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Prostate cancer (PC) is a leading cause of cancer-related deaths in men worldwide. Interleukin-30 (IL-30) is a PC progression driver, and its suppression would be strategic for fighting metastatic disease. Biocompatible lipid nanoparticles (NPs) were loaded with CRISPR-Cas9gRNA to delete the human IL30 (hIL30) gene and functionalized with anti-PSCA-Abs (Cas9hIL30-PSCA NPs). Efficiency of the NPs in targeting IL-30 and the metastatic potential of PC cells was examined in vivo in xenograft models of lung metastasis, and in vitro by using two organ-on-chip (2-OC)-containing 3D spheroids of IL30 + PC-endothelial cell co-cultures in circuit with either lung-mimicking spheroids or bone marrow (BM)-niche-mimicking scaffolds. Cas9hIL30-PSCA NPs demonstrated circulation stability, genome editing efficiency, without off-target effects and organ toxicity. Intravenous injection of three doses/13 days, or five doses/20 days, of NPs in mice bearing circulating PC cells and tumor microemboli substantially hindered lung metastasization. Cas9hIL30-PSCA NPs inhibited PC cell proliferation and expression of IL-30 and metastasis drivers, such as CXCR2, CXCR4, IGF1, L1CAM, METAP2, MMP2, and TNFSF10, whereas CDH1 was upregulated. PC-Lung and PC-BM 2-OCs revealed that Cas9hIL30-PSCA NPs suppressed PC cell release of CXCL2/GROβ, which was associated with intra-metastatic myeloid cell infiltrates, and of DKK1, OPG, and IL-6, which boosted endothelial network formation and cancer cell migration. Development of a patient-tailored nanoplatform for selective CRISPR-mediated IL-30 gene deletion is a clinically valuable tool against PC progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The development of selective and nontoxic immunotherapy targeting prostate cancer (PC) is challenging. Interleukin (IL)30 plays immunoinhibitory and oncogenic roles in PC, and its tumor-specific suppression may have significant clinical implications. CRISPR/Cas9-mediated IL30 gene deletion in PC xenografts using anti-PSCA antibody-driven lipid nanocomplexes (Cas9gRNA-hIL30-PSCA NxPs) revealed significant genome editing efficiency and circulation stability without off-target effects or organ toxicity. Biweekly intravenous administration of Cas9gRNA-hIL30-PSCA NxPs to PC-bearing mice inhibited tumor growth and metastasis and improved survival. Mechanistically, Cas9gRNA-hIL30-PSCA NxPs suppressed ANGPTL 1/2/4, IL1β, CCL2, CXCL1/6, SERPINE1-F1, EFNB2, PLG, PF4, VEGFA, VEGFD, ANG, TGFβ1, EGF and HGF expression in human PC cells while upregulated CDH1, DKK3 and PTEN expression, leading to low proliferation and extensive ischemic necrosis. In the syngeneic PC model, IL30-targeting immunoliposomes downregulated NFKB1 expression and prevented intratumoral influx of CD11b + Gr-1 + MDCs, Foxp3 + Tregs, and NKp46 + RORγt + ILC3, and prolonged host survival by inhibiting tumor progression. This study serves as a proof of principle that immunoliposome-based targeted delivery of Cas9gRNA-IL30 represent a potentially safe and effective strategy for PC treatment., (© 2024. The Author(s).)

Background: Despite a multimodal approach including surgery, chemo- and radiotherapy, the 5-year event-free survival rate for rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in childhood, remains very poor for metastatic patients, mainly due to the selection and proliferation of tumour cells driving resistance mechanisms. Personalised medicine-based protocols using new drugs or targeted therapies in combination with conventional treatments have the potential to enhance the therapeutic effects, while minimizing damage to healthy tissues in a wide range of human malignancies, with several clinical trials being started. In this study, we analysed, for the first time, the antitumour activity of SFX-01, a complex of synthetic d, l-sulforaphane stabilised in alpha-cyclodextrin (Evgen Pharma plc, UK), used as single agent and in combination with irradiation, in four preclinical models of alveolar and embryonal RMS. Indeed, SFX-01 has shown promise in preclinical studies for its ability to modulate cellular pathways involved in inflammation and oxidative stress that are essential to be controlled in cancer treatment., Methods: RH30, RH4 (alveolar RMS), RD and JR1 (embryonal RMS) cell lines as well as mouse xenograft models of RMS were used to evaluate the biological and molecular effects induced by SFX-01 treatment. Flow cytometry and the modulation of key markers analysed by q-PCR and Western blot were used to assess cell proliferation, apoptosis, autophagy and production of intracellular reactive oxygen species (ROS) in RMS cells exposed to SFX-01. The ability to migrate and invade was also investigated with specific assays. The possible synergistic effects between SFX-01 and ionising radiation (IR) was studied in both the in vitro and in vivo studies. Student's t-test or two-way ANOVA were used to test the statistical significance of two or more comparisons, respectively., Results: SFX-01 treatment exhibited cytostatic and cytotoxic effects, mediated by G2 cell cycle arrest, apoptosis induction and suppression of autophagy. Moreover, SFX-01 was able to inhibit the formation and the proliferation of 3D tumorspheres as monotherapy and in combination with IR. Finally, SFX-01, when orally administered as single agent, displayed a pattern of efficacy at reducing the growth of tumour masses in RMS xenograft mouse models; when combined with a radiotherapy regime, it was observed to act synergistically, resulting in a more positive outcome than would be expected by adding each exposure alone., Conclusions: In summary, our results provide evidence for the antitumour properties of SFX-01 in preclinical models of RMS tumours, both as a standalone treatment and in combination with irradiation. These forthcoming findings are crucial for deeper investigations of SFX-01 molecular mechanisms against RMS and for setting up clinical trials in RMS patients in order to use the SFX-01/IR co-treatment as a promising therapeutic approach, particularly in the clinical management of aggressive RMS disease., (© 2024. The Author(s).)

