Killing Pathway Research Articles

Apoptin Regulates Apoptosis and Autophagy by Modulating Reactive Oxygen Species (ROS) Levels in Human Liver Cancer Cells.

Apoptin is a protein that specifically induces apoptosis in tumor cells. The anti-tumorigenic functions of Apoptin, including autophagy activation and its interaction with apoptosis, have not been precisely elucidated. Here we investigate the main pathways of apoptin-mediated killing of human liver cancer cells, as well as its putative role in autophagy and apoptosis. The anti-proliferative effect of apoptin in liver cancer cells was analyzed in vitro by crystal violet staining and MTS detection, and also in vivo using a tumor-based model. The main pathway related to apoptin-induced growth inhibition in vitro was evaluated by flow cytometry and fluorescence staining. The relationship between apoptosis and autophagy on apoptin-treating cells was analyzed using apoptosis and autophagy inhibitors, mitochondrial staining, Annexin V-FITC/PI flow detection, LC3 staining, and western blotting. The effect of ROS toward the apoptosis and autophagy of apoptin-treating cells was also evaluated by ROS detection, Annexin V-FITC/PI flow detection, LC3 staining, and western blotting. Inhibition of apoptosis in apoptin-treating liver cancer cells significantly reduced the autophagy levels in vitro. The overall inhibition increased from 12 h and the effect was most obvious at 48 h. Inhibition of autophagy could increase apoptin-induced apoptosis of cells in a time-dependent manner, reaching its peak at 24 h. Apoptin significantly alters ROS levels in liver cancer cells, and this effect is directly related to apoptosis and autophagy. ROS appears to be the key factor linking apoptin-induced autophagy and apoptosis through the mitochondria in liver cancer cells. Therefore, evaluating the interaction between apoptin-induced apoptosis and autophagy is a promising step for the development of alternate tumor therapies.

Sulphur doped carbon dots enhance photodynamic therapy via PI3K/Akt signalling pathway.

ObjectivesPhotodynamic therapy (PDT) is a promising approach for cancer treatment, and the underlying signalling pathway changes has been carried out for studying the PDT mechanisms, but is majorly limited to organic photosensitizers (PSs). For the emerging nano‐PSs typically possessing higher 1O2 quantum yield, few mechanistic studies were carried out, which limited their further applications in clinical therapeutics. PI3K/Akt signalling pathway, a most frequently activated signalling network in cancers, could promote cancer cell survival, but was seldom reported in previous PDT studies mediated by nano‐PSs.Materials and MethodsSulphur doped carbon dots (S‐CDs) was prepared via a hydrothermal synthetic route and was characterized by transmission electron microscopy, X‐ray photoelectron spectroscopy and so on. CCK‐8 assay and Annexin V/PI staining were performed to demonstrate the death of cancer cells, Western blot, RT‐PCR and immunofluorescence were employed to explore the underlying mechanism, and variation of PI3K/Akt and other signalling pathways was detected by Western blot.ResultsS‐CDs was successfully synthesized, and it was much more efficient compared with classic organic PSs. S‐CDs could induce cancer cell death through mitochondria mediated cell apoptosis with the imbalance of Bcl‐2 family proteins and caspase cascade via several signalling pathways. Low concentration of S‐CDs could effectively inhibit PI3K/Akt pathway and promote p38/JNK pathway, on one way inhibiting cancer cell survival and on the other way promoting cell apoptosis.ConclusionsHerein, we found that S‐CDs acted as an inhibitor of the PI3K/Akt pathway for efficient cancer cell killing, thus yielding in a higher PDT performance over the existing photosensitizers.

Biocompatible Fluorescent Nanodiamonds as Multifunctional Optical Probes for Latent Fingerprint Detection.

There is an immense literature on detection of latent fingerprints (LFPs) with fluorescent nanomaterials because fluorescence is one of the most sensitive detection methods. Although many fluorescent probes have been developed for latent fingerprint detection, many challenges remain, including the low selectivity, complicated processing, high background, and toxicity of nanoparticles used to visualize LFPs. In this study, we demonstrate biocompatible, efficient, and low background LFP detection with poly(vinylpyrrolidone) (PVP) coated fluorescent nanodiamonds (FNDs). PVP-coated FND (FND@PVP) is biocompatible at the cellular level. They neither inhibit cellar proliferation nor induce cell death via apoptosis or other cell killing pathways. Moreover, they do not elicit an immune response in cells. PVP coating enhances the physical adhesion of FND to diverse substrates and in particular results in efficient binding of FND@PVP to fingerprint ridges due to the intrinsic amphiphilicity of PVP. Clear, well-defined ridge structures with first, second, and third-level of LFP details are revealed within minutes by FND@PVP. The combination of this binding specificity and the remarkable optical properties of FND@PVP permits the detection of LPFs with high contrast, efficiency, selectivity, sensitivity, and reduced background interference. Our results demonstrate that background-free imaging via multicolor emission and dual-modal imaging of FND@PVP nanoparticles have great potential for high-resolution imaging of LFPs.

