H4 Cells Research Articles

Peripheral Blood Mononuclear Cells of Alzheimer's Disease Patients Control CCL4 and CXCL10 Levels in a Human Blood Brain Barrier Model.

Alzheimer's disease (AD) is accompanied by a neuroinflammation triggering chemoattractant signals towards peripheral blood mononuclear cells (PBMCs), which in turn could reduce amyloid plaques after transmigration through the blood brain barrier (BBB). But the chemotactic environment remains unclear. To analyze five chemokines known to be involved in AD in three different cellular models to better understand the cellular and molecular interactions in the BBB. Chemokines (CCL-2, 4 and 5, CXCL10 and CX3CL1) were measured in isolated cells, a BBB model without PBMCs (H4 and hCMEC/D3 cells, a neuroglioma and human endothelial cells, respectively) and in a complete BBB model with PBMCs from AD patients at a moderate stage. In one set of experiments, H4 cells were treated with Aβ42. CCL2 and CCL5 significantly increased in hCMEC/D3 and H4 cells in the complete BBB model. In turn, the rate of CCL2 increased in PBMCs whereas for CCL5, it decreased. CXCL10 increased in all cellular actors in the complete BBB model, compared to isolated cells. For CCL4, PBMCs induced a robust increase in H4 and hCMEC/D3. In turn, the level of CCL4 decreased in PBMCs. Furthermore, PBMCs triggered a significant increase in CX3CL1 in hCMEC/D3. Surprisingly, no effect of Aβ42 was observed in the complete BBB model. These findings highlight the interest of a BBB model in order to explore chemokine production. For the first time, results showed that PBMCs from patients with AD can control the production of CCL4 and CXCL10 in a human BBB model.

Implications of fALS Mutations on Sod1 Function and Oligomerization in Cell Models

Among the familial forms of amyotrophic lateral sclerosis (fALS), 20% are associated with the Cu,Zn-superoxide dismutase (Sod1). fALS is characterized by the accumulation of aggregated proteins and the increase in oxidative stress markers. Here, we used the non-invasive bimolecular fluorescence complementation (BiFC) assay in human H4 cells to investigate the kinetics of aggregation and subcellular localization of Sod1 mutants. We also studied the effect of the different Sod1 mutants to respond against oxidative stress by following the levels of reactive oxygen species (ROS) after treatment with hydrogen peroxide. Our results showed that only 30% of cells transfected with A4VSod1 showed no inclusions while for the other Sod1 mutants tested (L38V, G93A and G93C), this percentage was at least 70%. In addition, we found that 10% of cells transfected with A4VSod1 displayed more than five inclusions per cell and that A4V and G93A Sod1 formed inclusions more rapidly than L38V and G93C Sod1. Expression of WTSod1 significantly decreased the intracellular oxidation levels in comparison with expression of fALS Sod1 mutants, suggesting the mutations induce a functional impairment. All fALS mutations impaired nuclear localization of Sod1, which is important for maintaining genomic stability. Consistently, expression of WTSod1, but not of fALS Sod1 mutants, reduced DNA damage, as measured by the comet assay. Altogether, our study sheds light into the effects of fALS Sod1 mutations on inclusion formation, dynamics, and localization as well as on antioxidant response, opening novel avenues for investigating the role of fALS Sod1 mutations in pathogenesis.

Arundic Acid Increases Expression and Function of Astrocytic Glutamate Transporter EAAT1 Via the ERK, Akt, and NF-κB Pathways.

Glutamate is the major excitatory neurotransmitter in the brain, but excessive synaptic glutamate must be removed to prevent excitotoxic injury and death. Two astrocytic glutamate transporters, excitatory amino acid transporter (EAAT) 1 and 2, play a major role in eliminating excess glutamate from the synapse. Dysregulation of EAAT1 contributes to the pathogenesis of multiple neurological disorders, such as Alzheimer's disease (AD), ataxia, traumatic brain injuries, and glaucoma. In the present study, we investigated the effect of arundic acid on EAAT1 to determine its efficacy in enhancing the expression and function of EAAT1, and its possible mechanisms of action. The studies were carried out in human astrocyte H4 cells as well as in human primary astrocytes. Our findings show that arundic acid upregulated EAAT1 expression at the transcriptional level by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Arundic acid increased astrocytic EAAT1 promoter activity, messenger RNA (mRNA)/protein levels, and glutamate uptake, while pharmacological inhibition of NF-κB or mutation on NF-κB binding sites in the EAAT1 promoter region abrogated these effects. Arundic acid increased NF-κB reporter activity and induced NF-κB nuclear translocation as well as its bindings to the EAAT1 promoter. Furthermore, arundic acid activated the Akt and ERK signaling pathways to enhance EAAT1 mRNA/protein levels. Finally, arundic acid attenuated manganese-induced decrease in EAAT1 expression by inhibiting expression of the transcription factor Ying Yang 1 (YY1). These results demonstrate that arundic acid increases the expression and function of EAAT1 via the Akt, ERK, and NF-κB signaling pathways, and reverses Mn-induced EAAT1 repression by inhibiting the Mn-induced YY1 activation.

Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100mM EtOH (in culture medium) for 2days. For interaction studies, cells were treated with memantine (4μM), EDTA (1mM), or BAPTA-AM (10μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

Abstract 3323: Anticancer activity of medicinal plant and neuroglioma

Abstract Neuroglioma, also called gliocytoma, is one of the common malignant tumors in central nervous system at present. Neuroglioma can make aggressive growth around brain tissue, so complete radical treatment can be realized by single excision. Nutritional and botanical treatments have been found useful in treating neuroglioma and other types of cancers. Vernonia amygdalina (VA) is a valuable medicinal plant that is widespread in East and West Africa. It has been reported to have not only diverse therapeutic effects for many tropical diseases, but has recently been shown to possess anti-cancer properties. Therefore, the goal of this research was to determine the therapeutic mechanisms of VA leaf extracts in the management of brain tumor. To achieve this goal, Human H4 neuroglioma cells were treated with different concentrations of Vernonia Amygdalina for 24 hr. Cell survival was determined by MTs assay. The extent of oxidative cell/tissue damage was determined by measuring malondialdehyde (lipid peroxidation biomarker) concentrations by spectrophotometry. Cell apoptosis was measured by flow cytometry assessment (Annexin V/PI assay). Data obtained from the MTS assay indicated that VA significantly (p < 0.05) reduced the viability of in H4 cells in a concentration-dependent manner. We detected a significant (p < 0.05) increase in malondialdehyde (MDA) concentrations in VA-treated glioblastoma cells compared to the control. Flow cytometry data showed a strong concentration-response relationship between VA exposure annexin V/PI positive cells. Taken together, our finding indicates that VA induced cytotoxicity and apoptosis in H4 cells is associated with the formation of MDA, a by-product of lipid peroxidation and biomarker of oxidative stress. At therapeutic concentrations, VA-induced cytotoxic and apoptotic effects in H4 cells is mediated by oxidative stress. Note: This abstract was not presented at the meeting. Citation Format: Michael Quincy Shivers. Anticancer activity of medicinal plant and neuroglioma [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 3323. doi:10.1158/1538-7445.AM2017-3323

EFFECTS OF CD2AP DEFICIENCY ON RECEPTOR-MEDIATED ENDOCYTOSIS AND APP PROCESSING IN HUMAN NEURONAL CELLS

Genome-wide association studies (GWAS) and next-generation studies (NGS) have identified >30 risk loci for late-onset Alzheimer's disease (LOAD). Several of these risk genes, including CD2AP, are involved in endocytosis. Abnormalities in endocytosis are strongly implicated in LOAD and are believed to occur in the early stages of the disease. CD2AP regulates endocytosis and is strongly associated with LOAD risk (p=5.2x10-11). Genetic variants at the CD2AP locus are non-coding and predicted to affect gene expression̤ We hypothesised that a decrease in CD2AP expression would influence endocytosis and subsequent Aβ production. To test this, we characterised the effects of CD2AP knockdown on endocytosis and APP metabolism in two human neuronal cell lines. Endogenous CD2AP expression was knocked down in H4 neuroglioma and SH-SY5Y neuroblastoma cells using siRNA. Rates of endocytosis were measured by flow cytometry. Expression levels of APP, Aβ40, sAPPα, sAPPβ and β-CTF were measured by ELISA. CD2AP knockdown in H4 cells caused a 54.3% increase in the rate of receptor-mediated endocytosis (p=0.026) and a 34.8% decrease in β-CTF protein levels (p=1x10-4). CD2AP knockdown in SH-SY5Y cells did not affect the rate of receptor-mediated endocytosis. However, we did observe a significant decrease in β-CTF protein levels. CD2AP may play a role in β-CTF production in an Aβ-dependent pathway. The effect of CD2AP deficiency on endocytosis appears to differ between neuroglioma and neuroblastoma cells.