The global rise of single-use throw-away plastic products has elicited a massive increase in the nano/microplastics (N/MPLs) exposure burden in humans. Recently, it has been demonstrated that disposable period products may release N/MPLs with usage, which represents a potential threat to women's health which has not been scientifically addressed yet. By using polyethyl ene (PE) particles (200 nm to 9 μm), we showed that acute exposure to a high concentration of N/MPLs induced cell toxicity in vaginal keratinocytes after effective cellular uptake, as viability and apoptosis data suggest, along with transmission electron microscopy (TEM) observations. The internalised N/MPLs altered the expression of junctional and adherence proteins and the organisation of the actin cortex, influencing the level of genes involved in oxidative stress signalling pathways and that of miRNAs related to epithelial barrier function. When the exposure to PE N/MPLs was discontinued or became chronic, cells were able to recover from the negative effects on viability and differentiation/proliferation gene expression in a few days. However, in all cases, PE N/MPL exposure prompted a sustained alteration of DNA methyltransferase and DNA demethylase expression, which might impact epigenetic regulation processes, leading to accelerated cell ageing and inflammation, or the occurrence of malignant transformation.

Nanomaterials are gaining increasing attention as innovative materials in medicine. Among nanomaterials, zinc oxide (ZnO) nanostructures are particularly appealing because of their opto-electrical, antimicrobial, and photochemical properties. Although ZnO is recognized as a safe material and the Zn ion (Zn 2+ ) concentration is strictly regulated at a cellular and systemic level, different studies have demonstrated cellular toxicity of ZnO nanoparticles (ZnO-NPs) and ZnO nanorods (ZnO-NRs). Recently, ZnO-NP toxicity has been shown to depend on the intracellular accumulation of ROS, activation of autophagy and mitophagy, as well as stabilization and accumulation of hypoxia-inducible factor-1α (HIF-1α) protein. However, if the same pathway is also activated by ZnO-NRs and how non-cancer cells respond to ZnO-NR treatment, are still unknown. To answer to these questions, we treated epithelial HaCaT and breast cancer MCF-7 cells with different ZnO-NR concentrations. Our results showed that ZnO-NR treatments increased cell death through ROS accumulation, HIF-1α and endothelial PAS domain protein 1 (EPAS1) activation, and induction of autophagy and mitophagy in both cell lines. These results, while on one side, confirmed that ZnO-NRs can be used to reduce cancer growth, on the other side, raised some concerns on the activation of a hypoxic response in normal cells that, in the long run, could induce cellular transformation.

Life on Earth has evolved in the presence of a gravity constraint. Any change in the value of such a constraint has important physiological effects. Gravity reduction (microgravity) alters the performance of muscle, bone and, immune systems among others. Therefore, countermeasures to limit such deleterious effects of microgravity are needed considering future Lunar and Martian missions. Our study aims to demonstrate that the activation of mitochondrial Sirtuin 3 (SIRT3) can be exploited to reduce muscle damage and to maintain muscle differentiation following microgravity exposure. To this effect, we used a RCCS machine to simulate microgravity on ground on a muscle and cardiac cell line. During microgravity, cells were treated with a newly synthesized SIRT3 activator, called MC2791 and vitality, differentiation, ROS and, autophagy/mitophagy were measured. Our results indicate that SIRT3 activation reduces microgravity-induced cell death while maintaining the expression of muscle cell differentiation markers. In conclusion, our study demonstrates that SIRT3 activation could represent a targeted molecular strategy to reduce muscle tissue damage caused by microgravity.