Reactive oxygen species play a dominant role in all pathways of rapid quinolone-mediated killing.

Quinolones have been thought to rapidly kill bacteria in two ways: (i) quinolone-topoisomerase-DNA lesions stimulate the accumulation of toxic reactive oxygen species (ROS); and (ii) the lesions directly cause lethal DNA breaks. Traditional killing assays may have underestimated the ROS contribution by overlooking the possibility that ROS continue to accumulate and kill cells on drug-free agar after quinolone removal. Quinolone-induced, ROS-mediated killing of Escherichia coli was measured by plating post-treatment samples on agar with/without anti-ROS agents. When E. coli cultures were treated with ciprofloxacin or moxifloxacin in the presence of chloramphenicol (to accentuate DNA-break-mediated killing), lethal activity, revealed by plating on quinolone-free agar, was inhibited by supplementing agar with ROS-mitigating agents. Moreover, norfloxacin-mediated lethality, observed with cells suspended in saline, was blocked by inhibitors of ROS accumulation and exacerbated by a katG catalase deficiency that impairs peroxide detoxification. Unlike WT cells, the katG mutant was killed by nalidixic acid or norfloxacin with chloramphenicol present and by nalidixic or oxolinic acid with cells suspended in saline. ROS accumulated after quinolone removal with cultures either co-treated with chloramphenicol or suspended in saline. Deficiencies in recA or recB reduced the protective effects of ROS-mitigating agents, supporting the idea that repair of quinolone-mediated DNA lesions suppresses the direct lethal effects of such lesions. ROS are the dominant factor in all modes of quinolone-mediated lethality, as quinolone-mediated primary DNA lesions are insufficient to kill without triggering ROS accumulation. ROS-stimulating adjuvants may enhance the lethality of quinolones and perhaps other antimicrobials.

Nigericin Promotes NLRP3-Independent Bacterial Killing in Macrophages.

Altered microbiota has been associated with a number of diseases, including inflammatory bowel diseases, diabetes, and cancer. This dysregulation is thought to relate the host inflammatory response to enteric pathogens. Macrophages play a key role in host response to microbes and are involved in bacterial killing and clearance. This process is partially mediated through the potassium efflux-dependent, cytosolic, PYCARD-containing inflammasome protein complex. Surprisingly, we discovered an alternative mechanism for bacterial killing, independent of the NLRP3 inflammasome/PYCARD. Using the NLRP3 inflammasome-deficient Raw 264.7 and PYCARD-deficient J77 macrophages, which both lack PYCARD, we found that the potassium efflux activator nigericin enhances bacterial killing. Macrophage response to nigericin was examined by RT gene profiling and subsequent qPCR, which demonstrated altered expression of a series of genes involved in the IL-18 bacterial killing pathway. Based on our results we propose a model of bacterial killing, unrelated to NLRP3 inflammasome activation in macrophage cells. Improving understanding of the molecular pathways driving bacterial clearance within macrophage cells will aid in the development of novel immune-targeted therapeutics in a number of diseases.

Methylation of selected CpG islands involved in the transcription of myeloperoxidase and superoxide dismutase 2 in neutrophils of periparturient and mid-lactation cows