Ganglioside and related-sphingolipid profiles are altered in a cellular model of Alzheimer's disease

Sphingolipid-related issues are increasingly discussed to contribute to the neuropathological process of Alzheimer's disease (AD). In this study, gangliosides and related-sphingolipids (ceramides, neutral glycosphingolipids and sphingomyelins) were analyzed in neuroglioma (H4) cells expressing the Swedish mutation of the human amyloid precursor protein (H4APPsw) and compared with those of wild-type control H4 cells. These cells were chosen since H4APPsw cells were previously reported to reproduce well some essential features of AD. We found that H4APPsw cells exhibited a striking elevation of the simplest ganglioside GM3, an abnormality that was consistently reported in AD patients and animal models of AD. Concomitantly, the levels of both lactosylceramide (the immediate metabolic precursor of GM3) and ganglioside GD1a increased, suggesting a deregulation in the biosynthesis of gangliosides in the H4APPsw cells. Moreover, while the total ceramide level remained unaltered in H4APPsw cells, a shift in ceramide composition from long chain - to very long chain fatty acid-ceramide species was recorded. Because sphingolipid alterations occurring in H4APPsw cells were similar to those observed in transgenic mice and in human brains, this cellular model might be useful to further explore the complex role of sphingolipids in AD pathogenesis.

Secretions of Bifidobacterium infantis and Lactobacillus acidophilus Protect Intestinal Epithelial Barrier Function.

The secreted metabolites of probiotics are cytoprotective to intestinal epithelium and have been shown to attenuate inflammation and reduce gut permeability. The present study was designed to determine the protective effects of probiotic conditioned media (PCM) from Bifidobacterium infantis (BCM) and Lactobacillus acidophilus (LCM) on interleukin (IL)-1β-induced intestinal barrier compromise. The epithelial barrier was determined by measuring the transepithelial electrical resistance (TER) across a Caco-2 cell monolayer using a Transwell model. The paracellular permeability was determined by fluorescein isothiocyanate-labeled dextran flux. The expression of tight junction (TJ) proteins and nuclear factor-kappa B (NF-κB) p65 were determined using Western blot and the distribution of NF-κB p65 was determined by immunofluorescence staining. BCM and LCM induced a dose-dependent increase in Caco-2 TER after 4 and 24 hours of incubation (P < 0.05). The maximal increase of Caco-2 TER occurred at 4 hours of treatment with a PCM concentration of 15%. Preincubation with BCM and LCM for 4 hours significantly prevented the decrease of Caco-2 TER induced by 24 hours of stimulation with 10 ng/mL IL-1β. BCM and LCM decreased paracellular permeability in both stimulated and unstimulated Caco-2 monolayers (P < 0.05). IL-1β stimulation decreased occludin expression and increased claudin-1 expression in Caco-2 cells (P < 0.05), which was prevented in cells treated with BCM or LCM. The changes of claudin-1 expression in H4 cells were similar to Caco-2 cells in response to PCM treatment and IL-1β stimulation; however, a similar response in occludin was not demonstrated. The IL-1β-induced nuclear translocation of NF-κB p65 in Caco-2 cells was prevented by pretreatment with both PCMs. BCM and LCM protected the intestinal barrier against IL-1β stimulation by normalizing the protein expression of occludin and claudin-1 and preventing IL-1β-induced NF-κB activation in Caco-2 cells, which may be partly responsible for the preservation of intestinal permeability.

Minocycline attenuates sevoflurane-induced cellinjury via activation of Nrf2.