Background: Metastatic prostate cancer (PC) is a leading cause of cancer death in men worldwide. Targeting of the culprits of disease progression is an unmet need. Interleukin (IL)-30 promotes PC onset and development, but whether it can be a suitable therapeutic target remains to be investigated. Here, we shed light on the relationship between IL30 and canonical PC driver genes and explored the anti-tumor potential of CRISPR/Cas9-mediated deletion of IL30., Methods: PC cell production of, and response to, IL30 was tested by flow cytometry, immunoelectron microscopy, invasion and migration assays and PCR arrays. Syngeneic and xenograft models were used to investigate the effects of IL30, and its deletion by CRISPR/Cas9 genome editing, on tumor growth. Bioinformatics of transcriptional data and immunopathology of PC samples were used to assess the translational value of the experimental findings., Results: Human membrane-bound IL30 promoted PC cell proliferation, invasion and migration in association with STAT1/STAT3 phosphorylation, similarly to its murine, but secreted, counterpart. Both human and murine IL30 regulated PC driver and immunity genes and shared the upregulation of oncogenes, BCL2 and NFKB1, immunoregulatory mediators, IL1A, TNF, TLR4, PTGS2, PD-L1, STAT3, and chemokine receptors, CCR2, CCR4, CXCR5. In human PC cells, IL30 improved the release of IGF1 and CXCL5, which mediated, via autocrine loops, its potent proliferative effect. Deletion of IL30 dramatically downregulated BCL2, NFKB1, STAT3, IGF1 and CXCL5, whereas tumor suppressors, primarily SOCS3, were upregulated. Syngeneic and xenograft PC models demonstrated IL30's ability to boost cancer proliferation, vascularization and myeloid-derived cell infiltration, which were hindered, along with tumor growth and metastasis, by IL30 deletion, with improved host survival. RNA-Seq data from the PanCancer collection and immunohistochemistry of high-grade locally advanced PCs demonstrated an inverse association (chi-squared test, p = 0.0242) between IL30 and SOCS3 expression and a longer progression-free survival of patients with IL30 Neg SOCS3 Pos PC, when compared to patients with IL30 Pos SOCS3 Neg PC., Conclusions: Membrane-anchored IL30 expressed by human PC cells shares a tumor progression programs with its murine homolog and, via juxtacrine signals, steers a complex network of PC driver and immunity genes promoting prostate oncogenesis. The efficacy of CRISPR/Cas9-mediated targeting of IL30 in curbing PC progression paves the way for its clinical use., (© 2022. The Author(s).)

Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to be solved in order to translate the use of MSC-EV detection in clinical settings. Even if flow cytometry (FC) has been largely applied for EV studies, the lack of consensus on protocols for FC detection of EVs generated controversy. Standard FC procedures, based on scatter measurements, only allows the detection of the "tip of the iceberg" of all EVs. We applied an alternative FC approach based on the use of a trigger threshold on a fluorescence channel. The EV numbers obtained by the application of the fluorescence triggering resulted significantly higher in respect to them obtained from the same samples acquired by placing the threshold on the side scatter (SSC) channel. The analysis of EV concentrations carried out by three different standardized flow cytometers allowed us to achieve a high level of reproducibility (CV < 20%). By applying the here-reported method highly reproducible results in terms of EV analysis and concentration measurements were obtained.

SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities., Competing Interests: The authors have declared that no competing interests exist.

Aim of the Study: To investigate, in vivo and in vitro, the fibroblast-to-myofibroblast transition in patients with hypermobile Ehlers-Danlos Syndrome (EDS). To analyze the dermis of patients with classical form of EDS (cEDS) and with hEDS, to identify qualitative and/or quantitative differences in ECM component and ultrastructural changes in collagen., Materials and Methods: Seven subjects, aged over 18, two with cEDS and five with hEDS underwent two skin biopsy. One sample was prepared for transmission electron microscopy (TEM), the other for immunofluorescence. The diameter of collagen fibers was measured with TEM. Fibrils were analyzed in four patients: the two with cEDS and two with hEDS. For each patient, the diameter of n=250 collagen fibrils was measured. αSMA was used as specific marker for myofibroblast to highlight their presence in vivo in the skin of patients with hEDS., Result: IF observation could not assess an increased expression of αSMA in hEDS patients, which showed no statistical difference compared to classic form patients. The major result from the analysis of TEM images is the clear difference in ECM composition between the two forms of EDS: ECM in hEDS is optically more dense and more prominently composed of elastic fibers., Conclusion: Our study provides the following important evidence: 1) the absence in vivo of dermal fibroblasts in patients with hEDS, demonstrated by αSMA negativity; 2) the presence of statistically significant changes in the diameter of collagen fibrils between the classic and the hypermobile forms.

Calcium signaling can elicit different pathways involved in an extreme variety of biological processes. Calcium levels must be tightly regulated in a spatial and temporal manner in order to be efficiently and properly utilized in the host physiology. The Ca 2+ -ATPase, encoded by pmr-1 gene, was first identified in yeast and localized to the Golgi and it appears to be involved in calcium homeostasis. PMR-1 function is evolutionary conserved from yeast to human, where mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. In this work, we used the Caenorhabditis elegans model system to gain insight into the downstream response elicited by the loss of pmr-1 gene. We found that pmr-1 knocked down animals not only showed defects in the oligosaccharide structure of glycoproteins at the cell surface but also were characterized by reduced susceptibility to bacterial infection. Although increased resistance to the infection might be related to lack of regular recognition of C. elegans surface glycoproteins by microbial agents, we provide genetic evidence that pmr-1 interfered nematodes mounted a stronger innate immune response to Gram-positive bacterial infection. Thus, our observations indicate pmr-1 as a candidate gene implicated in mediating the worm's innate immune response.