It is generally accepted that intracellular killing of microorganisms by production of reactive oxygen species (ROS) in the phagosome of the neutrophil is an important arm of innate defense. High-producing dairy cows are prone to periparturient metabolic and infectious diseases. Both myeloperoxidase (MPO) activity and ROS production decrease the day of parturition. Several studies have demonstrated changes in the expression of genes involved in, for example, metabolism and defense in the circulating neutrophil during peripartum. In this study, we wanted to further characterize the periparturient neutrophil in terms of its oxidative killing capacity by analyzing the oxidative burst at 3 levels. First, the ROS phenotype was evaluated using chemiluminescence. The cows (sampled within 24 h after parturition and at 135 d in milk) showed a significantly slower production of ROS at parturition. Both primiparous (n = 13) and multiparous (n = 12) cows were included in this study, but parity did not affect the kinetics of ROS production. Second, the expression of 11 genes involved in ROS production was measured in the same cows: cytochrome b-245 α and β chain (CYBA, CYBB; coding for membrane-bound constituents of NADPH oxidase); neutrophil cytosolic factors 1, 2, and 4 (NCF1, NCF2, and NCF4); Rac family small GTPase 1 and 2 (RAC1 and RAC2; coding for regulatory proteins of NADPH oxidase); superoxide dismutase 2 (SOD2); catalase (CAT); myeloperoxidase (MPO; coding for enzymes involved in metabolizing downstream ROS); and spleen-associated tyrosine kinase (SYK; involved in signaling). During peripartum, a shift in expression in the oxidative killing pathway was observed, characterized by a downregulation of MPO and a simultaneous upregulation of the genes coding for NADPH oxidase. Third, as total DNA methylation is known to change during pregnancy, we investigated whether the observed differences were due to different methylation patterns. Promotor regions initiate transcription of particular genes; therefore, we analyzed the methylation status in annotated CpG islands of MPO and SOD2, 2 genes with a significant difference in expression between both lactation stages. The differences in methylation of these CpG islands were nonsignificant. High-throughput techniques may be necessary to obtain more detailed information on the total DNA methylation dynamics in bovine neutrophils and increase our understanding of how gene expression is controlled in neutrophils.

IPLA2 Activation Mediates Granular Exocytosis and Corrects Microbicidal Defects in ROS-Deficient and CGD Human Neutrophils.

The ubiquitous calcium-independent phospholipase A2 enzyme (iPLA2) is inhibited by calmodulin binding and known to be responsible for phospholipid remodeling housekeeping functions including granule exocytosis-associated membrane fusion in normal human neutrophils. We evaluate in human neutrophils the iPLA2 secretagogue effects using normal neutrophils, where reactive oxygen species (ROS) generation has been blocked by diphenyleneiodonium, as well as in neutrophils from chronic granulomatous disease (CGD) patients. Neutrophils were pretreated with W7, a calmodulin inhibitor known to activate iPLA2 and exocytosis of granules, and vesicles as well as intra- and extra-microbicidal activity against Staphylococcus aureus and Aspergillus fumigatus were evaluated. W7 increases exocytosis of primary, secondary, and tertiary granules and vesicles and improves neutrophil microbicidal activity against S. aureus and A. fumigatus. In neutrophils, calmodulin-mediated iPLA2 inhibition controls granule and vesicle exocytosis in the phagosome and in the extracellular microenvironment. Relieving iPLA2 inhibition results in increased exocytosis of primary, secondary, and tertiary granules and secretory vesicles with correction of defective intracellular and extracellular microbicidal activity. In CGD patients presenting ROS defective production, this increase in the non-oxidative killing pathway partially corrects their microbicidal defects.

Evaluating CD8+ T Cell Responses In Vitro.

The 51Cr-release assay described in the 1960s has been for decades the gold standard cytolytic assay but for safety reasons is no longer in use in many laboratories. Whereas other radioactive tests were later on described, they never ousted the 51Cr-release assay. More thorough understanding of CTL biology and killing pathways has more recently resulted in the design of reliable nonradioactive tests to analyze CD8+ T cell responses. Allowing for real-time evaluation of target cell viability, some of them are particularly attractive and are described here together with traditional assays.

The Goddess Athena as Symbol of Phronesis in Porphyry’s On the Cave of the Nymphs

AbstractOn the Cave of the Nymphs, an allegorical exegesis of Homer’s description of the cave of the nymphs at Odyssey 13.102-112, a passage quoted in full at the beginning of the treatise after the briefest possible indication of the project on which Porphyry is embarking, has been generally given little attention in discussions of Neoplatonic philosophy, as it is deemed to be of little importance for establishing Porphyrian doctrine. However, the treatise contains significant philosophical thoughts on the relationship between the soul and body, embodiment, demonology, and the concept of salvation of soul, which are compatible with his other works, especially On Abstinence from Killing Animals (De Abstinentia) and Pathways to the Intelligible (Sententiae). The concept of salvation of soul is found in Porphyry’s identification of the goddess Athena with phronesis, along with the olive tree, while Odysseus represents the soul descending into genesis, but will return back to his fatherland.In this context, this paper will explore the role and meaning of phronesis, namely the goddess Athena, in the process of the soul’s journey towards the intelligible realm and show the relevance of the Neoplatonic doctrine of virtues, particularly the cathartic virtues, in Sententia 32 to Porphyry’s reading of Homer’s image of Odysseus under guidance of the goddess Athena. Phronesis inspires the soul to incline towards the level of Intellect that is, away from damaging influences of the body to which the soul is enslaved and which confuses it with desires, passions, fears and illusory impressions, and prevents it from attaining the intelligible realm, whereas the body and its desires lead us to conflict and unjust behaviour in order to gain wealth, status, power, and pleasure.

