Cell Compartment Research Articles

Breaking the barriers: Overcoming cancer resistance by targeting the NLRP3 inflammasome.

Inflammation has a pivotal role in the initiation and progression of various cancers, contributing to crucial processes such as metastasis, angiogenesis, cell proliferation and invasion. Moreover, the release of cytokines mediated by inflammation within the tumour microenvironment (TME) has a crucial role in orchestrating these events. The activation of inflammatory caspases, facilitated by the recruitment of caspase-1, is initiated by the activation of pattern recognition receptors on the immune cell membrane. This activation results in the production of proinflammatory cytokines, including IL-1β and IL-18, and participates in diverse biological processes with significant implications. The NOD-Like Receptor Protein 3 (NLRP3) inflammasome holds a central role in innate immunity and regulates inflammation through releasing IL-1β and IL-18. Moreover, it interacts with various cellular compartments. Recently, the mechanisms underlying NLRP3 inflammasome activation have garnered considerable attention. Disruption in NLRP3 inflammasome activation has been associated with a spectrum of inflammatory diseases, encompassing diabetes, enteritis, neurodegenerative diseases, obesity and tumours. The NLRP3 impact on tumorigenesis varies across different cancer types, with contrasting roles observed. For example, colorectal cancer associated with colitis can be suppressed by NLRP3, whereas gastric and skin cancers may be promoted by its activity. This review provides comprehensive insights into the structure, biological characteristics and mechanisms of the NLRP3 inflammasome, with a specific focus on the relationship between NLRP3 and tumour-related immune responses, and TME. Furthermore, the review explores potential strategies for targeting cancers via NLRP3 inflammasome modulation. This encompasses innovative approaches, including NLRP3-based nanoparticles, gene-targeted therapy and immune checkpoint inhibitors.

Transcriptomics combined with physiological analysis provided new insights into the enhancing Dendrobium nobile resilience to Cu stress through Ce modulation

In the face of escalating environmental challenges, understanding the mechanisms through which plants can resist heavy metal stress is paramount. This study delves into the efficacy of cerium (Ce), a rare earth element, in mitigating copper (Cu) toxicity in Dendrobium nobile Lindl., an orchid plant with significant medicinal and ornamental value. Through a comprehensive experimental approach that encompasses physiological, biochemical, and molecular analyses, we demonstrate that Ce application notably enhances D. nobile's tolerance to Cu stress. The investigation revealed that Ce supplementation significantly ameliorated the adverse effects of Cu on plant growth, evidenced by a notable decrease in Cu accumulation within the plant tissues. At the molecular level, transcriptome sequencing uncovered the upregulation of genes involved in antioxidant defense mechanisms, heavy metal chelation, and stress response signaling pathways. Key findings include the enhanced activities of antioxidant enzymes such as superoxide dismutase, peroxidase, and catalase, alongside an increase in the levels of antioxidants and phytochelatins, which collectively contribute to the detoxification of Cu. Furthermore, Ce was found to influence the subcellular distribution of Cu, promoting its sequestration in less harmful cellular compartments. Our study provides novel insights into the role of Ce in modulating plant responses to heavy metal stress, highlighting its potential as a mitigative agent against Cu toxicity. The elucidation of specific gene regulations and pathway activations offers a foundation for future research aimed at enhancing plant resilience to environmental stresses through biotechnological interventions.

Molecular and microbiological methods for the identification of nonreplicating Mycobacterium tuberculosis.

