AbstractBackgroundImmune response changes have been reported in amyotrophic lateral sclerosis (ALS), but their clinical relevance remains undetermined. Therefore, we aim to evaluate the relationships between blood leukocyte subpopulations and prognosis of ALS.MethodsA longitudinal cohort of 288 ALS patients with up to 5 years of follow-up during 2015-2020 were recruited at the only tertiary referral center for ALS in Stockholm, Sweden. Routine differential leukocyte counts, and determination of lymphocyte subpopulations including an extended T cell panel with flow cytometry, collected at diagnosis and at regular intervals thereafter. The primary outcome was risk of death (alternatively use of invasive ventilation) after diagnosis of ALS. The secondary outcomes included repeatedly measured functional status - through Amyotrophic Lateral Sclerosis Functional Rating Scale-revised (ALSFRS-R) score and disease progression rate. Cox model was used to evaluate the associations between leukocytes and risk of death. Generalized estimating equation model (GEE) was used to assess the correlation between leukocytes and ALSFRS-R score and disease progression rate.ResultsThe counts of leukocytes, neutrophils and monocytes increased gradually over time since diagnosis and were negatively correlated with ALSFRS-R score, but not associated with risk of death or disease progression rate. Focusing on lymphocyte subpopulations, increasing counts of natural killer (NK) cells (HR=0.61, 95% CI= [0.42-0.88] per SD increase) and proportions of Th2-diffrentiated CD4+ central memory (CM) T cells (HR=0.64, 95% CI= [0.48-0.85] per SD increase) were correlated with a lower risk of death. Increasing proportions of CD4+ effector memory cells re-expressing CD45RA (EMRA) T cells (HR=1.39, 95% CI= [1.01-1.92] per SD increase) and CD8+ T cells (HR=1.38, 95% CI= [1.03-1.86] per SD increase) were associated with a higher risk of death. None of the lymphocyte subpopulations was correlated with ALSFRS-R score or disease progression rate.ConclusionOur findings suggest a dual role of immune responses in ALS prognosis, where neutrophils and monocytes primarily reflect functional status whereas NK cells and different T lymphocyte populations act as prognostic markers for survival. The findings also provide insights for cell-specific treatment for ALS.FundingThis work was supported by the European Research Council (ERC) Starting Grant (MegaALS, No. 802091), the Swedish Research Council (No. 2019-01088), Karolinska Institutet (Strategic Research Area in Epidemiology and Senior Researcher Award), and China Scholarship Council.

SummaryIntroductionNew strategies for weight loss and weight maintenance in humans are needed. Human brown adipose tissue (BAT) can stimulate energy expenditure and may be a potential therapeutic target for obesity and type 2 diabetes. However, whether exercise training is an efficient stimulus to activate and recruit BAT remains to be explored. This study aimed to evaluate whether regular exercise training affects cold‐stimulated BAT metabolism and, if so, whether this was associated with changes in plasma metabolites.MethodsHealthy sedentary men (n = 11; aged 31 [SD 7] years; body mass index 23 [0.9] kg m−2; VO2 max 39 [7.6] mL min−1 kg−1) participated in a 6‐week exercise training intervention. Fasting BAT and neck muscle glucose uptake (GU) were measured using quantitative [18F]fluorodeoxyglucose positron emission tomography–magnetic resonance imaging three times: (1) before training at room temperature and (2) before and (3) after the training period during cold stimulation. Cervico‐thoracic BAT mass was measured using MRI signal fat fraction maps. Plasma metabolites were analysed using nuclear magnetic resonance spectroscopy.ResultsCold exposure increased supraclavicular BAT GU by threefold (p < 0.001), energy expenditure by 59% (p < 0.001) and plasma fatty acids (p < 0.01). Exercise training had no effect on cold‐induced GU in BAT or neck muscles. Training increased aerobic capacity (p = 0.01) and decreased visceral fat (p = 0.02) and cervico‐thoracic BAT mass (p = 0.003). Additionally, training decreased very low‐density lipoprotein particle size (p = 0.04), triglycerides within chylomicrons (p = 0.04) and small high‐density lipoprotein (p = 0.04).ConclusionsAlthough exercise training plays an important role for metabolic health, its beneficial effects on whole body metabolism through physiological adaptations seem to be independent of BAT activation in young, sedentary men.

