Genes In Whole Blood Research Articles

The effect of antiretroviral treatment on selected genes in whole blood from HIV-infected adults sensitised by Mycobacterium tuberculosis.

HIV-1 co-infection is a leading cause of susceptibility to tuberculosis (TB), with the risk of TB being increased at all stages of HIV-1 infection. Antiretroviral treatment (ART) is the most effective way to reduce the risk of TB in HIV-1 co-infected people. Studying protective, ART-induced, immune restoration in HIV-1 infected individuals sensitised by Mycobacterium tuberculosis (Mtb) can thus help identify mechanisms of protection against TB. In order to understand ART-mediated prevention of TB in HIV-1 infected adults, we investigated the expression of 30 genes in whole blood from HIV-1 infected patients during the first 6 months of ART-induced immune reconstitution. The 30 selected genes were previously described to be differentially expressed between sorted Mtb specific central and effector memory CD4 T cells. HIV-1 infected persons sensitised by Mtb were recruited in Khayelitsha, South Africa, when initiating ART. RNA was extracted from whole blood at initiation and 1, 3 and 6 months of ART. qRT-PCR was used to determine gene expression and three reference ‘housekeeping’ genes were used to calculate the fold change in the expression of each gene relative to day 0 of ART. Results were assessed longitudinally. We observed a decrease in the expression of a number of genes at 6 months of ART, reflecting a decrease in immune activation. However, following correction for multiple comparisons and increasing CD4 counts, only the decrease in CD27 gene expression remained statistically significant. While not statistically significant, a number of genes also showed increased expression at various timepoints, illustrating the broad regeneration of the T cell pool in HIV-1 infected adults on ART. Our findings generate hypotheses underlying ART- induced protective immune reconstitution and may pave the way for future studies to evaluate ART mediated prevention of TB in HIV-1 infected persons.

Productive and physiological responses of feeder cattle supplemented with Yucca schidigera extract during feedlot receiving.

This experiment evaluated the effects of supplementing a saponin-containing feed ingredient, manufactured from purified extract of Yucca schidigera [Micro-Aid (MA); DPI Global; Porterville, CA], on performance, health, and physiological responses of receiving cattle. A total of 105 recently weaned Angus x Hereford calves (75 steers and 30 heifers), originating from eight cow-calf operations, were obtained from an auction facility on day -2 and road transported (800 km; 12 h) to the experimental facility. Immediately after arrival on day -1, shrunk BW was recorded and calves were grouped with free-choice access to grass hay, mineral supplement, and water. On day 0, calves were ranked by sex, source, and shrunk BW, and allocated to one of 21 pens (5 calves/pen; being one or two heifers within each pen). Pens were assigned to receive a total mixed ration (TMR) and one of three treatments (as-fed basis): (1) 1 g/calf daily of MA (M1; n = 7), (2) 2 g/calf daily of MA (M2; n = 7), or (3) no MA supplementation (CON; n = 7). Calves received the TMR to yield 15% (as-fed basis) orts, and treatments were top-dressed from days 0 to 59. Calves were assessed for bovine respiratory disease (BRD) signs and TMR intake was recorded for each pen daily. Calves were vaccinated against BRD pathogens on days 0 and 21. Final shrunk BW was recorded on day 60, and blood samples were collected on days 0, 2, 6, 10, 14, 21, 28, 34, 45, and 59. ADG was greater (P = 0.03) in M2 vs. M1 and CON (1.53, 1.42, and 1.42 kg/day, respectively), and similar (P = 0.95) between M1 and CON calves. No treatment effects were detected for TMR intake (P = 0.52), whereas feed efficiency was greater (P ≤ 0.05) in M2 vs. M1 and CON calves (213, 200, and 204 g/kg, respectively) and similar (P = 0.40) between M1 and CON calves. No treatment effects were detected (P = 0.39) for diagnosis of BRD signs. The number of antimicrobial treatments required upon BRD diagnosis was greater (P ≤ 0.01) in CON vs. M1 and M2 (1.40, 1.05, and 1.10 treatments, respectively), and similar (P = 0.60) between M1 and M2 calves. No other treatment effects were detected (P ≥ 0.23), including circulating concentrations of hormones and metabolites, serum antibody titers to BRD pathogens, and mRNA expression of innate immunity genes in whole blood. Collectively, results from this experiment suggest that MA supplementation at 2 g/animal daily enhances performance and response to BRD treatment in high-risk cattle during feedlot receiving.

