Alpha Release Research Articles

In Vitro and In Vivo Short-Term Pulmonary Toxicity of Differently Sized Colloidal Amorphous SiO₂.

In vitro prediction of inflammatory lung effects of well-dispersed nanomaterials is challenging. Here, the in vitro effects of four colloidal amorphous SiO2 nanomaterials that differed only by their primary particle size (9, 15, 30, and 55 nm) were analyzed using the rat NR8383 alveolar macrophage (AM) assay. Data were compared to effects of single doses of 15 nm and 55 nm SiO2 intratracheally instilled in rat lungs. In vitro, all four elicited the release of concentration-dependent lactate dehydrogenase, β-glucuronidase, and tumor necrosis factor alpha, and the two smaller materials also released H2O2. All effects were size-dependent. Since the colloidal SiO2 remained well-dispersed in serum-free in vitro conditions, effective particle concentrations reaching the cells were estimated using different models. Evaluating the effective concentration–based in vitro effects using the Decision-making framework for the grouping and testing of nanomaterials, all four nanomaterials were assigned as “active.” This assignment and the size dependency of effects were consistent with the outcomes of intratracheal instillation studies and available short-term rat inhalation data for 15 nm SiO2. The study confirms the applicability of the NR8383 AM assay to assessing colloidal SiO2 but underlines the need to estimate and consider the effective concentration of such well-dispersed test materials.

High mobility group box 1 (HMGB1) acts as an “alarmin” to promote acute myeloid leukaemia progression

ABSTRACTHigh mobility group box 1 (HMGB1) is a non-histone protein localised in the cell nucleus, where it interacts with DNA and promotes nuclear transcription events. HMGB1 levels are elevated during acute myeloid leukaemia (AML) progression followed by participation of this protein in triggering signalling events in target cells as a pro-inflammatory stimulus. This mechanism was hypothesised to be employed as a survival pathway by malignant blood cells and our aims were therefore to test this hypothesis experimentally. Here we report that HMGB1 triggers the release of tumour necrosis factor alpha (TNF-α) by primary human AML cells. TNF-α induces interleukin 1 beta (IL-1β) production by healthy leukocytes, leading to IL-1β-induced secretion of stem cell factor (SCF) by competent cells (for example endothelial cells). These results were verified in mouse bone marrow and primary human AML blood plasma samples. In addition, HMGB1 was found to induce secretion of angiogenic vascular endothelial growth factor (VEGF) and this process was dependent on the immune receptor Tim-3. We therefore conclude that HMGB1 is critical for AML progression as a ligand of Tim-3 and other immune receptors thus supporting survival/proliferation of AML cells and possibly the process of angiogenesis.

Salmonella enterica Serovar Typhimurium Alters the Extracellular Proteome of Macrophages and Leads to the Production of Proinflammatory Exosomes.

ABSTRACTSalmonella enterica serovar Typhimurium is a Gram-negative bacterium, which can invade and survive within macrophages. Pathogenic salmonellae induce the secretion of specific cytokines from these phagocytic cells and interfere with the host secretory pathways. In this study, we describe the extracellular proteome of human macrophages infected with S. Typhimurium, followed by analysis of canonical pathways of proteins isolated from the extracellular milieu. We demonstrate that some of the proteins secreted by macrophages upon S. Typhimurium infection are released via exosomes. Moreover, we show that infected macrophages produce CD63+ and CD9+ subpopulations of exosomes at 2 h postinfection. Exosomes derived from infected macrophages trigger the Toll-like receptor 4-dependent release of tumor necrosis factor alpha (TNF-α) from naive macrophages and dendritic cells, but they also stimulate secretion of such cytokines as RANTES, IL-1ra, MIP-2, CXCL1, MCP-1, sICAM-1, GM-CSF, and G-CSF. Proinflammatory effects of exosomes are partially attributed to lipopolysaccharide, which is encapsulated within exosomes. In summary, we show for the first time that proinflammatory exosomes are formed in the early phase of macrophage infection with S. Typhimurium and that they can be used to transfer cargo to naive cells, thereby leading to their stimulation.

