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Gardner, R.

Publications and source records attributed to Gardner, R..

15 recordsLinked to original sources

Senescence-directed nanotherapy ameliorates fibrosis and overcomes immune exclusion in cancer

Fibrotic remodeling of tissues and tumors establishes immunosuppressive microenvironments that drive organ dysfunction and, in cancer, limit response to immunotherapy. Senescent-like cells are conserved drivers of fibrosis and therapeutic targets, yet their functional heterogeneity complicates therapeutic intervention. Here, we show that P-selectin is expressed by a subset of senescent-like cells in fibrotic tissues and tumors. Leveraging fucoidan-based nanoparticles that bind P-selectin, we developed senescence-modulating nanoparticles (SMNPs) to selectively target these disease-associated states. SMNPs exerted potent antifibrotic and immunomodulatory effects while improving therapeutic index. Mechanistically, we identified a pathogenic, immunosuppressive macrophage population as a functional target in vivo. In fibrotic tumors, niche remodeling restored immune infiltration and sensitized tumors to immune-checkpoint-based therapies. These findings establish SMNPs as a generalizable strategy to target pathogenic senescent cell subsets across fibrosis and cancer.

cancer biology↗

Lung-Targeting Interleukin-10 mRNA Lipid Nanoparticles Ameliorate Acute Lung Injury

Acute respiratory distress syndrome (ARDS) is the most severe manifestation of acute lung injury (ALI), characterized by diffuse pulmonary inflammation, impaired gas exchange, and high morbidity and mortality. Despite its clinical significance, no specific or effective pharmacological therapies are currently available for its treatment. In this study, we developed a lung -targeted mRNA-sulfonium lipid nanoparticle (mRNA/sLNP) delivery system for the treatment of ALI in a mouse model. We first optimized sulfonium lipid structures, and the optimized sLNP was comprehensively characterized and subsequently loaded with interleukin-10 (IL-10) mRNA. In a lipopolysaccharide (LPS)-induced ALI mousemodel, IL-10/sLNPdemonstrated both prophylactic and therapeutic efficacy, significantly attenuating pulmonary and systemic inflammation, restoring barrier integrity, and reducing tissue injury.

bioengineering↗

Structural Homology and Electrostatic Potential Comparisons of Epitope Pair Candidates for Molecular Mimicry Triggering of Type 1 Diabetes Mellitus

BackgroundMolecular mimicry, where foreign and self-peptides contain similar epitopes, can induce autoimmune responses. Identifying potential molecular mimics and studying their properties is key to understanding the onset of autoimmune diseases such as type 1 diabetes mellitus (T1DM). Previous work identified pairs of infectious epitopes (EINF) and T1DM epitopes (ET1D) that demonstrated sequence homology; however, structural homology was not considered. Correlating sequence homology with structural properties is important for translational investigation of potential molecular mimics. This work compares sequence homology with structural homology by calculating the structures and electrostatic potential surfaces of the epitope pairs identified in previous work from our laboratory. ResultsFor each pair of EINF and ET1D, the root mean square deviations (RMSD) were calculated between their predicted structures and their electrostatic potentials. Structures were predicted using the AlphaFold software program. Of the 53 epitope pairs considered here, only 10 did not exhibit any matching (i.e. less than 3 residues overlap). When considering all residues the RMSD ranges from 0.33 [A] to 11.66 [A] with an average of 2.68 [A]. Twenty-two pairs (42%) have RMSD of less than 1.5 [A] and 30 (58%) less than 3 [A]. ConclusionsMost of the EINF/ET1D pairs selected by sequence homology show similar structural and electrostatic distributions, indicating that the EINF may also bind to the same protein targets, i.e. the major histocompatibility complex molecules, for T1DM, leading to molecular mimicry onset of the disease. These findings suggest that searching for epitope pairs using sequence homology, a much less computationally demanding approach, leads to strong candidates for molecular mimicry that should be considered for further study. But structure homology, electrostatic potential calculations and full docking calculations may be necessary to advance the in-silico molecular mimicry predictions, which may be useful to select the most promising candidates for experimental studies.

