FT-ICR-MS-based metabolomics: A deep dive into plant metabolism.

Metabolomics involves the identification and quantification of metabolites to unravel the chemical footprints behind cellular regulatory processes and to decipher metabolic networks, opening new insights to understand the correlation between genes and metabolites. In plants, it is estimated the existence of hundreds of thousands of metabolites and the majority is still unknown. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a powerful analytical technique to tackle such challenges. The resolving power and sensitivity of this ultrahigh mass accuracy mass analyzer is such that a complex mixture, such as plant extracts, can be analyzed and thousands of metabolite signals can be detected simultaneously and distinguished based on the naturally abundant elemental isotopes. In this review, FT-ICR-MS-based plant metabolomics studies are described, emphasizing FT-ICR-MS increasing applications in plant science through targeted and untargeted approaches, allowing for a better understanding of plant development, responses to biotic and abiotic stresses, and the discovery of new natural nutraceutical compounds. Improved metabolite extraction protocols compatible with FT-ICR-MS, metabolite analysis methods and metabolite identification platforms are also explored as well as new in silico approaches. Most recent advances in MS imaging are also discussed.

Immunolabelling perturbs the endogenous and antibody-conjugated elemental concentrations during immuno-mass spectrometry imaging.

Immuno-mass spectrometry imaging uses lanthanide-conjugated antibodies to spatially quantify biomolecules via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The multi-element capabilities allow for highly multiplexed analyses that may include both conjugated antibodies and endogenous metals to reveal relationships between disease and chemical composition. Sample handling is known to perturb the composition of the endogenous elements, but there has been little investigation into the effects of immunolabelling and coverslipping. Here, we used cryofixed muscle sections to examine the impact of immunolabelling steps on the concentrations of a Gd-conjugated anti-dystrophin primary antibody, and the endogenous metals Cu and Zn. Primary antibody incubation resulted in a decrease in Zn, and an increase in Cu. Zn was removed from the cytoplasm where it was hypothesised to be more labile, whereas concentrated locations of Zn remained in the cell membrane in all samples that underwent the immunostaining process. Cu increased in concentration and was found mostly in the cell membrane. The concentration of the Gd-conjugated antibody when compared to the standard air-dried sample was not significantly different when coverslipped using an organic mounting medium, whereas use of an aqueous mounting medium significantly reduced the concentration of Gd. These results build on the knowledge of how certain sample handling techniques change elemental concentrations and distributions in tissue sections. Immunolabelling steps impact the concentration of endogenous elements, and separate histological sections are required for the quantitative analysis of endogenous elements and biomolecules. Additionally, coverslipping tissue sections for complementary immunohistochemical/immunofluorescent imaging may compromise the integrity of the elemental label, and organic mounting media are recommended over aqueous mounting media.

Preformulation evaluation of selumetinib for topical application: skin distribution and photodegradation analysis using MALDI imaging and LC-MS/MS.

Understanding drug behavior within the skin, especially for photosensitive compounds, is crucial for developing effective and safe topical therapies. This study employs Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to investigate the skin permeation and photostability of selumetinib, a MEK inhibitor used in treating type 1 neurofibromatosis (NF1). The highest amounts of selumetinib in the skin sections were obtained when using the gel formulation, suggesting that it is to be preferred to cream formulations to achieve higher permeation of the drug. Our study also revealed that selumetinib is amenable to photodegradation in ex vivo skin explants, and yields one main degradation product, whose degradation is likely triggered by hydrogen abstraction. MALDI-MSI results showed selumetinib and its degradation product concentrate in skin appendages, indicating these structures might serve as drug reservoirs, potentially prolonging retention and efficacy. This study demonstrates that combining MALDI-MSI with LC/MS-MS can highly contribute to the characterization of the fate of photosensitive compounds in the skin, an essential prerequisite to the development of compound-specific photoprotective measures. It will also pave the way for innovative topical delivery strategies for NF1 treatment.

Detection of proteomic alterations at different stages in a Huntington's disease mouse model via matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging.