T Helper 1 Cellular Immunity Toward Recoverin Is Enhanced in Patients With Active Autoimmune Retinopathy

Autoimmune retinopathy (AIR) causes rapidly progressive vision loss that is treatable but often is confused with other forms of retinal degeneration including retinitis pigmentosa (RP). Measurement of anti-retinal antibodies (ARA) by Western blot is a commonly used laboratory assay that supports the diagnosis yet does not reflect current disease activity. To search for better diagnostic indicators, this study was designed to compare immune biomarkers and responses toward the retinal protein, recoverin, between newly diagnosed AIR patients, slow progressing RP patients and healthy controls. All individuals had measurable anti-recoverin IgG and IgM antibodies by ELISA regardless of disease status or Western blot results. Many AIR patients had elevated anti-recoverin IgG1 levels and a strong cellular response toward recoverin dominated by IFNγ. RP patients and controls responded to recoverin with a lower IFNγ response that was balanced by IL-10 production. Both AIR and RP patients displayed lower levels of total peripheral blood mononuclear cells that were due to reductions of CD4+ TH cells. A comparison of messenger RNA (mRNA) for immune-related genes in whole blood of AIR patients versus RP patients or controls indicated lower expression of ATG5 and PTPN22 and higher expression of several genes involved in TH cell signaling/transcription and adhesion. These data indicate that an immune response toward recoverin is normal in humans, but that in AIR patients the balance shifts dramatically toward higher IFNγ production and cellular activation.

Abstract 3674: Pharmacodynamic assessment in whole blood for the BET bromodomain inhibitor CPI-0610 of target engagement in patients with progressive lymphoma

Abstract Defining the relationship between small molecule exposure, clinical efficacy and target engagement via pharmacodynamic (PD) biomarkers is a key element of phase 1 clinical development. CPI-0610 is an orally bioavailable inhibitor of the bromodomain and extra-terminal (BET) domain family of proteins currently being evaluated in a number of phase I trials. To support the clinical development of CPI-0610, we sought to identify BET target genes in whole blood that function as PD markers for target engagement in patients. The utilization of whole blood rather than tumor samples for PD assessments allows for a high level of compliance for sample collection and assessment of serial samples to understand the kinetics of optimal gene regulation. This is important for the mechanism of action of BET bromodomain inhibitors, as it has been shown that target gene suppression is rapidly reversible upon removal of drug. A preliminary list of BET target genes was first identified after ex vivo treatment of healthy donor blood samples with a BET inhibitor followed by transcriptional profiling. Further characterization was carried out using blood samples recovered from in vivo studies in mice, as well as rat and dog toxicology studies for CPI-0610. Blood samples collected from patients with progressive lymphoma participating in a Phase I clinical study (NCT01949883) were analyzed for changes in the BET target genes and correlated with CPI-0610 dose, pharmacokinetic (PK) exposure and clinical activity. This enabled further refinement of the BET target gene signature to identify the most robust gene expression changes in patient samples. IL8 and CCR1 were the most strongly regulated genes, showing an exposure-dependent downregulation in patient samples. Using these target genes, we were able to identify a minimum threshold of exposure required for BET target engagement as well as show that BET target engagement can be achieved at doses below the maximum tolerated dose. Furthermore, ≥50% downregulation of IL8 was consistent with anti-lymphoma activity in patients from our trial. CCR2, FN1, CSF1R and THBS1 were identified and validated preclinically but in patient samples were difficult to interpret due to lack of robust regulation or low basal levels of expression. In summary, a BET target gene signature assay using whole blood samples was established and implemented in a Phase I lymphoma study which demonstrated that CPI-0610 regulates direct BET target genes in blood cells in a robust and dose-dependent manner. Citation Format: Jennifer A. Mertz, Kristie A. Blum, Anas Younes, Jeremy S. Abramson, Michael B. Maris, Ian W. Flinn, Andre Goy, Darrell R. Borger, Michael R. Cooper, Robert J. Sims. Pharmacodynamic assessment in whole blood for the BET bromodomain inhibitor CPI-0610 of target engagement in patients with progressive lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3674.

Discovery and translation of a target engagement marker for AMP-activated protein kinase (AMPK).

