Exhausted CD4⁺ T Cells during Malaria Exhibit Reduced mTORc1 Activity Correlated with Loss of T-bet Expression

CD4⁺ T cell functional inhibition (exhaustion) is a hallmark of malaria and correlates with impaired parasite control and infection chronicity. However, the mechanisms of CD4⁺ T cell exhaustion are still poorly understood. In this study, we show that Ag-experienced (Ag-exp) CD4⁺ T cell exhaustion during Plasmodium yoelii nonlethal infection occurs alongside the reduction in mammalian target of rapamycin (mTOR) activity and restriction in CD4+ T cell glycolytic capacity. We demonstrate that the loss of glycolytic metabolism and mTOR activity within the exhausted Ag-expCD4⁺ T cell population during infection coincides with reduction in T-bet expression. T-bet was found to directly bind to and control the transcription of various mTOR and metabolism-related genes within effector CD4⁺ T cells. Consistent with this, Ag-expTh1 cells exhibited significantly higher and sustained mTOR activity than effector T-bet- (non-Th1) Ag-expT cells throughout the course of malaria. We identified mTOR to be redundant for sustaining T-bet expression in activated Th1 cells, whereas mTOR was necessary but not sufficient for maintaining IFN-γ production by Th1 cells. Immunotherapy targeting PD-1, CTLA-4, and IL-27 blocked CD4⁺ T cell exhaustion during malaria infection and was associated with elevated T-bet expression and a concomitant increased CD4⁺ T cell glycolytic metabolism. Collectively, our data suggest that mTOR activity is linked to T-bet in Ag-expCD4⁺ T cells but that reduction in mTOR activity may not directly underpin Ag-expTh1 cell loss and exhaustion during malaria infection. These data have implications for therapeutic reactivation of exhausted CD4⁺ T cells during malaria infection and other chronic conditions

Nanostructured Bimetallic Block Copolymers as Precursors to Magnetic FePt Nanoparticles

Phase-separated block copolymers (BCPs) that function as precursors to arrays of FePt nanoparticles (NPs) are of potential interest for the creation of media for the next-generation high-density magnetic data storage devices. A series of bimetallic BCPs has been synthesized by incorporating a complex containing Fe and Pt centers into the coordinating block of four different poly­(styrene-<i>b</i>-4-vinylpyridine)­s (PS-<i>b</i>-P4VPs, <b>P1–P4</b>). To facilitate phase separation for the resulting metalated BCPs (<b>PM1–PM4</b>), a loading of the FePt-bimetallic complex corresponding to ca. 20% was used. The bulk and thin-film self-assembly of these BCPs was studied by transmission electron microscopy (TEM) and atomic force microscopy, respectively. The spherical and cylindrical morphologies observed for the metalated BCPs corresponded to those observed for the metal-free BCPs. The products from the pyrolysis of the BCPs in bulk were also characterized by TEM, powder X-ray diffraction, and energy-dispersive X-ray spectroscopy, which indicated that the FePt NPs formed exist in an fct phase with average particle sizes of ca. 4–8 nm within a carbonaceous matrix. A comparison of the pyrolysis behavior of the metalated BCP (<b>PM3</b>), the metalated <b>P4VP</b> homopolymer (<b>PM5</b>), and the molecular model organometallic complex revealed the importance of using a nanostructured BCP approach for the synthesis of ferromagnetic FePt NPs with a smaller average NP size and a close to 1:1 Fe/Pt stoichiometric ratio

Dysregulation of glucose metabolism is an early event in sporadic Parkinson's disease

AbstractUnlike most other cell types, neurons preferentially metabolize glucose via the pentose phosphate pathway (PPP) to maintain their antioxidant status. Inhibiting the PPP in neuronal cell models causes cell death. In rodents, inhibition of this pathway causes selective dopaminergic cell death leading to motor deficits resembling parkinsonism. Using postmortem human brain tissue, we characterized glucose metabolism via the PPP in sporadic Parkinson's disease (PD), Alzheimer's disease (AD), and controls. AD brains showed increased nicotinamide adenine dinucleotide phosphate (NADPH) production in areas affected by disease. In PD however, increased NADPH production was only seen in the affected areas of late-stage cases. Quantifying PPP NADPH-producing enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase by enzyme-linked immunosorbent assay, showed a reduction in the putamen of early-stage PD and interestingly in the cerebellum of early and late-stage PD. Importantly, there was no decrease in enzyme levels in the cortex, putamen, or cerebellum of AD. Our results suggest that down-regulation of PPP enzymes and a failure to increase antioxidant reserve is an early event in the pathogenesis of sporadic PD

Exhausted CD4+ T Cells during Malaria Exhibit Reduced mTORc1 Activity Correlated with Loss of T-bet Expression

CD4&lt;sup&gt;+&lt;/sup&gt; T cell functional inhibition (exhaustion) is a hallmark of malaria and correlates with impaired parasite control and infection chronicity. However, the mechanisms of CD4&lt;sup&gt;+&lt;/sup&gt; T cell exhaustion are still poorly understood. In this study, we show that Ag-experienced (&lt;i&gt;Ag-exp&lt;/i&gt;) CD4&lt;sup&gt;+&lt;/sup&gt; T cell exhaustion during &lt;i&gt;Plasmodium yoelii&lt;/i&gt; nonlethal infection occurs alongside the reduction in mammalian target of rapamycin (mTOR) activity and restriction in CD4&lt;sup&gt;+&lt;/sup&gt; T cell glycolytic capacity. We demonstrate that the loss of glycolytic metabolism and mTOR activity within the exhausted &lt;i&gt;Ag-exp&lt;/i&gt;CD4&lt;sup&gt;+&lt;/sup&gt; T cell population during infection coincides with reduction in T-bet expression. T-bet was found to directly bind to and control the transcription of various mTOR and metabolism-related genes within effector CD4&lt;sup&gt;+&lt;/sup&gt; T cells. Consistent with this, &lt;i&gt;Ag-exp&lt;/i&gt;Th1 cells exhibited significantly higher and sustained mTOR activity than effector T-bet- (non-Th1) &lt;i&gt;Ag-exp&lt;/i&gt;T cells throughout the course of malaria. We identified mTOR to be redundant for sustaining T-bet expression in activated Th1 cells, whereas mTOR was necessary but not sufficient for maintaining IFN-&#x3B3; production by Th1 cells. Immunotherapy targeting PD-1, CTLA-4, and IL-27 blocked CD4&lt;sup&gt;+&lt;/sup&gt; T cell exhaustion during malaria infection and was associated with elevated T-bet expression and a concomitant increased CD4&lt;sup&gt;+&lt;/sup&gt; T cell glycolytic metabolism. Collectively, our data suggest that mTOR activity is linked to T-bet in &lt;i&gt;Ag-exp&lt;/i&gt;CD4&lt;sup&gt;+&lt;/sup&gt; T cells but that reduction in mTOR activity may not directly underpin &lt;i&gt;Ag-exp&lt;/i&gt;Th1 cell loss and exhaustion during malaria infection. These data have implications for therapeutic reactivation of exhausted CD4&lt;sup&gt;+&lt;/sup&gt; T cells during malaria infection and other chronic conditions

An open-source solution for advanced imaging flow cytometry data analysis using machine learning

Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information. As a result, the quality, reproducibility and rigour of results are limited by the skill, experience and ingenuity of the data analyst. Here, we describe a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated and corrected raw image files (.rif) data files from an imaging flow cytometer (the proprietary .cif file format) are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analysed using cutting-edge machine learning and clustering approaches using “user-friendly” platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data set. It will help to reveal otherwise unappreciated populations of cells based on features that may be hidden to the human eye that include subtle measured differences in label free detection channels such as bright-field and dark-field imagery

N-Acetyl Cysteine May Support Dopamine Neurons in Parkinson\u27s Disease: Preliminary Clinical and Cell Line Data.

BACKGOUND: The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson\u27s disease (PD). METHODS: The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD, using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study, patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson\u27s Disease Rating Scale (UPDRS) to measure clinical symptoms. RESULTS: The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC, consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; p CONCLUSIONS: The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients, and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT02445651

Melatonin and Other Tryptophan Metabolites Produced by Yeasts: Implications in Cardiovascular and Neurodegenerative Diseases

Yeast metabolism produces compounds derived from tryptophan, which are found in fermented beverages, such as wine and beer. Melatonin and serotonin, in particular, may play a significant role due to their bioactivity in humans. Indeed, the former is a neurohormone related to circadiam rhythms, which also has a putative protective effect against degenerative diseases. Serotonin, on the other hand, is a neurotransmitter itself, in addition to being a precursor of melatonin synthesis. This paper summarizes data reported on fermented beverages, to evaluate dietary intake. Additionally, the article reviews observed effects of yeast amino acid metabolites on the prevention of neurodegenerative diseases (Alzheimer’s and Parkinson’s) and angiogenesis, focusing on evidence of the molecular mechanism involved and identification of molecular target