Huntington's disease (HD) is a progressive and irreversible neurodegenerative disease leading to the inability to carry out daily activities and for which no cure exists. The underlying mechanisms of the disease have not been fully elucidated yet. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) allows the spatial information of proteins to be obtained upon the tissue sections without homogenisation. In this study, we aimed to examine proteomic alterations in the brain tissue of an HD mouse model with MALDI-MSI coupled to LC-MS/MS system. We used 3-, 6- and 12-month-old YAC128 mice representing pre-stage, mild stage and pathological stage of the HD and their non-transgenic littermates, respectively. The intensity levels of 89 proteins were found to be significantly different in YAC128 in comparison to their control mice in the pre-stage, 83 proteins in the mild stage, and 82 proteins in the pathological stage. Among them, Tau, EF2, HSP70, and NogoA proteins were validated with western blot analysis. In conclusion, the results of this study have provided remarkable new information about the spatial proteomic alterations in the HD mouse model, and we suggest that MALDI-MSI is an excellent technique for identifying such regional proteomic changes and could offer new perspectives in examining complex diseases.

Ion mobility mass spectrometry in the omics era: Challenges and opportunities for metabolomics and lipidomics.

Researchers worldwide are taking advantage of novel, commercially available, technologies, such as ion mobility mass spectrometry (IM-MS), for metabolomics and lipidomics applications in a variety of fields including life, biomedical, and food sciences. IM-MS provides three main technical advantages over traditional LC-MS workflows. Firstly, in addition to mass, IM-MS allows collision cross-section values to be measured for metabolites and lipids, a physicochemical identifier related to the chemical shape of an analyte that increases the confidence of identification. Second, IM-MS increases peak capacity and the signal-to-noise, improving fingerprinting as well as quantification, and better defining the spatial localization of metabolites and lipids in biological and food samples. Third, IM-MS can be coupled with various fragmentation modes, adding new tools to improve structural characterization and molecular annotation. Here, we review the state-of-the-art in IM-MS technologies and approaches utilized to support metabolomics and lipidomics applications and we assess the challenges and opportunities in this growing field.

Effects of In Utero PFOS Exposure on Epigenetics and Metabolism in Mouse Fetal Livers.

Prenatal exposure to perfluorooctanesulfonate (PFOS) increases fetus' metabolic risk; however, the investigation of the underlying mechanism is limited. In this study, pregnant mice in the gestational days (GD, 4.5-17.5) were exposed to PFOS (0.3 and 3 µg/g of body weight). At GD 17.5, PFOS perturbed maternal lipid metabolism and upregulated metabolism-regulating hepatokines (Angptl4, Angptl8, and Selenop). Mass-spectrometry imaging and whole-genome bisulfite sequencing revealed, respectively, selective PFOS localization and deregulation of gene methylation in fetal livers, involved in inflammation, glucose, and fatty acid metabolism. PCR and Western blot analysis of lipid-laden fetal livers showed activation of AMPK signaling, accompanied by significant increases in the expression of glucose transporters (Glut2/4), hexose-phosphate sensors (Retsat and ChREBP), and the key glycolytic enzyme, pyruvate kinase (Pk) for glucose catabolism. Additionally, PFOS modulated the expression levels of PPARα and PPARγ downstream target genes, which simultaneously stimulated fatty acid oxidation (Cyp4a14, Acot, and Acox) and lipogenesis (Srebp1c, Acaca, and Fasn). Using human normal hepatocyte (MIHA) cells, the underlying mechanism of PFOS-elicited nuclear translocation of ChREBP, associated with a fatty acid synthesizing pathway, was revealed. Our finding implies that in utero PFOS exposure altered the epigenetic landscape associated with dysregulation of fetal liver metabolism, predisposing postnatal susceptibility to metabolic challenges.

Detection of early proteomic alterations in 5xFAD Alzheimer's disease neonatal mouse model via MALDI-MSI.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder, characterized by memory deficit and dementia. AD is considered a multifactorial disorder where multiple processes like amyloid-beta and tau accumulation, axonal degeneration, synaptic plasticity, and autophagic processes plays an important role. In this study, the spatial proteomic differences in the neonatal 5xFAD brain tissue were investigated using MALDI-MSI coupled to LC-MS/MS, and the statistically significantly altered proteins were associated with AD. Thirty-five differentially expressed proteins (DEPs) between the brain tissues of neonatal 5xFAD and their littermate mice were detected via MALDI-MSI technique. Among the 35 proteins identified, 26 of them were directly associated with AD. Our results indicated a remarkable resemblance in the protein expression profiles of neonatal 5xFAD brain when compared to AD patient specimens or AD mouse models. These findings showed that the molecular alterations in the AD brain existed even at birth and that some proteins are neurodegenerative presages in neonatal AD brain. HIGHLIGHTS: Spatial proteomic alterations in the 5xFAD mouse brain compared to the littermate. 26 out of 35 differentially expressed proteins associated with Alzheimer's disease (AD). Molecular alterations and neurodegenerative presages in neonatal AD brain. Alterations in the synaptic function an early and common neurobiological thread.