ObjectiveImmunotherapy using vitamin D (vitD(3)) and phenylbutyrate (PBA) may support standard drug regimens used to treat infectious diseases. We investigated if vitD(3) + PBA enhanced clinical recovery from pulmonary tuberculosis (TB). MethodsA randomized controlled trial was conducted in Addis Ababa, Ethiopia. Patients with smear-positive or smear-negative TB received daily oral supplementation with 5000 IU vitD(3) and 2 x 500 mg PBA or placebo for 16 weeks, together with 6-month chemotherapy. Primary end-point: reduction of a clinical composite TB score at week 8 compared with baseline using modified intention-to-treat (mITT, n = 348) and per-protocol (n = 296) analyses. Secondary end-points: primary and modified TB scores (week 0, 4, 8, 16, 24), sputum conversion, radiological findings and plasma 25(OH)D-3 concentrations. ResultsMost subjects had low baseline plasma 25(OH)D-3 levels that increased gradually in the vitD(3) + PBA group compared with placebo (P < 0.0001) from week 0 to 16 (mean 34.7 vs. 127.4 nmol L-1). In the adjusted mITT analysis, the primary TB score was significantly reduced in the intervention group at week 8 (-0.52, 95% CI -0.93, -0.10; P = 0.015) while the modified TB score was reduced at week 8 (-0.58, 95% CI -1.02, -0.14; P = 0.01) and 16 (-0.34, 95% CI -0.64, -0.03; P = 0.03). VitD(3) + PBA had no effect on longitudinal sputum-smear conversion (P = 0.98). Clinical adverse events were more common in the placebo group (24.3%) compared with the vitD(3) + PBA group (12.6%). ConclusionDaily supplementation with vitD(3) + PBA may ameliorate clinical TB symptoms and disease-specific complications, while the intervention had no effect on bacterial clearance in sputum.

Mapping structural connectivity in healthy adults for the Human Connectome Project (HCP) benefits from high quality, high resolution, multiband (MB)-accelerated whole brain diffusion MRI (dMRI). Acquiring such data at ultrahigh fields (7T and above) can improve intrinsic signal-to-noise ratio (SNR), but suffers from shorter T2 and T2(⁎) relaxation times, increased B1(+) inhomogeneity (resulting in signal loss in cerebellar and temporal lobe regions), and increased power deposition (i.e. specific absorption rate (SAR)), thereby limiting our ability to reduce the repetition time (TR). Here, we present recent developments and optimizations in 7T image acquisitions for the HCP that allow us to efficiently obtain high quality, high resolution whole brain in-vivo dMRI data at 7T. These data show spatial details typically seen only in ex-vivo studies and complement already very high quality 3T HCP data in the same subjects. The advances are the result of intensive pilot studies aimed at mitigating the limitations of dMRI at 7T. The data quality and methods described here are representative of the datasets that will be made freely available to the community in 2015.

With the increasing interest in clinical trials with regulatory T cells (Tregs), immunological profiling of prospective target groups and standardized procedures for Treg isolation are needed. In this study, flow cytometry was used to assess peripheral blood lymphocyte profiles of young healthy individuals and patients undergoing haemodialysis treatment. Tregs obtained from the former may be used in haematopoietic stem cell transplantation and Tregs from the latter in the prevention of kidney transplant rejection. FOXP3 mRNA expression with accompanying isoform distribution was also assessed by the quantitative reverse transcriptase polymerase chain reaction. Flow-cytometric gating strategies were systematically analysed to optimize the isolation of Tregs. Our findings showed an overall similar immunological profile of both cohorts in spite of great differences in both age and health. Analysis of flow-cytometric gating techniques highlighted the importance of gating for both CD25high and CD127low expression in the isolation of FOXP3-positive cells. This study provides additional insight into the immunological profile of young healthy individuals and uraemic patients as well as in-depth analysis of flow-cytometric gating strategies for Treg isolation, supporting the development of Treg therapy using cells from healthy donors and uraemic patients.

Resonant inelastic x-ray scattering spectra excited in the immediate vicinity of the core-level ionization thresholds of N-2 have been recorded. Final states of well-resolved symmetry-selected Rydberg series converging to valence-level ionization thresholds with vibrational excitations are observed. The results are well described by a quasi-two-step model which assumes that the excited electron is unaffected by the radiative decay. This threshold dynamics simplifies the interpretation of resonant inelastic x-ray scattering spectra considerably and facilitates characterization of low-energy excited final states in molecular systems.