Distinct Roles of Sensory Neurons in Mediating Pathogen Avoidance and Neuropeptide-Dependent Immune Regulation

Increasing evidence implies an extensive and universal interaction between the immune system and thenervous system. Previous studies showed that OCTR-1, a neuronal G-protein-coupled receptor (GPCR) analogous to human norepinephrine receptors, functions in sensory neurons to control the gene expression of both microbial killing pathways and the unfolded protein response (UPR) in Caenorhabditis elegans. Here, we found that OCTR-1-expressing neurons, ASH, are involved in controlling innate immune pathways. In contrast, another group of OCTR-1-expressing neurons, ASI, was shown to promote pathogen avoidance behavior. We also identified neuropeptide NLP-20 and AIA interneurons, which are responsible for the integration of conflicting cues and behaviors, as downstream components of the ASH/ASI neural circuit. These findings provide insights into a neuronal network involved inregulating pathogen defense mechanisms in C.elegans and might have broad implications for the strategies utilized by metazoans to balance theenergy-costly immune activation and behavioral response.

Immune responses to invasive aspergillosis: new understanding and therapeutic opportunities.

Invasive aspergillosis is a worldwide disease that primarily affects immune-compromised patients, agricultural workers with corneal abrasions, individuals with structural lung disease, and patients with primary immune deficiency. The critical function of the immune system is to prevent the germination of airborne conidia into tissue-invasive hyphae. This review covers recent advances that shape our understanding of anti-Aspergillus immunity at the molecular and cellular level. Host defense against conidia and hyphae occurs via distinct molecular mechanisms that involve intracellular and extracellular killing pathways, as well as cooperation between different myeloid cell subsets. The strength and efficacy of the host response is shaped by the tissue microenvironment. In preclinical models of disease, host immune augmentation strategies have yielded benefits, yet translating these insights into therapeutic strategies in humans remains challenging. Although advances in early diagnostic strategies and in antifungal drugs have ameliorated clinical outcomes of invasive aspergillosis, further improvements depend on gaining deeper insight into and translating advances in anti-Aspergillus immunity.

Cholesterol-lowering drug, in combination with chromium chloride, induces early apoptotic signals in intracellular L. donovani amastigotes, leading to death.

Leishmania establishes a successful parasitism by evading both oxidative and non-oxidative killing pathways, and its drug resistance against the currently available therapeutics demands for a safe and cheap drug. Since the parasite synthesizes ergosterol instead of cholesterol, using the same biochemical pathway and enzymes, an inhibitor of HMG-CoA-Reductase, Lovastatin, has been tried for its anti-Leishmanial effect. Lovastatin, being an inhibitor of HMG-CoA-Reductase, inhibits infection by cholesterol depletion, while chromium chloride complexes, at their higher concentrations, are reported to exhibit cytotoxicity. In intracellular amastigotes, cytotoxicity has been checked by assessing various manifestation of cell death, viz. DNA fragmentation, AnnexinV-FITC binding and JC-1 fluorescence ratio. Release of hydrogen peroxide (HPO) and nitric oxide (NO) has been assessed in live cell. Lovastatin and CrCl3.6H2O in combination has appeared to be ineffective on promastigotes but has induced cytotoxic effect on the intracellular amastigotes through up-regulation of cellular signalling mechanisms. CrCl 3.6H2O stimulates generation of NO, leading to reduction of the number of intracellular amastigote, while Lovastatin shows HPO-mediated killing of the same, keeping the host cell unaffected. This novel therapeutic approach, involving two known safe compounds in suboptimal doses, may resolve human visceral Leishmaniasis.