Chronic tuberculosis (TB) disease, which requires months-long chemotherapy with multiple antibiotics, is defined by diverse pathological manifestations and bacterial phenotypes. Targeting drug-tolerant bacteria in the host is critical to achieving a faster and durable cure for TB. In order to facilitate this field of research, we need to consider the physiology of persistent MTB during infection, which is often associated with the nonreplicating (NR) state. However, the traditional approach to quantifying bacterial burden through colony enumeration alone only informs on the abundance of live bacilli at the time of sampling, and provides an incomplete picture of the replicative state of the pathogen and the extent to which bacterial replication is balanced by ongoing cell death. Modern approaches to profiling bacterial replication status provide a better understanding of inter- and intra-population dynamics under different culture conditions and in distinct host microenvironments. While some methods use molecular markers of DNA replication and cell division, other approaches take advantage of advances in the field of microfluidics and live-cell microscopy. Considerable effort has been made over the past few decades to develop preclinical in vivo models of TB infection and some are recognized for more closely recapitulating clinical disease pathology than others. Unique lesion compartments presenting different environmental conditions produce significant heterogeneity between Mycobacterium tuberculosis populations within the host. While cellular lesion compartments appear to be more permissive of ongoing bacterial replication, caseous foci are associated with the maintenance of M. tuberculosis in a state of static equilibrium. The accurate identification of nonreplicators and where they hide within the host have significant implications for the way novel chemotherapeutic agents and regimens are designed for persistent infections.

CTLA4 blockade abrogates KEAP1/STK11-related resistance to PD-(L)1 inhibitors

For patients with advanced non-small-cell lung cancer (NSCLC), dual immune checkpoint blockade (ICB) with CTLA4 inhibitors and PD-1 or PD-L1 inhibitors (hereafter, PD-(L)1 inhibitors) is associated with higher rates of anti-tumour activity and immune-related toxicities, when compared with treatment with PD-(L)1 inhibitors alone. However, there are currently no validated biomarkers to identify which patients will benefit from dual ICB1,2. Here we show that patients with NSCLC who have mutations in the STK11 and/or KEAP1 tumour suppressor genes derived clinical benefit from dual ICB with the PD-L1 inhibitor durvalumab and the CTLA4 inhibitor tremelimumab, but not from durvalumab alone, when added to chemotherapy in the randomized phase III POSEIDON trial3. Unbiased genetic screens identified loss of both of these tumour suppressor genes as independent drivers of resistance to PD-(L)1 inhibition, and showed that loss of Keap1 was the strongest genomic predictor of dual ICB efficacy—a finding that was confirmed in several mouse models of Kras-driven NSCLC. In both mouse models and patients, KEAP1 and STK11 alterations were associated with an adverse tumour microenvironment, which was characterized by a preponderance of suppressive myeloid cells and the depletion of CD8+ cytotoxic T cells, but relative sparing of CD4+ effector subsets. Dual ICB potently engaged CD4+ effector cells and reprogrammed the tumour myeloid cell compartment towards inducible nitric oxide synthase (iNOS)-expressing tumoricidal phenotypes that—together with CD4+ and CD8+ T cells—contributed to anti-tumour efficacy. These data support the use of chemo-immunotherapy with dual ICB to mitigate resistance to PD-(L)1 inhibition in patients with NSCLC who have STK11 and/or KEAP1 alterations.

Rapid and effective protocol to measure glutathione peroxidase activity

Abstract Background Intracellular hydroperoxides are harmful reactive oxygen species that can cause damage. Cells depend on antioxidant enzymes such as glutathione peroxidase (GPx) to prevent their accumulation. GPx is present in all cell compartments, including the cytosol, mitochondria, and peroxisomes. This article will present a rapid and reliable method for measuring GPx activity, which has been rigorously tested and proven dependable. Results The enzyme samples are incubated in a phosphate buffer containing specific concentrations of glutathione (GSH) and peroxide and then incubated for a suitable time to react before Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) is added. This method, designed for ease of use, is effective and straightforward. When GSH reacts with DTNB, mixed disulfides (GS-S-DTNB) are formed, where the thiol group replaces one of the nitrobenzoic acid moieties in DTNB. The release of the nitrobenzoic acid group as nitro-2-thiobenzoate anion (TNB−) creates a yellow anion that can be measured spectrophotometrically at a wavelength of 412 nm. The intensity of the yellow color is directly proportional to the unreacted glutathione concentration. GPx activity was linked to decreased absorbance of the TNB− anion, providing a sensitive and relatively specific method for detecting thiol groups in various biological samples. Conclusion This study utilized a Box–Behnken design (BBD) to apply to response surface modeling (RSM) to determine the best conditions for measuring GPx activity. The efficiency of the new protocol was validated by comparing GPx activity in matched samples using the Bland–Altman plot analysis and reference methods. The new protocol was just as accurate as the reference one, with a correlation coefficient of 0.9991. Graphical abstract