immunology↗

Transcriptomic analysis reveals new reparative mechanisms of SCF and GCSF - reduced neuropathology in aged APPPS1 mice

Alzheimers Disease (AD) is a neurodegenerative disease characterized by amyloid plaque deposition, tau hyperphosphorylation, neuroinflammation, and cognitive decline. Our previous studies showed that combined treatment with stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) reduces AD pathology in APP/PS1 mice. This study aimed to explore the molecular mechanism underlying SCF+G-CSFs therapeutic effects using transcriptomic analysis. Aged APP/PS1 mice received daily subcutaneous injections of SCF+G-CSF or vehicle for 12 days. RNA was extracted from brain tissue on day 13 for gene chip analysis. Age-matched wild-type (WT) mice served as controls. Data were analyzed using TAC, STRING v12.0, Reactome, and ShinyGO 0.77. A total of 45,037 differentially expressed genes (DEGs) were detected. Twenty-seven DEGs met a [≥]2-fold threshold in SCF+G-CSF-treated vs. vehicle-treated APP/PS1 mice; 89 DEGs met this threshold in APP/PS1 vs. WT mice. SCF+G-CSF treatment upregulated six immune-related genes (S100a8, S100a9, Ngp, Lcn2, Ltf, and Camp) associated with amyloid clearance, immune cell recruitment, and repair. Pathway analysis showed downregulation of IL-2, IL-4, IL-7, and EGFR1, and upregulation of IL-17 signaling, suggesting modulation of both innate and adaptive immunity. Notably, SCF+G-CSF downregulated several oncogenes, including Cbl, Akap9, Kcnq1ot1, and Snhg11, highlighting an overlap between cancer and AD-related pathways. SCF+G-CSF also promoted NADPH oxidase activation via Rho GTPases and showed >400-fold enrichment in metal ion sequestration, indicating potential metal chelation effects. These findings suggest that SCF+G-CSF treatment modifies immune and metabolic pathways, reduces AD pathology, and highlights new therapeutic targets involving inflammation, metal homeostasis, and oncogenic signaling.

neuroscience↗

Is Britain still Great for Pine Marten? A Habitat Suitability Assessment

ContextSpecies distribution models are used to predict habitat suitability for a species, by quantifying the environmental characteristics that allow a species to occupy a geographical area. The abundance and range of pine marten (Martes martes) has declined substantially in Great Britain, with remaining populations restricted to Scotland. ObjectivesHere, we perform species distribution modelling using BIOMOD2 platform to determine habitat suitability, and inform the identification of potential reintroduction sites for pine marten in Great Britain. MethodsUsing a global range dataset of 4,189 occurrences and seven environmental variables, ensemble species distribution models were used to predict habitat suitability across Europe at 1 km resolution and Great Britain at 100 m resolution. ResultsAcross the extent of both Europe and Britain, results indicate high suitability in areas with woody vegetation cover in low topographic positions, and notably low in urban areas and extensive areas of arable land. In Britain, high habitat suitability is identified across substantial areas in the South East of England, parts of South West of England, East Yorkshire and Gloucestershire, with pockets of suitable habitat along the West Coast of Britain. The results indicate that elevation and land cover are important drivers of suitability. ConclusionHabitat suitability modelling at a high resolution of 100m proves effective for informing potential reintroduction sites for pine marten in Britain. We also demonstrate the importance of using occurrence data from pine martens global range to predict optimal habitat suitability.

zoology↗

Chlamydia muridarum Causes Persistent Subclinical Infection and Elicits Innate and Adaptive Immune Responses in C57BL/6J, BALB/cJ and J:ARC(S) Mice Following Exposure to Shedding Mice