Minocycline has been demonstrated to exert neuroprotective effects in various experimental models. In the present study, we investigated the mechanisms underlying the protective effects of minocycline on cell injury induced by the inhalation of the anesthetic, sevoflurane. In our in vivo experiments using rats, minocycline attenuated sevoflurane-induced neuronal degeneration and apoptosis in the rat hippocampus, and this effect was associated with the minocycline-mediated suppression of oxidative stress in the hippocampus. In in vitro experiments, minocycline inhibited sevoflurane-induced apoptosis and the production of reactive oxygen species (ROS) in H4 human neuroglioma cells. In addition, minocycline suppressed the sevoflurane-induced upregulation of interleukin (IL)-6 and the activation of the nuclear factor-κB (NF-κB) signaling pathway in H4 cells. Furthermore, we found that nuclear factor E2-related factor 2 (Nrf2), an activator of the stress response, was upregulated and activated upon sevoflurane treatment both in the rat hippocampus and in H4 cells. In addition, minocycline further augmented the upregulation and activation of Nrf2 when used in conjunction with sevoflurane. Moreover, the knockdown of Nrf2 in H4 cells by small interfering RNA (siRNA) diminished the cytoprotective effect of minocycline, and attenuated the inhibitory effect of minocycline on ROS production, IL-6 upregulation and the activation of the NF-κB signaling pathway. On the whole, our findings indicate that minocycline may exert protective effects against sevoflurane-induced cell injury via the Nrf2-modulated antioxidant response and the inhibition of the activation of the NF-κB signaling pathway.

A moderate metal-binding hydrazone meets the criteria for a bioinorganic approach towards Parkinson's disease: Therapeutic potential, blood-brain barrier crossing evaluation and preliminary toxicological studies

Alzheimer's and Parkinson's diseases share similar amyloidogenic mechanisms, in which metal ions might play an important role. In this last neuropathy, misfolding and aggregation of α-synuclein (α-Syn) are crucial pathological events. A moderate metal-binding compound, namely, 8-hydroxyquinoline-2-carboxaldehyde isonicotinoyl hydrazone (INHHQ), which was previously reported as a potential ‘Metal-Protein Attenuating Compound’ for Alzheimer's treatment, is well-tolerated by healthy Wistar rats and does not alter their major organ weights, as well as the tissues' reduced glutathione and biometal levels, at a concentration of 200mgkg−1. INHHQ definitively crosses the blood-brain barrier and can be detected in the brain of rats so late as 24h after intraperitoneal administration. After 48h, brain clearance is complete. INHHQ is able to disrupt, in vitro, anomalous copper-α-Syn interactions, through a mechanism probably involving metal ions sequestering. This compound is non-toxic to H4 (human neuroglioma) cells and partially inhibits intracellular α-Syn oligomerization. INHHQ, thus, shows definite potential as a therapeutic agent against Parkinson's as well.

Human fetal intestinal epithelial cells metabolize and incorporate branched chain fatty acids in a structure specific manner

BackgroundBranched chain fatty acids (BCFA) are constituents of gastrointestinal (GI) tract in healthy newborn human infants, reduce the incidence of necrotizing enterocolitis (NEC) in a neonatal rat model, and are incorporated into small intestine cellular lipids in vivo. We hypothesize that BCFA are taken up, metabolized and incorporated into human fetal cells in vitro. MethodsHuman H4 cells, a fetal non-transformed primary small intestine cell line, were incubated with albumin-bound non-esterified anteiso-17:0, iso-16:0, iso-18:0 and/or iso-20:0, and FA profiles in lipid fractions were analyzed. ResultsAll BCFA were readily incorporated as major constituents of cellular lipids. Anteiso-17:0 was preferentially taken up, and was most effective among BCFA tested in displacing normal (n-) FA. The iso BCFA were preferred in reverse order of chain length, with iso-20:0 appearing at lowest level. BCFA incorporation in phospholipids (PL) followed the same order of preference, accumulating 42% of FA as BCFA with no overt morphological signs of cell death. Though cholesterol esters (CE) are at low cellular concentration among lipid classes examined, CE had the greatest affinity for BCFA, accumulating 65% of FA as BCFA. BCFA most effectively displaced lower saturated FA. Iso-16:0, iso-18:0 and anteiso-17:0 were both elongated and chain shortened by ±C2. Iso-20:0 was chain shortened to iso-18:0 and iso-16:0 but not elongated. ConclusionsNontransformed human fetal intestinal epithelial cells incorporate high levels of BCFA when they are available and metabolize them in a structure specific manner. These findings imply that specific pathways for handling BCFA are present in the lumen-facing cells of the human fetal GI tract that is exposed to vernix-derived BCFA in late gestation.