BackgroundActivation of the AMP-activated protein kinase (AMPK) is an attractive approach for the treatment of type 2 diabetes. AMPK activation reduces glucose levels in animal models of type 2 diabetes by increasing glucose uptake in skeletal muscles and reducing hepatic glucose production. Furthermore, AMPK activation ameliorates hepatic steatosis in animal models. For the clinical development of AMPK activators it is essential to have a reliable target engagement marker for appropriate dose finding and to support proof of clinical principle. While the activation of AMPK by quantification of the phosphorylation of AMPK at Thr172 in target tissues can be assessed pre-clinically, this is not feasible in clinical studies. Therefore, we attempted to identify and translate a peripheral target engagement biomarker downstream of AMPK activation for clinical use in blood samples.MethodsFor pharmacological activation of AMPK, two AMPK activators were synthesized (compound 1 and 2). A compound with structural similarities but no pharmacological effect on AMPK phosphorylation was synthesized as negative control (compound 3). Whole blood from healthy volunteers was incubated with an AMPK activator for up to 6 hours and mRNA sequencing was performed. Additionally, human PBMCs were isolated to evaluate Thr172-phosphorylation of AMPK in Western blots. In order to enable identification of translatable biomarker candidates, blood samples from HanWistar rats treated for two weeks with an AMPK activator were also subjected to mRNA sequencing. Furthermore, concentration-response curves for four biomarker candidates were recorded in human blood samples using Nanostring nCounter technology. Finally, ZDF rats were treated with increasing doses of compound 2 for five weeks to investigate the glucose-lowering efficacy. To investigate changes of mRNA expression of two selected biomarker candidates in this ZDF rat study, qRT-PCR was performed.ResultsPharmacological activation of AMPK in human PBMCs revealed an increase in Thr172-phosphorylation of AMPK, confirming target engagement in these blood cells. RNA sequencing of human blood samples identified 608 deregulated genes after AMPK activation. Additionally, AMPK activation led to deregulation of 367 genes in whole blood from HanWistar rats which mapped to the respective human genes. 22 genes out of the intersection of genes deregulated in both species are proposed as potential translatable target engagement biomarker candidates. The most prominent genes were transmembrane glycoprotein NMB (GPNMB, osteoactivin), calcium-binding protein A9 (S100A9), peptidoglycan recognition protein (PGLYRP1) and Ras homolog gene family, member B (RHOB). Specificity for AMPK was shown by testing inactive compound 3 in HanWistar rats. The exposure-effect relationship for GPNMB was investigated in a subchronic study in diabetic ZDF rats. GPNMB showed a dose-dependent up-regulation both acutely and after subchronic dosing. GPNMB up-regulation correlated with an increased Thr172-phosphorylation of AMPK in liver and quadriceps muscle in rats.ConclusionGPNMB has been identified as a translatable target engagement biomarker for use in clinical studies.

Integrative omics to detect bacteremia in patients with febrile neutropenia.

BackgroundCancer chemotherapy-associated febrile neutropenia (FN) is a common condition that is deadly when bacteremia is present. Detection of bacteremia depends on culture, which takes days, and no accurate predictive tools applicable to the initial evaluation are available. We utilized metabolomics and transcriptomics to develop multivariable predictors of bacteremia among FN patients.MethodsWe classified emergency department patients with FN and no apparent infection at presentation as bacteremic (cases) or not (controls), according to blood culture results. We assessed relative metabolite abundance in plasma, and relative expression of 2,560 immunology and cancer-related genes in whole blood. We used logistic regression to identify multivariable predictors of bacteremia, and report test characteristics of the derived predictors.ResultsFor metabolomics, 14 bacteremic cases and 25 non-bacteremic controls were available for analysis; for transcriptomics we had 7 and 22 respectively. A 5-predictor metabolomic model had an area under the receiver operating characteristic curve of 0.991 (95%CI: 0.972,1.000), 100% sensitivity, and 96% specificity for identifying bacteremia. Pregnenolone steroids were more abundant in cases and carnitine metabolites were more abundant in controls. A 3-predictor gene expression model had corresponding results of 0.961 (95%CI: 0.896,1.000), 100%, and 86%. Genes involved in innate immunity were differentially expressed.ConclusionsClassifiers derived from metabolomic and gene expression data hold promise as objective and accurate predictors of bacteremia among FN patients without apparent infection at presentation, and can provide insights into the underlying biology. Our findings should be considered illustrative, but may lay the groundwork for future biomarker development.

Gene expression profiles of immune-regulatory genes in whole blood of cattle with a subclinical infection of Mycobacterium avium subsp. paratuberculosis.

Johne’s disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), resulting in inflammation of intestines and persistent diarrhea. The initial host response against MAP infections is mainly regulated by the Th1 response, which is characterized by the production of IFN-γ. With the progression of disease, MAP can survive in the host through the evasion of the host’s immune response by manipulating the host immune response. However, the host response during subclinical phases has not been fully understood. Immune regulatory genes, including Th17-derived cytokines, interferon regulatory factors, and calcium signaling-associated genes, are hypothesized to play an important role during subclinical phases of Johne’s disease. Therefore, the present study was conducted to analyze the expression profiles of immune regulatory genes during MAP infection in whole blood. Different expression patterns of genes were identified depending on the infection stages. Downregulation of IL-17A, IL-17F, IL-22, IL-26, HMGB1, and IRF4 and upregulation of PIP5K1C indicate suppression of the Th1 response due to MAP infection and loss of granuloma integrity. In addition, increased expression of IRF5 and IRF7 suggest activation of IFN-α/β signaling during subclinical stages, which induced indoleamine 2,3-dioxygenase mediated depletion of tryptophan metabolism. Increased expression of CORO1A indicate modulation of calcium signaling, which enhanced the survival of MAP. Taken together, distinct host gene expression induced by MAP infection indicates enhanced survival of MAP during subclinical stages.

