Baseline and time-updated factors in preclinical development of anionic dendrimers as successful anti-HIV-1 vaginal microbicides.

Although a wide variety of topical microbicides provide promising in vitro and in vivo efficacy, most of them failed to prevent sexual transmission of human immunodeficiency virus type 1 (HIV-1) in human clinical trials. In vitro, ex vivo, and in vivo models must be optimized, considering the knowledge acquired from unsuccessful and successful clinical trials to improve the current gaps and the preclinical development protocols. To date, dendrimers are the only nanotool that has advanced to human clinical trials as topical microbicides to prevent HIV-1 transmission. This fact demonstrates the importance and the potential of these molecules as microbicides. Polyanionic dendrimers are highly branched nanocompounds with potent activity against HIV-1 that disturb HIV-1 entry. Herein, the most significant advancements in topical microbicide development, trying to mimic the real-life conditions as closely as possible, are discussed. This review also provides the preclinical assays that anionic dendrimers have passed as microbicides because they can improve current antiviral treatments' efficacy. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

New Approaches to Dendritic Cell-Based Therapeutic Vaccines Against HIV-1 Infection.

Due to the success of combined antiretroviral therapy (cART) in recent years, the pathological outcome of Human Immunodeficiency Virus type 1 (HIV-1) infection has improved substantially, achieving undetectable viral loads in most cases. Nevertheless, the presence of a viral reservoir formed by latently infected cells results in patients having to maintain treatment for life. In the absence of effective eradication strategies against HIV-1, research efforts are focused on obtaining a cure. One of these approaches is the creation of therapeutic vaccines. In this sense, the most promising one up to now is based on the establishing of the immunological synapse between dendritic cells (DCs) and T lymphocytes (TL). DCs are one of the first cells of the immune system to encounter HIV-1 by acting as antigen presenting cells, bringing about the interaction between innate and adaptive immune responses mediated by TL. Furthermore, TL are the end effector, and their response capacity is essential in the adaptive elimination of cells infected by pathogens. In this review, we summarize the knowledge of the interaction between DCs with TL, as well as the characterization of the specific T-cell response against HIV-1 infection. The use of nanotechnology in the design and improvement of vaccines based on DCs has been researched and presented here with a special emphasis.

G1-S4 or G2-S16 carbosilane dendrimer in combination with Platycodin D as a promising vaginal microbicide candidate with contraceptive activity.

PURPOSE: HIV-1 and herpes simplex virus type-2 (HSV-2) represent two of the most relevant sexually transmitted diseases (STDs) worldwide. Moreover, each year there are >200 million pregnancies worldwide, and more than half are unintended. Continued high rates of unintended pregnancies and spread of HIV-1 and HSV-2 require new approaches to address these problems. G1-S4 and G2-S16 dendrimers emerge as potential candidates for the development of a topical microbicide due to their safety and effectivity against HIV-1 and HSV-2 infection, both in vitro and in vivo. Our goal is to develop a dual topical microbicide to prevent the transmission of STDs and unintended pregnancies. Platycodin D (PD) was selected for its great spermicidal activity, topical application, and biocompatibility. MATERIALS AND METHODS: Toxicology and inhibitory profile of G1-S4/PD and G2-S16/PD were evaluated in vitro and in vivo. Spermicidal activity was assessed by a computer-assisted sperm analysis system (CASA). RESULTS: G1-S4/PD and G2-S16/PD presented >95% of HIV-1 inhibition in TZM-bl cells and peripheral blood mononuclear cells. CASA assessment determined that 0.25 mM of PD with therapeutic concentrations of G1-S4 or G2-S16 was able to induce 100% immobilization of the sperm in 30 seconds. To evaluate the toxicity in vivo, a vaginal toxicity assay was performed in BALB/c mice. No significant changes or damage to the vaginal epithelium after 7 consecutive days of application were observed. CONCLUSION: Our data indicate that G1-S4/PD and G2-S16/PD combinations are promising candidates to be developed for vaginal microbicides with contraceptive activity.

Elevated liver stiffness is linked to increased biomarkers of inflammation and immune activation in HIV/hepatitis C virus-coinfected patients.