Background: Current approaches aimed at inducing immunogenic cell death (ICD) to incite an immune response against cancer neoantigens are based on the use of chemotherapeutics and other agents. Results are hampered by issues of efficacy, combinatorial approaches, dosing and toxicity. Here, we adopted a strategy based on the use of an immunomolecule that overcomes pharmachemical limitations., Methods: Cytofluorometry, electron microscopy, RT-PCR, western blotting, apotome immunofluorescence, MLR and xenografts., Results: We report that an ICD process can be activated without the use of pharmacological compounds. We show that in Kras-mut/TP53-mut colorectal cancer cells the 15 kDa βGBP cytokine, a T cell effector with onco-suppressor properties and a potential role in cancer immunosurveillance, induces key canonical events required for ICD induction. We document ER stress, autophagy that extends from cancer cells to the corresponding xenograft tumours, CRT cell surface shifting, ATP release and evidence of dendritic cell activation, a process required for priming cytotoxic T cells into a specific anticancer immunogenic response., Conclusions: Our findings provide experimental evidence for a rationale to explore a strategy based on the use of an immunomolecule that as a single agent couples oncosuppression with the activation of procedures necessary for the induction of long term response to cancer.

In this review, we propose that paraganglioma is a fundamentally organized, albeit aberrant, tissue composed of neoplastic vascular and neural cell types that share a common origin from a multipotent mesenchymal-like stem/progenitor cell. This view is consistent with the pseudohypoxic footprint implicated in the molecular pathogenesis of the disease, is in harmony with the neural crest origin of the paraganglia, and is strongly supported by the physiological model of carotid body hyperplasia. Our immunomorphological and molecular studies of head and neck paragangliomas demonstrate in all cases relationships between the vascular and the neural tumor compartments, that share mesenchymal and immature vasculo-neural markers, conserved in derived cell cultures. This immature, multipotent phenotype is supported by constitutive amplification of NOTCH signaling genes and by loss of the microRNA-200s and -34s, which control NOTCH1 , ZEB1 , and PDGFRA in head and neck paraganglioma cells. Importantly, the neuroepithelial component is distinguished by extreme mitochondrial alterations, associated with collapse of the ΔΨm. Finally, our xenograft models of head and neck paraganglioma demonstrate that mesenchymal-like cells first give rise to a vasculo-angiogenic network, and then self-organize into neuroepithelial-like clusters, a process inhibited by treatment with imatinib.

Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.

The given and family names of two co-authors were incorrect in the published article. The correct spelling should read as: Sampath Chandra Prasad and Vinagolu K Rajasekhar.

Platelet multidrug resistance protein4 (MRP4)-overexpression has a role in reducing aspirin action. Aspirin in vivo treatment enhances platelet MRP4 expression and MRP4 mediated transport inhibition reduces platelet function and delays thrombus formation. The aim of our work was to verify whether MRP4 expression is enhanced in platelets obtained from patients under chronic aspirin treatment and whether it correlates with residual platelet reactivity. We evaluated changes on mRNA and protein-MRP4 expression and platelet aggregation in four populations: healthy volunteers (HV), aspirin-free control population (CTR), patients who started the treatment less than one month ago (ASA<1 month patients) and aspirinated patients who started the treatment more than two months ago (ASA>2 months patients). In platelets obtained from ASA>2 months patients, it was found a statistically significant MRP4 enhancement of both mRNA and protein expression compared to HV, CTR and ASA<1 month patients. Platelets obtained from ASA>2 months patients that present high levels of platelet MRP4, have higher serum TxB 2 levels and collagen-induced platelet aggregation compared to patient with low levels of MRP4 in platelets. In addition collagen induced platelet aggregation is higher in in vitro aspirinated platelets obtained from patients with high levels of MRP4 patients compared to those obtained from patients with low MRP4 levels. We can assert that, in patients under chronic aspirin treatment, platelets that present high MRP4 levels have an increase of residual platelet reactivity, which is due in part to incomplete COX-1 inhibition, and in part to COX-1-independent mechanism.

PEL cells relay on the constitutive activation of STAT3 for their survival, thus its inhibition by AG490 leads to apoptotic cell death. In this study, we found that the cytotoxic activity of AG490 correlated with the reduction of HSP70 and its master regulator HSF1 that, based on knocking-down experiments, was found to play a pro-survival role in PEL cells. To counteract the pro-death effect mediated by HSF1/HSP70 down-regulation, AG490 induced a complete autophagy, whose inhibition potentiated its cytotoxic effect against PEL cells. AG490 as well as HSF1 siRNA reduced the expression of Mcl-1, a Bcl-2 family member that negatively regulates apoptosis and autophagy. These results suggest that STAT3 inhibition, by down-regulating the expression of HSF1/HSP70, reduces Mcl-1 and leads to both apoptosis and autophagy induction in PEL cells., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Circulating tumor cells (CTCs) are often undetected through the immunomagnetic epithelial cell adhesion molecule (EpCAM)-based CellSearch(®) System in breast and colorectal cancer (CRC) patients treated with bevacizumab (BEV), where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro., Methods: The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch(®), untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch(®)., Results: Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins (CK), while no evidence of epithelial to mesenchymal transition (EMT) in treated cells was observed. The recovery rate of cells through CellSearch(®) was gradually reduced in course of treatment with BEV, being 84%, 70% and 40% at 1, 2 and 3 months, respectively., Conclusions: We hypothesize that BEV may prevent CellSearch(®) from capturing CTCs through altering EpCAM isoforms.

Aim: The aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα)., Methods: The effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV)., Results: In DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment., Conclusions: The present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients., (© 2014 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Unlabelled: Autophagy is a catabolic pathway that helps cells to survive under stressful conditions. Cells also use autophagy to clear microbiological infections, but microbes have learned how to manipulate the autophagic pathway for their own benefit. The experimental evidence obtained in this study suggests that the autophagic flux is blocked at the final steps during the reactivation of Epstein-Barr virus (EBV) from latency. This is indicated by the level of the lipidated form of LC3 that does not increase in the presence of bafilomycin and by the lack of colocalization of autophagosomes with lysosomes, which correlates with reduced Rab7 expression. Since the inhibition of the early phases of autophagy impaired EBV replication and viral particles were observed in autophagic vesicles in the cytoplasm of producing cells, we suggest that EBV exploits the autophagic machinery for its transportation in order to enhance viral production. The autophagic block was not mediated by ZEBRA, an immediate-early EBV lytic gene, whose transfection in Ramos, Akata, and 293 cells promoted a complete autophagic flux. The block occurred only when the complete set of EBV lytic genes was expressed. We suggest that the inhibition of the early autophagic steps or finding strategies to overcome the autophagic block, allowing viral degradation into the lysosomes, can be exploited to manipulate EBV replication., Importance: This study shows, for the first time, that autophagy is blocked at the final degradative steps during EBV replication in several cell types. Through this block, EBV hijacks the autophagic vesicles for its intracellular transportation and enhances viral production. A better understanding of virus-host interactions could help in the design of new therapeutic approaches against EBV-associated malignancies., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

There is concern about the possible toxicity of palladium nanoparticles (Pd-NP), as they are released in the environment through many applications. We previously studied the toxicity of Pd-NP at high concentrations; here we address the possible toxicity of Pd-NP at low, subtoxic doses. In particular, we have exposed normal human PBMC entering into the first in vitro mitotic division to Pd-NP and to Pd(IV) ions to evaluate ROS generation and cell cycle progression. We have measured a statistically significant increase of intracellular ROS in Pd(IV) exposed cells, but not in Pd-NP exposed cells. TEM revealed accumulation of lipid droplets and autophagic and mitophagic vacuoles, which appeared more conspicuous in cells exposed to Pd(IV) ions than to Pd-NP. Pd-NP were visible in the cytoplasm of Pd-NP exposed cells. Pd-NP addition was associated with a significant increase of cells within the G0/G1-phase and a significant reduction in GS- and G2/M-phases. Cells exposed to Pd(IV) ions showed a significant amplification of these cell cycle alterations. These results suggest that ions, per se or released by NPs, are the true inducers of Pd toxicity. It will be essential to verify whether the observed disturbance represents a temporary response or might result in permanent alterations.

Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The "NOTCH signaling pathway" was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.

Understanding the mechanisms of autophagy induction and its role during chemotherapeutic treatments is of fundamental importance in order to manipulate it to improve the outcome of chemotherapy. In particular whether the bortezomib-induced autophagy plays a pro-survival or pro-death role is still controversial. In this study we investigated if bortezomib induced endoplasmic reticulum (ER) stress and activated autophagy in Primary Effusion Lymphoma (PEL) cells and how they influenced cell survival. We found that bortezomib induced up-regulation of the pro-survival and pro-death ER stress molecules BIP and CHOP and activated c-Jun NH2-terminal kinase (JNK), resulting in Bcl-2 phosphorylation and induction of autophagy. JNK and autophagy activation played a pro-survival role in this setting, thus their inhibition increased the bortezomib cytotoxic effect and PARP cleavage in PEL cells. Based on our results we suggest that the combination of bortezomib with JNK or autophagy inhibitors could be exploited to improve the outcome of therapy of this aggressive B cell lymphoma.

Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-μ, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation.

Thirty-eight new (4-(4-substituted-phenyl/2,4-disubstituted-phenyl)-thiazol-2-yl)hydrazine derivatives were synthesized in good yield and assayed for their in vitro anti-Candida activity, compared to topical and systemic antifungal drugs, against twenty-two clinical isolates of Candida spp. The concurrent presence of aliphatic chains or cycloaliphatic rings at N1-hydrazine and a 4-methyl/4-methoxyphenyl at C4 position of the thiazole nucleus exhibited an interesting anti-Candida inhibitory activity. Moreover, some of the most active compounds showed synergistic antifungal effects and lower cell toxicity when combined with clotrimazole., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Adaptation to endoplasmic reticulum (ER) stress relies on activation of the unfolded protein response (UPR) and induction of autophagy. Indeed, cells die if ER stress is not countered by the UPR. Here we show in U937 cells that the ER stressors tunicamycin and thapsigargin cause increased expression of c-Jun N-terminal kinase 2 (JNK2), which allows regulation of the UPR, whose silencing or pharmacological inhibition delays BiP (immunoglobulin heavy-chain binding protein) upregulation, and causes earlier and greater expression of CCAAT/enhancer-binding protein-homologous protein (CHOP). Furthermore, we show that pharmacological inhibition or silencing of JNK2 causes accumulation of both p62 and the acidic compartment, caspase 3 activation and apoptosis. Our results reveal that JNK2 prevents accumulation of the acidic compartment in U937 cells undergoing autophagic flux and, by this mechanism, it keeps stressed cells alive. Our findings highlight a potential role for JNK2 in tumor cell survival, senescence and neurodegenerative diseases, in which ER stress, autophagy and lysosome activity are known to interplay.