Cystatins, cysteine peptidase inhibitors, as regulators of immune cell cytotoxicity

Cystatins comprise a superfamily of evolutionarily related proteins, present in all living organisms, from protozoa to mammals. They act as inhibitors of cysteine peptidases although they can also function independently of their inhibitory function. Cysteine cathepsins are implicated in various physiological and pathological processes. In the immune response they are involved in antigen processing and presentation, the cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), migration and adhesion of immune cells, cytokine and growth factor regulation and toll-like receptor signalling. Cystatins are probably involved in the regulation of all these processes; importantly, cystatin F has a crucial role in the regulation of immune cell cytotoxicity. NK cells and CTLs exploit the granzyme/perforin pathway for target cell killing, with perforin and granzymes as crucial effector molecules. Granzymes are synthesized as inactive pro-granzymes and need to be proteolytically activated by cathepsins C and H. Cystatin F is the main regulator of the activity of cathepsins C and H in cytotoxic cells and, consequently, regulates their cytotoxicity. The role of cystatins and cysteine cathepsins in the immune response is presented, with emphasis on their role in the regulation of cytotoxicity of NK cells and CTLs.

In Vitro Comparison of ADCC and CAR Sensitivity of Adult HER-2+ B-ALL Using the NK-92 Human Cell Line Transduced with a Human CD16 (ADCC) or an Anti-HER2 Chimeric Antigen Receptor (CAR)

Introduction: Prognosis of acute lymphoblastic leukemia (ALL) in adults remains dismal. Recent advances have consisted of targeted therapies with monoclonal antibodies and chimeric antigen receptor (CAR) T lymphocytes (anti-CD19 CAR T cells), showing very encouraging results. However, relapse still occur after this new therapeutics and increasing the armamentarium for ALL is still required. Her2 is expressed in above one third of adult B-ALL (Chevallier,Haematologica 2009). The anti-HER2 antibodytrastuzumab has been tested prospectively in such patients, showing a low efficacy (Chevallier, Blood 2012).The present work was designed to compare, in vitro, thelysis sensibility of B-ALL HER2+ mediated by two mechanisms of HER2 recognition based on antibody (Ab) specificity: firstly, when the anti-HER2 monoclonalAbtrastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (antibodydependant cellular cytotoxicity, ADCC) andsecondly, when HER2-positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (anti-HER2 CAR).Methods: Frozen samples from 7 HER2+ B-ALL adults were available, including onesample expressing also CD20. Characteristics of Patients and controls are presented in Table 1.Three human breast-cancer cell lines (BCCL) expressing different validated (HercepTest, Dako) levels of HER2 (BT474 (HER2 3+), MCF-7 (HER2 0-/1+) and MDA-MB-231 (HER2 0-/1+) were used as positive control. Leukemic HER2-negative B-ALL cells from two patients were used as negative control, including one patient with CD20+ expression. The level of HER2 expression by HER2+ B-ALL was determined by fluorescence-activated cell sorter (FACS) and compared to the HER2 expressing BCCL.Cytotoxic activity was assessed at a 30-to-1 effector-to-target ratio in a 4 hours 51Cr-release assay.We used the same cytotoxic effector lymphocytes (the human NK cell line NK-92), armed with eitheraFcγRIIIa/FcεRIγ receptor (referred to as NK-92CD16) or with an anti-HER2/FcεRIγ CAR receptor (referred to as NK-92CAR) as previously described (Clémenceau, JImmunol Research 2015). For ADCC/CAR assays, target cells were pre-incubated withtrastuzumab and /or rituximab at 10µg/ml each.The study was approved by our local review board and twoalivepatients gave informed consent.Results:The level of HER2 expression by HER2+ B-ALL (n=7) was highly variable with a mean relative fluorescence intensity ratio (RFI) of 13 (range 5 to 30). According to the HercepTestranking used for BCCL lines (see above) all B-ALL would be classified as HER2 0-/1+. In vitro HER2+ BCCL were efficiently lysed by anti-HER2 NK-92CAR in this 4 hours 51Cr-release assay and this direct pathway of killing by CAR was always more efficient than the indirect pathway mediated by ADCC. In addition, thelysis level was correlated to HER2 level expression.Trastuzumabalone has no effect on the spontaneous 51Cr-release of B-ALL cells (not shown). Anti-HER2 CAR mediatedlysis was observed for 5 out of 6 HER2+ B-ALL cells (83%, 1 case (#5) non interpretable). Although lysis levels were low, they werespecificsince the CAR mediatedlysis was inhibited when the B-ALL were pre-incubated with 10 µg/mltrastuzumab (not shown). In contrast, for these 5 HER2+ B-ALL, no anti-HER2 ADCClysis was observed. These results confirmed that in these experimental conditions, HER2 targeting by CAR is more efficient than by ADCC. For the two HER2- B-ALL, no ADCC or CARlysis were observed.Finally, for the two B-ALL expressing CD20, anti-CD20 ADCClysiscan be observed demonstrating that B-ALL cells are not intrinsically resistant to ADCCcytoxicity. Regarding the unique case of B-ALL expressing both CD20/HER2 antigens, only anti-CD20 ADCClysiswas observed. Results are presented in Table 2.Conclusion:Together, these preliminary results suggest that for the CD20-negative and HER2-positive B-ALLs, one cannot rely on ADCC as a mechanism of target celllysis, while the interest of an anti-HER2-CAR approach deserve further studies to better correlate the level of HER2 expression and the sensitivity tolysisby HER2-specific CAR. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Neuronal GPCR OCTR-1 regulates innate immunity by controlling protein synthesis in Caenorhabditis elegans