OBJECTIVES: Immune dysregulation is a hallmark of HIV and hepatitis C virus (HCV) infections. We aimed to evaluate the relationship between liver stiffness measurement (LSM) and biomarkers of T-cell activation, bacterial translocation, inflammation, endothelial dysfunction, and coagulopathy in HIV/HCV-coinfected patients. DESIGN: Cross-sectional study. METHODS: We studied 238 HIV/HCV-coinfected patients, 32 healthy controls, and 39 HIV-monoinfected patients. Patients were stratified according to LSM into four groups: less than 12.5, 12.5-25, 25-40, and more than 40 kPa. T-cell subsets were measured using flow cytometry and plasma biomarkers using immunoassays. RESULTS: HIV/HCV-coinfected patients had higher biomarker levels of immune activation in peripheral blood [T-cell activation (CD4CD38 and CD8CD38), bacterial translocation (soluble CD14), inflammation [IL-1b, IL-6, IL-8, IL-18, IFN-γ-inducible protein 10 (IP-10)] endothelial dysfunction [soluble vascular cell adhesion molecule 1 (sVCAM1), soluble intercellular cell adhesion molecule 1 (sICAM1), and soluble tumor necrosis factor receptor 1 (sTNFR1)], and coagulopathy (plasminogen activator inhibitor-1)] than healthy controls and HIV-monoinfected patients. Moreover, in HIV/HCV-coinfected patients, a direct relationship between LSM and immune activation [T-cell activation (CD8CD38 bacterial translocation (lipopolysaccharide), inflammation (IL-8, IP-10), endothelial dysfunction (sVCAM1, sICAM1, and sTNFR1), and coagulopathy (D-dimer)] was found. Subsequently, patients were stratified into different fibrosis stages, finding that patients with cirrhosis who had LSM at least 40 kPa showed higher biomarker values of immune activation [T-cell activation (CD4CD38 and CD8CD38), bacterial translocation (lipopolysaccharide), inflammation (IL-8, IL-6, IP-10), endothelial dysfunction (sVCAM1, sICAM1, and sTNFR1), and coagulopathy (D-dimer)] than patients from the other three groups (<12.5, 12.5-25, and 25-40 kPa). CONCLUSION: T-cell activation, bacterial translocation, inflammation, endothelial dysfunction, and coagulopathy increased with the severity of liver fibrosis in HIV/HCV-coinfected patients, particularly in patients who had LSM at least 40 kPa.

New anionic carbosilane dendrons functionalized with a DO3A ligand at the focal point for the prevention of HIV-1 infection.

Novel third-generation polyanionic carbosilane dendrons with sulfonate or carboxylate end-groups and functionalized with a DO3A ligand at the focal point, and their corresponding copper complexes, have been prepared as antiviral compounds to prevent HIV-1 infection. The topology enables the compound to have an excellent chelating agent, DO3A, while keeping anionic peripheral groups for a therapeutic action. In this study, the cytotoxicity and anti-HIV-1 abilities of carboxylate- (5) or sulfonate-terminated (6) dendrons containing DO3A and their copper complexes (7 or 8) were evaluated. All compounds showed low cytotoxicity and demonstrated potent and broad-spectrum anti-HIV-1 activity in vitro. We also assessed the mode of antiviral action on the inhibition of HIV-1 through a panel of different in vitro antiviral assays. Our results show that copper-free dendron 6 protects the epithelial monolayer from short-term cell disruption. Copper-free dendrons 5 and 6 exert anti-HIV-1 activity at an early stage of the HIV-1 lifecycle by binding to the envelope glycoproteins of HIV-1 and by interacting with the CD4 cell receptor and blocking the binding of gp120 to CD4, and consequently HIV-1 entry. These findings show that copper-free dendrons 5 and 6 have a high potency against HIV-1 infection, confirming their non-specific ability and suggesting that these compounds deserve further study as potential candidate microbicides to prevent HIV-1 transmission.

Isolation Methods of Peripheral Blood Mononuclear Cells in Spanish Biobanks: An Overview.

The Spanish Hematic Derivatives Group, consisting of 26 biobanks, was established in 2011. We describe here the viability results of our publically available standard operating procedure to freeze and thaw peripheral blood mononuclear cells (PBMCs). Our protocol maximizes PBMC viability while avoiding where possible interbiobank and intrabiobank assay variability.

Mechanistic Studies of Viral Entry: An Overview of Dendrimer-Based Microbicides As Entry Inhibitors Against Both HIV and HSV-2 Overlapped Infections.