A Novel Microfluidic Cell Co-culture Platform for the Study of the Molecular Mechanisms of Parkinson's Disease and Other Synucleinopathies.

Although, the precise molecular mechanisms underlying Parkinson's disease (PD) are still elusive, it is now known that spreading of alpha-synuclein (aSyn) pathology and neuroinflammation are important players in disease progression. Here, we developed a novel microfluidic cell-culture platform for studying the communication between two different cell populations, a process of critical importance not only in PD but also in many biological processes. The integration of micro-valves in the device enabled us to control fluid routing, cellular microenvironments, and to simulate paracrine signaling. As proof of concept, two sets of experiments were designed to show how this platform can be used to investigate specific molecular mechanisms associated with PD. In one experiment, naïve H4 neuroglioma cells were co-cultured with cells expressing aSyn tagged with GFP (aSyn-GFP), to study the release and spreading of the protein. In our experimental set up, we induced the release of the contents of aSyn-GFP producing cells to the medium and monitored the protein's diffusion. In another experiment, H4 cells were co-cultured with N9 microglial cells to assess the interplay between two cell lines in response to environmental stimuli. Here, we observed an increase in the levels of reactive oxygen species in H4 cells cultured in the presence of activated N9 cells, confirming the cross talk between different cell populations. In summary, the platform developed in this study affords novel opportunities for the study of the molecular mechanisms involved in PD and other neurodegenerative diseases.

Anti-Campylobacter activity of resveratrol and an extract from waste Pinot noir grape skins and seeds, and resistance of Camp.jejuni planktonic and biofilm cells, mediated via the CmeABC efflux pump.

To define anti-Campylobacter jejuni activity of an extract from waste skins and seeds of Pinot noir grapes (GSS), resveratrol and possible resistance mechanisms, and the influence of these on Camp.jejuni morphology. Using gene-specific knock-out Camp.jejuni mutants and an efflux pump inhibitor, we showed CmeABC as the most active efflux pump for extrusion across the outer membrane of GSS extract and resveratrol. Using polystyrene surface and pig small intestine epithelial (PSI) and human foetal small intestine (H4) cell lines, GSS extract shows an efficient inhibition of adhesion of Camp.jejuni to these abiotic and biotic surfaces. Low doses of GSS extract can inhibit Camp.jejuni adhesion to polystyrene surfaces and to PSI and H4 cells, and can thus modulate Camp.jejuni invasion and intracellular survival. An understanding of the activities of GSS extract and resveratrol as bacterial growth inhibitors and the specific mechanisms of cell accumulation is crucial for our understanding of Camp.jejuni resistance. GSS extract inhibition of Camp.jejuni adhesion to abiotic and biotic surfaces provides a further step towards the application of new innovative strategies to control Campylobacter contamination and infection via the food chain.

Specific connectivity between photoreceptors and horizontal cells in the zebrafish retina.

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

Characterization of FRM-36143 as a new γ-secretase modulator for the potential treatment of familial Alzheimer's disease.

BackgroundFamilial Alzheimer’s disease (FAD) is caused by mutations in the amyloid precursor protein (APP) or presenilin (PS). Most PS mutations, which account for the majority of FAD cases, lead to an increased ratio of longer to shorter forms of the amyloid beta (Aβ) peptide. The therapeutic rationale of γ-secretase modulators (GSMs) for Alzheimer’s disease is based on this genetic evidence as well as on enzyme kinetics measurements showing changes in the processivity of the γ-secretase complex. This analysis suggests that GSMs could potentially offset some of the effects of PS mutations on APP processing, thereby addressing the root cause of early onset FAD. Unfortunately, the field has generated few, if any, molecules with good central nervous system (CNS) drug-like properties to enable proof-of-mechanism studies.MethodWe characterized the novel GSM FRM-36143 using multiple cellular assays to determine its in vitro potency and off-target activity as well as its potential to reverse the effect of PS mutations. We also tested its efficacy in vivo in wild-type mice and rats.ResultsFRM-36143 has much improved CNS drug-like properties compared to published GSMs. It has an in vitro EC50 for Aβ42 of 35 nM in H4 cells, can reduce Aβ42 to 58 % of the baseline in rat cerebrospinal fluid, and also increases the non-amyloidogenic peptides Aβ37 and Aβ38. It does not inhibit Notch processing, nor does it inhibit 24-dehydrocholesterol reductase (DHCR24) activity. Most interestingly, it can reverse the effects of presenilin mutations on APP processing in vitro.ConclusionsFRM-36143 possesses all the characteristics of a GSM in terms of Aβ modulation Because FRM-36143 was able to reverse the effect of PS mutations, we suggest that targeting patients with this genetic defect would be the best approach at testing the efficacy of a GSM in the clinic. While the amyloid hypothesis is still being tested with β-site APP-cleaving enzyme inhibitors and monoclonal antibodies in sporadic AD, we believe it is not a hypothesis for FAD. Since GSMs can correct the molecular defect caused by PS mutations, they have the promise to provide benefits to the patients when treated early enough in the course of the disease.

