Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. Although their potential as cancer biomarkers has been promising, the identification ...and quantification of EVs in clinical samples remains challenging. Here we describe a sensitive and rapid analytical technique for profiling circulating EVs directly from blood samples of patients with colorectal cancer. EVs are captured by two types of antibodies and are detected by photosensitizer-beads, which enables us to detect cancer-derived EVs without a purification step. We also show that circulating EVs can be used for detection of colorectal cancer using the antigen CD147, which is embedded in cancer-linked EVs. This work describes a new liquid biopsy technique to sensitively detect disease-specific circulating EVs and provides perspectives in translational medicine from the standpoint of diagnosis and therapy.
Multipotent mesenchymal stromal cells (MSCs) ameliorate a wide range of diseases in preclinical models, but the lack of clarity around their mechanisms of action has impeded their clinical utility. ...The therapeutic effects of MSCs are often attributed to bioactive molecules secreted by viable MSCs. However, we found that MSCs underwent apoptosis in the lung after intravenous administration, even in the absence of host cytotoxic or alloreactive cells. Deletion of the apoptotic effectors BAK and BAX prevented MSC death and attenuated their immunosuppressive effects in disease models used to define MSC potency. Mechanistically, apoptosis of MSCs and their efferocytosis induced changes in metabolic and inflammatory pathways in alveolar macrophages to effect immunosuppression and reduce disease severity. Our data reveal a mode of action whereby the host response to dying MSCs is key to their therapeutic effects; findings that have broad implications for the effective translation of cell-based therapies.
To explore whether a cross-talk exists between PARP inhibition and PD-L1/PD-1 immune checkpoint axis, and determine whether blockade of PD-L1/PD-1 potentiates PARP inhibitor (PARPi) in tumor ...suppression.
Breast cancer cell lines, xenograft tumors, and syngeneic tumors treated with PARPi were assessed for PD-L1 expression by immunoblotting, IHC, and FACS analyses. The phospho-kinase antibody array screen was used to explore the underlying mechanism of PARPi-induced PD-L1 upregulation. The therapeutic efficacy of PARPi alone, PD-L1 blockade alone, or their combination was tested in a syngeneic tumor model. The tumor-infiltrating lymphocytes and tumor cells isolated from syngeneic tumors were analyzed by CyTOF and FACS to evaluate the activity of antitumor immunity in the tumor microenvironment.
PARPi upregulated PD-L1 expression in breast cancer cell lines and animal models. Mechanistically, PARPi inactivated GSK3β, which in turn enhanced PARPi-mediated PD-L1 upregulation. PARPi attenuated anticancer immunity via upregulation of PD-L1, and blockade of PD-L1 resensitized PARPi-treated cancer cells to T-cell killing. The combination of PARPi and anti-PD-L1 therapy compared with each agent alone significantly increased the therapeutic efficacy
Our study demonstrates a cross-talk between PARPi and tumor-associated immunosuppression and provides evidence to support the combination of PARPi and PD-L1 or PD-1 immune checkpoint blockade as a potential therapeutic approach to treat breast cancer.
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Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP ...inhibitors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with niraparib (MK-4827) > olaparib (AZD-2281) >> veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specific DNA repair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair, the Fanconi anemia pathway, polymerase β, and FEN1 are critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.
Among the features that distinguish type 1 innate lymphoid cells (ILC1s) from natural killer (NK) cells is a gene signature indicative of 'imprinting' by cytokines of the TGF-β family. We studied ...mice in which ILC1s and NK cells lacked SMAD4, a signal transducer that facilitates the canonical signaling pathway common to all cytokines of the TGF-β family. While SMAD4 deficiency did not affect ILC1 differentiation, NK cells unexpectedly acquired an ILC1-like gene signature and were unable to control tumor metastasis or viral infection. Mechanistically, SMAD4 restrained non-canonical TGF-β signaling mediated by the cytokine receptor TGFβR1 in NK cells. NK cells from a SMAD4-deficient person affected by polyposis were also hyper-responsive to TGF-β. These results identify SMAD4 as a previously unknown regulator that restricts non-canonical TGF-β signaling in NK cells.
Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how ...TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.
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► Triacylglyceride (TG) synthesis is coupled with lipid droplet (LD) growth ► Two LD populations exist: growing LDs, containing TG enzymes, and small LDs ► Specific TG synthesis enzymes move from the ER to LDs through membrane bridges ► LD localization of TG enzymes mediates expansion of a subset of LDs
Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. Wilfling et al. identify two LD populations distinct in their capacity for localized triacylglycerol (TG) synthesis, which is coupled to LD growth. TG synthesis enzymes relocate via membrane bridges from the ER to the LD surface to mediate LD expansion.
The aim of this study is to evaluate the relationship between antinuclear antibody (ANA) titer and specificity, as well as the relationship between the number of positive-autoantibodies (AAbs) in ...antinuclear antibodies (ANAs) and specificity for systemic lupus erythematosus (SLE), so as to explore their significance in the diagnosis of SLE. A total of 1297 patients with ANA results was enrolled in this study, including 148 patients with SLE patients. The sensitivity, specificity, sensitive likelihood ratio and specific likelihood ratio of indicators in SLE were determined by receiver-operator characteristic (ROC) curve after measurement of ANA and ANAs by indirect immunofluorescence (IIF) and immunoblotting, respectively. ROC analysis showed that the specificity of ANA titer ≥ 1 +, ≥ 2 + and ≥ 3 + for SLE was estimated to be 81.29%, 90.69% and 96.52% respectively, with a increased titer-specific likelihood ratio (5.16, 9.29 and 19.60, respectively). The specificity of the number of positive-AAbs ≥ 1, ≥ 2 and ≥ 3 in ANAs for SLE was estimated to be 80.42%, 94.95% and 99.3% respectively, with a increased number-specific likelihood ratio (4.8, 15.26 and 72.48, respectively). The estimated sensitivity of the number of positive-AAbs ≥ 3, AnuA and anti-rRNP was higher than that of anti-Sm (p < 0.01) (50.68%, 41.89% and 31.76% vs. 16.89%, respectively), while there was no significant difference in their specificity (99.3%, 99.74% and 99.56% vs. 99.74%, respectively) (p > 0.05). High titers of ANA and the presence of multiple AAbs in ANAs are highly specific for SLE and highly suggestive of SLE. The likelihood of SLE can be assessed by ANA titer and the number of positive-AAbs in ANAs.
The DNA damage response can be initiated in response to a variety of stress signals that are encountered during physiological processes or in response to exogenous cues, such as ionizing radiation or ...DNA-damaging therapeutic agents. A number of methods have been developed to examine the morphological, biochemical, and molecular changes that take place during the DNA damage response. When cells are exposed to ionizing radiation or DNA-damaging chemotherapeutic agents, double-stranded breaks (DSBs) are generated that rapidly result in the phosphorylation of histone H2A variant H2AX. Because phosphorylation of H2AX at Ser 139 (γ-H2AX) is abundant, fast, and correlates well with each DSB, it is the most sensitive marker that can be used to examine the DNA damage produced and the subsequent repair of the DNA lesion. γ-H2AX can be detected by immunoblotting and immunostaining using microscopic or flow cytometric detection. Since γ-H2AX can be also generated during DNA replication, as a consequence of apoptosis, or as it is found associated with residual DNA damage, it is important to determine the kinetics, number, size, and morphology of γ-H2AX-associated foci. This chapter describes a few standard protocols that we have successfully used in our laboratory for a number of experimental systems, primarily hematologic and epithelial cells grown in culture.
The myokine irisin increases cortical bone mass Colaianni, Graziana; Cuscito, Concetta; Mongelli, Teresa ...
Proceedings of the National Academy of Sciences - PNAS,
09/2015, Letnik:
112, Številka:
39
Journal Article
Recenzirano
Odprti dostop
It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. ...Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 μg kg⁻¹. We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 μg kg⁻¹ per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes,OpnandSost,but notUcp1orPparγexpression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulatedAtf4, Runx2, Osx, Lrp5, β-catenin, Alp,andCol1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursorFndc5was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressedFndc5,albeit at low levels. Furthermore, muscle fibers from r-irisin–injected mice displayed enhanced Fndc5 positivity, and irisin inducedFdnc5mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle–bone connectivity.
The nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome is a key regulator of innate immune responses, and its aberrant activation is ...implicated in the pathogenesis of many diseases such as Alzheimer’s disease and type 2 diabetes. Targeting the NLRP3 inflammasome could hold promise to combat these complex diseases, but therapies specifically inhibiting the NLRP3 inflammasome have not been developed for patient treatment. The current study aimed to identify food-borne exosome-like nanoparticles (ELNs) that inhibit NLRP3 inflammasome activity. Nine vegetables or fruits were selected to extract ELNs, which were examined for their inhibitory effects on activation of the NLRP3 inflammasome in primary macrophages. Although most of the tested ELNs posed minimal impacts, the ELNs from ginger rhizomes (G-ELNs) strongly inhibited NLRP3 inflammasome activation. The G-ELNs contained lipids, proteins, and RNAs and were easily taken up by macrophages. G-ELN treatment suppressed pathways downstream of inflammasome activation including caspase1 autocleavage, interleukin (IL)-1β and IL-18 secretion, and pyroptotic cell death. Apoptotic speck protein containing a caspase recruitment domain (ASC) oligomerization and speck formation assays indicated that G-ELNs blocked assembly of the NLRP3 inflammasome. The lipids in G-ELNs, rather than the RNAs or proteins, were responsible for the inhibitory activity observed. Together, the data suggested G-ELNs as new potent agents that block NLRP3 inflammasome assembly and activation. The unique features of G-ELNs including biomolecule protection and tissue bioavailability should facilitate the development of G-ELN-based therapy to target the NLRP3 inflammasome in the disease settings.
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