Upon pathogen infection, microbial killing pathways and cellular stress pathways are rapidly activated by the host innate immune system. These pathways must be tightly regulated because insufficient or excessive immune responses have deleterious consequences. Increasing evidence indicates that the nervous system regulates the immune system to confer coordinated protection to the host. However, the precise mechanisms of neural-immune communication remain unclear. Previously we have demonstrated that OCTR-1, a neuronal G protein-coupled receptor, functions in the sensory neurons ASH and ASI to suppress innate immune responses in non-neural tissues against Pseudomonas aeruginosa in Caenorhabditis elegans. In the current study, by using a mass spectrometry-based quantitative proteomics approach, we discovered that OCTR-1 regulates innate immunity by suppressing translation and the unfolded protein response (UPR) pathways at the protein level. Functional assays revealed that OCTR-1 inhibits specific protein synthesis factors such as ribosomal protein RPS-1 and translation initiation factor EIF-3.J to reduce infection-triggered protein synthesis and UPR. Translational inhibition by chemicals abolishes the OCTR-1-controlled innate immune responses, indicating that activation of the OCTR-1 pathway is dependent on translation upregulation such as that induced by pathogen infection. Because OCTR-1 downregulates protein translation activities, the OCTR-1 pathway could function to suppress excessive responses to infection or to restore protein homeostasis after infection.

Mitochondrial ROS and the Effectors of the Intrinsic Apoptotic Pathway in Aging Cells: The Discerning Killers!

It has become clear that mitochondrial reactive oxygen species (mtROS) are not simply villains and mitochondria the hapless targets of their attacks. Rather, it appears that mitochondrial dysfunction itself and the signaling function of mtROS can have positive effects on lifespan, helping to extend longevity. If events in the mitochondria can lead to better cellular homeostasis and better survival of the organism in ways beyond providing ATP and biosynthetic products, we can conjecture that they act on other cellular components through appropriate signaling pathways. We describe recent advances in a variety of species which promoted our understanding of how changes of mtROS generation are part of a system of signaling pathways that emanate from the mitochondria to impact organism lifespan through global changes, including in transcriptional patterns. In unraveling this, many old players in cellular homeostasis were encountered. Among these, maybe most strikingly, is the intrinsic apoptotic signaling pathway, which is the conduit by which at least one class of mtROS exercise their actions in the nematode Caenorhabditis elegans. This is a pathway that normally contributes to organismal homeostasis by killing defective or otherwise unwanted cells, and whose various compounds have also been implicated in other cellular processes. However, it was a surprise that that appropriate activation of a cell killing pathway can in fact prolong the lifespan of the organism. In the soma of adult C. elegans, all cells are post-mitotic, like many of our neurons and possibly some of our immune cells. These cells cannot simply be killed and replaced when showing signs of dysfunction. Thus, we speculate that it is the ability of the apoptotic pathway to pull together information about the functional and structural integrity of different cellular compartments that is the key property for why this pathway is used to decide when to boost defensive and repair processes in irreplaceable cells. When this process is artificially stimulated in mutants with elevated mtROS generation or with drug treatments it leads to lifespan prolongations beyond the normal lifespan of the organism.