The electronic structure of the amino acid L-cysteine in an aqueous environment was studied using resonant inelastic soft X-ray scattering (RIXS) in a 2D map representation and analyzed in the framework of a "building block" approach. The element selectivity of RIXS allows a local investigation of the electronic structure of the three functional groups of cysteine, namely, the carboxyl, amino, and thiol groups, by measuring at the O K, N K, and S L2,3 edges, respectively. Variation of the pH value allows an investigation of molecules with protonated and deprotonated functional groups, which can then be compared with simple reference molecules that represent the isolated functional groups. We find that such building blocks can provide an excellent description of X-ray emission spectroscopy (XES) and RIXS spectra, but only if all nearest-neighbor atoms are included. This finding is analogous to the building block principle commonly used in X-ray absorption spectroscopy. The building blocks show a distinct spectral character (fingerprint) and allow a comprehensive interpretation of the cysteine spectra. This simple approach opens the path to investigate the electronic structure of more complex biological molecules in aqueous solutions using XES and RIXS.

Tuberculosis ( TB ) is an airborne infectious disease that kills almost two million individuals every year. Multidrug‐resistant ( MDR ) TB is caused by strains of Mycobacterium tuberculosis ( M. tb ) resistant to isoniazid and rifampin, the backbone of first‐line antitubercular treatment. MDR TB affects an estimated 500 000 new patients annually. Genetic analysis of drug‐resistant MDR ‐ TB showed that airborne transmission of undetected and untreated strains played a major role in disease outbreaks. The need for new TB vaccines and faster diagnostics, as well as the development of new drugs, has recently been highlighted. The major problem in terms of current TB research and clinical demands is the increasing number of cases of extensively drug‐resistant and ‘treatment‐refractory’ TB . An emerging scenario of adjunct host‐directed therapies is intended to target pulmonary TB where inflammatory processes can be deleterious and lead to immune exhaustion. ‘Target‐organ‐saving’ strategies may be warranted to prevent damage to infected tissues and achieve focused, clinically relevant and long‐lasting anti‐ M. tb cellular immune responses. Candidates for such interventions may be biological agents or already approved drugs that can be ‘re‐purposed’ to interfere with biologically relevant cellular checkpoints. Here, we review current concepts of inflammation in TB disease and discuss candidate pathways for host‐directed therapies to achieve better clinical outcomes.

The ordering of supersaturated cubic titanium aluminum nitride (c-Ti0.35Al0.65N) coatings is probed from room temperature up to and above the point of spinodal decomposition, using Near Edge X-ray Absorption Fine-structure (NEXAFS) and first principles calculations. The measured and calculated nitrogen (N) K spectra suggest that unoccupied N p states hybridize with Ti d states. When temperature is raised the N p–Ti d overlap decreases, whereas hybridization between N p and Al p tends to increase. The observed spectral changes with temperature together with calculations of defect heat of formation suggest a depletion of N in the surroundings of Ti in c-Ti1−xAlxN and/or in the formed c-TiN.

Functional neuroimaging studies suggest that the anterior, mid, and posterior division of the insula subserve different functions in the perception of pain. The anterior insula (AI) has predominantly been associated with cognitive-affective aspects of pain, while the mid and posterior divisions have been implicated in sensory-discriminative processing. We examined whether this functional segregation is paralleled by differences in (1) structural and (2) resting state connectivity and (3) in correlations with pain-relevant psychological traits. Analyses were restricted to the 3 insular subdivisions and other pain-related brain regions. Both type of analyses revealed largely overlapping results. The AI division was predominantly connected to the ventrolateral prefrontal cortex (structural and resting state connectivity) and orbitofrontal cortex (structural connectivity). In contrast, the posterior insula showed strong connections to the primary somatosensory cortex (SI; structural connectivity) and secondary somatosensory cortex (SII; structural and resting state connectivity). The mid insula displayed a hybrid connectivity pattern with strong connections with the ventrolateral prefrontal cortex, SII (structural and resting state connectivity) and SI (structural connectivity). Moreover, resting state connectivity revealed strong connectivity of all 3 subdivisions with the thalamus. On the behavioural level, AI structural connectivity was related to the individual degree of pain vigilance and awareness that showed a positive correlation with AI-amygdala connectivity and a negative correlation with AI-rostral anterior cingulate cortex connectivity. In sum, our findings show a differential structural and resting state connectivity for the anterior, mid, and posterior insula with other pain-relevant brain regions, which might at least partly explain their different functional profiles in pain processing. (C) 2014 The Authors. Published by Elsevier B.V.

A detailed study of inelastic x-ray scattering from the ground state to the 3Σg(3σ(g)(-1)3s(g)1) state of the O2 molecule is presented. The observed angular anisotropy shows that the vibrational excitations within this final state are strongly dependent on the polarization of the incident radiation. The analysis demonstrates that this is a manifestation of interference between resonant and direct nonresonant inelastic x-ray scattering. This interference provides a new tool to monitor nuclear dynamics by relative rotation of the polarization vectors of the incident and scattered photons.