Tandem Mass Spectrometry Imaging Enables High Definition for Mapping Lipids in Tissues.

Mass spectrometry imaging (MSI) of lipids in biological tissues is useful for correlating molecular distribution with pathological results, which could provide useful information for both biological research and disease diagnosis. It is well understood that the lipidome could not be clearly deciphered without tandem mass spectrometry analysis, but this is challenging to achieve in MSI due to the limitation in sample amount at each image spot. Here we develop a multiplexed MS2 imaging (MS2 I) method that can provide MS2 images for 10 lipid species or more for each sampling spot, providing spatial structural lipidomic information. Coupling with on-tissue photochemical derivatization, imaging of 20 phospholipid C=C location isomers is also realized, showing enhanced molecular images with high definition in structure for mouse brain and human liver cancer tissue sections. Spatially mapped t-distributed stochastic neighbor embedding has also been adopted to visualize the tumor margin with enhancement by structural lipidomic information.

A Caged In-Source Laser-Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI-MS Imaging.

Insufficient vacuum stability of matrix chemicals is a major limitation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo-cleavable caged molecule 4,5-dimethoxy-2-nitrobenzyl-2,5-dihydroxyacetophenone (DMNB-2,5-DHAP) and employed it for lipid MALDI-MSI of mouse brain tissue sections. DMNB-2,5-DHAP is vacuum-stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built-in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5-DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum-stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI-2 laser-induced postionization.

A mass spectrometry imaging and lipidomic investigation reveals aberrant lipid metabolism in the orthotopic mouse glioma.

Lipids perform multiple biological functions and reflect the physiology and pathology of cells, tissues, and organs. Here, we sought to understand lipid content in relation to tumor pathology by characterizing phospholipids and sphingolipids in the orthotopic mouse glioma using MALDI MS imaging (MSI) and LC-MS/MS. Unsupervised clustering analysis of the MALDI-MSI data segmented the coronal tumoral brain section into 10 histopathologically salient regions. Heterogeneous decrease of the common saturated phosphatidylcholines (PCs) in the tumor was accompanied by the increase of analogous PCs with one or two additional fatty acyl double bonds and increased lyso-PCs. Polyunsaturated fatty acyl-PCs and ether PCs highlighted the striatal tumor margins, whereas the distributions of other PCs differentiated the cortical and striatal tumor parenchyma. We detected a reduction of SM d18:1/18:0 and the heterogeneous mild increase of SM d18:1/16:0 in the tumor, whereas ceramides accumulated only in a small patch deep in the tumoral parenchyma. LC-MS/MS analyses of phospholipids and sphingolipids complemented the MALDI-MSI observation, providing a snapshot of these lipids in the tumor. Finally, the proposed mechanisms responsible for the tumoral lipid changes were contrasted with our interrogation of gene expression in human glioma. Together, these lipidomic results unveil the aberrant and heterogeneous lipid metabolism in mouse glioma where multiple lipid-associated signaling pathways underline the tumor features, promote the survival, growth, proliferation, and invasion of different tumor cell populations, and implicate the management strategy of a multiple-target approach for glioma and related brain malignancies.

Spatial Multiomics of Lipids, N-Glycans, and Tryptic Peptides on a Single FFPE Tissue Section.

Mass spectrometry imaging (MSI) is an emerging technology that is capable of mapping various biomolecules within their native spatial context, and performing spatial multiomics on formalin-fixed paraffin-embedded (FFPE) tissues may further increase the molecular characterization of pathological states. Here we present a novel workflow which enables the sequential MSI of lipids, N-glycans, and tryptic peptides on a single FFPE tissue section and highlight the enhanced molecular characterization that is offered by combining the multiple spatial omics data sets. In murine brain and clear cell renal cell carcinoma (ccRCC) tissue, the three molecular levels provided complementary information and characterized different histological regions. Moreover, when the spatial omics data was integrated, the different histopathological regions of the ccRCC tissue could be better discriminated with respect to the imaging data set of any single omics class. Taken together, these promising findings demonstrate the capability to more comprehensively map the molecular complexity within pathological tissue.

Mass spectrometry detection of inhaled drug in distal fibrotic lung.