This review provides an overview of the development of different dendrimers, mainly polyanionic, against human immunodeficiency virus (HIV) and genital herpes (HSV-2) as topical microbicides targeting the viral entry process. Vaginal topical microbicides to prevent sexually transmitted infections such as HIV and HSV-2 are urgently needed. To inhibit HIV/HSV-2 entry processes, new preventive targets have been established to maximize the current therapies against wild-type and drug-resistant viruses. The entry of HIV/HSV-2 into target cells is a multistep process that triggers a cascade of molecular interactions between viral envelope proteins and cell surface receptors. Polyanionic dendrimers are highly branched nanocompounds with potent activity against HIV/HSV-2. Inhibitors of each entry step have been identified with regard to generations and surface groups, and possible roles for these agents in anti-HIV/HSV-2 therapies have also been discussed. Four potential binding sites for impeding HIV infection (HSPG, DC-SIGN, GSL, and CD4/gp120 inhibitors) and HSV-2 infection (HS, gB, gD, and gH/gL inhibitors) exist according to their mechanisms of action and structures. This review clarifies that inhibition of HIV/HSV-2 entry continues to be a promising target for drug development because nanotechnology can transform the field of HIV/HSV-2 prevention by improving the efficacy of the currently available antiviral treatments.

Polyanionic carbosilane dendrimers prevent hepatitis C virus infection in cell culture.

Hepatitis C virus (HCV) infection is a major biomedical problem worldwide. Although new direct antiviral agents (DAAs) have been developed for the treatment of chronic HCV infection, the potential emergence of resistant virus variants and the difficulties to implement their administration worldwide make the development of novel antiviral agents an urgent need. Moreover, no effective vaccine is available against HCV and transmission of the virus still occurs particularly when prophylactic measures are not taken. We used a cell-based system to screen a battery of polyanionic carbosilane dendrimers (PCDs) to identify compounds with antiviral activity against HCV and show that they inhibit effective virus adsorption of major HCV genotypes. Interestingly, one of the PCDs irreversibly destabilized infectious virions. This compound displays additive effect in combination with a clinically relevant DAA, sofosbuvir. Our results support further characterization of these molecules as nanotools for the control of hepatitis C virus spread.

Effect of Several HIV Antigens Simultaneously Loaded with G2-NN16 Carbosilane Dendrimer in the Cell Uptake and Functionality of Human Dendritic Cells.

Dendrimers are highly branched, star-shaped, and nanosized polymers that have been proposed as new carriers for specific HIV-1 peptides. Dendritic cells (DCs) are the most-potent antigen-presenting cells that play a major role in the development of cell-mediated immunotherapy due to the generation and regulation of adaptive immune responses against HIV-1. This article reports on the associated behavior of two or three HIV-derived peptides simultaneously (p24/gp160 or p24/gp160/NEF) with cationic carbosilane dendrimer G2-NN16. We have found that (i) immature DCs (iDCs) and mature (mDCs) did not capture efficiently HIV peptides regarding the uptake level when cells were treated with G2-NN16-peptide complex alone; (ii) the ability of DCs to migrate was not depending on the peptides presence; and (iii) with the use of molecular dynamic simulation, a mixture of peptides decreased the cell uptake of the other peptides (in particular, NEF hinders the binding of more peptides and is especially obstructing of the binding of gp160 to G2-NN16). The results suggest that G2-NN16 cannot be considered as an alternative carrier for delivering two or more HIV-derived peptides to DCs.

Development of water-soluble polyanionic carbosilane dendrimers as novel and highly potent topical anti-HIV-2 microbicides.

The development of topical microbicide formulations for vaginal delivery to prevent HIV-2 sexual transmission is urgently needed. Second- and third-generation polyanionic carbosilane dendrimers with a silicon atom core and 16 sulfonate (G2-S16), napthylsulfonate (G2-NS16) and sulphate (G3-Sh16) end-groups have shown potent and broad-spectrum anti-HIV-1 activity. However, their antiviral activity against HIV-2 and mode of action have not been probed. Cytotoxicity, anti-HIV-2, anti-sperm and antimicrobial activities of dendrimers were determined. Analysis of combined effects of triple combinations with tenofovir and raltegravir was performed by using CalcuSyn software. We also assessed the mode of antiviral action on the inhibition of HIV-2 infection through a panel of different in vitro antiviral assays: attachment, internalization in PBMCs, inactivation and cell-based fusion. Vaginal irritation and histological analysis in female BALB/c mice were evaluated. Our results suggest that G2-S16, G2-NS16 and G3-Sh16 exert anti-HIV-2 activity at an early stage of viral replication inactivating the virus, inhibiting cell-to-cell HIV-2 transmission, and blocking the binding of gp120 to CD4, and the HIV-2 entry. Triple combinations with tenofovir and raltegravir increased the anti-HIV-2 activity, consistent with synergistic interactions (CIwt: 0.33-0.66). No vaginal irritation was detected in BALB/c mice after two consecutive applications for 2 days with 3% G2-S16. Our results have clearly shown that G2-S16, G2-NS16 and G3-Sh16 have high potency against HIV-2 infection. The modes of action confirm their multifactorial and non-specific ability, suggesting that these dendrimers deserve further studies as potential candidate microbicides to prevent vaginal/rectal HIV-1/HIV-2 transmission in humans.