Exploring the Wnt signaling pathway in schizophrenia and bipolar disorder

The Wnt signaling pathway plays a crucial role in neurodevelopment and in regulating the function and structure of the adult nervous system. Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental disorders with evidence of subtle neurodevelopmental, structural and functional neuronal abnormalities. We aimed to elucidate the role of aberrant regulation of the Wnt system in these disorders by evaluating plasma levels of secreted Wnt modulators in patients (SCZ = 551 and BD = 246) and healthy controls (HCs = 639) using enzyme immune-assay. We also investigated the expression of 141 Wnt-related genes in whole blood in a subsample (SCZ = 338, BD = 241, and HCs = 263) using microarray analysis. Both SCZ and BD had dysregulated mRNA expression of Wnt-related genes favoring attenuated canonical (beta-catenin-dependent) signaling, and there were also indices of enhanced non-canonical Wnt signaling. In particular, FZD7, which may activate all Wnt pathways, but favors non-canonical signaling, and NFATc3, a downstream transcription factor and readout of the non-canonical Wnt/Ca2+ pathway, were significantly increased in SCZ and BD (p < 3 × 10−4). Furthermore, patients had lower plasma levels of soluble dickkopf 1 and sclerostin (p < 0.01) compared with HC. Our findings suggest that SCZ and BD are characterized by abnormal Wnt gene expression and plasma protein levels, and we propose that drugs targeting the Wnt pathway may have a role in the treatment of severe mental disorders.

Comparative analysis of gene expression in maternal peripheral blood and monocytes during spontaneous preterm labor

Preterm birth is the leading cause of newborn death worldwide, and is associated with significant cognitive and physiological challenges in later life. There is a pressing need to define the mechanisms that initiate spontaneous preterm labor, and for development of novel clinical biomarkers to identify high-risk pregnancies. Most preterm birth studies utilize fetal tissues, and there is limited understanding of the transcriptional changes that occur in mothers undergoing spontaneous preterm labor. Earlier work revealed that a specific population of maternal peripheral leukocytes (macrophages/monocytes) play an active role in the initiation of labor. Thus, we hypothesized that there are dynamic gene expression changes in maternal blood leukocytes during preterm labor. Using next-generation sequencing we aim to characterize the transcriptome in whole blood leukocytes and peripheral monocytes of women undergoing spontaneous preterm labor compared to healthy pregnant women who subsequently delivered at full term. RNA sequencing was performed in both whole blood and peripheral monocytes from women who underwent preterm labor (24-34 weeks of gestation, N= 20) matched for gestational age to healthy pregnant controls (N= 30). All participants were a part of the Ontario Birth Study cohort (Toronto, Ontario, Canada). We identified significant differences in expression of 262 genes in peripheral monocytes and 184 genes in whole blood of women who were in active spontaneous preterm labor compared to pregnant women of the same gestational age not undergoing labor, with 43 of these genes differentially expressed in both whole blood and peripheral monocytes. ADAMTS2 expression was significantly increased in women actively undergoing spontaneous preterm labor, which we validated through digital droplet reverse transcriptase polymerase chain reaction. Intriguingly, we have also identified a number of gene sets including signaling by stem cell factor-KIT, nucleotide metabolism, and trans-Golgi network vesicle budding, which exhibited changes in relative gene expression that was predictive of preterm labor status in both maternal whole blood and peripheral monocytes. This study is the first to investigate changes in both whole blood leukocytes and peripheral monocytes of women actively undergoing spontaneous preterm labor through robust transcript measurements from RNA sequencing. Our unique study design overcame confounding based on gestational age by collecting blood samples from women matched by gestational age, allowing us to study transcriptomic changes directly related to the active preterm parturition. We performed RNA profiling using whole genome sequencing, which is highly sensitive and allowed us to identify subtle changes in specific genes. ADAMTS2 expression emerged as a marker of prematurity within peripheral blood leukocytes, an accessible tissue that plays a functional role in signaling during the onset of labor. We identified changes in relative gene expression in a number of gene sets related to signaling in monocytes and whole blood of women undergoing spontaneous preterm labor compared to controls. These genes and pathways may help identify potential targets for the development of novel drugs for preterm birth prevention.

Thyroid State Regulates Gene Expression in Human Whole Blood.

Despite the well-recognized clinical features resulting from insufficient or excessive thyroid hormone (TH) levels in humans, it is largely unknown which genes are regulated by TH in human tissues. To study the effect of TH on human gene expression profiles in whole blood, mainly consisting of T3 receptor (TR) α-expressing cells. We performed next-generation RNA sequencing on whole blood samples from eight athyroid patients (four females) on and after 4 weeks off levothyroxine replacement. Gene expression changes were analyzed through paired differential expression analysis and confirmed in a validation cohort. Weighted gene coexpression network analysis (WGCNA) was applied to identify thyroid state-related networks. We detected 486 differentially expressed genes (fold-change >1.5; multiple testing corrected P value < 0.05), of which 76% were positively and 24% were negatively regulated. Gene ontology (GO) enrichment analysis revealed that three biological processes were significantly overrepresented, of which the process translational elongation showed the highest fold enrichment (7.3-fold, P = 1.8 × 10-6). WGCNA analysis independently identified various gene clusters that correlated with thyroid state. Further GO analysis suggested that thyroid state affects platelet function. Changes in thyroid state regulate numerous genes in human whole blood, predominantly TRα-expressing leukocytes. In addition, TH may regulate gene transcripts in platelets.