High-dimensional Immune Profiles and Machine Learning May Predict Acute Myeloid Leukemia Relapse Early following Transplant.

Identification of early immune signatures associated with acute myeloid leukemia (AML) relapse following hematopoietic stem cell transplant (HSCT) is critical for patient outcomes. We analyzed PBMCs from 58 patients with AML undergoing HSCT, focusing on T cell subsets and functional profiles. High-dimensional flow cytometry coupled with Uniform Manifold Approximation and Projection dimensionality reduction and PhenoGraph clustering revealed distinct changes in CD4+ and CD8+ T cell populations in 16 patients who relapsed within 1 y of HSCT. We observed increased IL-2, IL-10, and IL-17-producing CD4+ T cells, alongside decreased CD8+ T cell function early in relapsing patients. Notably, relapsing patients exhibited increased TCF-1intermediate cells, which lacked granzyme B or IFN-γ production in the CD4+ T cell compartment. We then developed a supervised machine learning algorithm that predicted AML relapse with 90% accuracy within 30 d after HSCT using high-throughput assays. The algorithm leverages condensed immune phenotypic data, alongside the ADASYN algorithm, for data balancing and 100 rounds of XGBoost supervised learning. This approach holds potential for detecting relapse-associated immune signatures months before clinical manifestation. Our findings demonstrate a distinct immunological signature potentially capable of predicting AML relapse as early as 30 d after HSCT.

A Novel Chimeric Oncolytic Virus Mediates a Multifaceted Cellular Immune Response in a Syngeneic B16 Melanoma Model

Background/Objectives: Oncolytic virotherapy is a promising approach in cancer immunotherapy. We have previously described a recombinant hybrid oncolytic virus (OV), VSV-NDV, which has a favorable safety profile and therapeutic immunogenicity, leading to direct oncolysis, abscopal effects, and prolonged survival in syngeneic in vivo tumor models. While OVs are known to mediate systemic anti-tumor immune responses, the detailed characterization of local and systemic immune responses to fusogenic oncolytic virotherapy remains unexplored. Methods and Results: We analyzed immune cell compartments in the spleen, blood, tumor-draining lymph nodes (TDLNs), and tumors over the course of VSV-NDV therapy in a bilateral syngeneic melanoma mouse model. Our results revealed significant local infiltration and activation of T lymphocytes in tumors and globally in the blood and spleen. Notably, in vivo CD8+ T cell depletion led to complete abrogation of the tumor response, highlighting the crucial role of T cells in promoting the therapeutic effects of oncolytic VSV-NDV. In vitro co-culture experiments enabled the interrogation of human immune cell responses to VSV-NDV-mediated oncolysis. Human peripheral blood mononuclear cells (PBMCs) were efficiently stimulated by exposure to VSV-NDV-infected cancer cells, which recapitulates the in vivo murine findings. Conclusions: Taken together, these data characterize a broad anti-tumor immune cell response to oncolytic VSV-NDV therapy and suggest that CD8+ T cells play a decisive role in therapeutic outcome, which supports the further development of this chimeric vector as a multimechanistic immunotherapy for solid cancers.