PURPOSE Signal dropout caused by coherent movement (subject or pulsatile movement) during the diffusion encoding is a problem for diffusion imaging. Any component of the movement that is co-linear with the diffusion gradient will lead to dropout, the magnitude of which will depend on factors such as phase-encode band width and if/how partial k-space acquisition is performed. These dropouts will typically affect a whole slice (in the case of subject movement) or a substantial part of the brain (for pulsatile effects) [1]. The consequence for modeling/tractography is that the dropout will be interpreted as high diffusivity along the direction of the diffusion gradient for the slice with dropout. Specifically for pulsatile effects this can cause a systematic overestimation of the diffusivity along the direction of high tissue velocity in basal-medial regions and bias fibre orientation estimation. Our proposed method will detect outliers in a slice-by-slice basis and, instead of “just” rejecting it propose a data-driven replacement for the slice. METHODS We have recently developed a method (EDDY) for simultaneously correcting for eddy current-induced distortions and subject movements [2]. It is based on comparing a Gaussian Process based prediction of the data with the observed data in the native scanner space. The sum-of-squares of the observed difference drives the estimation of the distortion and movement parameters. If observation-minus-prediction has a negative nonzero sum (across voxels) it is indicative of dropout being present in the observed data. Each slice of each diffusion weighted volume yields one such number which allows us to convert them to z-scores and build an empirical distribution of slice differences. The z-scores can be thresholded at an arbitrary level (4 in our examples) to define an outlier. The outlier slice can then be replaced by its prediction for the remaining iterations of EDDY and also for the generation of the final pre-processed data. RESULTS The method has proven to be very sensitive at detecting outliers. When “simulating dropout” by multiplying a slice by some number < 1 it has been able to reliably detect “whole slice” dropout of as little as 2%. The figure to the right shows examples of outliers that were detected and replaced in a dataset with 66 slices and 300 volumes from the HCP project [3,4]. DISCUSSION Unlike previous methods, such as RESTORE [5], our method considers a summary statistic from an entire slice when deciding if a point is an outlier or not. This means that we can achieve higher sensitivity than is possible when considering voxels in isolation. It also makes sense as the nature of the artifact means that it will affect whole slices or at least substantial parts of the brain in a given slice. This is possible because the comparison is performed in the space of the original acquisition, i.e. the observed slice has not been interpolated or processed in any other way. If instead the movement/distortion correction is performed prior to, and independent of, the outlier detection affected and unaffected voxels will be mixed through the interpolation leading to a loss of sensitivity. In addition a severe slice dropout can bias the estimation of movement/distortion. We therefore believe that it is important that the two corrections are incorporated into a framework that performs them simultaneously. Another novel aspect of our work is that it detects “outliers” using a non-parametric Gaussian Process framework rather than a specific model (e.g. the diffusion tensor). That means that it is datadriven and not coupled to a specific model and can therefore represent the signal from voxels with complex fibre patterns whereas the diffusion model might instead “detect” outlier points as demonstrated in the figure above. As the outlier detection is not tied to fibre orientation estimation the method can provide corrected data useable by all HARDI approaches, for example parametric [6] and non-parametric [7], and single and multi-shell [8]. REFERENCES 1. Jones & Pierpaoli, 2004, ISMRM:225. 2. Andersson et al., 2012, ISMRM:2426. 3. Van Essen et al., 2013, NeuroImage:80:62-79. 4. Sotiropoulos et al., 2013, NeuroImage 80:125-143. 5. Chang et al., 2005, MRM:53:1088-1095. 6. Behrens et al., 2007, NeuroImage 34:144-155. 7. Descoteaux et al., 2007, MRM, 58:497-510. 8. Wu & Alexander, 2007, NeuroImage 36:617-629. Diffusion gradient Basal Cranial