Intrathecal delivery of a palmitoylated peptide targeting Y382-384 within the P2X7 receptor alleviates neuropathic pain.

Pain hypersensitivity resulting from peripheral nerve injury depends on pathological microglial activation in the dorsal horn of the spinal cord. This microglial activity is critically modulated by P2X7 receptors (P2X7R) and ATP stimulation of these receptors produces mechanical allodynia, a defining feature of neuropathic pain. Peripheral nerve injury increases P2X7R expression and potentiates its cation channel function in spinal microglia. Here, we report a means to preferentially block the potentiation of P2X7R function by delivering a membrane permeant small interfering peptide that targets Y382-384, a putative tyrosine phosphorylation site within the P2X7R intracellular C-terminal domain. Intrathecal administration of this palmitoylated peptide (P2X7R379-389) transiently reversed mechanical allodynia caused by peripheral nerve injury in both male and female rats. Furthermore, targeting Y382-384 suppressed P2X7R-mediated release of cytokine tumor necrosis factor alpha and blocked the adoptive transfer of mechanical allodynia caused by intrathecal injection of P2X7R-stimulated microglia. Thus, Y382-384 site-specific modulation of P2X7R is an important microglial mechanism in neuropathic pain.

A Proteomic-Based Approach to Study the Mechanism of Cytotoxicity Induced by Interleukin-1\u03b1 and Cycloheximide

The exposure of HeLa cells to interleukin-1 alpha (IL-1α) in the presence of cycloheximide (CHX) leads to the release of active tumor necrosis factor alpha (TNF-α), eliciting cytocidal effect on these cells. A mass spectrometry (MS)-based analysis of the qualitative proteomic profiles of the HeLa cells treated only with IL-1α, CHX or simultaneously with IL-1α and CHX, in comparison to an untreated control, enabled to distinguish protein candidates possibly involved in this process. Among them protein disulphide isomerase (PDI) seemed to be particularly interesting for further research. Therefore, we focused on quantitative changes of PDI levels in HeLa cells subjected to IL-1α and CHX. Enzyme-linked immunosorbent assay (ELISA) was employed for determination of PDI concentrations in the investigated, differently treated HeLa cells. The obtained results confirmed up-regulation of PDI only in the cells stimulated with IL-1α alone. In contrary, the PDI levels in HeLa cells exposed to both IL-1α and CHX, where apoptotic process was intensive, did not increase significantly. Finally, we discuss how different expression levels of PDI together with other proteins, which were detected in this study, may influence the induction of cytotoxic effect and modulate sensitivity to cytotoxic action of IL1.Graphical

Pregnancy-induced changes in metabolome and proteome in ovine uterine flushings†

Mass spectrometry (MS) approaches were used herein to identify metabolites and proteins in uterine flushings (UF) that may contribute to nourishing the conceptus. Ovine uteri collected on Day 12 of the estrous cycle (n = 5 ewes exposed to vasectomized ram) or Days 12 (n = 4), 14 (n = 5), or 16 (n = 5) of pregnancy (bred with fertile ram) were flushed using buffered saline. Metabolites were extracted using 80% methanol and profiled using ultraperformance liquid chromatography (LC) tandem mass spectrometry. The proteome was examined by digestion with trypsin, followed by the analysis of peptides with LC-MS/MS. Metabolite profiling detected 8510 molecular features of which 9 were detected only in UF from Day 14-16 pregnant ewes that function in fatty acid transport (carnitines), hormone synthesis (androstenedione like), and availability of nutrients (valine). Proteome analysis detected 783 proteins present by Days 14-16 of pregnancy in UF, 7 of which are as follows: annexin (ANX) A1, A2, and A5; calcium-binding protein (S100A11); profilin 1; trophoblast kunitz domain protein 1 (TKDP); and interferon tau (IFNT). These proteins function in endocytosis, exocytosis, calcium signaling, and inhibition of prostaglandins (annexins and S100A11); protecting against maternal proteases (TKDP); remodeling cytoskeleton (profilin 1); and altering uterine release of prostaglandin F2 alpha as well as inducing IFNT-stimulated genes in the endometrium and the corpus luteum (IFNT). Identifying metabolites and proteins produced by the uterus and conceptus advances our understanding of embryo/maternal signaling and provides insights into possible the causes of reproductive failure.