Bryostatin activates HIV-1 latent expression in human astrocytes through a PKC and NF-ĸB-dependent mechanism.

Multiple studies have shown that HIV-1 patients may develop virus reservoirs that impede eradication; these reservoirs include the central nervous system (CNS). Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. To broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as a latent HIV-1 activator. We used primary astrocytes, NHA cells, and astrocytoma cells U-87. Infected cells with HIV-1(NL4.3) were treated with bryostatin alone or in combination with different inhibitors. HIV-1 production was quantified by using ELISA. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of the LTR promoter with the active NF-κB member p65/relA. To confirm the NF-κB role, Western blot and confocal microscopy were performed. Bryostatin reactivates latent viral infection in the NHA and U87 cells via activation of protein kinase C (PKC)-alpha and -delta, because the PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. No alteration in cell proliferation was found. Moreover, bryostatin strongly stimulated LTR transcription by activating the transcription factor NF-κB. Bryostatin could be a beneficial adjunct to the treatment of HIV-1 brain infection.

Establishment and Replenishment of the Viral Reservoir in Perinatally HIV-1-infected Children Initiating Very Early Antiretroviral Therapy.

BACKGROUND: Combination antiretroviral therapy (cART) generally suppresses the replication of the human immunodeficiency virus type 1 (HIV-1) but does not cure the infection, because proviruses persist in stable latent reservoirs. It has been proposed that low-level proviral reservoirs might predict longer virologic control after discontinuation of treatment. Our objective was to evaluate the impact of very early initiation of cART and temporary treatment interruption on the size of the latent HIV-1 reservoir in vertically infected children. METHODS: This retrospective study included 23 perinatally HIV-1-infected children who initiated very early treatment within 12 weeks after birth (n = 14), or early treatment between week 12 and 1 year (n = 9). We measured the proviral reservoir (CD4(+) T-cell-associated HIV-1 DNA) in blood samples collected beyond the first year of sustained virologic suppression. RESULTS: There is a strong positive correlation between the time to initiation of cART and the size of the proviral reservoir. Children who initiated cART within the first 12 weeks of life showed a proviral reservoir 6-fold smaller than children initiating cART beyond this time (P < .01). Rapid virologic control after initiation of cART also limits the size of the viral reservoir. However, patients who underwent transient treatment interruptions showed a dramatic increase in the size of the viral reservoir after discontinuation. CONCLUSIONS: Initiation of cART during the first 12 weeks of life in perinatally HIV-1-infected children limits the size of the viral reservoir. Treatment interruptions should be undertaken with caution, as they might lead to fast and irreversible replenishment of the viral reservoir.

Specific legislation on biobanks in Spain.

Spain has enacted specific legislation concerning biobanks. This legislation regulates how biobanks should be set up, how they should operate, and the requirements they need to comply with. The main objective of this legislation is to keep a good balance between scientific progress and respect for the rights and freedom of individuals participating in research. Therefore, this legislation lays down a series of basic principles, for instance, the principle to inform donors accurately i) on the deposit of samples in terms of the objectives and implications of their donation and on the need to obtain written consents; ii) on the obligation to establish consistent procedures to guarantee the confidentiality of personal data associated with and obtained from biological samples; iii) on the concept of free sample donation either by donors or by biobanks; iv) on the need for consistent procedures to deposit samples and data in biobanks; and v) for acts of donation and data for research projects to be performed correctly. Although this Spanish legislation fulfills its objectives, it has some drawbacks; mainly it overprotects research participants. This issue should be analyzed in future revisions of the legislation.

Triple combination of carbosilane dendrimers, tenofovir and maraviroc as potential microbicide to prevent HIV-1 sexual transmission.

AIM: To research the synergistic activity by triple combinations of carbosilane dendrimers with tenofovir and maraviroc as topical microbicide. METHODS: Cytotoxicity, anti-HIV-1 activity, vaginal irritation and histological analysis of triple combinations were determined. Analysis of combined effects and the median effective concentration were performed using CalcuSyn software. RESULTS: Combinations showed a greater broad-spectrum anti-HIV-1 activity than the single-drug, and preserved this activity in acid environment or seminal fluid. The strongest combinations were G2-STE16/G2-S24P/tenofovir, G2-STE16/G2-S16/maraviroc and G2-STE16/tenofovir/maraviroc at 2:2:1, 10:10:1 10:5:1 ratios, respectively. They demonstrated strong synergistic activity profile due to the weighted average combination indices varied between 0.06 and 0.38. No irritation was detected in female BALB/c mice. CONCLUSION: The three-drug combination increases their antiviral potency and act synergistically as potential microbicide.

In vivo delivery of siRNA to the brain by carbosilane dendrimer.

Nanotechnology offers a new platform for therapeutic delivery of antiretrovirals to the central nervous system (CNS). Nanoformulated antiretroviral drugs offer multifunctionality, that is, the ability to package multiple diagnostic and therapeutic agents in the same nanocompose, along with the added provisions of site-directed delivery, delivery across the blood-brain-barrier (BBB), and controlled release of therapeutics. We studied the viability of dendrimers and dendriplexes in human primary astrocytes, as well as their uptake by these astrocytes. Functional validation was performed by using specific siRNA against HIV-1 Nef to interfere to HIV-1 infectivity. A high efficiency in Nef silencing, reducing HIV-1 infectivity was observed in astrocytes treated with dendriplexes compared with control or siRandom treated astrocytes. More interestingly, we studied the biodistribution of the second generation of carbosilane dendrimer loaded with FITC (2G-(SNMe3I)11-FITC) in vivo, in BALB/c mice. Dendriplexes were inoculated into BALB/c mice by the retro-orbital venous plexus, and their localization was determined after 1 and 24h post-injection. Dendriplexes were detected inside the brain by a sensitive imaging system of fluorescent imaging in vivo (IVIS Lumina), and by confocal microscopy analysis of sections of OCT-embedded tissues. The 2G-(SNMe3I)11-FITC dendrimer transported efficiently siRNA into the brain, crossing the BBB. Moreover, this dendrimer successfully delivered and transfected siRNA to HIV-infected human primary astrocytes and achieved gene silencing without causing cytotoxicity. These results highlight the potential of this nanoformulation in the treatment of neurological disorders.

Nanotech-derived topical microbicides for HIV prevention: the road to clinical development.

More than three decades since its discovery, HIV infection remains one of the most aggressive epidemics worldwide, with more than 35 million people infected. In sub-Saharan Africa, heterosexual transmissions represent nearly 80% of new infections, with 50% of these occurring in women. In an effort to stop the dramatic spread of the HIV epidemic, new preventive treatments, such as microbicides, have been developed. Nanotechnology has revolutionized this field by designing and engineering novel highly effective nano-sized materials as microbicide candidates. This review illustrates the most recent advances in nanotech-derived HIV prevention strategies, as well as the main steps required to translate promising in vitro results into clinical trials.

The EPIICAL project: an emerging global collaboration to investigate immunotherapeutic strategies in HIV-infected children.

The EPIICAL (Early-treated Perinatally HIV-infected Individuals: Improving Children's Actual Life with Novel Immunotherapeutic Strategies) project arises from the firm belief that perinatally infected children treated with suppressive antiretroviral therapy (ART) from early infancy represent the optimal population model in which to study novel immunotherapeutic strategies aimed at achieving ART-free remission. This is because HIV-infected infants treated within 2-3 months of life have a much reduced viral reservoir size, and rarely show HIV-specific immunity but preserve normal immune development. The goal of EPIICAL is the establishment of an international collaboration to develop a predictive platform using this model to select promising HIV therapeutic vaccine candidates, leading to prioritisation or deprioritisation of novel immunotherapeutic strategies. To establish this platform, the EPIICAL Consortium aims to: develop predictive models of virological and immunological dynamics associated with response to early ART and to treatment interruption using available data from existing cohorts/studies of early-treated perinatally HIV-infected children; optimise methodologies to better characterise immunological, virological and genomic correlates/profiles associated with viral control; test novel immunotherapeutic strategies using in vivo proof-of-concept (PoC) studies with the aim of inducing virological, immunological and transcriptomic correlates/profiles equivalent to those defined by the predictive model. This approach will strengthen the capacity for discovery, development and initial testing of new therapeutic vaccine strategies through the integrated efforts of leading international scientific groups, with the aim of improving the health of HIV-infected individuals.

