Apoptotic Blebs Research Articles

Anagallis arvensis Induces Apoptosis in HL-60 Cells Through ROS-Mediated Mitochondrial Pathway

The present study was taken up to evaluate the apoptosis inducing ability of alcoholic extract of whole plant of Anagallis arvensis (AAE) in HL-60 cells. We observed time and concentration dependent decrease in cell viability after treatment with AAE. Fluorescent staining and scanning electron micrographs of treated HL-60 cells demonstrated chromatin condensation, nuclear fragmentation and formation of apoptotic blebs. There was a marked increase in hypodiploid population of cells as observed by cell cycle analysis. Annexin V-FITC/PI also depicted the presence of apoptotic cells. Anti-apoptotic protein Bcl-2 was observed to be decreased by 62% at 20 µg/ml concentration and a significant increase in ROS production up to 6.9-fold was observed in time dependent manner. In addition, alteration in mitochondrial membrane potential was observed, which was followed by cytochrome c release to cytoplasm. Activated levels of mitochondrial downstream pathway protein namely Caspase-3 and 9, were detected in treated HL-60 cells by colorimetric analysis. DNA ladder formation, a biochemical hallmark of apoptosis was also observed in treated HL-60 cells. The results of the present study support the apoptotic potential of AAE and probability of its promising role in development as effective anticancer agent against leukemia cells.

Apoptotic blebs from Leishmania major-infected macrophages as a new approach for cutaneous leishmaniasis vaccination

We focused on apoptotic blebs from Leishmania major-infected macrophages as a vaccine for cutaneous leishmaniasis. Apoptosis was induced in L. major-infected J774A.1 cells in order to prepare apoptotic blebs. Test groups of BALB/c mice were immunized with these at doses of 1 × 106, 5 × 106 or 1 × 107 blebs. An immunization control group received Leishmania lysate antigens. The results showed that as the number of apoptotic bodies increased, the lymphocyte proliferation index increased, and this was proportional to IFN-γ level in the test groups. Additionally, the difference of IFN-γ, IL-4, IFN-γ/IL-4 ratio, or total IgG (p < 0.0001) in all groups was statistically significant compared to the negative control group. The highest IFN-γ (514.0 ± 40.92 pg/mL) and IFN-γ/IL-4 ratio (2.94 ± 0.22) were observed in the group that received 1 × 107 apoptotic blebs. The highest levels of IL-4 (244.6 ± 38.8 pg/mL) and total IgG (5626 ± 377 μg/mL) were observed in the immunization control group. Reflecting these data, no lesions were observed in any of the groups vaccinated with apoptotic blebs after 12 weeks. In summary, the use of apoptotic blebs from L. major-infected macrophages is protective against the challenge with L. major in this animal model.

Tumour-suppressive effect of oestrogen receptor β in colorectal cancer patients, colon cancer cells, and a zebrafish model.

Oestrogen receptor β (ERβ) has been suggested to have anti-proliferative and anti-tumour effects in breast and prostate cancer cells, but other studies have indicated its tumour-promoting effects. Understanding the complex effects of this receptor in different contexts requires further study. We reported that high ERβ expression is independently associated with improved prognosis in female colorectal cancer (CRC) patients. Herein, we investigated the possible anti-tumour effect of ERβ and its selective agonist. CRC patients with high ERβ expression had significantly higher levels of membrane-associated β-catenin, cysteinyl leukotriene receptor 2 (CysLT2 R), and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which have anti-tumour effects, but lower levels of nuclear β-catenin, cysteinyl leukotriene receptor 1 (CysLT1 R), and cyclooxygenase-2 (COX-2), which have tumour-promoting effects. These interesting findings were further supported by two different publicly available CRC mRNA datasets that showed a significant positive correlation between ERβ expression and 15-PGDH and CysLT2 R expression and a negative correlation between ERβ expression and β-catenin, CysLT1 R, and COX-2 expression. We next evaluated ERβ expression in three different colon cancer mouse models; ERβ expression was negatively correlated with tumourigenesis. Furthermore, treatment with the ERβ-agonist ERB-041 reduced CysLT1 R, active β-catenin, and COX-2 levels but increased phospho-β-catenin, CysLT2 R, and 15-PGDH levels in HCT-116, Caco-2, and SW-480 colon cancer cells compared to vehicle-treated cells. Interestingly, ERB-041-treated cells showed significantly decreased migration, survival, and colonosphere formation and increased apoptotic activity, as indicated by increased CASPASE-3 and apoptotic blebs. Finally, patients with low ERβ expression had significantly more distant metastasis at the time of diagnosis than patients with high ERβ expression. Therefore, we studied the effects of ERB-041-treated colon cancer cells in a zebrafish xenograft model. We found significantly less distant metastasis of ERB-041-treated cells compared to vehicle-treated cells. These results further support ERβ's anti-tumour role in CRC and the possible use of its agonist in CRC patients. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Coordinated changes in cell membrane and cytoplasm during maturation of apoptotic bleb.