Importance of Autophagy in Mediating Human Immunodeficiency Virus (HIV) and Morphine-Induced Metabolic Dysfunction and Inflammation in Human Astrocytes.

Under physiological conditions, the function of astrocytes in providing brain metabolic support is compromised under pathophysiological conditions caused by human immunodeficiency virus (HIV) and opioids. Herein, we examined the role of autophagy, a lysosomal degradation pathway important for cellular homeostasis and survival, as a potential regulatory mechanism during pathophysiological conditions in primary human astrocytes. Blocking autophagy with small interfering RNA (siRNA) targeting BECN1, but not the Autophagy-related 5 (ATG5) gene, caused a significant decrease in HIV and morphine-induced intracellular calcium release. On the contrary, inducing autophagy pharmacologically with rapamycin further enhanced calcium release and significantly reverted HIV and morphine-decreased glutamate uptake. Furthermore, siBeclin1 caused an increase in HIV-induced nitric oxide (NO) release, while viral-induced NO in astrocytes exposed to rapamycin was decreased. HIV replication was significantly attenuated in astrocytes transfected with siRNA while significantly induced in astrocytes exposed to rapamycin. Silencing with siBeclin1, but not siATG5, caused a significant decrease in HIV and morphine-induced interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) release, while secretion of IL-8 was significantly induced with rapamycin. Mechanistically, the effects of siBeclin1 in decreasing HIV-induced calcium release, viral replication, and viral-induced cytokine secretion were associated with a decrease in activation of the nuclear factor kappa B (NF-κB) pathway.

Abstract 5569: The impact of ADI-PEG20 on PDL1 expression in ASS1 deficient uveal melanoma

Abstract Uveal melanoma (UM), involving the iris, choroid and ciliary body, is the commonest intraocular tumor in adults. Half of patients develop metastasis with a high mortality despite currently available systemic therapies including immune checkpoint blockade. Studies show that while CTLA4 antagonism has a modest effect in UM, PD1/PDL1 blockade is largely ineffective whereas trials of combination checkpoint blockade are yet to report. We confirmed previously that deficiency of argininosuccinate synthetase 1 (ASS1), a key enzyme involved in arginine synthesis, sensitizes UM cells to arginine deprivation using pegylated arginine deiminase or ADI-PEG20. Early trials of ADI-PEG20 in UM have shown safety and efficacy in the clinic and thus we tested the potential for a combined immunometabolic strategy. ASS1-deficient UM cell lines (OMM1, OMM2.5, MEL270) were analyzed for the immune checkpoint protein PDL1 along with their sensitivity to arginine depletion. We also tested metastatic UM for ASS1 and PDL1 expression, and the impact of ADI-PEG20 treatment using samples from a current clinical trial (NCT02029690). We showed that ASS1 and PDL1 protein expression were absent in the 3 UM cell lines and in a majority of primary tumors (75/102 for ASS1 and 83/102 for PDL1; 5% threshold of expression) and all metastatic tumor biopises (n=16/16 for both). Transfection of ASS1 in OMM1, OMM2.5, MEL270 led to an increase in PDL1 expression by qPCR, western blotting and FACS, which was reversible following knockdown of ASS1. Induction of PDL1 expression by ASS1 was accompanied by interferon (IFN)-alpha and beta (but not IFN-gamma) release into the cell supernatant, and was abrogated using the pan-STAT inhibitor ruxolitinib. Next, PDL1 expression was significantly increased in the 3 ASS1-negative UM cell lines with ADI-PEG20 treatment by 24hrs and was associated with Type I IFN signaling which waned along with PDL1 expression by 48hrs. The ADI-PEG 20 induction of PDL1 was abolished using ruxolitinib, indicating that the upregulation of Type I IFNs is critical for regulation of the PDL1 checkpoint protein. While analysis of UM biopsies of patients progressing on ADI-PEG20 revealed upregulation of ASS1 (n=2/2; and thus resistance to ADI-PEG20) a concomitant increase in PDL1 was not observed (n=0/2). Collectively, our data show that ASS1 is absent in a majority of patient biopsies of primary and metastatic UM tumors and is tightly correlated with PDL1 expression. UM cells lines displayed sensitivity to ADI-PEG20, which upregulated levels of PDL1 expression via Type 1 interferon signaling that may enhance the currently limited efficacy of checkpoint blockade in UM. Further studies are ongoing of the IFN-mediated signaling between ASS1 and PDL1 in UM in response to arginine deprivation with ADI-PEG20. Citation Format: Ramsay S. Khadeir, Melissa M. Phillips, Mandeep Sagoo, Victoria Cohen, Caroline Thuang, Peter W. Szlosarek. The impact of ADI-PEG20 on PDL1 expression in ASS1 deficient uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5569. doi:10.1158/1538-7445.AM2017-5569