9343 Protection Against Type 1 Diabetes Development In Mice With 4E-BP2 Deletion

Abstract Disclosure: V. Pita Grisanti: None. F. Pecanha: None. R. Louzada: None. M. Blandino: None. C. Jaramillo: None. N. Arenas: None. A. Bayer: None. E. Bernal-Mizrachi: None. Type 1 diabetes is an autoimmune disease characterized by β-cell destruction promoted by autoreactive T cells that acquire an effector inflammatory phenotype. 4E-BP1/2 proteins are translational repressors and downstream targets of mTORC1, a key regulator of metabolism. mTORC1 signaling disruption is implicated in human diseases including diabetes. Activation of 4E-BP2/eIF4E pathway by 4E-BP2 deletion promotes translation initiation, which induces β-cell expansion and proliferation and is crucial for the regulation of adaptive immunity. However, the involvement of 4E-BP2 in type 1 diabetes development has not been explored. Therefore, this study aimed to determine the role of 4E-BP2/eIF4E signaling in type 1 diabetes prevention by regulation of β-cell survival and immune modulation. To investigate the role of 4E-BP2/eIF4E axis in diabetes pathogenesis, we used the non-obese diabetic (NOD) mouse model as a type 1 diabetes model, and generated mice with global deletion of 4E-BP2 in the NOD background (4E-BP2KONOD). We assessed type 1 diabetes development, glucose homeostasis, pancreas morphometry and immune responses in male and female 4E-BP2KONOD and control NOD mice. We found that 4E-BP2KONOD exhibited reduced diabetes incidence in male but not female mice. Reduction in diabetes incidence in 4E-BP2KONOD male mice was associated with comparable degree of insulitis and β-cell proliferation, and with preserved β-cell mass compared to the control NOD mice. Glucose-stimulated insulin secretion of the islets, assessed in vitro by static incubation, was significantly higher in the 4E-BP2KONOD compared to the NOD control. Autoimmune responses were also evaluated by assessment of insulitis and characterization of T cell compartments, which showed a decrease in splenic CD8+ cytotoxic T cells and an increase in pancreatic regulatory T cell (Treg) infiltration mainly by increased Treg survival in 4E-BP2KONOD mice compared to control NOD. Finally, adoptive transfer studies demonstrated that lymphocytes derived from male 4E-BP2KONOD mice were less diabetogenic than those derived from control NOD mice. In conclusion, this study showed that activation of 4E-BP2/eIF4E axis in 4E-BP2KONOD male mice protects against type 1 diabetes development by dampening autoimmune responses and preserving β-cell mass. Targeting 4E-BP2 may provide a potential treatment for type 1 diabetes. Presentation: 6/1/2024

Immunometabolic Mechanisms of LANCL2 in CD4+ T Cells and Phagocytes Provide Protection from Systemic Lupus Erythematosus.

Lanthionine synthetase C-like 2 (LANCL2) is an immunoregulatory therapeutic target for autoimmune diseases. NIM-1324 is an investigational new drug aimed at addressing the unmet clinical needs of patients with systemic lupus erythematosus (SLE) by targeting the LANCL2 immunometabolic pathway. In R848 and bm12 adoptive transfer models of systemic inflammation that share pathologies with SLE, Lancl2-/- mice experienced greater mortality, increased spleen weight, and reduced CD25hi FOXP3+ CD4+ regulatory T cells compared with the wild type. Conversely, treatment with NIM-1324 in the wild type increased CD25hi FOXP3+ regulatory T cells while reducing inflammatory IL-17+ and IL-21+ CD4+ T cell subsets in the spleen. In traditional mouse models of SLE (NZB/W F1 and MRL/lpr), oral treatment with NIM-1324 protected against weight loss and proteinuria, decreased anti-dsDNA titers, and provided similar changes to the CD4+ T cell compartment in the spleen. The pharmacological activation of LANCL2 by NIM-1324 rescued hypocomplementemia, reduced kidney histopathological scores, and decreased blood IFN response genes and inflammatory cytokines. The loss of LANCL2 in phagocytes impairs phagosome processing, leading to increased uptake of material and inflammatory cytokine production, yet decreased markers of endosomal maturation, phagosome turnover, and lysozyme activity. Treatment with NIM-1324 increases metabolic and lysozyme activity in the phagosome, providing support for increased markers of early phagosome function. This efficacy translated to human PBMCs from patients with SLE, because ex vivo treatment with NIM-1324 resulted in reduced levels of IFN-α, IL-6, and IL-8. Consequently, the activation of LANCL2 effectively modulates CD4+ T cell differentiation and phagocyte activation, supporting immune tolerance in SLE.

The structural biology and dynamics of malate dehydrogenases.

Malate dehydrogenase (MDH) enzymes catalyze the reversible oxidoreduction of malate to oxaloacetate using NAD(P) as a cofactor. This reaction is vital for metabolism and the exchange of reducing equivalents between cellular compartments. There are more than 100 structures of MDH in the Protein Data Bank, representing species from archaea, bacteria, and eukaryotes. This conserved family of enzymes shares a common nucleotide-binding domain, substrate-binding domain, and subunits associate to form a dimeric or a tetrameric enzyme. Despite the variety of crystallization conditions and ligands in the experimental structures, the conformation and configuration of MDH are similar. The quaternary structure and active site dynamics account for most conformational differences in the experimental MDH structures. Oligomerization appears essential for activity despite each subunit having a structurally independent active site. There are two dynamic regions within the active site that influence substrate binding and possibly catalysis, with one of these regions adjoining the subunit interface. In this review, we introduce the reader to the general structural framework of MDH highlighting the conservation of certain features and pointing out unique differences that regulate MDH enzyme activity.

The effect of biochar amendment on Cd accumulation in Bidens pilosa L: Changing Cd subcellular distribution, cell wall polysaccharide Cd-binding capacity and composition

Biochar can reduce Cd uptake by plants, thereby reducing its biotoxicity, but the mechanisms involved at the subcellular level have not been thoroughly elucidated. In this work, we explored the effect of maize straw biochar on Cd accumulation by Bidens pilosa L. and its mechanism at subcellular levels. After 90 days of potting experiment, the subcellular fractions were extracted by differential centrifugation, and the polysaccharide fractions of root cell walls were extracted by leaching centrifugation, and then the Cd content of each fraction was determined. Results showed that Cd was preferentially distributed in cell walls of three organs. Additionally, biochar addition resulted in a greater distribution of Cd from cell wall to soluble fractions and organelles in stems. These results suggested that cell wall immobilization and intracellular compartmentalization were critical detoxification mechanisms tackling Cd stress with biochar addition of Bidens pilosa L. Pectin was the main sink where Cd was stored. And galacturonic acid content in pectin occupied the highest ratio among the three polysaccharide fractions. After biochar addition, in hemicellulose the change of Cd content was consistent with the change of galacturonic acid content. These results suggested that the galacturonic acid in hemicellulose played an important role in Cd binding. Biochar addition reduced the bioavailability of soil Cd and improved the growth environment, thus inducing Bidens pilosa L. to change the composition of root cell wall in response to Cd stress.

Therapeutic delivery of recombinant glucocerebrosidase enzyme-containing extracellular vesicles to human cells from Gaucher disease patients

BackgroundGaucher disease (GD) is one of the most common types of lysosomal storage diseases (LSDs) caused by pathogenic variants of lysosomal β-glucocerebrosidase gene (GBA1), resulting in the impairment of Glucocerebrosidase (GCase) enzyme function and the accumulation of a glycolipid substrate, glucosylceramide (GlcCer) within lysosomes. Current therapeutic approaches such as enzyme replacement therapy and substrate reduction therapy cannot fully rescue GD pathologies, especially neurological symptoms. Meanwhile, delivery of lysosomal enzymes to the endocytic compartment of affected human cells is a promising strategy for treating neuropathic LSDs.ResultHere, we describe a novel approach to restore GCase enzyme in cells from neuropathic GD patients by producing extracellular vesicle (EVs)-containing GCase from cells overexpressing GBA1 gene. Lentiviral vectors containing modified GBA1 were introduced into HEK293T cells to produce a stable cell line that provides a sustainable source of functional GCase enzyme. The GBA1-overexpressing cells released EV-containing GCase enzyme, that is capable of entering into and localizing in the endocytic compartment of recipient cells, including THP-1 macrophage, SH-SY5Y neuroblastoma, and macrophages and neurons derived from induced pluripotent stem cells (iPSCs) of neuropathic GD patients. Importantly, the recipient cells exhibit higher GCase enzyme activity.ConclusionThis study presents a promising therapeutic strategy to treat severe types of LSDs. It involves delivering lysosomal enzymes to the endocytic compartment of human cells affected by conditions such as GDs with neurological symptoms, as well as potentially other neurological disorders impacting lysosomes.

The physiological significance of plasma-accessible carbonic anhydrase in the respiratory systems of fishes.

Carbonic anhydrase (CA) activity is ubiquitously found in all vertebrate species, tissues and cellular compartments. Most species have plasma-accessible CA (paCA) isoforms at the respiratory surfaces, where the enzyme catalyzes the conversion of plasma bicarbonate to carbon dioxide (CO2) that can be excreted by diffusion. A notable exception are the teleost fishes that appear to lack paCA at their gills. The present review: (i) recapitulates the significance of CA activity and distribution in vertebrates; (ii) summarizes the current evidence for the presence or absence of paCA at the gills of fishes, from the basal cyclostomes to the derived teleosts and extremophiles such as the Antarctic icefishes; (iii) explores the contribution of paCA to organismal CO2 excretion in fishes; and (iv) the functional significance of its absence at the gills, for the specialized system of O2 transport in most teleosts; (v) outlines the multiplicity and isoform distribution of membrane-associated CAs in fishes and methodologies to determine their plasma-accessible orientation; and (vi) sketches a tentative time line for the evolutionary dynamics of branchial paCA distribution in the major groups of fishes. Finally, this review highlights current gaps in the knowledge on branchial paCA function and provides recommendations for future work.

How does NaCl enhance Cd tolerance in Inula crithmoides L? Insights into Cd uptake, compartmentalization, and chelation

This study investigated the effect of NaCl on the uptake, translocation, compartmentalization, and chelation of cadmium (Cd) in the halophyte Inula crithmoides. Seedlings were subjected hydroponically for 21 days to 25 and 50 μM Cd applied alone or combined with 100 mM NaCl. Findings revealed that, Cd alone induced intense chlorosis and necrosis and altered plant development resulting in diminished biomass production. However, NaCl alleviated Cd-induced toxicity by increasing biomass accumulation, associated with restoration of photosynthesis activity. At the level of whole plant, NaCl reduced Cd concentration in different organs as well as its translocation toward the shoots. At the cellular level, Na Cl changed the cell-compartmentalization of Cd in the shoots and roots by inducing a preferential accumulation into the soluble fraction (vacuole). NaCl increased the chelation of Cd to chloride and nitrate. As compared to Cd alone, salt addition to Cd-treated plants enhanced significantly succinic acid concentration in the leaves suggesting a possible role of this acid in Cd-chelation. Globally, NaCl alleviated Cd-induced phytotoxicity in this halophyte by reducing Cd absorption, translocation and increased Cd fixation to organic acids as well as through the changes in Cd2+ cell compartmentalization. Obtained data suggested that this fast growing halophyte could be used to rehabilitate Cd polluted saline soils.

Cryosectioning and immunofluorescence of C. elegans reveals endogenous polyphosphate in intestinal endo-lysosomal organelles

Polyphosphate (polyP) is a ubiquitous polyanion present throughout the tree of life. While polyP's widely varied functions have been interrogated in single-celled organisms, little is known about the cellular distribution and function of polyP in multicellular organisms. To study polyP in metazoans, we developed the nematode Caenorhabditis elegans as a model system. We designed a high-throughput, longitudinal-orientation cryosectioning method that allowed us to scrutinize the intracellular localization of polyP in fixed C.elegans using fluorescent polyP probes and co-immunostaining targeting appropriate marker proteins. We discovered that the vast majority of polyP is localized within the endo-lysosomal compartments of the intestinal cells and is highly sensitive toward the disruption of endo-lysosomal compartment generation and food availability. This study lays the groundwork for further mechanistic research of polyPs in multicellular organisms and provides a reliable method for immunostaining hundreds of fixed worms in a single experiment.

Immunomodulatory role of tumor microenvironment on oncological outcomes in advanced laryngeal cancer

BackgroundThe study evaluated the prognostic impact of the immune microenvironment in LSCC with markers of major immune cells to identify the key determinants of short-term disease-free survival (ST DFS) and reveal factors related to disease progression.MethodsThe study cohort included 61 patients who underwent total laryngectomy, 83.6% of whom were male with a mean age of 64.3 years at the time of surgery. Twenty-five patients had long term DFS (over 5 years), 8 – had moderate DFS (between 2 and 5 years), and 28 had short-term DFS (less than 2 years). Immunohistochemical staining and evaluation were performed on samples collected after the laryngectomy.ResultsThe samples’ assessment revealed that the mean expression of all analysed markers was the highest both in stroma and the tumor compartment for short term DFS (ST DFS) patients. Analysis confirmed that a high stromal density of CD8 cells (p = 0.038) significantly correlated with DFS, and that the increased presence of CD57 cells (p = 0.021) was significantly associated with ST DFS. Moreover, the high density of CD68 cells in the tumor epithelial compartment had a negative prognostic impact on DFS (p = 0.032). Analysis of overall survival in the studied cohort with Kaplan-Meyer curves revealed that a high stromal density of CD68 cells was a significant negative predictor of OS (p = 0.008).ConclusionsThe observed associations of CD68 cells infiltration with progression and prognosis in patients with LSCC provide potential screening and therapeutic opportunities for patients with unfavourable outcomes.

Altered B-cell, plasma cell, and antibody immune profiles in blood of patients with systemic mastocytosis

Systemic mastocytosis (SM) is a heterogeneous disease characterized by an expansion of KIT-mutated constitutively activated mast cells (MCs) that release MC mediators, which might act on the tumor microenvironment including other immune cells. To investigate the blood distribution of B-cell, plasma cell (PC), and antibody isotype compartments in patients with SM. We used spectral flow cytometry and the EuroFlow Immunomonitoring panel and Lymphocyte Screening Tube to quantify B cells, PCs, and their subsets in blood of 108 patients with SM (35 bone marrow mastocytosis [BMM] cases, 64 indolent SM [ISM] cases, 9 aggressive SM [ASM] cases) versus 117 age-matched healthy donors and paired bone marrow samples of 31 patients with SM versus 17 controls, respectively. In parallel, IgM, IgD, IgG, IgA, and IgE plasma levels were measured. Compared with healthy donors, patients with SM showed increased immature B-cell production in bone marrow (P= .003) associated with greater release of pre-germinal center immature (P< .001) and naive CD5+ B lymphocytes (P< .001) to blood, but a pronounced decrease in PC counts of all different IgH isotypes and subclasses (P≤ .001) together with overall increased IgM (P= .001) and IgD (P< .001) plasma levels. Different immune profiles were found per diagnostic subtype of disease with progressively greater counts in blood of immature B lymphocytes together with decreased IgMD+, IgG2+, IgA1+, and IgA2+ memory B cells (P≤ .032) and elevated IgM (P= .017) plasma levels in cases of ASM, increased IgM (P= .001) and IgD (P= .001) plasma levels in ISM cases, and exacerbated IgE (P< .001) with decreased IgG (P= .008) plasma levels in BMM cases. Our results reveal a significant dysregulation of the B-cell and PC compartments in blood of patients with SM, consistent with distinctly altered antibody isotype profiles in plasma of patients with BMM versus ISM versus ASM.