Apoptotic cells form membrane blebs, but little is known about how the formation and dynamics of membrane blebs are regulated. The size of blebs gradually increases during the progression of apoptosis, eventually forming large extracellular vesicles called apoptotic bodies that have immune-modulating activities. In this study, we investigated the molecular mechanism involved in the differentiation of blebs into apoptotic blebs by comparing the dynamics of the bleb formed during cell migration and the bleb formed during apoptosis. We revealed that the enhanced activity of ROCK1 is required for the formation of small blebs in the early phase of apoptosis, which leads to the physical disruption of nuclear membrane and the degradation of Lamin A. In the late phase of apoptosis, the loss of asymmetry in phospholipids distribution caused the enlargement of blebs, which enabled translocation of damage-associated molecular patterns to the bleb cytoplasm and maturation of functional apoptotic blebs. Thus, changes in cell membrane dynamics are closely linked to cytoplasmic changes during apoptotic bleb formation.

Cytotoxicity of Euphorbia peplus Extract on MCF7 Breast Cancer Cells

In this study, the effect of Euphorbia peplus aqueous extract on human breast cancer cell line MCF7 was examined. The short and long term cytotoxicity were evaluated using sulphorhodamine B and clonogenic assays respectively. Scanning and transmission electron microscopy were employed to examine Euphorbia peplus-induced ultrastructural changes in MCF7 cells. The sulphorhodamine B assay revealed that Euphorbia peplus inhibits the growth of MCF7 with an IC50 of 30.32 μg/ml. The clonogenic assay proved that Euphorbia peplus' growth inhibitory effect is long lasting. The ultrastructural examination demonstrated that Euphorbia peplus extract induces MCF7 cell death. Scanning electron microscopy showed apoptotic blebbing. Transmission electron microscopy displayed cellular shrinkage, the formulation of apoptotic bodies, mitochondrial changes, nuclear shrinkage, chromatin condensation, autophagic vacuoles, and necrotic changes. In summary, Euphorbia peplus has displayed growth inhibitory activity against MCF7 cells and induces cell death predominantly via apoptosis and could be exploited as a breast cancer treatment after further evaluation.

Functional Validation of DownRegulated MicroRNAs in HeLa Cells Treated with Polyalthia longifolia Leaf Extract Using Different Microscopic Approaches: A Morphological Alteration-Based Validation.

Several microscopy methods have been developed to assess the morphological changes in cells in the investigations of the mode of cell death in response to a stimulus. Our recent finding on the treatment of the IC50 concentration (26.67 μg/mL) of Polyalthia longifolia leaf extract indicated the induction of apoptotic cell death via the regulation of miRNA in HeLa cells. Hence, the current study was conducted to validate the function of these downregulated microRNAs in P. longifolia-treated HeLa cells using microscopic approaches. These include scanning electron microscope (SEM), transmission electron microscope (TEM), and acridine orange/propidium iodide (AO/PI)-based fluorescent microscopy techniques by observing the morphological alterations to cells after transfection with mimic miRNA. Interestingly, the morphological changes observed in this study demonstrated the apoptotic hallmarks, for instance, cell blebbing, cell shrinkage, cytoplasmic and nuclear condensation, vacuolization, cytoplasmic extrusion, and the formation of apoptotic bodies, which proved the role of dysregulated miRNAs in apoptotic HeLa cell death after treatment with the P. longifolia leaf extract. Conclusively, the current study proved the crucial role of downregulated miR-484 and miR-221-5p in the induction of apoptotic cell death in P. longifolia-treated HeLa cells using three approaches-SEM, TEM, and AO/PI-based fluorescent microscope.