A growing number of studies indicate that cell death can be either immunogenic or not, depending on its modalities, the type and the activation state of the cells, and finally, the environment where it happens. Increased understanding of the immunogenicity of cancer cell death will significantly improve the outcome of chemotherapeutic treatments.

To understand how cytotoxic agent-induced cancer cell death affects the immune system is of fundamental importance to stimulate immune response to counteract the high mortality due to cancer. Here we compared the immunogenicity of Primary Effusion Lymphoma (PEL) cell death induced by anticancer drug Bortezomib (Velcade) and Tyrphostin AG 490, a Janus Activated Kinase 2/signal trasducer and activator of transcription-3 (JAK2/STAT3) inhibitor. We show that both treatments were able to induce PEL apoptosis with similar kinetics and promote dendritic cells (DC) maturation. The surface expression of molecules involved in immune activation, namely calreticulin (CRT), heat shock proteins (HSP) 90 and 70 increased in dying cells. This was correlated with DC activation. We found that PEL cell death induced by Bortezomib was more effective in inducing uptake by DC compared to AG 490 or combination of both drugs. However the DC activation induced by all treatments was completely inhibited when these cells were pretreated with a neutralizing antiboby directed against the HSP90/70 and CRT common receptor, CD91. The activation of DC by Bortezomib and AG 490 treated PEL cells, as seen in the present study, might have important implications for a combined chemo and immunotherapy in such patients.

Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization.

Objective: To investigate the effects of palladium (Pd) nanoparticles on cytokine release from peripheral blood mononuclear cells (PBMCs) of control or Pd-sensitized nonatopic women., Methods: TNF-α, IL-5, IL-10 and IFN-γ release and/or expression from PBMCs incubated in presence of 5 to 10 nm Pd nanoparticles or Pd salt (potassium hexachloropalladate) were determined by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction analysis. Transmission electronmicroscopy was performed., Results: In lipopolysaccharide-stimulated PBMCs from controls, Pd salt inhibited IFN-γ and IL-10 release, whereas Pd nanoparticles enhanced IFN-γ release and inhibited TNF-α secretion. In lipopolysaccharide-stimulated PBMCs from Pd-sensitized women showing high IFN-γ release, Pd nanoparticles inhibited TNF-α release and Pd salt IL-10 release. TNF-α and IFN-γ release and messenger RNA expression were correlated. Transmission electronmicroscopy demonstrated uptake of nanoparticles in the endocytic compartment and activation of autophagy., Conclusions: Palladium ions and nanoparticles exert different effects in vitro on the expression and release of cytokines., ((C)2011The American College of Occupational and Environmental Medicine)

We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28. Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA. P38 is expressed and constitutively secreted, with increase after ER stress, in the KMS11 myeloma line and in the breast cancer lines MCF7 and SKBr3, but not in the non-tumorigenic breast epithelial MCF10A line. P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress. Consistently with the presence of p38 and p28 in culture media, morphological studies of SKBr3 and KMS11 cells detect N-terminal SEL1L immunolabeling in secretory/degradative compartments and extracellularly-released membrane vesicles. Our findings suggest that the two new SEL1L variants are engaged in endosomal trafficking and secretion via vesicles, which could contribute to relieve ER stress in tumorigenic cells. P38 and p28 could therefore be relevant as diagnostic markers and/or therapeutic targets in cancer.

SEL1L-A, a transmembrane glycoprotein residing in the endoplasmic reticulum (ER), is a component of the ER-associated degradation (ERAD) pathway. Alternative splicing generates two smaller SEL1L isoforms, -B and -C, that lack the SEL1L-A membrane-spanning region but retain some sel-1-like repeats, known to be involved in multi-protein interactions and signal transduction. In this study the functional characteristics of SEL1L-B and -C were investigated in human cell models. We show that these two isoforms are induced upon ER stress and activation of the unfolded protein response, together with SEL1L-A. Using transient transfection experiments (based on wild-type and mutant SEL1L constructs) combined with several biochemical tests we show that SEL1L-B and, more prominently, SEL1L-C are secreted glycoproteins. Although SEL1L-C is in monomeric form, SEL1L-B is engaged in intramolecular/intermolecular disulfide bonds. Both isoforms localize in secretory and degradative cellular compartments and in areas of cell-cell contact. However, whereas SEL1L-B is mainly associated with membranes, SEL1L-C shows the typical intralumenal localization of soluble proteins and is present in intercellular spaces. Furthermore, because of its peroxisomal domain, SEL1L-C localizes to peroxisomes. Both SEL1L-B and -C are involved in sorting and exporting unassembled Ig-mu(s) but do not affect two other ERAD substrates, the null Hong Kong variant of alpha(1)-antitrypsin, and mutant alpha(1)-AT Z. Overall these findings suggest that SEL1L-B and -C participate to novel molecular pathways that, in parallel with ERAD, contribute to the disposure of misfolded/unfolded or orphan proteins through degradation or secretion.