Chlamydia muridarum (Cm) has reemerged as a moderately prevalent infectious agent in research mouse colonies. Despite its experimental use, few studies evaluate Cms effects on immunocompetent mice following its natural route of infection. A Cm field isolate was administered (orogastric gavage) to 8-week-old female BALB/cJ (C) mice. After confirming shedding (through 95d), these mice were cohoused with naive C57BL/6J (B6), C, and Swiss (J:ARC[S]) mice (n=28/strain) for 30 days. Cohoused mice (n=3-6 exposed and 1-6 control/strain) were evaluated 7, 14, 21, 63, 120, and 180 days post-cohousing (DPC) via hemograms, serum biochemistry analysis, fecal qPCR, histopathology, and Cm MOMP immunohistochemistry. Immunophenotyping was performed on spleen (B6, C, S; n=6/strain) and intestines (B6; n=6) at 14 and 63 DPC. Serum cytokine concentrations were measured (B6; n=6 exposed and 2 control) at 14 and 63 DPC. All B6 mice were shedding Cm by 3 through 180 DPI. One of 3 C and 1 of 6 S mice began shedding Cm at 3 and 14 DPC, respectively, with the remaining shedding thereafter. Clinical pathology was nonremarkable. Minimal-to-moderate enterotyphlocolitis and gastrointestinal associated lymphoid tissue (GALT) hyperplasia was observed in 15 and 47 of 76 Cm-infected mice, respectively. Cm antigen was frequently detected in GALT-associated surface intestinal epithelial cells. Splenic immunophenotyping revealed increased monocytes and shifts in T cell population subsets in all strains/timepoints. Gastrointestinal immunophenotyping (B6) revealed sustained increases in total inflammatory cells and elevated cytokine production in innate lymphoid cells and effector T cells (large intestine). Elevated concentrations of pro-inflammatory cytokines were detected in the serum (B6). Results demonstrate that while clinical disease was not appreciated, 3 commonly utilized strains of mice are susceptible to chronic enteric Cm infection which may alter various immune responses. Considering the widespread use of mice to model GI disease, institutions should consider excluding Cm from their colonies.

microbiology↗

Single cell RNA sequencing reveals immunomodulatory mechanism of stem cell factor and granulocyte colony-stimulating factor treatment in the brains of aged APP/PS1 mice

Alzheimers disease (AD) leads to progressive neurodegeneration and dementia. AD primarily affects older adults with neuropathological changes including amyloid-beta (A{beta}) deposition, neuroinflammation, and neurodegeneration. We have previously demonstrated that systemic treatment with combined stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF), reduces A{beta} load, increases A{beta} uptake by activated microglia and macrophages, reduces neuroinflammation, and restores dendrites and synapses in the brains of aged APPswe/PS1dE9 (APP/PS1) mice. However, the mechanisms underlying SCF+G-CSF-enhanced brain repair in aged APP/PS1 mice remain unclear. This study used a transcriptomic approach to identify potential mechanisms by which SCF+G-CSF treatment modulates microglia and peripheral myeloid cells to mitigate AD pathology in the aged brain. After injections of SCF+G-CSF for 5 consecutive days, single-cell RNA sequencing was performed on CD11b+ cells isolated from the brains of 28-month-old APP/PS1 mice. The vast majority of cell clusters aligned with transcriptional profiles of microglia in various activation states. However, SCF+G-CSF treatment dramatically increased a cell population showing upregulation of marker genes related to peripheral myeloid cells. Flow cytometry data also revealed an SCF+G-CSF-induced increase of cerebral CD45high/CD11b+ active phagocytes. SCF+G-CSF treatment robustly increased the transcription of genes implicated in immune cell activation, including gene sets that regulate inflammatory processes and cell migration. Expression of S100a8 and S100a9 were robustly enhanced following SCF+G-CSF treatment in all CD11b+ cell clusters. Moreover, the topmost genes differentially expressed with SCF+G-CSF treatment were largely upregulated in S100a8/9-positive cells, suggesting a well-conserved transcriptional profile related to SCF+G-CSF treatment in resident and peripherally derived CD11b+ immune cells. This S100a8/9-associated transcriptional profile contained notable genes related to pro-inflammatory and anti-inflammatory responses, neuroprotection, and A{beta} plaque inhibition or clearance. Altogether, this study reveals immunomodulatory effects of SCF+G-CSF treatment in the aged brain with AD pathology, which will guide future studies to further uncover the therapeutic mechanisms.

