Metabolic profiling of an Echinostoma caproni infection in the mouse for biomarker discovery

Background

Metabolic profiling holds promise with regard to deepening our understanding of infection biology and disease states. The objectives of our study were to assess the global metabolic responses to an Echinostoma caproni infection in the mouse, and to compare the biomarkers extracted from different biofluids (plasma, stool, and urine) in terms of characterizing acute and chronic stages of this intestinal fluke infection.

Methodology/Principal Findings

Twelve female NMRI mice were infected with 30 E. caproni metacercariae each. Plasma, stool, and urine samples were collected at 7 time points up to day 33 post-infection. Samples were also obtained from non-infected control mice at the same time points and measured using ¹H nuclear magnetic resonance (NMR) spectroscopy. Spectral data were subjected to multivariate statistical analyses. In plasma and urine, an altered metabolic profile was already evident 1 day post-infection, characterized by reduced levels of plasma choline, acetate, formate, and lactate, coupled with increased levels of plasma glucose, and relatively lower concentrations of urinary creatine. The main changes in the urine metabolic profile started at day 8 post-infection, characterized by increased relative concentrations of trimethylamine and phenylacetylglycine and lower levels of 2-ketoisocaproate and showed differentiation over the course of the infection.

Conclusion/Significance

The current investigation is part of a broader NMR-based metabonomics profiling strategy and confirms the utility of this approach for biomarker discovery. In the case of E. caproni, a diagnosis based on all three biofluids would deliver the most comprehensive fingerprint of an infection. For practical purposes, however, future diagnosis might aim at a single biofluid, in which case urine would be chosen for further investigation, based on quantity of biomarkers, ease of sampling, and the degree of differentiation from the non-infected control group.     

Increased lipolysis but diminished gene expression of lipases in subcutaneous adipose tissue of healthy young males with intrauterine growth retardation

Intrauterine growth retardation (IUGR) is associated with a central fat distribution and risk of developing type 2 diabetes in adults when exposed to a sedentary Western lifestyle. Increased lipolysis is an early defect of metabolism in IUGR subjects, but the sites and molecular mechanisms involved are unknown. Twenty IUGR and 20 control (CON) subjects, aged 20-30 years, were studied before and after 10 days of bed rest using the glucose clamp technique combined with measurements of in vivo metabolism by microdialysis technique and blood flow by (133)Xe washout technique in subcutaneous abdominal (SCAAT) and femoral (SCFAT) adipose tissue. Additionally, mRNA expression of lipases was evaluated in biopsies from SCAAT. Lipolysis in SCAAT was substantially higher in IUGR than in CON subjects despite markedly lower mRNA expression of lipases. Blood flow was higher in IUGR compared with CON in both SCAAT and SCFAT. Whole body insulin sensitivity did not differ between groups and decreased after bed rest. After bed rest, SCAAT lipolysis remained higher in IUGR compared with CON, and SCFAT lipolysis decreased in CON but not in IUGR. Prior to the development of whole body insulin resistance, young men with IUGR are characterized by increased in vivo adipose tissue lipolysis and blood flow with a paradoxically decreased expression of lipases compared with CON, and 10 days of physical inactivity underlined the baseline findings. Subjects with IUGR exhibit primary defects in adipose tissue metabolism.     

Discovery of Infection Associated Metabolic Markers in Human African Trypanosomiasis

Human African trypanosomiasis (HAT) remains a major neglected tropical disease in Sub-Saharan Africa. As clinical symptoms are usually non-specific, new diagnostic and prognostic markers are urgently needed to enhance the number of identified cases and optimise treatment. This is particularly important for disease caused by Trypanosoma brucei rhodesiense, where indirect immunodiagnostic approaches have to date been unsuccessful. We have conducted global metabolic profiling of plasma from T.b.rhodesiense HAT patients and endemic controls, using ¹H nuclear magnetic resonance (NMR) spectroscopy and ultra-performance liquid chromatography, coupled with mass spectrometry (UPLC-MS) and identified differences in the lipid, amino acid and metabolite profiles. Altogether 16 significantly disease discriminatory metabolite markers were found using NMR, and a further 37 lipid markers via UPLC-MS. These included significantly higher levels of phenylalanine, formate, creatinine, N-acetylated glycoprotein and triglycerides in patients relative to controls. HAT patients also displayed lower concentrations of histidine, sphingomyelins, lysophosphatidylcholines, and several polyunsaturated phosphatidylcholines. While the disease metabolite profile was partially consistent with previous data published in experimental rodent infection, we also found unique lipid and amino acid profile markers highlighting subtle but important differences between the host response to trypanosome infections between animal models and natural human infections. Our results demonstrate the potential of metabolic profiling in the identification of novel diagnostic biomarkers and the elucidation of pathogenetic mechanisms in this disease.     