Abstract 279: c-Jun N-terminal kinase is involved in an oxidative stress- and vacuole-associated cell death pathway in triple negative breast cancer

Abstract Breast cancer is the most prevalent type of cancer among women. Triple negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer. It is the most challenging subtype to treat mainly due its aggressive nature and lack of targets such as HER2 (human epidermal growth factor 2), PR (progesterone receptor), and ER (estrogen receptor) found in other subtypes. We have shown that inhibiting both EGFR (epidermal growth factor receptor)/HER2 with lapatinib and JNK with JNK-IN-8 causes synergistic cell death in 11 out of 12 TNBC cell lines. EGFR is expressed in up to 90% tumors and JNK activation is linked to TNBC phenotype, metastasis, and stem cell character, among others. Synergism was achieved in the resistant, low-EGFR expressing cell line when a FGFR inhibitor (BGJ398) was used instead of lapatinib in combination with JNK-IN-8. Combination drug effects were accompanied by elevated oxidative stress. Suppression of oxidative stress restored cell viability in cells treated with the drug combination. JNK-IN-8 treatment alone or in combination with lapatinib induced massive cytoplasmic vacuolization with little apoptotic blebbing. The vacuoles were shown to be membrane-derived and acidic using Lucifer Yellow and Acridine Orange, respectively. Pre-treatment of cells with bafilomycin A1 (lysosome inhibitor), 5-(N-Ethyl-N-isopropyl) amiloride (macropinocytosis inhibitor), or N-acetylcysteine (antioxidant) reduced vacuolization. A similar effect was observed when cells were serum-starved. JNK1 knockout mouse mammary tumor cells, in comparison with JNK2 knockout, were resistant to drug synergism and displayed significantly less oxidative stress and vacuolization. Our studies demonstrated that concomitant blockade of JNK and individual tumor-appropriate targets induce synergistic cell death in TNBC cells in response to oxidative stress. Moreover, JNK blockade resulted in the formation of serum-dependent, acidic vacuoles that were reversible by lysosome or macropinocytosis inhibition. Loss-of-function studies indicated that JNK1 was the key isoform responsible for the drug response in TNBC cells. These findings suggest that JNK is involved in a novel cell death pathway and is a robust target in combination therapies against TNBC. Citation Format: Milad Soleimani, Ritvik Bhattacharjee, Uma Giri, Tamer S. Kaoud, Kevin N. Dalby, S. Gail Eckhardt, Carla L. Van Den Berg. c-Jun N-terminal kinase is involved in an oxidative stress- and vacuole-associated cell death pathway in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 279.

Reactive and destructive changes of Peyer’s patches in rats with experimental burn disease under infusion of detoxification solutions