Central role of Galectin-3 at the cross-roads of cardiac inflammation and fibrosis: Implications for heart failure and transplantation

Cardiac inflammation and fibrosis are central pathogenic mechanisms leading to heart failure. Transplantation is still the treatment of choice for many patients undergoing end-stage heart failure who remain symptomatic despite optimal medical therapy. In spite of considerable progress, the molecular mechanisms linking inflammation, fibrosis and heart failure remain poorly understood. Galectin-3 (GAL3), a chimera-type member of the galectin family, has emerged as a critical mediator implicated in cardiac inflammatory, vascular and fibrotic processes through modulation of different cellular compartments including monocytes and macrophages, fibroblasts, endothelial cells and vascular smooth muscle cells via glycan-dependent or independent mechanisms. GAL3-driven circuits may hierarchically amplify cytokine production and function, immune cell activation and fibrosis cascades, influencing a wide range of cardiovascular disorders. Thus, GAL3 emerges as a potential therapeutic target to counteract aberrant inflammation and fibrosis during heart failure and a potential biomarker of heart failure and clinical outcome of heart transplantation.

A distributed integral control mechanism for regulation of cholesterol concentration in the human retina.

Tight homeostatic control of cholesterol concentration within the complex tissue microenvironment of the retina is the hallmark of a healthy eye. By contrast, dysregulation of biochemical mechanisms governing retinal cholesterol homeostasis likely contributes to the aetiology and progression of age-related macular degeneration (AMD). While the signalling mechanisms maintaining cellular cholesterol homeostasis are well-studied, a systems-level description of molecular interactions regulating cholesterol balance within the human retina remains elusive. Here, we provide a comprehensive overview of all currently-known molecular-level interactions involved in cholesterol regulation across the major compartments of the human retina, encompassing the retinal pigment epithelium (RPE), photoreceptor cell layer, Müller cell layer and Bruch's membrane. We develop a comprehensive chemical reaction network (CRN) of these interactions, involving 71 molecular species, partitioned into 10 independent subnetworks. These subnetworks collectively ensure robust homeostasis of 14 forms of cholesterol across distinct retinal cellular compartments. We provide mathematical evidence that three independent antithetic integral feedback controllers tightly regulate ER cholesterol in retinal cells, with additional independent mechanisms extending this regulation to other forms of cholesterol throughout the retina. Our novel mathematical model of retinal cholesterol regulation provides a framework for understanding the mechanisms of cholesterol dysregulation in diseased eyes and for exploring potential therapeutic strategies.

Fission of double-membrane tubes under tension

The division of a cellular compartment culminates with the scission of a highly constricted membrane neck. Scission requires lipid rearrangements, topology changes, and transient formation of nonbilayer intermediate structures driven by curvature stress. Often, a side effect of this stress is pore formation, which may lead to content leakage and thus breaching of the membrane barrier function. In single-membrane systems, leakage is avoided through the formation of a hemifusion (HF) intermediate, whose structure is still a subject of debate. The consequences of curvature stress have not been explored in double-membrane systems, such as the mitochondrion. Here, we combine experimental and theoretical approaches to study neck constriction and scission driven by tension in biomimetic lipid systems, namely single- and double-membrane nanotubes (sNTs and dNTs), respectively. In sNTs, constriction by high tension gives rise to a metastable HF intermediate (seen as stalk or worm-like micelle), whereas poration is universally slower in a simple neck. In dNTs, high membrane tension causes sequential rupture of each membrane. In contrast, low tension leads to the HF of both membranes, which may lead to a leaky fusion pathway, or may progress to further fusion of the two membranes along a number of transformation pathways. These findings provide a new mechanistic basis for fundamental cellular processes.