II we investigated how these adaptations can be “memorized” after a period of detraining. We found increased levels of phosphorylation of key genes in previously trained muscle and identified differences in gene expression of PGC-1α and other genes important for myogenesis, suggesting potential mechanisms for a “muscle memory”. In a cross-sectional investigation in paper III, using global transcriptome analysis, gene ontology and genome-scale metabolic modelling we show that life-long high-level adaptation to endurance exercise is very different from the adaptation following life-long high-level resistance training, particularly in pathways related to the prevention of metabolic diseases, such as type 2 diabetes, and that differences between resistance training and sedentary behavior is comparably small. Furthermore, we found significant sex differences between untrained men and women and that these differences were markedly smaller comparing long-term trained men and women. We also showed that metabolically impaired individuals who submit to short-term endurance training become more similar to long-term endurance trained subjects and identified potential exercise-responsive genes. In paper IV we identified acute exercise-specific patterns of differential gene expression and identified important transcription factor motifs that contribute to these differences in long-term trained athletes. We showed that acute resistance exercise results in generally larger numbers of differentially expressed genes compared to acute endurance exercise and identified amongst others HIF1A and MYFfamily motifs as highly relevant to endurance and resistance exercise respectively. Furthermore, we identified groups of candidate genes that are especially relevant to these transcription factors and show that these genes are functionally closely connected. We also demonstrate that endurance trained athletes handle the metabolic stress of energy production differently than strength trained athletes and untrained subjects, surprisingly by a largescale downregulation of metabolites and enzymes engaged in energy production processes immediately following acute endurance exercise, confirming the uniqueness of endurance athletes proposed in paper III. In paper V we investigated how high-level long-term training modulates the response of circulating immune cells to acute endurance and resistance exercise. We show that lifelong high-level endurance athletes increase their numbers of circulating monocytes to a significantly larger extent and that the recovery of numbers of macrophages is significantly lower compared to untrained controls. Additionally, we show significant differences between the immune system response to acute endurance or resistance exercise. Furthermore, we cross-referenced immune cell concentrations in circulating plasma with the expression of immune cell marker genes in skeletal muscle and cytokine signaling in blood and demonstrated a higher enrichment of immune cell mobility related functional groups of genes in untrained control subjects compared to long-term trained athletes and a generally higher coordination of these functional groups of genes in response to acute endurance exercise compared to acute resistance exercise across all groups. In conclusion we show that life-long trained endurance athletes handle metabolic challenges in a unique way and have a resting transcriptome largely different to strength trained and control individuals. Furthermore, we suggest the phosphorylation of proteins related to protein synthesis as potential molecular mechanism for a muscle memory effect. Finally, we show, that long-term training does not blunt STARS pathway-based signal translation of mechanical contraction

Modulación de la expresión transcripcional de citoquinas de respuesta inmune innata y adaptativa en células mononucleares de sangre periférica humana tratadas con fucoidan de Lessonia trabeculata Villouta & Santelices 1986

Evalúa la expresión transcripcional de citoquinas moduladoras de la respuesta inmune innata y adaptativa en células mononucleares humanas (PBMC) tratadas con fucoidan del alga parda Lessonia trabeculata. El fucoidan de Lessonia trabeculata fue proporcionado por la empresa PSW S.A y caracterizado fitoquímicamente por el Laboratorio de Bioquímica y Biología Molecular de la Universidad Nacional Agraria La Molina. Las PBMC se trataron con diferentes concentraciones de fucoidan de Lessonia trabeculata (FLt, 80.84% pureza) y como controles se emplearon fucoidan de Fucus vesiculosus (FFv, 95% pureza) y lipopolisacárido de E. coli. La citotoxicidad se evaluó mediante el ensayo de reducción metabólica del bromuro de 3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol (MTT). Para evaluar la expresión transcripcional de las citoquinas de la inmunidad innata: IL-8, IL-12 (p35 y p40) y TGF- y las citoquinas de inmunidad adaptativa; IL-2 e IL-10 se empleó la reacción en cadena de la polimerasa convencional (PCR convencional). El tratamiento de cultivos de PBMC a las concentraciones de 10 y 100 μg/ml de FLt y FFv no ocasionó efecto citotóxico; por el contrario, FLt a 100 μg/ml incrementó significativamente el porcentaje de viabilidad celular respecto al control sin fucoidan (p<0.05). Respecto a la expresión transcripcional, el tratamiento con FLt a 10 y 100 μg/ml incrementó el ARNm IL-10 (p<0.0001) y ARNm IL-12p40 (p<0.05) respectivamente en relación al control sin fucoidan. Se concluye que el fucoidan de L. trabeculata a 10 y 100 μg/ml modula la expresión transcripcional de IL-12 de inmunidad innata e IL-10 de inmunidad adaptativa.Universidad Nacional Mayor de San Marcos (Lima). Vicerrectorado de Investigación y PosgradoTesi

The effects of the citrullinating enzyme, peptidylarginine deiminase, on the activation of T cells

Rheumatoid arthritis (RA) and periodontitis (PID) are two chronic inflammatory diseases associated with the modification of self-proteins by citrullinating peptidyl arginine deiminase (PAD) enzymes, leading to a loss of tolerance by the immune system. The main goal of this study was to explore the action of PAD enzymemediated citrullination on T cell membrane proteins and gene expression in relation to the T cell phenotype in PID. Effects on cells of the adaptive immune system have been less well studied in PID and the data obtained here shows that citrullination of peripheral blood mononuclear cells (PBMC) by PAD enzymes impairs T cell activation. Microarray studies showed that PAD enzyme treatment led to the dysregulation of genes involved in glucose and amino acid metabolism in PBMC. Real time quantitative polymerase chain reaction (RT-QPCR) in CD4 and CD8 T cells from PID patients showed a trend towards down-regulation of hexokinase 3 and up-regulation of argininosuccinate synthase1. Also, proteomic and genomic studies in PBMC implicated the involvement of the complement system in the impairment of the T cell response by PAD enzymes. Taken together, the results obtained here support a potential link between T cell surface citrullination and metabolic asynchrony in T cells and may offer an explanation for the lack of immune suppression in PID