The pathogenesis of burn immune dysfunction and burn enteropathy needs further clarification given that the cellular lesions of lymphoid tissue associated with mucous membranes are the least studied. The purpose of the study was to establish reactive and destructive changes in Peyer’s patches of rats after burn injury of the skin with the use of intravenous infusion of isotonic sodium chloride solution and combined colloid-hyperosmolar solutions. White male rats weighing 160-200 g at 6 months of age were divided into 4 groups (18 animals in each group): I, II, III – rats with burn skin injury (grade II-III burn with an area of 23% of body surface area and the development of moderate-severity shock state) which was administered a separate intravenous infusion once a day for the first 7 days of the experiment with isotonic sodium chloride, lactoprotein with sorbitol and HAES-LX-5%, in each case at a dose of 10 ml/kg; IV – intact animals. The material was collected from rats under deep thiopental intraperitoneal anesthesia after 1, 3, 7, 14, 21 and 30 days after burn injury. Biopsies from Peyer’s patches for histological and electron microscopic examination were processed using conventional methods. Investigation of histological preparations stained with hematoxylin-eosin was performed on an Olympus BX51 microscope. Ultrathin sections were contrasted on copper support meshes with uranyl acetate and lead citrate according to Reynolds and studied using a PEM-125K electron microscope. Electron and light microscopy data indicate that intravenous infusion of colloidal-hyperosmolar solutions (lactoprotein with sorbitol and HAES-LX-5%) promotes suppression of inflammatory response, inhibits necrosis, and optimizes lymphoid apoptosis at Peyer’s patches of rats with experimental burn disease caused by burn injury to the skin of 21-23% of the body surface. Apoptotic lymphocytes and their apoptotic bodies are effectively phagocytosed by macrophages and are digestible in heterophagolysosomes. The apoptotic altered dendritic cells in Peyer’s patches are characterized by osmiophilic cytoplasm and a nucleus with high electron density amorphous nucleoplasm. In the cytoplasm are located mitochondria with enlightened matrix and destroyed cristae, irregularly expanded tubules of variable configuration of a granular endoplasmic reticulum with electronically transparent content and numerous derivatives of their vacuole transformation, which are sharply darkened. The fusion of vacuoles leads to the formation of large electron-luminous cavities filled with various residues of compacted degraded cellular structures. Vacualization promotes site segmentation of condensed cytoplasm of apoptotic dendritic cells and formation of apoptotic blebs, which are subject to entrapment and subsequent sequential degradation with the participation of neighboring macrophages. The structural changes of the organelles of the protein-synthesizing apparatus found in Peyer’s plaque cells in rats with experimental burn disease can be regarded as a manifestation caused by functional overload of the granular endoplasmic reticulum (ER-stress). The consequence of optimal development of ER-stress and subsequent unfolded protein response is the apoptotic degradation of the corresponding cell, the course of which is modified by the use of colloid-hyperosmolar solutions.

Extracellular Vesicles and Their Potential Use in Monitoring Cancer Progression and Therapy: The Contribution of Proteomics.

Extracellular Vesicles (EVs) are small membrane-enclosed particles released by cells and able to vehiculate information between them. The term EVs categorizes many and different vesicles based on their biogenesis and release pathway, such as exosomes (Exo), ectosomes, or shedding microvesicles (SMVs), apoptotic blebs (ABs), and other EVs subsets, generating a heterogeneous group of components able to redistribute their cargo into the entire organism. Moreover EVs are becoming increasingly important in monitoring cancer progression and therapy, since they are able to carry specific disease biomarkers such as Glypican-1, colon cancer-associated transcript 2, CD63, CD24, and many others. The importance of their biological role together with their heterogeneity prompted researchers to adopt and standardize purification methods able to isolate EVs for characterizing their cargo. In this way, mass spectrometry (MS)-based proteomics approaches are emerging as promising tool for the identification and quantification of EVs protein cargoes, but this technique resulted to be deeply influenced by the low quality of the isolation techniques. This review presents the state-of-the-art of EVs isolation, purification, and characterization for omics studies, with a particular focus to their potential use in monitoring cancer progression and therapy.

ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly.

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

Two morphological changes of cancer cells under natural killer cell killing

Abstract Natural killer (NK) cells use granule exocytosis to kill cancer cells, which involves perforin-mediated delivery of granzymes from NK cells to cancer cells. Once inside the cancer cells, granzyme B, a member of serine protease family, activates caspase-3, which activates the serine/threonine kinase Rho-associated coiled coil–containing protein kinase (ROCK) I, an effector of the small GTPase Rho. Activated ROCK I increases the phosphorylation of myosin regulatory light chain and thus stimulates actomyosin contraction, required for apoptotic membrane blebbing. Ultimately, cancer cells under NK cell attack show rounding, shrinkage, and membrane rupture, which are classical morphological changes observed at the onset of apoptosis. Although much has been learned about these morphological changes, a fundamental question of how melanoma cell death is induced by NK cells remains unanswered. Do all melanoma cell lines undergo the classical dying process? If not, is there another dying process? To address this simple issue, we examined the dying of B16 melanoma cell lines under NK cell attack by using time-lapse imaging. Upon contact with NK cells, B16-F10 cells rounded and most of them showed membrane rupture (98 min); however, B16 parent cells showed writhing and delayed membrane rupture (235 min). This morphological difference depended on the expression levels of myosin regulatory light chain 9 (MYL9) but not activating ligands (CD112, CD155, Rae-1, and MULT-1), SPI, FasL, or PD-L1. Taken together, our data show that melanoma cells show two distinct types of morphological changes upon contact with NK cells and suggest that a strategy to decrease MYL9 expression by melanoma cells may improve the efficacy of NK cell–based immunotherapy.