Abstract B35: Molecular and phenotypic profiling of pancreatic cancer cachexia in novel murine models and patients

Abstract Background: Patients with pancreatic ductal adenocarcinoma (PDAC) suffer the highest rates of cachexia, with ~85% experiencing muscle and fat loss. Weight loss and muscle loss correlate with mortality in PDAC. Patients with cachexia experience fatigue, weakness and reduced quality of life. Cachexia impairs response to surgery, chemotherapy and radiation therapy. Currently the only effective therapy is removal of the tumor. However, pre-clinical studies demonstrate that slowing muscle or fat loss prolongs function and life, even absent effects on tumor growth. Thus anti-cachexia therapies should increase quality of life, clinical response and survival. Most cachexia research has been conducted using two classic non-PDAC mouse models, LLC lung and C26 colon cancer cell lines implanted s.c. Little genomic data are available in patients, with only 3 small gene expression studies in cachexia, none specific to PDAC. Thus whether mouse models faithfully reflect human PDAC cachexia is unknown. Methods: Mice: Cell lines derived from the KrasG12D;Trp53R172H;Pdx1-Cre (KPC) genetically engineered mouse model (GEMM) were injected orthotopically. Elastase-Cre and Pdx1-Cre KPC mice were generated. Mice were followed for tumor growth and metastasis, weight loss, body composition, strength, and muscle and fat wasting versus matched controls. A subset of models was characterized for serum cytokine levels and muscle transcriptomes. Patients: Patients undergoing surgery for PDAC (n=24) or benign conditions (n=12) donated clinical data, blood, muscle, subcutaneous adipose tissue and tumor (PDAC). Body composition was measured from CT scans. Whole blood, muscle and fat gene expression were profiled using the Ion AmpliSeq Transcriptome Panel and analyzed with Partech, Ingenuity, GSEA and NextBio. Results: Mice: We validated 9 novel murine models of PDAC cachexia. These models demonstrated considerable diversity in cachexia severity (latency to cachexia), body composition, tumor histology and metastasis, blood cytokine levels, and muscle gene expression. Orthotopic PDAC cell line and KPC GEMM models were more similar to each other than to LLC or C26 by phenotype and muscle gene expression. IL-6 and Activin, known to be elevated in human PDAC cachexia, were consistently elevated across mouse models. Inhibition of either reduced PDAC cachexia. Patients: PDAC and control groups had similar BMI, although 6-month history of weight loss and BMI-adjusted weight loss category were both greater for the PDAC group (p&amp;lt;0.0001). PDAC patients displayed reduced skeletal muscle and subcutaneous adipose tissue volume (p&amp;lt;0.05). Ampliseq (false discovery rate &amp;lt;0.05) revealed 23 differentially expressed genes in whole blood, 906 in muscle, and 2962 in adipose tissue, with ~250 overlapping genes in muscle and fat. The top predicted biofunctions increased by PDAC in both tissues were organismal death, followed by anomaly of musculoskeletal system, and hypoplasia; the most reduced biofunctions were cell movement, migration, and size of body. The top common canonical pathways increased were EIF2 signaling, and G1/S checkpoint regulation, while the most decreased were signaling from integrins, RhoGTPases, and leukocyte extravasation. Comparing mouse muscle to human muscle, “organismal death” was oppositely regulated and decreased in mouse. However, common pathways included EIF2 signaling and G1/S checkpoint regulation (increased) and cardiac hypertrophy signaling (decreased). At the gene level, substantial differences were observed between mouse and human muscle. Conclusions: Mouse models of PDAC are superior for modeling PDAC cachexia. In patients, the muscle and fat transcriptomes show a cachexia signature even in early stage (resectable) disease. Comparative molecular phenotyping of mouse and human PDAC cachexia reveal considerable overlap in pathways, albeit clear differences in gene level analysis. Thus careful characterization and application of models is warranted. Citation Format: Teresa A. Zimmers, Yanlin Jiang, Jianguo Liu, Eugene Ceppa, Atilla Nakeeb, Michael House, Nicholas Zyromski, C. Max Schmidt, Katharyn Hannaford, Daniel Schloss, Cynthia Brooks, Milan Radovich, Yunlong Lui, Guanglong Jiang, Stephen F. Konieczny, Patrick Schweickert, Brad Hancock, Joshua Kayes, Xiaoling Zhong, Christophe Poirier, Ernie Au, Andrea Bonetto, Safi Shahda, Marc Kohli, Bert O’Neil, Marianne Pons, Leonidas Koniaris.{Authors}. Molecular and phenotypic profiling of pancreatic cancer cachexia in novel murine models and patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B35.