Neural Inhibition of Dopaminergic Signaling Enhances Immunity in a Cell-Non-autonomous Manner

The innate immune system is the front line of host defense against microbial infections, but its rapid and uncontrolled activation elicits microbicidal mechanisms that have deleterious effects [1, 2]. Increasing evidence indicates that the metazoan nervous system, which responds to stimuli originating from both the internal and the external environment, functions as a modulatory apparatus that controls not only microbial killing pathways but also cellular homeostatic mechanisms [3-5]. Here we report thatdopamine signaling controls innate immune responses through a D1-like dopamine receptor, DOP-4, in Caenorhabditis elegans. Chlorpromazine inhibition of DOP-4 in the nervous system activates a microbicidal PMK-1/p38 mitogen-activated protein kinase signaling pathway that enhances host resistance against bacterial infections. The immune inhibitory function of dopamine originates in CEP neurons and requires active DOP-4 in downstream ASG neurons. Our findings indicate that dopamine signaling from the nervous system controls immunity in a cell-non-autonomous manner and identifies the dopaminergic system as a potential therapeutic target for not only infectious diseases but also a range of conditions that arise as a consequence of malfunctioning immune responses.

Uptake of poly-dispersed single-walled carbon nanotubes and decline of functions in mouse NK cells undergoing activation

The interaction of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNT) with NK cells undergoing activation was examined. Exposure to AF-SWCNT during NK activation in vitro by interleukin (IL)-2, and in vivo by Poly(I:C) significantly lowered cytotoxic activity generated against YAC-1 tumor cells. Recoveries of spleen NK1.1+ cells as well as the activated subset of NK cells (NK1.1+CD69+ cells) were significantly reduced by the AF-SWCNT exposure. The proportion of apoptotic NK cells (NK1.1+ phosphatidylserine+) in the spleen cell preparations activated in vitro was also significantly elevated. Expression levels of CD107a [for assessing NK cell degranulation] as well as of FasL marker [mediating non-secretory pathway of NK cell killing] were significantly lower in cells exposed to AF-SWCNT during the activation phase. Intracellular levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the cells were also significantly reduced. Fluorescent AF-SWCNT (FAF-SWCNT) were internalized by the NK cells and uptake was significantly greater in activated cells. Confocal microscopy indicated the internalized FAF-SWCNT were localized to the cytoplasm of the NK cells. These results indicated that AF-SWCNT were internalized by NK cells and caused a general down-regulation of a variety of parameters associated with NK cell cytotoxicity and other cellular functions.

Interferon-γ Autoantibodies as Predisposing Factor for Nontuberculous Mycobacterial Infection

Interferon-γ Autoantibodies as Predisposing Factor for Nontuberculous Mycobacterial Infection

Abrus Agglutinin, a type II ribosome inactivating protein inhibits Akt/PH domain to induce endoplasmic reticulum stress mediated autophagy-dependent cell death.

Abrus agglutinin (AGG), a type II ribosome-inactivating protein has been found to induce mitochondrial apoptosis. In the present study, we documented that AGG-mediated Akt dephosphorylation led to ER stress resulting the induction of autophagy-dependent cell death through the canonical pathway in cervical cancer cells. Inhibition of autophagic death with 3-methyladenine (3-MA) and siRNA of Beclin-1 and ATG5 increased AGG-induced apoptosis. Further, inhibiting apoptosis by Z-DEVD-FMK and N-acetyl cysteine (NAC) increased autophagic cell death after AGG treatment, suggesting that AGG simultaneously induced autophagic and apoptotic death in HeLa cells. Additionally, it observed that AGG-induced autophagic cell death in Bax knock down (Bax-KD) and 5-FU resistant HeLa cells, confirming as an alternate cell killing pathway to apoptosis. At the molecular level, AGG-induced ER stress in PERK dependent pathway and inhibition of ER stress by salubrinal, eIF2α phosphatase inhibitor as well as siPERK reduced autophagic death in the presence of AGG. Further, our in silico and colocalization study showed that AGG interacted with pleckstrin homology (PH) domain of Akt to suppress its phosphorylation and consequent downstream mTOR dephosphorylation in HeLa cells. We showed that Akt overexpression could not augment GRP78 expression and reduced autophagic cell death by AGG as compared to pcDNA control, indicating Akt modulation was the upstream signal during AGG's ER stress mediated autophagic cell death. In conclusion, we established that AGG stimulated cell death by autophagy might be used as an alternative tumor suppressor mechanism in human cervical cancer. © 2016 Wiley Periodicals, Inc.