neuroscience↗

Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

Cell-intrinsic mechanisms of immunogenicity in ovarian cancer (OC) are not well understood. The presence of damaging mutations in the SWI/SNF chromatin remodeling complex, such as the SMARCA4 (BRG1) catalytic subunit, has been associated with improved response to ICB, however the mechanism by which this occurs is unclear. The aim of this current study was to examine the alterations in tumor cell-intrinsic and extrinsic immune signaling caused by SMARCA4 loss. Using OC models with loss-of-function mutations in SMARCA4, we found that SMARCA4 loss resulted in increased cancer cell-intrinsic immunogenicity, characterized by upregulation of long-terminal RNA repeats such as endogenous retroviruses, increased expression of interferon-stimulated genes, and upregulation of antigen presentation machinery. Notably, this response was dependent on IRF3 signaling, but was independent of the type I interferon receptor. Mice inoculated with cancer cells bearing SMARCA4 loss demonstrated increased activation of cytotoxic T cells and NK cells in the tumor microenvironment as well as increased infiltration with activated dendritic cells. These results were recapitulated when animals bearing SMARCA4-proficient tumors were treated with a BRG1 inhibitor, suggesting that modulation of chromatin remodeling through targeting SMARCA4 may serve as a strategy to reverse immune evasion in OC.

cancer biology↗

Conserved degronome features governing quality control-associated proteolysis

The eukaryotic proteome undergoes constant surveillance by quality control systems that either sequester, refold, or eliminate aberrant proteins by ubiquitin-dependent mechanisms. Ubiquitin-conjugation necessitates the recognition of degradation determinants, termed degrons, by their cognate E3 ubiquitin-protein ligases. To learn about the distinctive properties of quality control degrons, we performed an unbiased peptidome stability screen in yeast. The search identified a large cohort of proteome-derived degrons, some of which exhibited broad E3 ligase specificity. Consequent application of a machine-learning algorithm established constraints governing degron potency, including the amino acid composition and secondary structure propensities. According to the set criteria, degrons with transmembrane domain-like characteristics are the most probable sequences to act as degrons. Similar quality control degrons were identified in viral and human proteins, suggesting conserved degradation mechanisms. Altogether, the emerging data indicate that transmembrane domain-like degron features have been preserved in evolution as key quality control determinants of proteins half-life.

cell biology↗

Acute ethanol stress induces sumoylation of conserved chromatin structural proteins in Saccharomyces cerevisiae