Recent phase II randomised trials in colorectal cancer failed to demonstrate any advantage of celecoxib combined with standard chemotherapy; some authors even reported that the addition of celecoxib to irinotecan and oxaliplatin in colon cancer results in an inferior response rate. This observation leads to the hypothesis that there are pharmacokinetic interactions between celecoxib and chemotherapeutic drugs. The aim of the study was to investigate the induction by celecoxib of some multidrug resistance proteins, MRP1, MRP2, MRP4 and MRP5, involved in the transport of irinotecan and 5-FU. WiDr and COLO-205 cells were treated with celecoxib at a clinically relevant concentration. A viability assay was performed by treating cells with chemotherapy alone and chemotherapy plus celecoxib. The expression of MRP1, MRP2, MRP4 and MRP5 was analysed by RT-PCR and Western blot analysis. The sub cellular localization of MRP4 and MRP5 was investigated by cryoimmunoelectron microscopy. In both cell lines celecoxib induced MRP4 and MRP5 over-expression at RNA and protein levels. No induction of MRP1 and MRP2 was observed in treated cells compared to controls. Cryoimmunoelectron microscopy showed increased MRP4 and MRP5 immunolabeling in celecoxib treated cells both at cytoplasmic level and along the plasma membrane. Our findings suggest that the low response rate observed in clinical trials using celecoxib added to 5-fluorouracil and irinotecan may reflect celecoxib-mediated extrusion of chemotherapeutic drugs from cancer cells through the up regulation of ATP-binding cassette proteins. Our findings, together with the results of clinical trials, may suggest that the combined use of celecoxib and drugs that are substrate for MRP4/MRP5 should be avoided.

The inherent toxicity of many metal compounds, together with their widespread environmental distribution, raises concerns of potential health hazards. Little is known about the impact of these important environmental toxicants on adult stem/progenitor cells, necessary for tissue homeostasis and repair. We recently reported that autophagy is implicated in the response of hematopoietic stem/progenitor cells to toxic concentrations of hexavalent chromium (Cr[VI]) and cadmium (Cd), two well known carcinogenic heavy metal cations. Autophagy may lead to cell death if carried out too extensively, but also acts as a survival pathway in cells under stress. In stem/progenitor cells, an autophagic phenotype could mitigate metal-induced toxicity, contributing to the conservation of tissue renewal capability. Given the key role of toxic damage to adult stem/progenitor cells in cancer, it is necessary to investigate whether autophagic responses modulate the carcinogenic potential of exposure to heavy metals during stem/progenitor cell differentiation.

Stem cells are a key target of environmental toxicants, but little is known about their toxicological responses. We aimed at developing an in-vitro model based on adult human stem cells to identify biomarkers of heavy metal exposure. To this end we investigated the responses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI]) and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood were exposed for 48 h to 0.1 microM and 10 microM Cr(VI) or Cd. Cultures treated with 10 microM Cr(VI) or Cd showed marked cell loss. Ultrastructural analysis of surviving cells revealed prominent autophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated with mitochondrial damage and replication, dilatation of the rough endoplasmic reticulum and Golgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did not show the morphologic hallmarks of apoptosis. Treatment with 0.1 microM Cr(VI) or Cd did not result in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulum and evidence of mitochondrial damage. We conclude that autophagy is implicated in the response of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd. Autophagy, which mediates cell survival and death under stress, deserves further evaluation to be established as biomarker of metal exposure.

The transfer of melanin from melanocytes to keratinocytes is upregulated by UV radiation and modulated by autocrine and paracrine factors. Among them, the keratinocyte growth factor (KGF/FGF7) promotes melanosome transfer acting on the recipient keratinocytes through stimulation of the phagocytic process. To search for possible differences in the melanosome uptake of keratinocytes from different skin color, we analyzed the uptake kinetics and distribution pattern of fluorescent latex beads in primary cultures of light and dark skin-derived keratinocytes stimulated with KGF and we compared the direct effect of KGF on the melanosome transfer in co-cultures of human primary melanocytes with light and dark keratinocytes. KGF-promoted melanosome transfer was more significant in light keratinocytes compared to dark, due to an increased expression of KGF receptor in light skin keratinocytes. Colocalization studies performed by confocal microscopy using FITC-dextran as a phagocytic marker and fluorescent beads as well as inhibition of particle uptake by cytochalasin D, revealed that beads internalization induced by KGF occurs via actin-dependent phagocytosis. 3D image reconstruction by fluorescence microscopy and ultrastructural analysis through transmission electron microscopy showed differences in the distribution pattern of the beads in light and dark keratinocytes, consistent with the different melanosome distribution in human skin.