Rap1 regulates E-cadherin-mediated cell-cell adhesion

The small GTPase Rap is best characterized as a critical regulator of integrin-mediated cell adhesion, although its mechanism of action is not understood. Rap also influences the properties of other cell-surface receptors and biological processes, although whether these are a consequence of effects on integrins is not clear. We show here that Rap also plays an important role in the regulation of cadherin-mediated cell-cell adhesion in epithelial cells. Expression of constitutively active Rap1A restored cadherin-mediated cell-cell contacts in mesenchymal Ras-transformed Madin-Darby canine kidney cells, resulting in reversion to an epithelial phenotype. Activation of endogenous Rap via the Rap exchange factor Epac1 also antagonized hepatocyte growth factor-induced disruption of adherens junctions. Inhibition of Rap signaling resulted in disruption of epithelial cell-cell contacts. Rap activity was required for adhesion of cells to recombinant E-cadherin extracellular domains, i.e. in the absence of integrin-mediated adhesion. These findings suggest that Rap signaling positively contributes to cadherin-mediated adhesion and that this occurs independently of effects on integrin-mediated adhesion. Our results imply that Rap may function in a broader manner to regulate the function of cell-surface adhesion receptors.     

Proteomic analysis of serum opsonins impacting biodistribution and cellular association of porous silicon microparticles

Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.     

Infection of myeloid dendritic cells with Listeria monocytogenes leads to the suppression of T cell function by multiple inhibitory mechanisms

Myeloid dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. In this study we provide evidence that myeloid DC respond to infection with Listeria monocytogenes with simultaneous induction of multiple stimulatory and inhibitory molecules. However, the overall impact of infected DC during T cell encounter results in suppression of T cell activation, indicating that inhibitory pathways functionally predominate. Inhibitory activity of infected DC is effected mainly by IL-10 and cyclooxygenase 2-mediated mechanisms, with soluble CD25 acting as an IL-2 scavenger as well as by the products of tryptophan catabolism. These inhibitory pathways are strictly TNF-dependent. In addition to direct infection, DC bearing this regulatory phenotype can be induced in vitro by a combination of signals including TNF, TLR2, and prostaglandin receptor ligation and by supernatants derived from the infected cells. Both infection-associated DC and other in vitro-induced regulatory DC are characterized by increased resistance to infection and enhanced bactericidal activity. Furthermore, myeloid DC expressing multiple regulatory molecules are identified in vivo in granuloma during listeriosis and tuberculosis. Based on the in vivo findings and the study of in vitro models, we propose that in granulomatous infections regulatory DC may possess dual function evolved to protect the host from disseminating infection via inhibition of granuloma destruction by T cells and control of pathogen spreading.     

Comparison of the results of surgical and non-surgical treatment of combat urogenital injuries in Bosnia war 1992-1995

Goal was to compare the results of surgical and non-surgical treatments of combat injuries of genitourinary system and to compare our data with data collected in the recent studies. The study was designed as a retrospective review of data collected in prospective databases. The data extracted from inpatients' medical records included demographics, mechanisms and type of injury, distribution of the lesions, clinical presentation features, applied diagnostic studies, treatment modalities, types of complication and results of treatment. Among 4.125 patients treated in the Mostar War Hospital, 111 had injury of genitourinary tract: 62 underwent a surgical and 49 non-surgical treatment. Mortality among operated patients was 16 (26%). Complications were noted in 47 patients (42%); in 33 (70%) were manifested as early complications, and 14 (30) as delayed ones (p = 0.006). Among the surgically treated patients, 40 (36%) had some complication, in comparison to 8 (7.2%) patients with complications among non-surgically treated patients; which represent a statistically significant difference (p < 0.05). In this study, there was a surprisingly high number of non-surgically treated patients, and this sub-group of UGT trauma patients had in some ways the superior treatment results in comparison with surgically treated patients. Conservatively treated patients had lower rate of complications, no mortality, and no patients with permanent disability.     

Ultrastructural study of the different zones of the rat liver acinus after portacaval anastomosis]

Liver atrophy is a main feature in rats with a porto caval shunt. Histological studies revealed small size hepatocytes. Ultrastructural differences between periportal and centrolobular zones were noticed, in particular, the dilatation of the nuclear envelope and of the rough endoplasmic reticulum which appeared dilated, desorganized and sometimes without ribosomes, was more pronounced in the periportal zone. Hepatocytes of this zone might be more sensitive to the decrease of O2 and/or hepatotrophic factors.