Stress is a ubiquitous part of life that disrupts cellular function and, if unresolved, can irreparably damage essential biomolecules and organelles. All organisms can experience stress in the form of unfavorable environmental conditions including exposure to extreme temperatures, hypoxia, reactive oxygen species, alcohol, or shifts in osmolarity. To survive, organisms must sense these changes then react and adapt. One highly conserved adaptive response to stress is through protein sumoylation, which is a post-translational modification by the small ubiquitin-like modifier (SUMO) protein. In this study, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae. Although ethanol induces protein sumoylation, the targets and roles of sumoylation are largely unknown. Here, we found that cells exhibit a transient sumoylation response after exposure of cells to [≤] 7.5% volume/volume ethanol. The response peaks at 15 minutes and resolves by 60 minutes, indicating that cells have an adaptive response to low concentrations of ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol stress with no resolution by 60 minutes. To identify key targets of ethanol-induced sumoylation, we performed mass spectrometry analyses and identified 18 proteins with increased sumoylation after acute ethanol exposure, with 15 identified as known chromatin-associated proteins. Two of these proteins are the chromatin structural proteins Smc5 and Smc6, which are sumoylated by the activity of the SUMO ligase Mms21. Ethanol-induced Smc5/6 sumoylation occurs during G1 and G2M phases of the cell cycle but is abrogated during S phase despite the fact that other proteins are sumoylated during this phase. Acute ethanol exposure leads to formation of Rad52 foci indicating DNA damage similar to that observed with the addition of methyl methanesulfonate (MMS), which is an alkylating agent that damages DNA. Whereas MMS exposure induces the intra-S phase DNA damage checkpoint as observed by Rad53 phosphorylation, ethanol exposure does not induce the intra-S phase checkpoint and prevents Rad53 phosphorylation when added with MMS. From these results, we propose that ethanol induces a structural change in chromatin, possibly through DNA damage, and this causes sumoylation of conserved chromatin-associated proteins, including Smc5 and Smc6.

cell biology↗

Can quantifying morphology and TMEM119 expression distinguish between microglia and infiltrating macrophages after ischemic stroke and reperfusion in male and female mice?

BackgroundIschemic stroke is an acquired brain injury with gender dependent outcomes. A persistent obstacle in understanding the sex-specific neuroinflammatory contributions to ischemic brain injury is distinguishing between resident microglia versus infiltrating macrophages--both phagocytes--and determining cell population specific contributions to injury evolution and recovery processes. Our purpose was to identify microglial and macrophage populations regulated by ischemic stroke using morphology analysis and the presence of microglia transmembrane protein 119 (TMEM119). Second, we examined sex and menopause differences in microglia/macrophage cell populations after an ischemic stroke. MethodsMale and female, premenopausal and postmenopausal, mice underwent either 60-min of middle cerebral artery occlusion and 24-h of reperfusion or sham surgery. The accelerated ovarian failure model was used to model post-menopause. Brain tissue was collected to quantify infarct area and for immunohistochemistry and western blot methods. Ionized calcium-binding adapter molecule, TMEM119, and confocal microscopy were used to analyze microglia morphology and TMEM119 area in ipsilateral brain regions. Western blot was used to quantify protein quantity. ResultsPost-stroke injury is increased in male and female post-menopause mice versus pre-menopause female mice (p<0.05) with differences primarily occurring in caudal sections. After stroke, microglia underwent a region, but not sex group, dependent transformation into less ramified cells (p<0.0001). However, the number of phagocytic microglia were increased in distal ipsilateral regions of postmenopausal mice versus the other sex groups (p<0.05). The number of TMEM119 positive cells was decreased in proximity to the infarct (p<0.0001) but without a sex group effect. Two key findings prevented distinguishing microglia from systemic macrophages. First, morphological data were not congruent with TMEM119 immunofluorescence data. Cells with severely decreased TMEM119 immunofluorescence were ramified, a distinguishing microglia characteristic. Second, whereas TMEM119 immunofluorescence area decreased in proximity to the infarcted area, TMEM119 protein quantity was unchanged in ipsilateral hemisphere regions using western blot methods. ConclusionsOur findings suggest that TMEM119 is not a stable microglia marker in male and female mice in the context of ischemic stroke. Until TMEM119 function in the brain is elucidated, its use to distinguish between cell populations following brain injury with cell infiltration is cautioned.

physiology↗

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naive tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type glycans. In overlay assays, these sugars were differentially recognized by the C-type lectins mannose receptor and DC-SIGN. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

biochemistry↗

A combined characterization of coelomic fluid cell types in the spiny starfish Marthasterias glacialis: inputs from flow cytometry and imaging