BACKGROUND: Currently the only available therapies for fibrotic Interstitial Lung Disease are administered systemically, often causing significant side effects. Inhaled therapy could avoid these but to date there is no evidence that drug can be effectively delivered to distal, fibrosed lung. We set out to combine mass spectrometry and histopathology with rapid sample acquisition using transbronchial cryobiopsy to determine whether an inhaled drug can be delivered to fibrotic, distal lung parenchyma in participants with Interstitial Lung Disease. METHODS: Patients with radiologically and multidisciplinary team confirmed fibrotic Interstitial Lung Disease were eligible for this study. Transbronchial cryobiopsies and endobronchial biopsies were taken from five participants, with Interstitial Lung Disease, within 70 min of administration of a single dose of nebulised ipratropium bromide. Thin tissue cryosections were analysed by Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry imaging and correlated with histopathology. The remainder of the cryobiopsies were homogenised and analysed by Liquid Chromatography-tandem Mass Spectrometry. RESULTS: Drug was detected in proximal and distal lung samples from all participants. Fibrotic regions were identified in research samples of four of the five participants. Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry imaging showed co-location of ipratropium with fibrotic regions in samples from three participants. CONCLUSIONS: In this proof of concept study, using mass spectrometry, we demonstrate for the first-time that an inhaled drug can deposit in distal fibrotic lung parenchyma in patients with Interstitial Lung Disease. This suggests that drugs to treat pulmonary fibrosis could potentially be administered by the inhaled route. Trial registration A prospective clinical study approved by London Camden and Kings Cross Research Ethics Committee and registered on clinicaltrials.gov (NCT03136120).

CD138 Is Expressed in Different Entities of Salivary Gland Cancer and Their Lymph Node Metastases and Therefore Represents a Potential Therapeutic Target.

Advanced salivary gland carcinomas (SGC) often lack therapeutic options. Agents targeting CD138 have recently shown promising results in clinical trials for multiple myeloma and a preclinical trial for triple-negative breast cancer. Immunohistochemistry for CD138 was performed for all patients who had undergone primary surgery for SGC with curative intent. Findings were validated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging. Overall, 111 primary SGC and 13 lymph node metastases from salivary duct carcinomas (SaDu) were evaluated. CD138 expression was found in 60% of all SGC with differing expression across entities (p < 0.01). A mean of 25.2% of the tumor cells in mucoepidermoid carcinoma (MuEp) were positive, followed by epithelial-myoepithelial carcinoma (20.9%), acinic cell carcinoma (16.0%), and SaDu (15.2%). High-/intermediate-grade MuEp showed CD138 expression in a mean of 34.8% of tumor cells. For SaDu, lymph node metastases showed CD138 expression in a mean of 31.2% of tumor cells which correlated with CD138 expression in their primaries (p = 0.01; Spearman's ρ = 0.71). MALDI-MS imaging confirmed the presence of the CD138 protein in SGC. No significant association was found between clinicopathological data, including progression-free survival (p = 0.50) and CD138 expression. CD138 is expressed in the cell membrane of different entities of SGC and SaDu lymph node metastases and therefore represents a potential target for CD138 targeting drugs.

Single-Photon-Induced Post-Ionization to Boost Ion Yields in MALDI Mass Spectrometry Imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a rapidly growing method in the life sciences. However, for many analyte classes, its sensitivity is limited due to poor ionization efficiencies. To mitigate this problem, we here introduce a novel post-ionization scheme based on single-photon induced chemical ionization using pulsed RF-Kr lamps. The fine-vacuum conditions of a dual ion-funnel ion source effectively thermalize the evolving MALDI plume and enable ample gas-phase reactions. Injected chemical dopants crucially support fragment-less ionization to [M+H]+ /[M-H]- species. Based on this interplay, numerous glycerophospho-, sphingo-, and further lipids, registered from mammalian tissue sections, were boosted by up to three orders of magnitude, similar to results obtained with laser-based post-ionization (MALDI-2). Experiments with deuterated matrix and dopant, however, indicated complex chemical ionization pathways different from MALDI-2.

Brain Lipid Dynamics in Amyloid Precursor Protein/Presenilin 1 Mouse Model of Early Alzheimer's Disease by Desorption Electrospray Ionization and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging Techniques.