Discovery of N-{4-[5-(4-Fluorophenyl)-3-methyl-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-acetamide (CBS-3595), a Dual p38α MAPK/PDE-4 Inhibitor with Activity against TNFα-Related Diseases.

The anti-inflammatory potential of p38 mitogen-activated protein kinase (MAPK) inhibitors was coincidentally expanded to a dual inhibition of p38α MAPK and phosphodiesterase 4 (PDE4), and the potential benefits arising from the blockage of both inflammation-related enzymes were thoroughly investigated. The most promising compound, CBS-3595 (1), was successively evaluated in in vitro experiments as well as in ex vivo and in vivo preclinical studies after administration of 1 to rodents, dogs, and monkeys. The resulting data clearly indicated a potent suppression of tumor necrosis factor alpha release. For reconfirming the findings of the animal studies when administering 1 to healthy human volunteers, a phase I clinical trial was conducted. Apart from further information regarding the pharmacokinetic and pharmacodynamic characteristics of 1, it was demonstrated that dual inhibition of p38α MAPK and PDE4 is able to synergistically attenuate the excessive anti-inflammatory response.

Long-term cytokine and growth factor release from equine platelet-rich fibrin clots obtained with two different centrifugation protocols

ObjectivesTo compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-β1) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240g/8min or 416g/10min. MethodsWhole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6h, 24h and then every 48h to 21days. Cytokines and GFs were measured by ELISA at 1h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included. ResultsThere were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p=0.01) effect of time was noted. All cytokines were detected after 6h of PRF clot culture until day 21. GF were detected at 1h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p=0.01) correlation was noticed between all the proteins evaluated. ConclusionsLeukocytes enmeshed in PRF clots were able to produce cytokines, TGF-β1 and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals.

Poly(ADP-ribose) polymerase-1 regulates microglia mediated decrease of endothelial tight junction integrity