Regulation of proliferation and differentiation in keratinocyte is a complex and dynamic process that involves activation of multiple signaling pathways triggered by different growth factors. Keratinocyte growth factor (KGF) is not only a potent mitogen, but differently from other growth factors, is a potent inducer of differentiation. The MAP kinase and AKT pathways are involved in proliferation and differentiation of many cell types including keratinocytes. We investigated here the role of KGF in modulating AKT and MAPK activity during differentiation of human keratinocytes. Our results show that the mechanisms of action of KGF are dose-dependent and that a sustained activation of the MAPK signaling cascade causes a negative regulation of AKT. We also demostrated increasing expression of KGFR substrates, such as PAK4 during keratinocyte differentiation parallel to the receptor upregulation.

Interaction of NK cells with autologous immature dendritic cells (iDCs) results in reciprocal activation. We have previously reported that NK cells trigger iDC to polarize and secrete IL-18; in turn, DC-activated NK cells secrete the nuclear protein/proinflammatory cytokine high mobility group box protein 1 (HMGB1), which induces DC maturation and prevents DC from lysis. However, activated NK cells can also kill iDC. To investigate whether effector and maturative properties may coexist or segregate in different NK subsets, human NK cell clones were generated and analyzed for their effects on iDC. We found that the ability of different NK cell clones to induce iDC maturation is unlinked to their phenotypic and cytolytic features but correlates with the relocation of HMGB1 from nucleus to cytoplasm. "Maturative" NK cell clones secrete HMGB1 spontaneously. It is interesting that secretion is strongly enhanced by engagement of the surface molecule NKp30 but only slightly induced by triggering of the activating NK receptor CD16. However, culturing freshly isolated NK cells for 1 week with low doses of anti-CD16 triggers the relocation of HMGB1 from nucleus to cytoplasm and its spontaneous secretion, resulting in a stronger maturation potential of the NK cells. Together, our data indicate that NK cells comprise functionally different subsets, endowed with different capacities to secrete HMGB1 and to induce maturation of autologous iDC. Nonetheless, maturation properties can be modulated by different stimuli. This suggests that depending on the environmental stimuli, NK/iDC interaction can lead to different outcomes, thus influencing immune response.

Melanogenesis and melanosome transfer from the melanocytes to the neighboring keratinocytes are induced by ultraviolet radiation and modulated by autocrine and paracrine factors. Keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) is a paracrine mediator of human keratinocyte growth and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of keratinocytes and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient keratinocytes and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-2, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the keratinocytes by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes.

Shape change is the earliest response of platelets to stimuli; it is mainly dependent upon Ca(2+)/calmodulin interaction subsequent to Ca(2+) mobilization and is mediated by myosin light chain kinase (MLCK) activation. It has been recently suggested that collagen itself is not able to elicit platelet shape change in the absence of ADP and thromboxane A(2) costimulation but is capable of inducing MLCK activation. Since we hypothesize that the morphological changes of the few platelets that adhere to collagen might not be revealed by turbidimetry, the aim of this study was to assess platelet shape change using transmission electron microscopy, in the absence of the amplificatory feedback pathways of ADP and thromboxane A(2). Our results demonstrated that only the platelets in contact with insoluble collagen fibers underwent a typical shape change, whereas those further away remained quiescent. Moreover, since cAMP enhances Ca(2+) mobilization in response to collagen, in the present study, we also investigated whether cAMP is involved in the inhibition of collagen-induced platelet shape change and MLC phosphorylation. Platelets were thus treated with iloprost (28 nm) prior to stimulation. Electron microscopy studies demonstrated that iloprost did not modify collagen-induced shape change, whereas immunoblotting studies showed a slight inhibition of MLC phosphorylation in the presence of enhanced cAMP levels. We can thus conclude that collagen is able to cause platelet shape change through activation of Ca(2+)/calmodulin-dependent MLCK, without the involvement of amplificatory pathways. Enhanced cytosolic cAMP levels do not inhibit collagen-induced platelet shape change but exert a weak inhibitory action on MLCK.

Blocking the activity of IL-1 beta has entered the clinical arena of treating autoimmune diseases. However, a successful outcome of this approach requires a clear definition of the mechanisms controlling IL-1 beta release. These are still unclear as IL-1 beta, lacking a secretory signal peptide, follows a nonclassical pathway of secretion. Here, we analyze the molecular mechanism(s) undergoing IL-1 beta processing and release in human monocytes and provide a unifying model for the regulated secretion of the cytokine. Our data show that in a first step, pro-caspase-1 and endotoxin-induced pro-IL-1 beta are targeted in part to specialized secretory lysosomes, where they colocalize with other lysosomal proteins. Externalization of mature IL-1 beta and caspase-1 together with lysosomal proteins is then facilitated by extracellular ATP. ATP triggers the efflux of K(+) from the cell, followed by Ca(2+) influx and activation of three phospholipases: phosphatidylcholine-specific phospholipase C and calcium-independent and -dependent phospholipase A(2). Whereas calcium-independent phospholipase A(2) is involved in processing, phosphatidylcholine-specific phospholipase C and calcium-dependent phospholipase A(2) are required for secretion. Dissection of the events that follow ATP triggering allowed to demonstrate that K(+) efflux is responsible for phosphatidylcholine-specific phospholipase C induction, which in turn allows the rise in intracellular free calcium concentration required for activation of phospholipase A(2). This activation is ultimately responsible for lysosome exocytosis and IL-1 beta secretion.