Rapid Identification of Bacterial Antibiotic Resistance by qPCR in Infants with Gram-Negative Septicaemia: A Proof-of-Concept Study

Background: Neonatal sepsis remains an important cause of neonatal morbidity and mortality. Tools to rapidly predict antibiotic resistance in neonatal sepsis would be extremely valuable. Objectives: To develop quantitative polymerase chain reaction (qPCR) primer/probe sets that can rapidly detect antibiotic resistance genes common to a neonatal unit, and to investigate the feasibility of direct detection of antibiotic resistance genes in whole blood of infants with Gram-negative septicaemia without first isolating the organism. Methods: Primer/probe sets were designed to detect genes that produce aminoglycoside-modifying enzymes or extended-spectrum β-lactamase. In phase 1, Gram-negative organisms isolated from neonatal clinical specimens within a 12-month period were analysed by qPCR to detect preselected genes. In phase 2, blood specimens of infants with Gram-negative septicaemia were subjected to qPCR analysis to detect antibiotic resistance genes for comparison against conventional antibiotic resistance profile results. Results: Two primer/probe sets showed promising diagnostic utilities for the prediction of antibiotic resistance; the diagnostic utilities (sensitivity, specificity, positive predictive value and negative predictive value) were 90.9, 96.4, 92.6 and 95.5%, respectively, for AAC3-2 [aac(3ʼ)-IIa/aacC3/aacC2, aac(3ʼ)-IIc/aacC2] to detect gentamicin resistance, and 59.3, 99.3, 94.1 and 92.6%, respectively, for BLA-C1 (bla_CTX-M-9, bla_CTX-M-14, bla_CTX-M-24, bla_CTX-M-27) to detect cephalosporin resistance. Twenty-six infants were tested in phase 2, and both gentamicin and cephalosporin resistance patterns were predicted with 100% sensitivity and 100% specificity by AAC3-2 and BLA-C1, respectively. Conclusions: qPCR with appropriately designed primer/probe sets can predict antibiotic resistance directly from neonatal blood, and it can substantially reduce the turnaround time for antibiotic resistance results.

MiRNA signatures and transcriptional regulation of their target genes in vitiligo

BackgroundmiRNAs are small non-coding RNA molecules that post-transcriptionally regulate gene expression. We have earlier reported the skin miRNA expression profiling in patients with non-segmental vitiligo. ObjectiveIn the present study, we show the expression of previously identified skin miRNAs signatures in blood and their target genes in whole blood and PBMCs as well as skin micro-environment of vitiligo patients and controls. MethodsmiRNA expression profiling in whole blood was performed using customized TaqMan® Low Density Array. We predicted the potential targets of differentially expressed miRNAs and investigated their expression levels in skin, whole blood and PBMCs from patients and controls using Real-time PCR. ResultsOur results showed miR-1, miR-184, miR-328, miR-383 and miR-577 hold similar pattern of expression as of skin, suggesting their potent eminence for being putative markers for vitiligo. In silico target prediction revealed miR-1 targets EDN1, G6PD, HSP60, HSP70, SERP1, SIRT1 & TYR; miR-184 targets EZR & LAMP1; miR-328 targets IL1B, POLH & TRPM1; miR-383 targets EDN1 & TYRP1; and miR-577 targets PTPN22 & TYRP1 which were corroborated by our validation study. ConclusionsIn conclusion, the present study for the first time provides new insights into the crucial role of miRNA regulated gene network involved in oxidative stress, autoimmunity and ER stress mediated pathogenesis of vitiligo.

Whole Blood RNA as a Source of Transcript-Based Nutrition- and Metabolic Health-Related Biomarkers.

Blood cells are receiving an increasing attention as an easily accessible source of transcript-based biomarkers. We studied the feasibility of using mouse whole blood RNA in this context. Several paradigms were studied: (i) metabolism-related transcripts known to be affected in rat tissues and peripheral blood mononuclear cells (PBMC) by fasting and upon the development of high fat diet (HFD)-induced overweight were assessed in whole blood RNA of fasted rats and mice and of HFD-fed mice; (ii) retinoic acid (RA)-responsive genes in tissues were assessed in whole blood RNA of control and RA-treated mice; (iii) lipid metabolism-related transcripts previously identified in PBMC as potential biomarkers of metabolic health in a rat model were assessed in whole blood in an independent model, namely retinoblastoma haploinsufficient (Rb+/-) mice. Blood was collected and stored in RNAlater® at -80°C until analysis of selected transcripts by real-time RT-PCR. Comparable changes with fasting were detected in the expression of lipid metabolism-related genes when RNA from either PBMC or whole blood of rats or mice was used. HFD-induced excess body weight and fat mass associated with expected changes in the expression of metabolism-related genes in whole blood of mice. Changes in gene expression in whole blood of RA-treated mice reproduced known transcriptional actions of RA in hepatocytes and adipocytes. Reduced expression of Fasn, Lrp1, Rxrb and Sorl1 could be validated as early biomarkers of metabolic health in young Rb+/- mice using whole blood RNA. Altogether, these results support the use of whole blood RNA in studies aimed at identifying blood transcript-based biomarkers of nutritional/metabolic status or metabolic health. Results also support reduced expression of Fasn, Lrp1, Rxrb and Sorl1 in blood cells at young age as potential biomarkers of metabolic robustness.

Genome-wide DNA methylation analysis in multiple tissues in primary Sjögren's syndrome reveals regulatory effects at interferon-induced genes

ObjectivesIncreasing evidence suggests an epigenetic contribution to the pathogenesis of autoimmune diseases, including primary Sjögren's Syndrome (pSS). The aim of this study was to investigate the role of DNA methylation...

Characterisation of haematological profiles and whole blood relative gene expression levels in Holstein-Friesian and Jersey bull calves undergoing gradual weaning

Haematological profiles indicate the health status of an animal and can be used to identify sub-clinical stress responses. The objectives of the study were to examine (i) the effect of breed and plane of nutrition, on haematological profiles of artificially reared Holstein-Friesian and Jersey bull calves in response to gradual weaning, and (ii) the effect of breed on immune response genes in bovine whole blood using real-time quantitative PCR. Holstein-Friesian and Jersey bull calves were group housed indoors and individually fed using an automatic feeder. They were allocated to a high, medium or low plane of nutrition, based on milk replacer (MR) and concentrate. The nutrition treatments were calculated using National Research Council guidelines in order to achieve a high, medium or low growth rate for each respective breed. During the weaning phase MR was gradually reduced over a 14-day (d) period (d −13 to d 0). Calves were blood sampled on d −14, −6, −3, 0, 1, 3, 8 and 14 relative to weaning (d 0) for subsequent haematological analysis. On d −14, 1 and 8, a subset of eight Holstein-Friesian calves randomly selected from the medium nutrition treatment and eight Jersey calves randomly selected from the high nutrition treatment, were blood sampled for gene expression profiling, targeting biomarkers of weaning stress. These two treatment groups were chosen to examine the effect of breed on expression of the genes of interest, as energy intake and animal performance were similar. There was no effect of breed×plane of nutrition interaction nor effect of plane of nutrition on any variable measured (P>0.05). Gradual weaning produced differential biological responses in the two breeds evidenced by breed×time interactions for lymphocyte, monocyte and red blood cell number, plasma haemoglobin and haptoglobin concentrations (P<0.05). The typical stress response consisting of neutrophilia and lymphopaenia was not observed for any treatment. An immune response to gradual weaning was observed as the relative gene expression level of the pro-apoptotic gene, Fas, increased on d 1 relative to d −14 (P<0.05). Relative gene expression levels were greater in Jersey calves compared with Holstein-Friesian for the pro-inflammatory cytokine CXCL8 (P=0.05) and the glucocorticoid receptor, GRα (P<0.05). The increased levels of these transcripts suggest that Jersey calves may have a more sensitive immune system compared with Holstein-Friesian.

A Single Meal Containing Raw, Crushed Garlic Influences Expression of Immunity- and Cancer-Related Genes in Whole Blood of Humans1–4

Background: Preclinical and epidemiologic studies suggest that garlic intake is inversely associated with the progression of cancer and cardiovascular disease.Objective: We designed a study to probe the mechanisms of garlic action in humans.Methods: We conducted a randomized crossover feeding trial in which 17 volunteers consumed a garlic-containing meal (100 g white bread, 15 g butter, and 5 g raw, crushed garlic) or a garlic-free control meal (100 g white bread and 15 g butter) after 10 d of consuming a controlled, garlic-free diet. Blood was collected before and 3 h after test meal consumption for gene expression analysis in whole blood. Illumina BeadArray was used to screen for genes of interest, followed by real-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) on selected genes. To augment human study findings, Mono Mac 6 cells were treated with a purified garlic extract (0.5 μL/mL), and mRNA was measured by qRT-PCR at 0, 3, 6, and 24 h.Results: The following 7 genes were found to be upregulated by garlic intake: aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT), hypoxia-inducible factor 1α (HIF1A), proto-oncogene c-Jun (JUN), nuclear factor of activated T cells (NFAT) activating protein with immunoreceptor tyrosine-based activation motif 1 (NFAM1), oncostatin M (OSM), and V-rel avian reticuloendotheliosis viral oncogene homolog (REL). Fold-increases in mRNA transcripts ranged from 1.6 (HIF1A) to 3.0 (NFAM1) (P < 0.05). The mRNA levels of 5 of the 7 genes that were upregulated in the human trial were also upregulated in cell culture at 3 and 6 h: AHR, HIF1A, JUN, OSM, and REL. Fold-increases in mRNA transcripts in cell culture ranged from 1.7 (HIF1A) to 12.1 (JUN) (P < 0.01). OSM protein was measured by ELISA and was significantly higher than the control at 3, 6, and 24 h (24 h: 19.5 ± 1.4 and 74.8 ± 1.4 pg/mL for control and garlic, respectively). OSM is a pleiotropic cytokine that inhibits several tumor cell lines in culture.Conclusion: These data indicate that the bioactivity of garlic is multifaceted and includes activation of genes related to immunity, apoptosis, and xenobiotic metabolism in humans and Mono Mac 6 cells. This trial is registered at clinicaltrials.gov as NCT01293591.

Dynamic expression of leukocyte innate immune genes in whole blood from horses with lipopolysaccharide-induced acute systemic inflammation.