ВНЕКЛЕТОЧНЫЕ МИКРОВЕЗИКУЛЯРНЫЕ ЧАСТИЦЫ В ПАТОГЕНЕЗЕ ТУБЕРКУЛЕЗА

The review describes main classes of extracellular microvesicular particles, mechanisms of their biogenesis and their potential role in the development of tuberculosis. Special attention is paid to apoptosis of macrophages infected with tuberculous mycobacteria, generation of apoptotic ectosome and involvement of the latter into the formation of anti-tuberculosis immune response. Conclusion: mechanisms of apoptotic blebbing, ectosomes and clearing of these particles possess the potential to become a new tool within pathogenetic therapy of tuberculosis.

Human Chorionic Gonadotropin Influences Systemic Autoimmune Responses.

Immunopathological outcomes in Systemic Lupus Erythematosus (SLE; or lupus) are believed to be autoantibody-mediated. Conditions which promote a Th2 skew (such as pregnancy) should encourage antibody production, worsening antibody-mediated diseases while ameliorating Th1/Th17-mediated diseases. Although an increased propensity toward autoreactivity can be observed in pregnant lupus patients and in pregnant lupus-prone mice, whether a unique human pregnancy-specific factor can contribute to such effects is unknown. This study assessed whether human chorionic gonadotropin (hCG, a pregnancy-specific hormone of diverse function) at physiological concentrations could mediate stimulatory influences on immune parameters in non-pregnant, lupus-prone mice, in light of the hormone's ameliorating effects on Th1-mediated autoimmunity in murine models. Results demonstrate that administration of hCG heightened global autoreactivity in such mice; antibodies to dsDNA, RNP68, Protein S, Protein C, β2-glycoprotein 1, and several phospholipids were enhanced, and hormone administration had adverse effects on animal survival. Specifically in splenic cell cultures containing cells derived from lupus-prone mice, hCG demonstrated synergistic effects with TLR ligands (up-modulation of costimulatory markers on B cells) as well as with TCR stimuli (enhanced proliferative responses, enhanced levels of cytokines, and the phosphorylation of p38). In both instances, enhanced synthesis of lupus-associated cytokines was observed, in addition to the heightened generation of autoantibodies reactive toward apoptotic blebs. These results suggest that selective transducive, proliferative, and differentiative effects of hCG on adaptive immune cells may drive autoreactive responses in a lupus environment, and may also potentially provide insights into the association between the presence of higher hCG levels (or the administration of hCG) with the presence (or appearance) of humoral autoimmunity.

Dynamic blebbing: A bottleneck to human embryonic stem cell culture that can be overcome by Laminin-Integrin signaling

This study characterizes dynamic and apoptotic blebbing in human embryonic stem cells (hESC), identifies dynamic blebbing as a bottleneck to successful cell attachment during passaging, and demonstrates that dynamic blebbing can be rapidly stopped by plating cells on recombinant human laminin. In freshly plated hESC, dynamic and apoptotic blebbing differed in time of occurrence, bleb retraction rate, mitochondrial membrane potential, and caspase 3&7 activation. While dynamic blebbing can be controlled with drugs that inhibit myosin II, these methods have off-target effects and are not suitable for clinical applications. Recombinant human laminin-521 or addition of laminin-111 to Matrigel provided a safe method to drastically decrease dynamic blebbing and improve cell attachment with proteins normally found in the inner cell mass. Inhibition of focal adhesion kinase, which is activated by binding of integrins to laminin, prolonged dynamic blebbing and inhibited attachment. These data show that hESC bind rapidly to laminins through an integrin, which activates focal adhesion kinase that in turn downregulates dynamic blebbing. Laminins enabled hESC to rapidly attach during passaging, improved plating efficiency, enabled passaging of single pluripotent stem cells, and avoided use of inhibitors that have non-specific off-target effects. These data provide a strategy for improving hESC culture using biologically safe recombinant human proteins.