Alzheimer's disease pathology includes, beside neuronal damage, reactive gliosis and reduced blood-brain barrier (BBB) integrity. Microglia are intimately associated with the BBB and upon AD pathology, pro-inflammatory responses of microglia could contribute to BBB damage. To study whether microglia can directly affect BBB integrity, the effects of amyloid beta (Aβ) -stimulated primary murine microglia on co-cultured mouse brain endothelial cells (bEnd3) and murine astrocyte cultures were assessed. We also assessed whether microglial phenotype modulation via poly(ADP-ribose) polymerase-1 (PARP-1) inhibition/ablation can reverse microglial impact on these BBB forming cells.Unstimulated microglia promoted expression of tight junction proteins (TJPs), zonula ocluden-1 (ZO-1) and occludin in co-cultured endothelia cells, whereas Aβ-stimulated microglia reduced endothelial expression of ZO-1 and occludin. Astrocytes co-cultured with microglia showed elevated glial fibrillary acidic protein (GFAP) expression, which was further increased if microglia had been stimulated with Aβ. Aβ induced microglial release of nitric oxide (NO) and tumour necrosis factor alpha (TNFα), which resulted in reduced endothelial expression of TJPs and increased paracellular permeability. Microglial PARP-1 inhibition attenuated these Aβ-induced events. These findings demonstrate that PARP-1 mediated microglial responses (NO and TNFα) can directly reduce BBB integrity by promoting TJP degradation, increasing endothelial cell permeability and inducing astrogliosis. PARP-1 as a modulator of microglial phenotype can prevent microglial BBB damaging events, and thus is a potential therapeutic target.

Tumor-derived fibulin-3 activates pro-invasive NF-κB signaling in glioblastoma cells and their microenvironment

Molecular profiling of glioblastomas has revealed the presence of key signaling hubs that contribute to tumor progression and acquisition of resistance. One of these main signaling mechanisms is the NF-κB pathway, which integrates multiple extracellular signals into transcriptional programs for tumor growth, invasion, and maintenance of the tumor-initiating population. We show here that an extracellular protein released by glioblastoma cells, fibulin-3, drives oncogenic NF-κB in the tumor and increases NF-κB activation in peritumoral astrocytes. Fibulin-3 expression correlates with a NF-κB-regulated “invasive signature” linked to poorer survival, being a possible tissue marker for regions of active tumor progression. Accordingly, fibulin-3 promotes glioblastoma invasion in a manner that requires NF-κB activation both in the tumor cells and their microenvironment. Mechanistically, we found that fibulin-3 activates the metalloprotease ADAM17 by competing with its endogenous inhibitor, TIMP3. This results in sustained release of soluble TNFα by ADAM17, which in turn activates TNF receptors and canonical NF-κB signaling. Taken together, our results underscore fibulin-3 as a novel extracellular signal with strong activating effect on NF-κB in malignant gliomas. Because fibulin-3 is produced de novo in these tumors and is absent from normal brain we propose that targeting the fibulin-3/NF-κB axis may provide a novel avenue to disrupt oncogenic NF-κB signaling in combination therapies for malignant brain tumors.

In Vitro and In Vivo Stimulation of Toll-Like Receptor 9 by CpG Oligodeoxynucleotides Incorporated Into Polypod-Like DNA Nanostructures

Cytosine-phosphate-guanine (CpG) DNA is known to increase the potency of vaccines. Here, in vitro and in vivo stimulation of toll-like receptor 9 by CpG DNA incorporated into polypod-like DNA nanostructures was evaluated by measuring the levels of tumor necrosis factor alpha released from macrophage-like RAW 264.7 cells and plasma interleukin (IL)-12p40 in vivo following intravenous injection into mice. Phosphodiester CpG1668 was selected as the CpG DNA, and tripodna and hexapodna, which were CpG1668-containing tripod and hexapod-like DNA nanostructures, respectively, were designed. CpG-tripodna and CpG-hexapodna induced tumor necrosis factor alpha release from RAW 264.7 cells about 10- and ∼30-fold higher than single-stranded CpG1668 (CpG-SS). Moreover, in all cases examined, plasma IL-12p40 concentrations increased after intravenous injection into mice, with peak levels depending on the samples and the doses. The area under the plasma concentration-time curves indicated that the CpG-hexapodna was approximately 20-fold more efficient in inducing IL-12p40 production than CpG-SS. The efficiency of CpG-tripodna and CpG-hexapodna to increase the potency of CpG-SS in vivo was comparable to that observed in cultured RAW 264.7 cells. These results provide experimental evidence that in vitro studies can be used to estimate the in vivo immunostimulatory activity of CpG DNA incorporated into DNA nanostructures.