Monocytes have long been considered a heterogeneous group of cells both in terms of morphology and function. In humans, three distinct subsets have been described based on their differential expression of the cell surface markers CD14 and CD16. However, the relationship between these subsets and the production of cytokines has for the most part been based on ELISA measurements, making it difficult to draw conclusions as to their functional profile on the cellular level. In this study, we have investigated lipoteichoic acid (LTA)‐ and lipopolysaccharide (LPS)‐induced cytokine secretion by monocytes using the FluoroSpot technique. This method measures the number of cytokine‐secreting cells on the single‐cell level and uses fluorescent detection, allowing for the simultaneous analysis of two cytokines from the same population of isolated cells. By this approach, human monocytes from healthy volunteers could be divided into several subgroups as IL‐1β, IL‐6, TNF‐α and MIP‐1β were secreted by larger populations of responding cells (25.9–39.2%) compared with the smaller populations of GM‐CSF (9.1%), IL‐10 (1.3%) and IL‐12p40 (1.2%). Furthermore, when studying co‐secretion in FluoroSpot, an intricate relationship between the monocytes secreting IL‐1β and/or IL‐6 and those secreting TNF‐α, MIP‐1β, GM‐CSF, IL‐10 and IL‐12p40 was revealed. In this way, dissecting the secretion pattern of the monocytes in response to TLR‐2 or TLR‐4 stimulation, several subpopulations with distinct cytokine‐secreting profiles could be identified.

Interleukin-7 (IL-7) and the IL-7 receptor (IL-7R) have been shown to be alternatively spliced in infectious diseases. We tested IL-7 and IL-7R splicing in a tuberculosis (TB)-vaccine/Mycobacterium tuberculosis (Mtb)-challenge model in non-human primates (NHPs). Differential IL-7 splicing was detected in peripheral blood mononuclear cells (PBMCs) from 15/15 NHPs showing 6 different IL-7 spliced isoforms. This pattern did not change after infection with virulent Mtb. We demonstrated increased IL-7 (6 exon) and IL-17 protein production in lung tissue along with concomitant decreased transforming growth factor-β (TGF-β) from NHPs (vaccinated with a recombinant BCG (rBCG)) who showed increased survival after Mtb challenge. IL-7 increased IL-17 and interferon-γ (IFN-γ) gene and protein expression in PBMCs. Mtb-infected NHPs showed differential IL-7R splicing associated with the anatomical location and tissue origin, that is, in lung tissue, hilus, axillary lymph nodes (LNs) and spleen. Differential splicing of the IL-7R was typical for healthy (non-Mtb infected) and for Mtb-infected lung tissue with a dominant expression of soluble IL-7R (sIL-7R) receptor lacking exon 6 (9:1 ratio of sIL-7R/cell-bound IL-7R). Differential ratios of cell-bound vs sIL-7R could be observed in hilus and axillary LNs from Mtb-infected NHPs with an inversed ratio of 1:9 (sIL-7R/cell-bound IL-7R) in spleen and PBMCs. Soluble IL-7R is exclusively present in lung tissue.

Resonant inelastic soft x-ray scattering (RIXS) spectra excited at the 1σ(g) → 3σ(u) resonance in gas-phase O2 show excitations due to the nuclear degrees of freedom with up to 35 well-resolved discrete vibronic states and a continuum due to the kinetic energy distribution of the separated atoms. The RIXS profile demonstrates spatial quantum beats caused by two interfering wave packets with different momenta as the atoms separate. Thomson scattering strongly affects both the spectral profile and the scattering anisotropy.

Resonant inelastic x-ray scattering spectra excited at the O1s(-1)pi* resonance of liquid acetone are presented. Scattering to the electronic ground state shows a resolved vibrational progression where the dominant contribution is due to the C-O stretching mode, thus demonstrating a unique sensitivity of the method to the local potential energy surface in complex molecular systems. For scattering to electronically excited states, soft vibrational modes and, to a smaller extent, intermolecular interactions give a broadening, which blurs the vibrational fine structure. It is predicted that environmental broadening is dominant in aqueous acetone.

Thermal treatment of supersaturated Ti1−xAlxN films (x≈0.67) with a dominant ternary cubic-phase were performed in the 700–1000°C range. Grazing incidence X-ray diffraction (GIXRD) shows that, for annealing temperatures up to 800°C, the film structure undergoes the formation of coherent cubic AlN (c-AlN) and TiN (c-TiN) nanocrystallites via spinodal decomposition and, at higher temperatures (⩾900°C), GIXRD shows that the c-AlN phase transforms into the thermodynamically more stable hexagonal AlN (h-AlN). X-ray absorption near-edge structure (XANES) at the Ti K-edge is consistent with spinodal decomposition taking place at 800°C, while Al K-edge and N K-edge XANES and X-ray emission data show the nucleation of the h-AlN phase at temperatures >800°C, in agreement with the two-step decomposition process for rock-salt structured TiAlN, which was also supported by X-ray diffraction patterns and first-principle calculations. Further, the resonant inelastic X-ray scattering technique near the N K-edge revealed that N2 is formed as a consequence of the phase transformation process.