Susan McRitchie

Antibiotic-mediated gut microbiome perturbation accelerates development of type 1 diabetes in mice.

The early life microbiome plays important roles in host immunological and metabolic development. Because the incidence of type 1 diabetes (T1D) has been increasing substantially in recent decades, we hypothesized that early-life antibiotic use alters gut microbiota, which predisposes to disease. Using non-obese diabetic mice that are genetically susceptible to T1D, we examined the effects of exposure to either continuous low-dose antibiotics or pulsed therapeutic antibiotics (PAT) early in life, mimicking childhood exposures. We found that in mice receiving PAT, T1D incidence was significantly higher, and microbial community composition and structure differed compared with controls. In pre-diabetic male PAT mice, the intestinal lamina propria had lower Th17 and Treg proportions and intestinal SAA expression than in controls, suggesting key roles in transducing the altered microbiota signals. PAT affected microbial lipid metabolism and host cholesterol biosynthetic gene expression. These findings show that early-life antibiotic treatments alter the gut microbiota and its metabolic capacities, intestinal gene expression and T-cell populations, accelerating T1D onset in non-obese diabetic mice.

Metabolomics Analysis of Hormone-Responsive and Triple-Negative Breast Cancer Cell Responses to Paclitaxel Identify Key Metabolic Differences

To date, no targeted therapies are available to treat triple negative breast cancer (TNBC), while other breast cancer subtypes are responsive to current therapeutic treatment. Metabolomics was conducted to reveal differences in two hormone receptor-negative TNBC cell lines and two hormone receptor-positive Luminal A cell lines. Studies were conducted in the presence and absence of paclitaxel (Taxol). TNBC cell lines had higher levels of amino acids, branched-chain amino acids, nucleotides, and nucleotide sugars and lower levels of proliferation-related metabolites like choline compared with Luminal A cell lines. In the presence of paclitaxel, each cell line showed unique metabolic responses, with some similarities by type. For example, in the Luminal A cell lines, levels of lactate and creatine decreased while certain choline metabolites and myo-inositol increased with paclitaxel. In the TNBC cell lines levels of glutamine, glutamate, and glutathione increased, whereas lysine, proline, and valine decreased in the presence of drug. Profiling secreted inflammatory cytokines in the conditioned media demonstrated a greater response to paclitaxel in the hormone-positive Luminal cells compared with a secretion profile that suggested greater drug resistance in the TNBC cells. The most significant differences distinguishing the cell types based on pathway enrichment analyses were related to amino acid, lipid and carbohydrate metabolism pathways, whereas several biological pathways were differentiated between the cell lines following treatment.

Metabolites as biomarkers of adverse reactions following vaccination: A pilot study using nuclear magnetic resonance metabolomics

An Adverse Event Following Immunization (AEFI) is an adverse reaction to a vaccination that goes above and beyond the usual side effects associated with vaccinations. One serious AEFI related to the smallpox vaccine is myopericarditis. Metabolomics involves the study of the low molecular weight metabolite profile of cells, tissues, and biological fluids, and provides a functional readout of the phenotype. Metabolomics may help identify a particular metabolic signature in serum of subjects who are predisposed to developing AEFIs. The goal of this study was to identify metabolic markers that may predict the development of adverse events following smallpox vaccination. Serum samples were collected from military personnel prior to and following receipt of smallpox vaccine. The study population included five subjects who were clinically diagnosed with myopericarditis, 30 subjects with asymptomatic elevation of troponins, and 31 subjects with systemic symptoms following immunization, and 34 subjects with no AEFI, serving as controls. Two-hundred pre- and post-smallpox vaccination sera were analyzed by untargeted metabolomics using 1H nuclear magnetic resonance (NMR) spectroscopy. Baseline (pre-) and post-vaccination samples from individuals who experienced clinically verified myocarditis or asymptomatic elevation of troponins were more metabolically distinguishable pre- and post-vaccination compared to individuals who only experienced systemic symptoms, or controls. Metabolomics profiles pre- and post-receipt of vaccine differed substantially when an AEFI resulted. This study is the first to describe pre- and post-vaccination metabolic profiles of subjects who developed an adverse event following immunization. The study demonstrates the promise of metabolites for determining mechanisms associated with subjects who develop AEFI and the potential to develop predictive biomarkers.

Preterm neonatal urinary renal developmental and acute kidney injury metabolomic profiling: An exploratory study

BackgroundAcute kidney injury (AKI) staging has been developed in the adult and pediatric populations, but these do not yet exist for the neonatal population. Metabolomics was utilized to uncover biomarkers of normal and AKI-associated renal function in preterm infants. The study comprised 20 preterm infants with an AKI diagnosis who were matched by gestational age and gender to 20 infants without an AKI diagnosis.MethodsUrine samples from pre-term newborn infants collected on day 2 of life were analyzed using broad-spectrum nuclear magnetic resonance (NMR) metabolomics. Multivariate analysis methods were used to identify metabolite profiles that differentiated AKI and no AKI, and to identify a metabolomics profile correlating with gestational age in infants with and without AKI.ResultsThere was a clear distinction between the AKI and no-AKI profiles. Two previously identified biomarkers of AKI, hippurate and homovanillate, differentiated AKI from no-AKI profiles. Pathway analysis revealed similarities to cholinergic neurons, prenatal nicotine exposure on pancreatic β cells, and amitraz-induced inhibition of insulin secretion. Additionally, a pH difference was noted. Both pH and the metabolites were found to be associated with AKI; however, only the metabotype was a significant predictor of AKI. Pathways for the no-AKI group that correlated uniquely with gestational age included aminoacyl-t-RNA biosynthesis, whereas pathways in the AKI group yielded potential metabolite changes in pyruvate metabolism.ConclusionsMetabolomics was able to differentiate the urinary profiles of neonates with and without an AKI diagnosis and metabolic developmental profiles correlated with gestational age. Further studies in larger cohorts are needed to validate these results.

Maternal Early Pregnancy Serum Metabolomics Profile and Abnormal Vaginal Bleeding as Predictors of Placental Abruption: A Prospective Study

Background & Objective Placental abruption, an ischemic placental disorder, complicates about 1 in 100 pregnancies, and is an important cause of maternal and perinatal morbidity and mortality worldwide. Metabolomics holds promise for improving the phenotyping, prediction and understanding of pathophysiologic mechanisms of complex clinical disorders including abruption. We sought to evaluate maternal early pregnancy pre-diagnostic serum metabolic profiles and abnormal vaginal bleeding as predictors of abruption later in pregnancy. Methods Maternal serum was collected in early pregnancy (mean 16 weeks, range 15 to 22 weeks) from 51 abruption cases and 51 controls. Quantitative targeted metabolic profiles of serum were acquired using electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS/MS) and the Absolute IDQ® p180 kit. Maternal sociodemographic characteristics and reproductive history were abstracted from medical records. Stepwise logistic regression models were developed to evaluate the extent to which metabolites aid in the prediction of abruption. We evaluated the predictive performance of the set of selected metabolites using a receiver operating characteristics (ROC) curve analysis and area under the curve (AUC). Results Early pregnancy vaginal bleeding, dodecanoylcarnitine/dodecenoylcarnitine (C12 / C12:1), and phosphatidylcholine acyl-alkyl C 38:1 (PC ae C38:1) strongly predict abruption risk. The AUC for these metabolites alone was 0.68, for early pregnancy vaginal bleeding alone was 0.65, and combined the AUC improved to 0.75 with the addition of quantitative metabolite data (P = 0.003). Conclusion Metabolomic profiles of early pregnancy maternal serum samples in addition to the clinical symptom, vaginal bleeding, may serve as important markers for the prediction of abruption. Larger studies are necessary to corroborate and validate these findings in other cohorts.

Pretreatment with indomethacin results in increased heat stroke severity during recovery in a rodent model of heat stroke

It has been suggested that medications can increase heat stroke (HS) susceptibility/severity. We investigated whether the nonsteroidal anti-inflammatory drug (NSAID) indomethacin (INDO) increases HS severity in a rodent model. Core temperature (Tc) of male, C57BL/6J mice (n = 45) was monitored continuously, and mice were given a dose of INDO [low dose (LO) 1 mg/kg or high dose (HI) 5 mg/kg in flavored treat] or vehicle (flavored treat) before heating. HS animals were heated to 42.4°C and euthanized at three time points for histological, molecular, and metabolic analysis: onset of HS [maximal core temperature (Tc,Max)], 3 h of recovery [minimal core temperature or hypothermia depth (HYPO)], and 24 h of recovery (24 h). Nonheated (control) animals underwent identical treatment in the absence of heat. INDO (LO or HI) had no effect on physiological indicators of performance (e.g., time to Tc,Max, thermal area, or cooling time) during heating or recovery. HI INDO resulted in 45% mortality rate by 24 h (HI INDO + HS group). The gut showed dramatic increases in gross morphological hemorrhage in HI INDO + HS in both survivors and nonsurvivors. HI INDO + HS survivors had significantly lower red blood cell counts and hematocrit suggesting significant hemorrhage. In the liver, HS induced cell death at HYPO and increased inflammation at Tc,Max, HYPO, and 24 h; however, there was additional effect with INDO + HS group. Furthermore, the metabolic profile of the liver was disturbed by heat, but there was no additive effect of INDO + HS. This suggests that there is an increase in morbidity risk with INDO + HS, likely resulting from significant gut injury.NEW & NOTEWORTHY This paper suggests that in a translational mouse model, NSAIDs may be counterindicated in situations that put an individual at risk of heat injury. We show here that a small, single dose of the NSAID indomethacin before heat stroke has a dramatic and highly damaging effect on the gut, which ultimately leads to increased systemic morbidity.

Impact of a western diet on the ovarian and serum metabolome.

OBJECTIVE The objective of this investigation was to determine differences in the profiles of endogenous metabolites (metabolomics) among ovaries and serum derived from Old World nonhuman primates fed prudent or Western diets. DESIGN A retrospective, observational study was done using archived ovarian tissue and serum from midlife cynomolgus monkeys (Macaca fasicularis). Targeted and broad spectrum metabolomics analysis was used to compare ovarian tissue and serum from monkeys that had been exposed to a prudent diet or a Western diet. Monkeys in the prudent diet group (n=13) were research naïve and had been exposed only to a commercial monkey chow diet (low in cholesterol and saturated fats, high in complex carbohydrates). Western diet monkeys (n=8) had consumed a diet that was high in cholesterol, saturated animal fats and soluble carbohydrates for 2 years prior to ovarian tissue and serum collection. OUTCOME MEASURES Metabolomic analyses were done on extracts of homogenized ovary tissue samples, and extracts of serum. Targeted analysis was conducted using the Biocrates p180 kit and broad spectrum analysis was conducted using UPLC-TOF-MS, resulting in the detection of 3500 compound ions. RESULTS Using metabolomics methods, which capture thousands of signals for metabolites, 64 metabolites were identified in serum and 47 metabolites were identified in ovarian tissue that differed by diet. Quantitative targeted analysis revealed 13 amino acids, 6 acrylcarnitines, and 2 biogenic amines that were significantly (p<0.05) different between the two diet groups for serum extracts, and similar results were observed for the ovary extracts. CONCLUSIONS These data demonstrate that dietary exposure had a significant impact on the serum and ovarian metabolome, and demonstrated perturbation in carnitine, lipids/fatty acid, and amino acid metabolic pathways.

Omics Technologies Used in Systems Biology

Abstract In this chapter, we provide information about a number of the leading and innovative “omics” approaches used in various disciplines including biology, environmental health sciences, and toxicological research. This chapter is divided into sections that cover technologies that enable genomics, transcriptomics, proteomics, and metabolomics-based approaches. Within each section we cover aspects of sample handling and the types of molecular and analytical methods used in capturing and preprocessing data. We briefly discuss data analysis as Chapter 5 covers data analysis in more detail. With changes in technology occurring rapidly, each section provides information that can be used as a starting point for further investigation.

Abstract 1836: Triple negative breast cancer: Metabolomics and flux analysis to identify targets for drug development

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA To date, no targeted therapies are clinically available to treat triple negative breast cancer (TNBC). This highly aggressive form of breast cancer (BCa) derives its name from the absence of estrogen receptor (ER+), progesterone receptor (PR+), and HER2/neu receptor (HER2+). These receptors are expressed in other subtypes of BCa (i.e. Luminal, HER2-enriched) and have been successfully targeted with therapeutics such as Tamoxifen and Herceptin. New therapeutics are needed to improve the prognosis for patients with TNBC. Metabolomics and metabolic flux analysis (MFA) were conducted to reveal differences in TNBC cell lines compared with hormone receptor-positive cell lines (ER+ and +/-HER2+) prior to and following treatment with chemotherapy drugs. Two triple-negative (MDA-MB-231 and MDA-MB-468) and two hormone-responsive (BT474 and MCF-7) cell lines were compared before and after treatment with Taxol®/Paclitaxel. The metabolic profiles were assessed using 1H NMR broad spectrum metabolomics, MFA using [U-13C]-glucose and [U-13C]-glutamine enriched media, and cytokine profiling of the media. Broad spectrum metabolomics analysis demonstrated differences in the metabolic profiles between the TNBC and hormone-responsive cell lines in the absence of treatment, with a higher level of amino acids (e.g. alanine, glutamate, glutamine, glutathione), short chain fatty acids (isobutyrate, β-hydroxybutyrate), and nucleotides and nucleotide sugars (ADP, ATP, UDP-glucose, UDP-glucuronate) in the TNBC cell lines. Further, MFA demonstrated that glucose utilization was greater in the TNBC cells (MDA-MB-468) compared to the hormone-responsive cells (MCF-7). In addition, 13C-labeled glycine and 13C-labeled glutathione were only observed in TNBC cell lines treated in the presence of [U-13C]-glucose. In response to Taxol treatment, more metabolites were altered in the hormone-responsive cell lines compared with the TNBC cells. Profiling of 80 inflammatory cytokines (RayBiotech arrays) also demonstrated different responses in secreted inflammatory signals following treatment. We observed significant upregulation in interleukins-4 and -16 in MDA-MB-468 cells, while there was a downregulation of osteoprotegerin in all four cell lines after treatment with Taxol. This approach holds promise for identifying biomarkers which may be leveraged for development of targeted treatments. Citation Format: Delisha A. Stewart, Jason Winnike, Susan McRitchie, Wimal Pathmasiri, Susan Sumner. Triple negative breast cancer: Metabolomics and flux analysis to identify targets for drug development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1836. doi:10.1158/1538-7445.AM2015-1836