Butenolides from a marine-derived fungus Aspergillus terreus with antitumor activities against pancreatic ductal adenocarcinoma cells.

Chemical study on the extract of a marine-derived fungus Aspergillus terreus yielded twelve butenolide derivatives, including three new compounds, namely asperlides A-C (1-3) and nine known butenolides (4-12). The structures of 1-3 were confirmed by comprehensive spectroscopic analysis, including HRESIMS, NMR spectroscopy, and calculated electronic circular dichroism (ECD). The cytotoxicity of the compounds was evaluated using PANC-1, HCC1806, HepG2, BEAS-2B and HT-29 cancer cells. The results showed that (+)-3',3'-di-(dimethylallyl)-butyrolactone II (4) and versicolactone B (6) exhibited the most potent cytotoxin of PANC-1 cell line, with the IC50 values of 5.3 and 9.4 μM, respectively. Morphological features of apoptosis were observed in 4 and 6-treated PANC-1 cells, including apoptotic body formation, membrane blebbing, cell shrinkage and nuclear condensation. Cell cycle analysis with propidium iodide staining exhibited that 4 inhibits proliferation of PANC-1 cells via the induction of G2/M and S phase arrest, while 6 could retard the PANC-1 cells via the induction of S phase arrest. Flow cytometric analysis suggested that treatment with 4 and 6 significantly induced PANC-1 cells apoptosis. These findings indicated that 4 and 6 might serve as a starting point for the development of an anticancer drug for the treatment of pancreatic ductal adenocarcinoma.

Structures and Activities of Tiahuramides A-C, Cyclic Depsipeptides from a Tahitian Collection of the Marine Cyanobacterium Lyngbya majuscula.

The structures of three new cyclic depsipeptides, tiahuramides A (1), B (2), and C (3), from a French Polynesian collection of the marine cyanobacterium Lyngbya majuscula are described. The planar structures of these compounds were established by a combination of mass spectrometry and 1D and 2D NMR experiments. Absolute configurations of natural and nonproteinogenic amino acids were determined through a combination of acid hydrolysis, derivitization with Marfey's reagent, and HPLC. The absolute configuration of hydroxy acids was confirmed by Mosher's method. The antibacterial activities of tiahuramides against three marine bacteria were evaluated. Compound 3 was the most active compound of the series, with an MIC of 6.7 μM on one of the three tested bacteria. The three peptides inhibit the first cell division of sea urchin fertilized eggs with IC50 values in the range from 3.9 to 11 μM. Tiahuramide B (2), the most potent compound, causes cellular alteration characteristics of apoptotic cells, blebbing, DNA condensation, and fragmentation, already at the first egg cleavage. The cytotoxic activity of compounds 1-3 was tested in SH-SY5Y human neuroblastoma cells. Compounds 2 and 3 showed an IC50 of 14 and 6.0 μM, respectively, whereas compound 1 displayed no toxicity in this cell line at 100 μM. To determine the type of cell death induced by tiahuramide C (3), SH-SY5Y cells were costained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. The double staining indicated that the cytotoxicity of compound 3 in this cell line is produced by necrosis.

Oxidized Hemoglobin Is Antigenic and Immunogenic in Lupus.

Hemolysis-associated anemia is characteristic of diseases such as atherosclerosis, lupus, malaria, and leishmaniasis; the toxic effects of free hemoglobin (Hb) have been extensively described. This study was based on the premise that release of this sequestered, inflammatory molecule can result in deleterious immunological consequences, particularly in the context of pre-existing lupus. IgG anti-Hb responses were detected in the sera of lupus patients. Lupus-prone mice exhibited heightened plasma Hb levels, and ferric (Fe3+) Hb triggered preferential release of lupus-associated cytokines from splenocytes derived from aging lupus-prone mice. Anti-Hb B cell precursor frequencies were heightened in such mice, which also expressed increased titers of anti-Hb antibodies in serum and in kidney eluates. Fe3+ Hb preferentially increased the functional maturation of bone marrow-derived dendritic cells (BMDCs) from lupus-prone mice, effects abrogated upon the inhibition of Stat3. Hb interacted with lupus-associated autoantigens extruded during apoptosis and coincubation of Hb and apoptotic blebs had additional maturation-inducing effects on lupus BMDCs. Immunization with Hb in lupus-prone mice induced antigen spreading to lupus-associated moieties; Hb-interacting autoantigens were preferentially targeted and increased complement deposition and glomerulosclerosis were observed. Hb therefore demonstrates both antigenicity and immunogenicity and triggers specific immuno-pathological effects in a lupus milieu.