BackgroundIn horses, insights into the innate immune processes in acute systemic inflammation are limited even though these processes may be highly important for future diagnostic and therapeutic advances in high-mortality disease conditions as the systemic inflammatory response syndrome (SIRS) and sepsis. Therefore, the aim of this study was to investigate the expression of 31 selected blood leukocyte immune genes in an equine model of acute systemic inflammation to identify significantly regulated genes and to describe their expression dynamics during a 24-h experimental period. Systemic inflammation was induced in 6 adult horses by the intravenous injection of 1 μg lipopolysaccharide (LPS) per kg btw. Sixteen blood samples were collected for each horse at predetermined intervals and analyzed by reverse transcription quantitative real-time PCR. Post-induction expression levels for each gene were compared with baseline levels.ResultsSystemic inflammation was confirmed by the presence of clinical and hematological changes which were consistent with SIRS. The clinical response to LPS was transient and brief as all horses except one showed unaltered general demeanor after 24 h. Twenty-two leukocyte genes were significantly regulated at at least one time point during the experimental period. By close inspection of the temporal responses the dynamic changes in mRNA abundance revealed a very rapid onset of both pro- and anti-inflammatory mediators and a substantial variation in both expression magnitudes and duration of changes between genes. A majority of the 22 significantly regulated genes peaked within the first 8 h after induction, and an on-going, albeit tightly controlled, regulation was seen after 24 h despite approximate clinical recovery.ConclusionsThis first broad study of gene expressions in blood leukocytes during equine acute LPS-induced systemic inflammation thoroughly characterized a highly regulated and dynamic innate immune response. These results provide new insights into the molecular mechanisms of equine systemic inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0450-5) contains supplementary material, which is available to authorized users.

Blood Genome-Wide Transcriptional Profiles Reflect Broad Molecular Impairments and Strong Blood-Brain Links in Alzheimer's Disease

To date, little is known regarding the etiology and disease mechanisms of Alzheimer's disease (AD). There is a general urgency for novel approaches to advance AD research. In this study, we analyzed blood RNA from female patients with advanced AD and matched healthy controls using genome-wide gene expression microarrays. Our data showed significant alterations in 3,944 genes (≥2-fold, FDR ≤1%) in AD whole blood, including 2,932 genes that are involved in broad biological functions. Importantly, we observed abnormal transcripts of numerous tissue-specific genes in AD blood involving virtually all tissues, especially the brain. Of altered genes, 157 are known to be essential in neurological functions, such as neuronal plasticity, synaptic transmission and neurogenesis. More importantly, 205 dysregulated genes in AD blood have been linked to neurological disease, including AD/dementia and Parkinson's disease, and 43 are known to be the causative genes of 42 inherited mental retardation and neurodegenerative diseases. The detected transcriptional abnormalities also support robust inflammation, profound extracellular matrix impairments, broad metabolic dysfunction, aberrant oxidative stress, DNA damage, and cell death. While the mechanisms are currently unclear, this study demonstrates strong blood-brain correlations in AD. The blood transcriptional profiles reflect the complex neuropathological status in AD, including neuropathological changes and broad somatic impairments. The majority of genes altered in AD blood have not previously been linked to AD. We believe that blood genome-wide transcriptional profiling may provide a powerful and minimally invasive tool for the identification of novel targets beyond Aβ and tauopathy for AD research.

Differential gene expression in obese pregnancy.

Maternal obesity is a major risk factor for maternal and perinatal complications, and is associated with increased risk of premature cardiovascular death in the offspring. We aimed to identify differentially expressed genes in obese and lean pregnancies. Pregnant women were recruited antenatally and whole-blood samples taken at gestational week 28. Women were classified as lean (BMI<25, n=5), overweight (BMI 25-30, n=11) or obese (BMI>30, n=4). Subcutaneous adipose tissue was obtained from a separate cohort of women undergoing elective Caesarean section (n=3 lean, n=6 overweight, n=9 obese). Total RNA was isolated and gene expression profiled by Illumina microarray technology. Differential gene expression was identified by Ingenuity Pathway Analysis and validated by TaqMan qRT-PCR. In whole blood, differential expression (p<0.05) of six genes (ANGPTL, COX7A2, EIF3A, PTS, CISD1 and GLRX) was identified between lean and non-lean pregnant women by microarray. When gene expression was studied in subcutaneous tissues, we observed a trend towards lower expression of the cytochrome c oxidase-subunit 7A2 gene (COX7A2) in overweight vs lean (p=0.057) and obese vs lean (p=0.06) women, whereas expression was similar between overweight and obese women (p=0.55). Other genes were not differentially expressed across groups. Maternal obesity is associated with differential expression of multiple genes in whole blood. In subcutaneous tissue at term, there was a trend towards reduced COX7A2 expression in obese women. COX7A2 is a mitochondrial protein, with key roles in steroidogenesis and oxidative stress regulation and could provide a link between inflammation and obesity-related pregnancy complications.