Coelomocytes is a generic name for a collection of cellular morphotypes, present in many coelomate animals, that has been reported as highly variable across echinoderm classes. The roles attributed to the major types of the free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. The main aim of the present study is the thorough characterization of coelomocytes present in the coelomic fluid of Marthasterias glacialis (class Asteroidea) through the combined use of flow cytometry (FC) and fluorescence plus transmission electron microscopy. Two coelomocyte populations (here named P1 and P2) were identified by flow cytometry and subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells showed two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the small P1 haploid cellular population was characterized by a low mitotic activity, relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. These last cells resemble stem-cell types present in other animals. P1 and P2 cells differ also in cell viability and cell cycle profiles. Additionally, two other morphotypes were only detected by fluorescence microscopy and a third one when using a combined microscopy/FC approach.

cell biology↗

Charting the emergent organotypic landscape of the mammalian gut endoderm at single-cell resolution

To comprehensively delineate the ontogeny of an organ system, we generated 112,217 singlecell transcriptomes representing all endoderm populations within the mouse embryo until midgestation. We employed graph-based approaches to model differentiating cells for spatio-temporal characterization of developmental trajectories. Our analysis reveals the detailed architecture of the emergence of the first (primitive or extra-embryonic) endodermal population and pluripotent epiblast. We uncover an unappreciated relationship between descendants of these lineages, before the onset of gastrulation, suggesting that mixing of extra-embryonic and embryonic endoderm cells occurs more than once during mammalian development. We map the trajectories of endoderm cells as they acquire embryonic versus extra-embryonic fates, and their spatial convergence within the gut endoderm; revealing them to be globally similar but retaining aspects of their lineage history. We observe the regionalized localization of cells along the forming gut tube, reflecting their extra-embryonic or embryonic origin, and their coordinate patterning into organ-specific territories along the anterior-posterior axis.

developmental biology↗

Gestational Age At Birth And Risk Of Intellectual Disability Without A Common Genetic Cause: Findings From The Stockholm Youth Cohort

BackgroundPreterm birth is linked to intellectual disability and there is evidence to suggest post-term birth may also incur risk. However, these associations have not yet been investigated in the absence of common genetic causes of intellectual disability (where risk associated with late delivery may be preventable) or with methods allowing stronger causal inference from non-experimental data. We aimed to examine risk of intellectual disability without a common genetic cause across the entire range of gestation, using a matched-sibling design to account for unmeasured confounding by shared familial factors.\n\nMethods and FindingsWe conducted a population-based retrospective study using data from the Stockholm Youth Cohort (n=499,621) and examined associations in a nested cohort of matched siblings (n=8,034). Children born at non-optimal gestational duration (before/after 40 weeks 3 days) were at greater risk of intellectual disability. Risk was greatest among those born extremely early (adjusted OR24 weeks=14.54 [95% CI 11.46-18.44]), lessening with advancing gestational age toward term (aOR32 weeks=3.59 [3.22-4.01]; aOR37 weeks=1.50 [1.38-1.63]); aOR38 weeks=1.26 [1.16-1.37]; aOR39 weeks=1.10 [1.04-1.17]) and increasing with advancing gestational age post-term (aOR42 weeks=1.16 [1.08-1.25]; aOR42 weeks=1.41 [1.21-1.64]; aOR44 weeks=1.71 [1.34-2.18]; aOR45 weeks=2.07 [1.47-2.92]). Associations persisted in a nested cohort of matched outcome-discordant siblings suggesting they were robust against confounding from shared genetic or environmental traits, although there may have been residual confounding by unobserved non-shared characteristics. Risk of intellectual disability was greatest among children showing evidence of fetal growth restriction, especially when birth occurred before or after term.\n\nConclusionsBirth at non-optimal gestational duration may be linked causally with greater risk of intellectual disability. The mechanisms underlying these associations need to be elucidated as they will be relevant to clinical practice concerning elective delivery within the term period and the mitigation of risk in children who are born post-term.

epidemiology↗