Targeting the stress oncoprotein LEDGF/p75 to sensitize chemoresistant prostate cancer cells to taxanes.

Prostate cancer (PCa) is associated with chronic prostate inflammation resulting in activation of stress and pro-survival pathways that contribute to disease progression and chemoresistance. The stress oncoprotein lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, promotes cellular survival against environmental stressors, including oxidative stress, radiation, and cytotoxic drugs. Furthermore, LEDGF/p75 overexpression in PCa and other cancers has been associated with features of tumor aggressiveness, including resistance to cell death and chemotherapy. We report here that the endogenous levels of LEDGF/p75 are upregulated in metastatic castration resistant prostate cancer (mCRPC) cells selected for resistance to the taxane drug docetaxel (DTX). These cells also showed resistance to the taxanes cabazitaxel (CBZ) and paclitaxel (PTX), but not to the classical inducer of apoptosis TRAIL. Silencing LEDGF/p75 effectively sensitized taxane-resistant PC3 and DU145 cells to DTX and CBZ, as evidenced by a significant decrease in their clonogenic potential. While TRAIL induced apoptotic blebbing, caspase-3 processing, and apoptotic LEDGF/p75 cleavage, which leads to its inactivation, in both taxane-resistant and -sensitive PC3 and DU145 cells, treatment with DTX and CBZ failed to robustly induce these signature apoptotic events. These observations suggested that taxanes induce both caspase-dependent and -independent cell death in mCRPC cells, and that maintaining the structural integrity of LEDGF/p75 is critical for its role in promoting taxane-resistance. Our results further establish LEDGF/p75 as a stress oncoprotein that plays an important role in taxane-resistance in mCRPC cells, possibly by antagonizing drug-induced caspase-independent cell death.

Innate and humoral recognition of the products of cell death: differential antigenicity and immunogenicity in lupus.

While apoptotic debris is believed to constitute the original antigenic insult in lupus (which is characterized by a time-dependent diversification of autoreactivity), whether such debris and autoantibodies specifically recognizing its constituents mediate differential effects on innate and humoral responses in lupus-prone mice is currently unknown. Apoptotic blebs (as opposed to cellular lysate) enhanced preferentially the maturation of dendritic cells (DCs) from bone marrow precursors drawn from lupus-prone mice. Murine, somatically mutated, apoptotic cell-reactive immunoglobulin (Ig)G monoclonal antibodies demonstrated enhanced recognition of DCs and also displayed a prominent lupus strain-specific bias in mediating DC maturation. Further, immunization of such antibodies specifically in lupus-prone mice resulted in widespread humoral autoreactivity; hypergammaglobulinaemia (a hallmark of systemic autoimmunity) was observed, accompanied by enhanced antibody titres to cellular moieties. Induced antibodies recognized antigens distinct from those recognized by the antibodies employed for immunization; in particular, nephritis-associated anti-double stranded (ds) DNA antibodies and neonatal lupus-associated anti-Ro60 antibodies were elicited by a non-dsDNA, non-Ro60 reactive antibody, and Sm was a favoured target. Further, only in lupus-prone mice did such immunization enhance the kinetics of humoral anti-self responses, resulting in the advanced onset of glomerulosclerosis. These studies reveal that preferential innate and humoral recognition of the products of cell death in a lupus milieu influence the indices associated with autoimmune pathology.

Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity

IntroductionPrimary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. MethodsWe isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. ResultsWe found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. DiscussionWhen compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication.