A novel role of AIM2 inflammasome-mediated pyroptosis in radiofrequency ablation of hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: The absence of melanoma 2 (AIM2) protein triggers the activation of the inflammasome cascade. It is unclear whether AIM2 plays a role in hepatocellular carcinoma (HCC) and radiofrequency ablation (RFA), which uses radiofrequency waves to treat tumors. In this study, we investigated if RFA could induce pyroptosis, also called cell inflammatory necrosis, in HCC through AIM2-inflammasome signaling in vivo and in vitro. MATERIALS AND METHODS: BALB/c nude mice were used to generate HepG2 or SMMC-7721 cell-derived tumor xenografts. HCC cells with knockdown or overexpression of AIM2 were created using short hairpin RNA (shRNA) and expression vector transfection, respectively, for functional and mechanistic studies. Downstream effects were examined using flow cytometry, qRT-PCR, ELISAs, and other molecular assays. RESULTS: RFA significantly suppressed tumor growth in HCC cell xenografts. Flow cytometry analysis revealed that RFA could induce pyroptosis. Furthermore, AIM2, NLRP3, caspase-1, γ-H2AX, and DNA-PKc had significantly greater expression levels in liver tissues from mice treated with RFA compared with those of the controls. Additionally, interleukin (IL)-1ß and IL-18 expression levels were significantly higher in the HCC cell-derived xenograft mice treated with RFA compared with those without RFA. Notably, a significantly greater effect was achieved in the RFA complete ablation group versus the partial ablation group. Knockdown or overexpression of AIM2 in HCC cells demonstrated that AIM2 exerted a role in RFA-induced pyroptosis. CONCLUSIONS: RFA can suppress HCC tumor growth by inducing pyroptosis via AIM2. Therefore, therapeutically intervening with AIM2-mediated inflammasome signaling may help improve RFA treatment outcomes for HCC patients.

Effect of Cannabis sativa L. extracts, phytocannabinoids and their acetylated derivates on the SHSY-5Y neuroblastoma cells' viability and caspases 3/7 activation.

BACKGROUND: There is a need for novel treatments for neuroblastoma, despite the emergence of new biological and immune treatments, since refractory pediatric neuroblastoma is still a medical challenge. Phyto cannabinoids and their hemisynthetic derivatives have shown evidence supporting their anticancer potential. The aim of this research was to examine Phytocannabinoids or hemisynthetic cannabinoids, which reduce the SHSY-5Y, neuroblastoma cell line's viability. METHODS: Hexane and acetyl acetate extracts were produced starting with Cannabis sativa L. as raw material, then, 9-tetrahidrocannabinol, its acid counterpart and CBN were isolated. In addition, acetylated derivatives of THC and CBN were synthesized. The identification and purity of the chemicals was determined by High Performance Liquid Chromatography and 1H y 13C Magnetic Nuclear Resonance. Then, the capacity to affect the viability of SHSY-5Y, a neuroblastoma cell line, was examined using the resazurin method. Finally, to gain insight into the mechanism of action of the extracts, phytocannabinoids and acetylated derivatives on the examined cells, a caspase 3/7 determination was performed on cells exposed to these compounds. RESULTS: The structure and purity of the isolated compounds was demonstrated. The extracts, the phytocannabinoids and their acetylated counterparts inhibited the viability of the SHSY 5Y cells, being CBN the most potent of all the tested molecules with an inhibitory concentration of 50 percent of 9.5 µM. CONCLUSION: Each of the evaluated molecules exhibited the capacity to activate caspases 3/7, indicating that at least in part, the cytotoxicity of the tested phytocannabinoids and their hemi-synthetic derivatives is mediated by apoptosis.

Pro-Apoptotic Activity and Cell Cycle Arrest of Caulerpa sertularioides against SKLU-1 Cancer Cell in 2D and 3D Cultures.

Cancer is a disease with the highest mortality and morbidity rate worldwide. First-line drugs induce several side effects that drastically reduce the quality of life of people with this disease. Finding molecules to prevent it or generate less aggressiveness or no side effects is significant to counteract this problem. Therefore, this work searched for bioactive compounds of marine macroalgae as an alternative treatment. An 80% ethanol extract of dried Caulerpa sertularioides (CSE) was analyzed by HPLS-MS to identify the chemical components. CSE was utilized through a comparative 2D versus 3D culture model. Cisplatin (Cis) was used as a standard drug. The effects on cell viability, apoptosis, cell cycle, and tumor invasion were evaluated. The IC50 of CSE for the 2D model was 80.28 µg/mL versus 530 µg/mL for the 3D model after 24 h of treatment exposure. These results confirmed that the 3D model is more resistant to treatments and complex than the 2D model. CSE generated a loss of mitochondrial membrane potential, induced apoptosis by extrinsic and intrinsic pathways, upregulated caspases-3 and -7, and significantly decreased tumor invasion of a 3D SKLU-1 lung adenocarcinoma cell line. CSE generates biochemical and morphological changes in the plasma membrane and causes cell cycle arrest at the S and G2/M phases. These findings conclude that C. sertularioides is a potential candidate for alternative treatment against lung cancer. This work reinforced the use of complex models for drug screening and suggested using CSE's primary component, caulerpin, to determine its effect and mechanism of action on SKLU-1 in the future. A multi-approach with molecular and histological analysis and combination with first-line drugs must be included.

Pro-Apoptotic Activity of Bioactive Compounds from Seaweeds: Promising Sources for Developing Novel Anticancer Drugs.

The process by which cancer cells evade or inhibit apoptosis is considered one of the characteristics of cancer. The ability of cancer cells to escape apoptosis contributes to tumor proliferation and promotes metastasis. The discovery of new antitumor agents is essential for cancer treatment due to the lack of selectivity of drugs and cellular resistance to anticancer agents. Several studies showed that macroalgae produce various metabolites with different biological activities among marine organisms. This review discusses multiple metabolites extracted from macroalgae and their pro-apoptotic effects through regulating apoptosis signaling pathway target molecules and the structure-activity relationship. Twenty-four promising bioactive compounds have been reported, where eight of these compounds exhibited values of maximum inhibitory concentration (IC50) of less than 7 µg/mL. Fucoxanthin was the only carotenoid reported that induced apoptosis in HeLa cells with an IC50 below 1 µg/mL. Se-PPC (a complex of proteins and selenylated polysaccharides) is the magistral compound because it is the only one with an IC50 of 2.5 µg/mL which regulates the primary proteins and critical genes of both apoptosis pathways. Therefore, this review will help provide the basis for further studies and the development of new anticancer drugs, both as single agents and adjuvants, decreasing the aggressiveness of first-line drugs and offering patients better survival and quality of life.

Rosmarinic acid decreases viability, inhibits migration and modulates expression of apoptosis-related CASP8/CASP3/NLRP3 genes in human metastatic melanoma cells.

Cutaneous melanoma is the most aggressive type of skin cancer; it is difficult to treat, and has been highlighted in recent years due to increasing numbers of cases worldwide. The use of antitumoral therapeutics for this neoplasm has been associated with severe side effects, low quality of life, and resistance. We aimed in this study to explore the effect of the phenolic compound rosmarinic acid (RA) on human metastatic melanoma cells. SK-MEL-28 melanoma cells were treated for 24 h with different concentrations of RA. In parallel, peripheral blood mononuclear cells (PBMCs) also were treated with RA under the same experimental conditions to verify the cytotoxic effect on non-tumoral cells. Then, we assessed cell viability and migration, levels of intracellular and extracellular reactive oxygen species (ROS), as well as nitric oxide (NOx), non-protein thiols (NPSH), and total thiol (PSH). Gene expression of the caspase 8, caspase 3 and NLRP3 inflammasome was evaluated by RT-qPCR. The enzymatic activity of the caspase 3 protein was assessed by a sensitive fluorescent assay. Fluorescence microscopy was employed to corroborate the effects of RA on melanoma cell viability, mitochondria transmembrane potential and apoptotic bodies formation. We found that RA potently reduces melanoma cell viability and migration after 24 h of treatment. On the other hand, it has no cytotoxic effect on non-tumoral cells. The fluorescence micrographics indicated that RA reduces transmembrane potential of mitochondria and induces apoptotic bodies formation. Moreover, RA significantly decreases intracellular and extracellular ROS levels, and increases the antioxidant defenders NPSH and PSH. A remarkable feature found in our study was that RA strongly upregulates the gene expression of the caspase 8 and caspase 3, and downregulates NLRP3 inflammasome expression. Similar to gene expression, RA greatly increases the enzymatic activity of caspase 3 protein. Taken together, we have shown for the first time that RA reduces cell viability and migration of human metastatic melanoma cells, in addition to modulates apoptosis-related gene expression. We suggest that RA may have the potential to be used in a therapeutic perspective, particularly for CM cell treatment.

Characterization of the anticonvulsant effect of dapsone on metabolic activity assessed by [18F]FDG -PET after kainic acid-induced status epilepticus in rats.

BACKGROUND: Development of effective drugs for epilepsy are needed, as nearly 30 % of epileptic patients, are resistant to current treatments. This study is aimed to characterize the anticonvulsant effect of dapsone (DDS), in the kainic acid (KA)-induced Status Epilepticus (SE) by recording the brain metabolic activity with an [18F]FDG-PET analysis. METHODS: Wistar rats received KA (10 mg/kg, i.p., single dose) to produce sustained seizures. [18F]FDG-PET and electroencephalographic (EEG) studies were then performed. DDS or vehicle were administered 30 min before KA. [18F]FDG uptake and EEG were evaluated at baseline, 2 and 25 h after KA injection. Likewise, caspase-8, 3 hippocampal activities and Fluoro-Jade B neuronal degeneration and Hematoxylin-eosin staining were measured 25 h after KA. RESULTS: PET data evaluated at 2 h showed hyper-uptake of [18F]FDG in the control group, which was decreased by DDS. At 25 h, hypo-uptake was observed in the control group and higher values due to DDS effect. EEG spectral power was increased 2 h after KA administration in the control group during the generalized tonic-clonic seizures, which was reversed by DDS, correlated with [18F]FDG-PET uptake changes. The values of caspases-8 activity decreased 48 and 43 % vs control group in the groups treated with DDS (12.5 y 25 mg/kg respectively), likewise; caspase-3 activity diminished by 57 and 53 %. Fewer degenerated neurons were observed due to DDS treatments. CONCLUSIONS: This study pinpoints the anticonvulsant therapeutic potential of DDS. Given its safety and effectiveness, DDS may be a viable alternative for patients with drug-resistant epilepsy.

Caracasine, An ent-kaurane Diterpene with Proapoptotic and Pro-differentiator Activity in Human Leukaemia Cell Lines.

BACKGROUND: Kaurane-type diterpenoids, obtained from various natural sources, have shown many biological activities, including anti-inflammatory and antitumor effects. Caracasine, an ent-kaurane diterpenoid isolated from the flowers of Croton micans, was shown to induce apoptosis in leukaemia cell lines. OBJECTIVE: The present study aimed to ascertain the compound's mechanism of cell death induction using two leukaemia cell lines, Jurkat E6.1 (T cell) and HL-60 (promyeloblast cells). METHODS: Cell death in Jurkat and HL60 cells were evaluated by flow cytometry for apoptosis with annexin-V/PI, mitochondrial membrane potential disturbance, changes in cell cycle, CD95 expression, caspase activation, Nuclear Factor kappa B inhibition, and differentiation into a neutrophil-like cell (dHL60). RESULTS: Caracasine (10 µM) increased the G0/G1 phase in Jurkat and arrested the cell cycle in the S phase in HL60. Caracasine increased CD95 expression (p<0.01 in Jurkat and p<0.05 in HL60) and caspase-8 activation (p<0.001 in Jurkat and p<0.05 in HL60). Caspase-9 was activated in both cell lines (p<0.001) along with the decline in mitochondrial Δψm (p<0.05 in Jurkat and p<0.001 in HL60). In HL60 cells, the kaurane induced neutrophil differentiation was assessed by CD40 expression and reactive oxygen species production. In Jurkat cells, caracasine inhibited the NF-κB pathway in cells pretreated with PHA to activate the NF-κB pathway, suggesting a possible role in inflammatory diseases. CONCLUSION: Caracasine induced apoptosis through the intrinsic and extrinsic pathways in both cell lines were evaluated which could be the leading structure for new anti-leukemic and anti-inflammatory drugs.

Apoptosis (programmed cell death) and its signals: a review / Apoptose, morte celular programada e seus sinais: uma revisão

Apoptosis is a sequential order of cell death occurring regularly to ensure a homeostatic balance between the rate of cell formation and cell death. However, a misplaced of this balancing function can contribute to an abnormal cell growth / proliferation or autoimmune disorders etc. Apoptosis is therefore said to be crucial from the point of development of an embryo throughout the growth of an organism contributing to the renewal of tissues and also the getting rid of inflammatory cells. This review seeks to elaborate on the recent overview of the mechanism involved in apoptosis, some element and signal contributing to its function and inhibition together with how their malfunction contribute to a number of cancer related cases.(AU)

A apoptose é uma ordem sequencial de morte celular que ocorre regularmente para garantir um equilíbrio homeostático entre a taxa de formação e a morte celular. No entanto, um desequilíbrio dessa função pode contribuir para um crescimento/proliferação celular anormal ou distúrbios autoimunes. A apoptose é, portanto, considerada crucial do ponto de desenvolvimento de um embrião ao longo do crescimento de um organismo que contribui para a renovação dos tecidos e também a eliminação de células inflamatórias. Esta revisão procura elaborar a recente visão geral do mecanismo envolvido na apoptose, alguns elementos e sinais que contribuem para sua função e inibição, além de como o mau funcionamento deles contribui para vários casos relacionados ao câncer.(AU)

Apoptosis (programmed cell death) and its signals - A review / Apoptose, morte celular programada e seus sinais: uma revisão

Abstract Apoptosis is a sequential order of cell death occurring regularly to ensure a homeostatic balance between the rate of cell formation and cell death. However, a misplaced of this balancing function can contribute to an abnormal cell growth / proliferation or autoimmune disorders etc. Apoptosis is therefore said to be crucial from the point of development of an embryo throughout the growth of an organism contributing to the renewal of tissues and also the getting rid of inflammatory cells. This review seeks to elaborate on the recent overview of the mechanism involved in apoptosis, some element and signal contributing to its function and inhibition together with how their malfunction contribute to a number of cancer related cases.

Resumo A apoptose é uma ordem sequencial de morte celular que ocorre regularmente para garantir um equilíbrio homeostático entre a taxa de formação e a morte celular. No entanto, um desequilíbrio dessa função pode contribuir para um crescimento/proliferação celular anormal ou distúrbios autoimunes. A apoptose é, portanto, considerada crucial do ponto de desenvolvimento de um embrião ao longo do crescimento de um organismo que contribui para a renovação dos tecidos e também a eliminação de células inflamatórias. Esta revisão procura elaborar a recente visão geral do mecanismo envolvido na apoptose, alguns elementos e sinais que contribuem para sua função e inibição, além de como o mau funcionamento deles contribui para vários casos relacionados ao câncer.

Insights into the activation of oral keratinocyte cell death by Candida albicans and Staphylococcus aureus biofilms.

Polymicrobial biofilms comprising Candida albicans and Staphylococcus aureus can increase the frequency and severity of oral diseases. This study assessed oral keratinocyte cell death, apoptosis and/or necrosis, promoted by soluble factors from single and dual biofilms of S. aureus and C. albicans. The soluble factors were obtained from the 16-h biofilm growth media. Cell viability was assessed by MTT and cell membrane damage by LDH. SEM was used for morphology changes. Assessment of apoptosis and necrosis was performed using annexin V and propidium iodide and caspases -2, -3, -6, -8 and -9. Statistical analysis was conducted with ANOVA and Tukey tests (α = 5%). Dual biofilms promoted the greatest harmful effect on oral cells, with a viability rate of 31.76%, damage to cell membranes and LDH released. Dual biofilms also induced higher percentages of necrotic cells (24.95%). Apoptosis was associated with caspases -2, -3, -6 and -8 activation.

A model for the regulation of apoptosis intrinsic pathway: The potential role of the transcriptional regulator E2F in the point of no return.

Apoptosis has been extensively characterized by both experimental approaches and model simulations. However, it is still not fully understood how the regulation occurs, especially in the intrinsic pathway, which can be activated by a great variety of signals. In addition, the conditions in which a point of no return could be reached remain elusive. In this work, we use differential equations models to approach these issues. Our starting point was the model for caspase activation of Legewie et al. (Legewie S, et al., PLoS Computational Biology 2006, 2(9): e120), which exhibits irreversible bistability. We added an activation module to this model, with the main events related to mitochondrial outer membrane permeabilization, which includes cytochrome C release by the mitochondria and its effects on caspase activation and respiratory chain disruption. This "Extended Legewie Model" (ELM) uses BAK as the apoptotic stimulus and active caspase 3 as a measure of apoptosis activation. Unexpectedly, in the extended model, BAK cannot trigger apoptosis activation using physiologically sound initial values of the variables, due to limitations in apoptosome concentration increase. Therefore, the next step was to find a regulatory mechanism, allowing apoptosis activation in the ELM, starting from physiological initial concentrations. For this aim, we performed a sensitivity analysis on the 61 parameters of the system, finding that those producing the most relevant changes in the qualitative behaviour were the rates of synthesis of caspase 3, caspase 9 and XIAP. Based on these results, the transcription factor E2F was included in the ELM because it directly regulates the rate of synthesis of caspase 3 and 9. Depending on the concentration of E2F, the ELM shows different qualitative behaviours. On one hand, for low E2F apoptosis is impossible and for high E2F apoptosis is inevitable. Therefore, if E2F is sufficiently increased, the point of no return is crossed. On the other hand, for intermediate values of E2F there is a bistable region where the fate of the system also depends on the concentration of BAK and other signalling species.

Adenosine A1 Receptor Agonist (R-PIA) before Pilocarpine Modulates Pro- and Anti-Apoptotic Factors in an Animal Model of Epilepsy.

We aimed to characterize the mechanisms involved in neuroprotection by R-PIA administered before pilocarpine-induced seizures. Caspase-1 and caspase-3 activities were assayed using fluorimetry, and cathepsin D, HSP-70, and AKT expression levels were assayed using Western Blot of hippocampal samples. R-PIA was injected before pilocarpine (PILO), and four groups were studied at 1 h 30 min and 7 days following initiation of status epilepticus (SE): PILO, R-PIA+PILO, SALINE, and R-PIA+SALINE. At 1 h 30 min, significantly higher activities of caspase-1 and -3 were observed in the PILO group than in the SALINE group. Caspase-1 and -3 activities were higher in the R-PIA+PILO group than in the PILO group. At 7 days following SE, caspase-1 and -3 activities were higher than in the initial post-seizure phase compared to the SALINE group. The pretreatment of rats receiving PILO significantly reduced caspase activities compared to the PILO group. Expression of HSP-70, AKT, and cathepsin D was significantly higher in the PILO group than in the SALINE. In the R-PIA+PILO group, the expression of AKT and HSP-70 was greater than in rats receiving only PILO, while cathepsin D presented decreased expression. Pretreatment with R-PIA in PILO-injected rats strongly inhibited caspase-1 and caspase-3 activities and cathepsin D expression. It also increased expression levels of the neuroprotective proteins HSP-70 and AKT, suggesting an important role in modulating the cellular survival cascade.

Exposure to hydrocarbons and chlorpyrifos alters the expression of nuclear receptors and antioxidant, detoxifying, and immune response proteins in the liver of the rainbow trout, Oncorhynchus mykiss.

The development of oil and gas production together with the fruit production in nearby areas of North Patagonia, Argentina, suggests aquatic pollution scenarios which include permanent oil pollution combined with short events of pesticides application. It has been reported that oil hydrocarbons activate the aryl hydrocarbon receptor (AhR) pathway in the rainbow trout, Oncorhynchus mykiss, and that the insecticide Chlorpyrifos (CPF) interacts with these effects. Thus, it is interesting to investigate whether hydrocarbons and insecticides, applied by separate or combined, can affect fish health and reproductive signaling by acting on different nuclear receptors' regulatory pathways. To study this kind of interactions, we exposed juvenile rainbow trout to water accommodated fraction (WAF) of crude oil (62 µg L-1 TPH) for 48 h and subsequently exposed the livers ex vivo to the insecticide Chlorpyrifos (CPF) (20 µg L-1) for 1 h. We analyzed the mRNA expression of nuclear receptors and proteins involved in detoxifying, antioxidant, immune and apoptosis responses by qRT-PCR. We also performed histopathological analysis. WAF induced the expression of the androgen (AR) and the Liver X receptor (LXR) by 8- and 3-fold, respectively. AR induction was reversed by subsequent exposure to CPF. The progesterone receptor (PR) and glucocorticoid receptor (GR) were increased 2-fold and 3-fold by WAF respectively, while estrogen and mineralocorticoid receptors were not affected. GR was also induced by CPF with an additive effect in the WAF-CPF treatment. The antioxidant genes, gamma glutamyl transferase (GGT), superoxide dismutase (SOD1) were induced by WAF (2-3-fold). WAF upregulated the ATP Binding Cassette Subfamily C Member 2 (ABCC2, MRP2) (4-fold) and downregulated alkaline phosphatase. WAF also induced the inflammatory interleukins (IL) IL-8, and IL-6 and the anti-inflammatory IL-10, while CPF induced the inflammatory tumor necrosis factor (-α) and IL-6, and activated the intrinsic apoptotic pathway through the induction of caspases 3 and 9. Both, WAF and CPF downregulated the expression of the extrinsic apoptosis initiator caspase 8 and the inflammatory caspase 1. In conclusion, WAF hydrocarbons alter O. mykiss endocrine regulation by inducing AR, PR and GR. The subsequent exposure to CPF reverses AR, suggesting a complex interaction of different pollutants in contaminated environments, WAF hydrocarbons alter liver metabolism by inducing the expression of LXR, GR, antioxidant and detoxifying enzymes, and both inflammatory and anti-inflammatory cytokines, and causing mild hepatic steatosis. CPF activates inflammatory and stress responses associated with the induction of inflammatory cytokines together with apoptosis initiator and executioner caspases.

Morin exhibits leukemic cellular apoptosis through caspase pathway.

The present study investigated the possibility of apoptosis-inducing activity in human leukemia U-937 and THP-1 cells by the flavonoid morin. The treatments were evaluated by using the MTT and LDH assays; analysis of mitochondrial membrane potential (ΔΨm) was evaluated by flow cytometry, cell death by apoptosis was confirmed by fluorescence microscopy and by assessing the activity of caspases-3 and -6. The data indicated that the flavonoid morin has promoted a decrease in cell viability in a concentration-dependent way for both of the cancerous cell lines. An increase in the percentage of cell death caused by apoptosis was associated to a potential alteration in the mitochondrial membrane (ΔΨm) suggesting the involvement of cell death in intrinsic apoptotic pathways. Activation of caspases-3 and -6 confirmed the presence of apoptotic activity from morin. The results reinforce the antileukemic potential of flavonol morin.

Validação da importância funcional das Natterinas na resposta imune inata em zebrafish por CRISPR/Cas9, um método de loss-of-function

Zebrafish presents Natterin-like isoforms that share identity with the Natterina family, an important aerolysin for membrane penetration and inflammasome activation. The aim of this project is to silence the natterin-like gene in zebrafish using the CRISPR/Cas9 depletion system to assess the molecule's action in the body's immune defense against pathogens and its homeostatic function. Initially, we verified the susceptibility of zebrafish to infection with pathogens that activate the canonical and non-canonical pathways of the inflammasome. Thus, zebrafish larvaes with 1 day post fertilization (dpf) were exposed for 2 h to the bacteria Staphylococcus aureus and to Salmonella typhimurium for analysis of the effect on survival, embryonic development, locomotor activity and activation of the inflammasome immediately after the infection. The larvaes showed defects in embryonic development and behavioral change, and responded to infection by S. aureus and S. typhimurium with the production of mature caspase-a. Then, after synthesizing the sgRNA complex (trcarRNA + natterin-like crRNA, XM_017356964 / 2) for the complementary target in the second exon of the natterin-like gene on chromosome 7 (ID: 795232) the complex was microinjected into 0 embryos hpf with Cas9 enzyme in different concentrations (50 μg / nL: 250 μg / nL, 50: 125, 25: 125 and 12.5: 125) for gene depletion and obtaining KO embryos. The complete absence of natterin-like in the entire body of embryos depleted by CRISPR/Cas9 was confirmed by in situ hybridization. All concentrations delayed embryonic development with changes in the body's morphology with anomalies development and induced persistent mortality up to 144 hpf, dose-dependent. Lastly, WT larvae and depleted larvae of the natterin-like gene by CRISPR/Cas9 (sgRNA 50 ng/μL: Cas9 250 ng/μL) were exposed to infection in 1 dpf by S. aureus and S. typhimurium for 15 min, 30 min, 1 h and 2 h. Immediately after infection, the effect on survival, embryonic development, locomotor activity and activation of the inflammasome was evaluated. The absence of natterin-like in larvae (1 dpf) altered the response to infection by S. aureus and S. typhimurium inducing changes in locomotor activity in zebrafish associated with the decreased production of caspase-a and caspase-b at all times of infection performed. Our results show that natterin-like is crucial for the embryonic development of zebrafish and acts as an effector molecule in the innate immune response against bacterial pathogens activating the inflammasome complex in zebrafish.

Zebrafish apresenta isoformas de Natterin-like que compartilham identidade com a família Natterina, uma aerolisina importante para penetração em membranas e ativação do inflamassoma. O objetivo deste projeto é silenciar o gene da natterin-like em zebrafish utilizando o sistema de depleção CRISPR/Cas9 para avaliar a ação da molécula na defesa imune do organismo contra patógenos e sua função homeostática. Inicialmente verificamos a suscetibilidade do zebrafish para infecção com patógenos que ativam as vias canônica e não-canonica do inflamassoma. Assim, larvas de zebrafish com 1 dia após fertilização (dpf) foram expostas por 2 h às bactérias Staphylococcus aureus e Salmonella typhimurium para análise da sobrevivência, desenvolvimento embrionário, atividade locomotora e ativação do inflamassoma. As larvas apresentaram defeitos no desenvolvimento embrionário e no comportamento locomotor e responderam à infecção por S. aureus (canônico) e S. typhimurium (não- canônico) com a produção de caspase-a madura. Em seguida, após sintetizar o complexo sgRNA (trcarRNA + crRNA natterin-like, XM_017356964/2) para o alvo complementar no segundo exon do gene da natterin-like gene no cromossomo 7 (ID: 795232) o complexo foi microinjetado em embrioes de 0 hpf com a enzima Cas9 em diferentes concentracoes (50 μg/nL:250 μg/nL, 50:125, 25:125 e 12.5:125) para a depleção do gene e obtenção de embrioes depletados KO. A ausência completa de natterin-like no corpo inteiro dos embriões depletados por CRISPR/Cas9 foi confirmada por hibridização in situ. Todas as concentracoes atrasaram o desenvolvimento embrionario com alterações na morfologia do corpo com desenvolvimento de anomalias e induziram mortalidade persistente ate 144 hpf, dose- dependente. Por fim, larvas WT e larvas depletadas do gene natterin-like por CRISPR/Cas9 (sgRNA 50 ng/μL: Cas9 250 ng/μL) foram expostas à infecção em 1 dpf por S. aureus e S. typhimurium durante 15 min, 30 min, 1 h e 2 h. Imediatamente após a infecção foi avaliado o efeito na sobrevivência, no desenvolvimento embrionário, atividade locomotora e na ativação do inflamassoma. A ausência de natterin-like em larvas (1 dpf) alterou a resposta à infecção por S. aureus e S. typhimurium induzindo aumento na mortalidade e hiperatividade locomotora, indicativo de altos níveis de estree ou ansiedade. Confirmamos que a suscetibilidade à infecção nas larvas depletadas de natterin-like está relacionada à expressão deficiente de caspase-a e caspase-b. Podemos concluir que a natterin-like é crucial para o controle da ativação do inflamassoma responsável por garantir uma resposta inflamatória eficiente contra patógenos, visando à eliminação bacteriana e a sobrevivência do próprio hospedeiro.

Canonical and Non-canonical Inflammasome Activation by Outer Membrane Vesicles Derived From Bordetella pertussis.

Outer Membrane Vesicles (OMVs) derived from different Gram-negative bacteria have been proposed as an attractive vaccine platform because of their own immunogenic adjuvant properties. Pertussis or whooping cough is a highly contagious vaccine-preventable respiratory disease that resurged during the last decades in many countries. In response to the epidemiological situation, new boosters have been incorporated into vaccination schedules worldwide and new vaccine candidates have started to be designed. Particularly, our group designed a new pertussis vaccine candidate based on OMVs derived from Bordetella pertussis (BpOMVs). To continue with the characterization of the immune response induced by our OMV based vaccine candidate, this work aimed to investigate the ability of OMVs to activate the inflammasome pathway in macrophages. We observed that NLRP3, caspase-1/11, and gasdermin-D (GSDMD) are involved in inflammasome activation by BpOMVs. Moreover, we demonstrated that BpOMVs as well as transfected B. pertussis lipooligosaccharide (BpLOS) induce caspase-11 (Casp11) and guanylate-binding proteins (GBPs) dependent non-canonical inflammasome activation. Our results elucidate the mechanism by which BpOMVs trigger one central pathway of the innate response activation that is expected to skew the adaptive immune response elicited by BpOMVs vaccination.

Study of Eosinophil Apoptosis Induced by Fasciola hepatica Excretory-Secretory Products.

The excretory-secretory products released by the liver fluke Fasciola hepatica (FhESP) are in close contact with the immune system and have different immunomodulatory effects associated with the parasite virulence. The control of the early immune response is crucial for the establishment of the fluke in the host. Related to this, eosinophils (Eo) are implicated as effector cells in helminthic infections, and the induction of Eo apoptosis has been demonstrated to be a remarkable immunoevasion mechanism induced by the parasite. In this chapter, we describe different techniques to assay Eo apoptosis triggered by FhESP as well as the mechanisms involved in this phenomenon.

Cucurbitacin IIb from Ibervillea sonorae Induces Apoptosis and Cell Cycle Arrest via STAT3 Inhibition.

BACKGROUND: Cucurbitacin IIb (CIIb) from Ibervillea sonorae has a high capacity to suppress cancer cell proliferation and induce apoptosis. This study investigated the molecular mechanisms related to the antiproliferative and apoptosis induction capacity of CIIb in HeLa cells. MATERIALS AND METHODS: The cell viability and anti-proliferative effect of CIIb were evaluated by using the trypan blue exclusion assay. The effect of CIIb on the mitochondrial membrane potential was determined by flow cytometry using JC-1. The activity of caspase-3 and caspase-9 was evaluated by flow cytometry using commercial kits. The effect of CIIb on the cell cycle was investigated using Fluorescence-Activated Cell Sorting (FACS) analysis. Western blot analysis was used to evaluate both the inhibitory effect of CIIb on the STAT3 signaling pathway and cyclin -B1, and DNA damage by the comet assay. RESULTS: CIIb triggers disruption of the mitochondrial membrane potential (Δψm) and consequently activated the caspases -3 and -9, as a result of the activation of the intrinsic pathway of the apoptosis. Likewise, the CIIbinduced cell cycle was arrested in S and G2/M after 24h of treatment. CIIb also reduced the expression of STAT3 and cyclin -B1. Finally, CIIb produced an antiproliferative effect at 48 and 72 h, inducing DNA damage. CONCLUSION: These results demonstrate CIIb-induced apoptosis and cell cycle arrest in HeLa through the inhibition of STAT3.

Chronic alcoholism associated with diabetes induced apoptosis in the corpus cavernosum of rats

Purpose To evaluate the effects of alcohol exposure and diabetes on apoptotic process in the corpus cavernosum. Methods Forty eight male Wistar rats were divided into four groups: control, diabetic, alcoholic and diabetic-alcoholic. Samples of the corpus cavernosum were prepared to study protein expression of apoptotic genes (Caspases-3 and 9) by immunohistochemistry and Real-Time PCR. Results The immunoreactivity of Caspases-3 and -9 was diffuse and higher in the treated groups though there was no significant difference between the experimental groups, only when compared with the control group. An increase was observed in the gene expression of Caspases-9 in the diabetic and ethanol-diabetic groups when compared with control and ethanol groups. Conclusions The association of these factors (ethanol and diabetes) probably can affect the apoptosis mechanism in lesions of the cavernous tissue in the rat penis. Both gene and protein expression of Caspase-9 in diabetic and ethanol-diabetic groups suggest the involvement of the apoptosis cascade from this study model.(AU)

Apoptosis (programmed cell death) and its signals - A review

Abstract Apoptosis is a sequential order of cell death occurring regularly to ensure a homeostatic balance between the rate of cell formation and cell death. However, a misplaced of this balancing function can contribute to an abnormal cell growth / proliferation or autoimmune disorders etc. Apoptosis is therefore said to be crucial from the point of development of an embryo throughout the growth of an organism contributing to the renewal of tissues and also the getting rid of inflammatory cells. This review seeks to elaborate on the recent overview of the mechanism involved in apoptosis, some element and signal contributing to its function and inhibition together with how their malfunction contribute to a number of cancer related cases.

Resumo A apoptose é uma ordem sequencial de morte celular que ocorre regularmente para garantir um equilíbrio homeostático entre a taxa de formação e a morte celular. No entanto, um desequilíbrio dessa função pode contribuir para um crescimento/proliferação celular anormal ou distúrbios autoimunes. A apoptose é, portanto, considerada crucial do ponto de desenvolvimento de um embrião ao longo do crescimento de um organismo que contribui para a renovação dos tecidos e também a eliminação de células inflamatórias. Esta revisão procura elaborar a recente visão geral do mecanismo envolvido na apoptose, alguns elementos e sinais que contribuem para sua função e inibição, além de como o mau funcionamento deles contribui para vários casos relacionados ao câncer.