Increased Skin Irritation by Hydroquinone and Rsetinoic Acid Used in Combination

BackgroundHydroquinone (HQ) is frequently combined with retinoic acid (RA) to enhance lightening efficacy, which may also affect skin irritancy. Although skin irritation leads to postinflammatory hyperpigmentation, little research has been performed to compare skin irritancy between each component and the combination.ObjectiveThis study was done to examine whether HQ-RA combination increased skin irritation induced by HQ or RA alone.MethodsPatch testing was performed using maximum therapeutic and higher concentrations of HQ and RA in 10 volunteers, and then, it was performed using their popular therapeutic concentrations and combination in the other 20 volunteers. In vitro irritation was also assessed in primary cultured normal human keratinocytes treated with 80% and 50% cell survival doses of HQ, 80% cell survival dose of RA, and their combination.ResultsThe combination in patch testing induced stronger erythema than the corresponding concentrations of HQ and RA, which was remarkable with use of combination of higher concentrations. In cultured keratinocytes, the RA combination significantly decreased cell viability, but increased cytotoxicity and extracellular interleukin 1 alpha release with corresponding doses of HQ.ConclusionThe results of patch tests and in vitro irritation assessment tests suggested that HQ and RA increased skin irritation when used in combination.

Hypoallergenic and Physicochemical Properties of the A2 β-Casein Fractionof Goat Milk.

Goat milk has a protein composition similar to that of breast milk and contains abundant nutrients, but its use in functional foods is rather limited in comparison to milk from other sources. The aim of this study was to prepare a goat A2 β-casein fraction with improved digestibility and hypoallergenic properties. We investigated the optimal conditions for the separation of A2 β-casein fraction from goat milk by pH adjustment to pH 4.4 and treating the casein suspension with calcium chloride (0.05 M for 1 h at 25°C). Selective reduction of β-lactoglobulin and αs-casein was confirmed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. The hypoallergenic property of A2 β-casein fraction was examined by measuring the release of histamine and tumor necrosis factor alpha from HMC-1 human mast cells exposed to different proteins, including A2 β-casein fraction. There was no significant difference in levels of both indicators between A2 β-casein treatment and the control (no protein treatment). The A2 β-casein fraction is abundant in essential amino acids, especially, branched-chain amino acids (leucine, valine, and isoleucine). The physicochemical properties of A2 β-casein fraction, including protein solubility and viscosity, are similar to those of bovine whole casein which is widely used as a protein source in various foods. Therefore, the goat A2 β-casein fraction may be useful as a food material with good digestibility and hypoallergenic properties for infants, the elderly, and people with metabolic disorders.

Defective Platelet Activation and Bleeding Complications upon Cholestasis in Mice

Background/Aims: Platelets are essential mediators of hemostasis to avoid excessive blood loss. Cirrhosis and chronic liver diseases are characterized by alterations in hemostasis. Alterations in the secondary hemostasis have been well studied, while defects in primary hemostasis, especially the consequences of cholestatic liver disease on platelet function are not well defined. Methods: After bile duct ligation (BDL) platelet activation and thrombus formation were analyzed in mice. Results: BDL in mice had a moderate effect on platelet counts; however, intrinsic platelet activation was strongly reduced upon activation of the collagen receptor GPVI at early time points. 7 days after bile duct ligation, platelets displayed an almost complete loss of activation with reduced agonist-triggered release of alpha and dense granules and expression of integrin α_IIbβ₃ on the platelet surface. This activation defects resulted in strongly reduced thrombus formation under flow, reduced platelet adhesion to fibrinogen and bleeding complications in BDL mice as measured by tail bleeding experiments. Mechanistically, elevated nitric oxide and prostacyclin levels induced phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), an established inhibitor of platelet activation. Furthermore increased tissue plasminogen activator in plasma of BDL mice led to enhanced plasmin levels that might be responsible for reduced glycoprotein expression of BDL platelets. Besides, high amounts of bile acids contribute to defective signal transduction as shown in platelets from mice fed with a cholic acid diet. Conclusions: Cholestatic liver disease induces multiple platelet activation defects and impairs thrombus formation responsible for bleeding complications at least in mice.

