While substantial efforts have been made to optimize and standardize fecal metabolomics for studies in adults, the development of a standard protocol to analyze infant feces is, however, still ...lagging behind. Here, we present the development of a hands-on and robust protocol for proton 1H NMR spectroscopy of infant feces. The influence of extraction solvent, dilution ratio, homogenization method, filtration, and duration of centrifugation on the biochemical composition of infant feces was carefully evaluated using visual inspection of 1H NMR spectra in combination with multivariate statistical modeling. The optimal metabolomics protocol was subsequently applied on feces from seven infants collected at 8 weeks, 4, and 9 months of age. Interindividual variation was exceeding the variation induced by different fecal sample preparation methods, except for filtration. We recommend extracting fecal samples using water with a dilution ratio of 1:5 feces-to-water to homogenize using bead beating and to remove particulates using centrifugation. Samples collected from infants aged 8 weeks and 4 months showed elevated concentrations of milk oligosaccharide derivatives and lactic acid, whereas short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) were higher in the 9 month samples. The established protocol enables hands-on and robust analyses of the infant gut metabolome. The wide-ranging application of this protocol will facilitate interlaboratory comparison of infants’ metabolic profiles and finally aid in a better understanding of infant gut health.
Volatile organic compounds (VOCs) in exhaled breath have the potential to be used as biomarkers for screening and diagnosis of diseases. Clinical studies are often complicated by both modifiable and ...non-modifiable factors influencing the composition of VOCs in exhaled breath. Small laboratory animal studies contribute in obtaining fundamental insight in alterations in VOC composition in exhaled breath and thereby facilitate the design and analysis of clinical research. However, long term animal experiments are often limited by invasive breath collection methods and terminal experiments. To overcome this problem, a novel device was developed for non-invasive breath collection in mice using glass nose-only restrainers thereby omitting the need of anesthetics. C57Bl/6 J mice were used to test reproducibility and different air sampling settings for air-flow (ml min
) and time (minutes). Exhaled air was collected on desorption tubes and analysed for VOCs by gas chromatography time-of-flight mass spectrometry (GC-tof-MS). In total 27 compounds were putatively identified and used to assess the variability of the VOC measurements in the breath collections. Best reproducibility is obtained when using an air flow of 185 ml min
and a collection time of 20 min. Due to the non-invasive nature of breath collections in murine models, this device has the potential to facilitate VOC research in relation to disturbed metabolism and or disease pathways.
The tumor microenvironment (TME) is a complex structure comprised of tumor, immune and stromal cells, vasculature, and extracellular matrix (ECM). During tumor development, ECM homeostasis is ...dysregulated. Collagen remodeling by matrix metalloproteinases (MMPs) generates specific collagen fragments, that can be detected in the circulation of cancer patients and correlate with poor disease outcome. Leukocyte-Associated Immunoglobulin-like Receptor-1 (LAIR-1) is an inhibitory collagen receptor expressed on immune cells in the TME and in the circulation. We hypothesized that in addition to ECM collagen, collagen fragments produced in cancer can mediate T cell immunosuppression through LAIR-1. Our analyses of TCGA datasets show that cancer patients with high tumor mRNA expression of MMPs, collagen I and LAIR-1 have worse overall survival. We show that
generated MMP1 or MMP9 collagen I fragments bind to and trigger LAIR-1. Importantly, LAIR-1 triggering by collagen I fragments inhibits CD3 signaling and IFN-γ secretion in a T cell line. LAIR-2 is a soluble homologue of LAIR-1 with higher affinity for collagen and thereby acts as a decoy receptor. Fc fusion proteins of LAIR-2 have potential as cancer immunotherapeutic agents and are currently being tested in clinical trials. We demonstrate that collagen fragment-induced inhibition of T cell function could be reversed by LAIR-2 fusion proteins. Overall, we show that collagen fragments produced in cancer can mediate T cell suppression through LAIR-1, potentially contributing to systemic immune suppression. Blocking the interaction of LAIR-1 with collagen fragments could be an added benefit of LAIR-1-directed immunotherapy.