Alzheimer's disease (AD) is closely associated with lipid metabolism dysfunction. However, space distribution and metabolism of aberrant lipids in the brain of early-stage AD mouse remain unclear. In our current work, a novel lipidomics method based on mass spectrometry imaging was developed to visually disclose molecular perturbation and characterize space distribution in the brain of double transgenic amyloid precursor protein/presenilin 1 mouse (2 and 3 months old). Significant changes were detected, including phosphatidylethanolamines, phosphatidylcholines, fatty acids, lysophospholipids, and glycerides in AD mouse brain. The results in this study suggest that these significantly altered lipid metabolic pathways (glycerophospholipid metabolism) may be implicated in early-stage AD. Our work deepens the understanding of the physio-pathologic mechanism of early-stage AD.

Effective processing and evaluation of chemical imaging data with respect to morphological features of the zebrafish embryo.

A workflow was developed and implemented in a software tool for the automated combination of spatially resolved laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) data and data on the morphology of the biological tissue. Making use of a recently published biological annotation software, FishImager automatically assigns the biological feature as regions of interest (ROIs) and overlays them with the quantitative LA-ICP-MS data. Furthermore, statistical tools including cluster algorithms can be applied to the elemental intensity data and directly compared with the ROIs. This is effectively visualized in heatmaps. This allows gaining statistical significance on distribution and co-localization patterns. Finally, the biological functions of the assigned ROIs can then be easily linked with elemental distributions. We demonstrate the versatility of FishImager with quantitative LA-ICP-MS data of the zebrafish embryo tissue. The distribution of natural elements and xenobiotics is analyzed and discussed. With the help of FishImager, it was possible to identify compartments affected by toxicity effects or biological mechanisms to eliminate the xenobiotic. The presented workflow can be used for clinical and ecotoxicological testing, for example. Ultimately, it is a tool to simplify and reproduce interpretations of imaging LA-ICP-MS data in many applications.

Assessing the reproducibility of labelled antibody binding in quantitative multiplexed immuno-mass spectrometry imaging.

Immuno-mass spectrometry imaging (iMSI) uses laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to determine the spatial expression of biomolecules in tissue sections following immunolabelling with antibodies conjugated to a metal reporter. As with all immunolabelling techniques, the binding efficiency of multiplexed staining can be affected by a number of factors including epitope blocking and other forms of steric hindrance. To date, the effects on the binding of metal-conjugated antibodies to their epitopes in a multiplexed analysis have yet to be quantitatively explored by iMSI. Here we describe a protocol to investigate the effects of multiplexing on reproducible binding using the muscle proteins, dystrophin, sarcospan, and myosin as a model, with antibodies conjugated with Maxpar® reagents before histological application to murine quadriceps sections using standard immunolabelling protocols and imaging with LA-ICP-MS. The antibodies were each individually applied to eight sections, and multiplexed to another eight sections. The average concentrations of the lanthanide analytes were determined, before statistical analyses found there was no significant difference between the individual and multiplexed application of the antibodies. These analyses provide a framework for ensuring reproducibility of antibody binding during multiplexed iMSI, which will allow quantitative exploration of protein-protein interactions and provide a greater understanding of fundamental biological processes during healthy and diseased states.

Mammalian ovarian lipid distributions by desorption electrospray ionization-mass spectrometry (DESI-MS) imaging.

Merging optical images of tissue sections with the spatial distributions of molecules seen by imaging mass spectrometry is a powerful approach to better understand the metabolic roles of the mapped molecules. Here, we use histologically friendly desorption electrospray ionization-mass spectrometry (DESI-MS) to map the lipid distribution in tissue sections of ovaries from cows (N = 8), sows (N = 3), and mice (N = 12). Morphologically friendly DESI-MS imaging allows the same sections to be examined for morphological information. Independent of the species, ovarian follicles, corpora lutea, and stroma could be differentiated by principal component analysis, showing that lipid profiles are well conserved among species. As examples of specific findings, arachidonic acid and the phosphatidylinositol PI(38:4), were both found concentrated in the follicles and corpora lutea, structures that promoted ovulation and implantation, respectively. Adrenic acid was spatially located in the corpora lutea, suggesting the importance of this fatty acid in the ovary luteal phase. In summary, lipid information captured by DESI-MS imaging could be related to ovarian structures and data were all conserved among cows, sows, and mice. Further application of DESI-MS imaging to either physiological or pathophysiological models of reproductive conditions will likely expand knowledge of the roles of specific lipids and pathways in ovarian activity and mammalian fertility. Graphical abstract Desorption electrospray ionization-mass spectrometry (DESI-MS) is performed directly from frozen ovarian tissue sections placed onto glass slides. Because the desorption and ionization process of small molecules is so gentle, the tissue architecture is preserved. The sample can then be stained and tissue morphology information can be overlaid with the chemical information obtained by DESI-MS.