The effect of P2Y12 inhibition on platelet activation assessed with aggregation- and flow cytometry-based assays

Patients on P2Y12 inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing, might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, the response to ADP does not necessarily reflect the effect of P2Y12 inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity toward other physiologically relevant agonists could provide more insight in the efficacy of P2Y12 inhibitors.The effect of in vitro and in vivo P2Y12 inhibition on αIIbβ3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha, and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y12 inhibitors was compared to VerifyNow results.P2Y12 inhibitors caused strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen-related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y12 inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y12-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed with flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition.Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response identifies different patients as nonresponders to P2Y12 inhibitors, compared to only ADP-induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.

Pyrethroid Insecticides Directly Activate Microglia Through Interaction With Voltage-Gated Sodium Channels

Microglia are considered to be the resident immune cells of the central nervous system and contribute significantly to ongoing neuroinflammation in a variety of neurodegenerative diseases. Recently, we and others identified that voltage-gated sodium channels (VGSC) are present on microglia cells and contribute to excessive accumulation of intracellular Na+ and release of major pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α). Based on this finding and the fact that pyrethroid pesticides act on VGSC, we hypothesized that exposure of microglia to the pyrethroid pesticides, permethrin and deltamethrin, would activate microglia and increase the release of TNF-α. BV2 cells or primary microglia were treated with 0-5 µM deltamethrin or permethrin in the presence or absence of tetrodotoxin (TTX), a VGSC blocker for 24-48 h. Both pyrethroids caused a rapid Na+ influx and increased accumulation of intracellular sodium [(Na+)i] in the microglia in a dose- and time-dependent manner, which was significantly reduced by TTX. Furthermore, deltamethrin and permethrin increased the release of TNF-α in a dose- and time-dependent manner, which was significantly reduced by pre-treatment of cells with TTX. These results demonstrate that pyrethroid pesticides may directly activate microglial cells through their interaction with microglial VGSC. Because neuroinflammation plays a key role in many neurodegenerative diseases, these data provide an additional mechanism by which exposure to pyrethroid insecticides may contribute to neurodegeneration.

Release of Tumor Necrosis Factor Alpha and Interleukin-1 Beta from THP-1 Cells Infected with Candida parapsilosis

Department of Microbiology and Immunology, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Division of Rheumatology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Granduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan Corresponding author: Jeng-Hsien Yen, Division of Rheumatology, Department of Internal Medicine, Kaohsiung Medical University Hospital, No. 100, Zih-yen 1st Road, Kaohsiung, Taiwan. E-mail: jehsye@kmu.edu.tw

The water-soluble non-starch polysaccharides from bananas display immunomodulatory properties on cultured macrophages

Some diet components, such as certain indigestible polysaccharides from edible plants, may interact with the gut-associated lymphoid tissue and improve the host immune response to pathogens. The non-starch polysaccharides (NSP) from bananas are non-digestible carbohydrates that resemble some immunomodulatory polysaccharides occurring in the cell wall of cereals. Based on this similarity, the effects of the water-soluble NSP from two banana cultivars (Nanicão and Thap Maeo) on the phagocytic activity, nitric oxide (NO) and cytokines produced by cultured macrophages were investigated. An investigation into the monosaccharide composition and the oligomers released by enzymatic hydrolysis of the ultra-filtered fraction above 50kDa and the heat-treated fraction of water-soluble NSP from both cultivars revealed they are mostly composed of mannan and galacturonans (homogalacturonan, xylogalacturonan and rhamnogalacturonan). The NSP tested were able to activate the macrophages, but the effects on the phagocytic activity and the release of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and nitric oxide production were dependent on the polysaccharide concentration and the cultivar origin. Despite some specific differences, the NSP from Nanicão and Thap Maeo banana fruits may be considered prospective food immunomodulators, which contribute to the promotion of a more responsive immune system.