In cyclic ion mobility (cIMS), ions are permitted to travel multiple passes around the drift cell, increasing the distance traveled and the relative separation between ions. This study tests the ...hypothesis that multiple passes around the cell can also result in improved precision when measuring an ion’s mobility and the collision cross section (TWCCS) derived therefrom. Experiments were performed with a diverse set of compounds, including 16 polycyclic aromatic hydrocarbons using gas chromatographic atmospheric pressure chemical ionization and a set of drug molecules by direct infusion electrospray ionization. The average periodic drift time, viz., the average time required for the ion to travel around the cIMS cell once, shifts dramatically, approaching part-per-million (ppm) precision as the number of passes increases to ∼100. Extrapolation of the precision of the CCS values with respect to the number of passes led to the prediction that the precision will reach 1000 ppm after 50 passes, 100 ppm after 100 passes, and <10 ppm after 150 passes. Experiments wherein the number of passes exceeded 100 produced TWCCS values having within-run precisions ranging between 15 and 117 ppm. The improved precision with an increasing number of passes may be a consequence of mitigating space-charge effects by allowing the ions to occupy a larger region of the cIMS cell. A method is proposed to enable practical measurements of TWCCS with ppm precision and is demonstrated to characterize an unknown drug mixture.
Collagen expression and structure in the tumour microenvironment are associated with tumour development and therapy response. Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a widely ...expressed inhibitory collagen receptor. LAIR-2 is a soluble homologue of LAIR-1 that competes for collagen binding. Multiple studies in mice implicate blockade of LAIR-1:collagen interaction in cancer as a promising therapeutic strategy. Here, we investigated the role of LAIR-1 in anti-tumour responses. We show that although LAIR-1 inhibits activation, proliferation, and cytokine production of mouse T cells in vitro
,
tumour outgrowth in LAIR-1-deficient mice did not differ from wild type mice in several in vivo tumour models. Furthermore, treatment with NC410, a LAIR-2-Fc fusion protein, did not result in increased tumour clearance in tested immunocompetent mice, which contrasts with previous data in humanized mouse models. This discrepancy may be explained by our finding that NC410 blocks human LAIR-1:collagen interaction more effectively than mouse LAIR-1:collagen interaction. Despite the lack of therapeutic impact of NC410 monotherapy, mice treated with a combination of NC410 and anti-programmed death-ligand 1 did show reduced tumour burden and increased survival. Using LAIR-1-deficient mice, we showed that this effect seemed to be dependent on the presence of LAIR-1. Taken together, our data demonstrate that the absence of LAIR-1 signalling alone is not sufficient to control tumour growth in multiple immunocompetent mouse models. However, combined targeting of LAIR-1 and PD-L1 results in increased tumour control. Thus, additional targeting of the LAIR-1:collagen pathway with NC410 is a promising approach to treating tumours where conventional immunotherapy is ineffective.
Background: Smoking is the main preventable lifestyle-related risk factor threatening human health. In this study, time trends in smoking behaviour between 1996 and 2005 among adolescents enrolled in ...secondary school were assessed. Methods: In 1996, 2001 and 2005, a survey was conducted in the south-eastern region of the Netherlands. All students in second and fourth year of secondary education (1996: n = 20 000; 2001: n = 27 500; 2005: n = 24 000) were asked to complete a questionnaire about their smoking behaviour. A simulation model was used to estimate lifetime health gains related to the observed trends. Results: In 1996, 2001 and 2005, the number of questionnaires analysed were 13 554 (68%), 20 767 (76%) and 17 896 (75%), respectively. The results show a decrease in ‘ever smoking’ as well as ‘current smoking’ between 1996 and 2005. Among second year high school students, current smoking prevalence decreased from 22.2% in 1996 to 8.0% in 2005 (Ptrend < 0.001). Among fourth year students, current smoking declined from 37.5% in 1996 to 22ċ0% in 2005 (Ptrend < 0.001). Time trends were not influenced by gender or educational level. Model projections show that if these students not take up smoking later in life, 11 500 new cases of COPD, 3400 new cases of lung cancer and 1800 new cases of myocardial infarction could be prevented for the Dutch 13-year-olds. Conclusion: This study found that, in the past decade, smoking prevalence among adolescents has declined by almost 50%, potentially resulting in a considerable reduction in new cases of COPD or lung cancer.
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