Chronic white matter lesion activity predicts clinical progression in primary progressive multiple sclerosis.

Chronic active and slowly expanding lesions with smouldering inflammation are neuropathological correlates of progressive multiple sclerosis pathology. T1 hypointense volume and signal intensity on T1-weighted MRI reflect brain tissue damage that may develop within newly formed acute focal inflammatory lesions or in chronic pre-existing lesions without signs of acute inflammation. Using a recently developed method to identify slowly expanding/evolving lesions in vivo from longitudinal conventional T2- and T1-weighted brain MRI scans, we measured the relative amount of chronic lesion activity as measured by change in T1 volume and intensity within slowly expanding/evolving lesions and non-slowly expanding/evolving lesion areas of baseline pre-existing T2 lesions, and assessed the effect of ocrelizumab on this outcome in patients with primary progressive multiple sclerosis participating in the phase III, randomized, placebo-controlled, double-blind ORATORIO study (n = 732, NCT01194570). We also assessed the predictive value of T1-weighted measures of chronic lesion activity for clinical multiple sclerosis progression as reflected by a composite disability measure including the Expanded Disability Status Scale, Timed 25-Foot Walk and 9-Hole Peg Test. We observed in this clinical trial population that most of total brain non-enhancing T1 hypointense lesion volume accumulation was derived from chronic lesion activity within pre-existing T2 lesions rather than new T2 lesion formation. There was a larger decrease in mean normalized T1 signal intensity and greater relative accumulation of T1 hypointense volume in slowly expanding/evolving lesions compared with non-slowly expanding/evolving lesions. Chronic white matter lesion activity measured by longitudinal T1 hypointense lesion volume accumulation in slowly expanding/evolving lesions and in non-slowly expanding/evolving lesion areas of pre-existing lesions predicted subsequent composite disability progression with consistent trends on all components of the composite. In contrast, whole brain volume loss and acute lesion activity measured by longitudinal T1 hypointense lesion volume accumulation in new focal T2 lesions did not predict subsequent composite disability progression in this trial at the population level. Ocrelizumab reduced longitudinal measures of chronic lesion activity such as T1 hypointense lesion volume accumulation and mean normalized T1 signal intensity decrease both within regions of pre-existing T2 lesions identified as slowly expanding/evolving and in non-slowly expanding/evolving lesions. Using conventional brain MRI, T1-weighted intensity-based measures of chronic white matter lesion activity predict clinical progression in primary progressive multiple sclerosis and may qualify as a longitudinal in vivo neuroimaging correlate of smouldering demyelination and axonal loss in chronic active lesions due to CNS-resident inflammation and/or secondary neurodegeneration across the multiple sclerosis disease continuum.

A Clearance Period after Soluble Lead Nanoparticle Inhalation Did Not Ameliorate the Negative Effects on Target Tissues Due to Decreased Immune Response.

The inhalation of metal (including lead) nanoparticles poses a real health issue to people and animals living in polluted and/or industrial areas. In this study, we exposed mice to lead(II) nitrate nanoparticles [Pb(NO3)2 NPs], which represent a highly soluble form of lead, by inhalation. We aimed to uncover the effects of their exposure on individual target organs and to reveal potential variability in the lead clearance. We examined (i) lead biodistribution in target organs using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) and atomic absorption spectrometry (AAS), (ii) lead effect on histopathological changes and immune cells response in secondary target organs and (iii) the clearance ability of target organs. In the lungs and liver, Pb(NO3)2 NP inhalation induced serious structural changes and their damage was present even after a 5-week clearance period despite the lead having been almost completely eliminated from the tissues. The numbers of macrophages significantly decreased after 11-week Pb(NO3)2 NP inhalation; conversely, abundance of alpha-smooth muscle actin (α-SMA)-positive cells, which are responsible for augmented collagen production, increased in both tissues. Moreover, the expression of nuclear factor κB (NF-κB) and selected cytokines, such as tumor necrosis factor alpha (TNFα), transforming growth factor beta 1 (TGFß1), interleukin 6(IL-6), IL-1α and IL-1ß , displayed a tissue-specific response to lead exposure. In summary, diminished inflammatory response in tissues after Pb(NO3)2 NPs inhalation was associated with prolonged negative effect of lead on tissues, as demonstrated by sustained pathological changes in target organs, even after long clearance period.