Potential of Ni(II)-NTA-modified poly(ethylene imine) glycopolymers as carrier system for future dendritic cell-based immunotherapy.

Dendritic cells (DCs) play a crucial role in the development of cell-mediated immunotherapy due to their ability to induce and maintain strong immune responses. In our study, we evaluated a biocompatible Ni(II)-NTA-modified poly(ethylene imine) dendritic glycopolymer (Ni(II)-NTA-DG) as new carrier system to increase the antigen uptake into iDCs for future DC-based immunotherapy. Ni(II)-NTA-DG led to an increase in His6-Gp160 uptake in monocytes and iDCs, where His6-Gp160 is localized in the early endosomal and lysosomal compartments. Ni(II)-NTA-DG and the formed polyplexes induced an activation of iDCs, showing an increasing expression of costimulatory molecules CD86, CD80, and proinflammatory cytokines IL-6 and IL-8. Beside no influencing effect of Ni(II)-NTA-DG and polyplexes on the maturation of antigen-bearing DCs, the mature peptide bearing DCs remained their ability to migrate along a gradient of CCR7 ligands. Thus, Ni(II)-NTA-DG with advancing biological properties is a promising carrier system for the future application in DC-based immunotherapy.

Low thymic output, peripheral homeostasis deregulation, and hastened regulatory T cells differentiation in children with 22q11.2 deletion syndrome.

OBJECTIVE: To perform an extensive analysis of the immune status of asymptomatic children with the 22q11.2 deletion syndrome, with special emphasis on the regulatory T cells (Treg) population. STUDY DESIGN: Analysis of thymic function, frequency and absolute counts of immune subsets, and phenotype of Treg were performed in 10 asymptomatic children bearing the 22q11.2 deletion and compared with 12 age-matched, healthy children. RESULTS: Children with 22q11.2 deletion syndrome showed a curtailed thymic output, lower T-cell levels, and a homeostatic deregulation in the CD4 T-cell compartment, characterized by a greater proliferative history in the naïve CD4 T-cell subset. Treg numbers were markedly reduced in children with 22q11.2 deletion syndrome, and remaining Treg showed mostly an activated phenotype. CONCLUSIONS: Reduced thymic output in children with 22q11.2 deletion syndrome could be related with an increased proliferation in the naïve CD4 T-cell compartment and the consequent Treg activation to ensure that T-cell expansion remains under control. Deregulated peripheral homeostasis and loss of suppressive capacity by Treg could compromise the integrity of T-cell immunity during adulthood and play a relevant role in the increased incidence of autoimmune diseases reported in patients with the 22q11.2 deletion syndrome.

TNF-Α may mediate inflammasome activation in the absence of bacterial infection in more than one way.

Members of the mammalian nucleotide binding domain, leucine-rich repeat (LRR)-containing receptor family of proteins are key modulators of innate immunity regulating inflammation. To date, microbial pathogen-associated molecules and toxins have been identified as key triggers of activation of inflammasomes. However, recently, environmental, and neurodegenerative stimuli have been identified that lead to IL-1ß release by means of inflammasomes. IL-1ß plays a crucial role during brain inflammation, and caspase-1 appears to be a key modulator of IL-1ß bioactivity and the consequent transcriptional regulation of gene expression within the brain during inflammation. We show here that exposure of a human neuroblastoma cell line (SK-N-MC cells) to TNF-α promotes ROS-mediated caspase-1 activation and IL-1ß secretion. The involvement of NF-κB in the regulation of IL-1ß synthesis is investigated through specific inhibition of this transcription factor. The effect of TNF-α was abolished in the presence of ROS inhibitors as NAC, or DPI. Remarkably, SK-N-MC cells do not respond to ATP stimulation in spite of P2X7R expression. These results provide a mechanism by which danger signals and particulate matter mediate inflammation via the inflammasome in the absence of microbial infection.