Anti-inflammatory effects of Bifidobacterium longum subsp infantis secretions on fetal human enterocytes are mediated by TLR-4 receptors.

The therapeutic and preventive application of probiotics for necrotizing enterocolitis (NEC) has been supported by more and more experimental and clinical evidence in which Toll-like receptor 4 (TLR-4) exerts a significant role. In immune cells, probiotics not only regulate the expression of TLR-4 but also use the TLR-4 to modulate the immune response. Probiotics may also use the TLR-4 in immature enterocytes for anti-inflammation. Here we demonstrate that probiotic conditioned media (PCM) from Bifidobacterium longum supp infantis but not isolated organisms attenuates interleukin-6 (IL-6) induction in response to IL-1β by using TLR-4 in a human fetal small intestinal epithelial cell line (H4 cells), human fetal small intestinal xenografts, mouse fetal small intestinal organ culture tissues, and primary NEC enterocytes. Furthermore, we show that PCM, using TLR-4, downregulates the mRNA expression of interleukin-1 receptor-associated kinase 2 (IRAK-2), a common adapter protein shared by IL-1β and TLR-4 signaling. PCM also reduces the phosphorylation of the activator-protein 1 (AP-1) transcription factors c-Jun and c-Fos in response to IL-1β stimulation in a TLR-4-dependent manner. This study suggests that PCM may use TLR-4 through IRAK-2 and via AP-1 to prevent IL-1β-induced IL-6 induction in immature enterocytes. Based on these observations, the combined use of probiotics and anti-TLR-4 therapy to prevent NEC may not be a good strategy.

Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: implications for Alzheimer's disease.

BackgroundPolymorphisms in the gene for phosphatidylinositol binding clathrin assembly protein (PICALM), an endocytic-related protein, are associated with a small, increased risk of developing Alzheimer’s disease (AD), strongly suggesting that changes in endocytosis are involved in the aetiology of the disease. We have investigated the involvement of PICALM in the processing of amyloid precursor protein (APP) to understand how PICALM could be linked to the development of AD. We used siRNA to deplete levels of PICALM, its isoforms and clathrin heavy chain in the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP. We then used Western blotting, ELISA and immunohistochemistry to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including β-amyloid (Aβ). Levels of functional endocytosis were quantified using ALEXA 488-conjugated transferrin and flow cytometry as a marker of clathrin-mediated endocytosis (CME).ResultsFollowing depletion of all the isoforms of PICALM by siRNA in H4 cells, levels of intracellular APP, intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 was not affected. Functional endocytosis was significantly reduced after both PICALM and clathrin depletion, highlighting the importance of PICALM in this process. However, depletion of clathrin did not affect APP but did reduce β-CTF levels. PICALM depletion altered the intracellular distribution of clathrin while clathrin reduction affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. Individual depletion of PICALM isoforms 1 and 2 did not affect APP levels while clathrin depletion had a differential effect on the isoforms, increasing isoform 1 while decreasing isoform 2 expression.ConclusionsThe depletion of PICALM in brain-derived cells has significant effects on the processing of APP, probably by reducing CME. In particular, it affects the production of β-CTF which is increasingly considered to be an important mediator in AD independent of Aβ. Thus a decrease in PICALM expression in the brain could be beneficial to slow or prevent the development of AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-016-0288-1) contains supplementary material, which is available to authorized users.

THE ROLE OF CD2AP IN APP PROCESSING

Genome-wide association studies (GWAS) have identified >20 risk loci for late-onset Alzheimer's disease (LOAD). Several of these risk genes are involved in endocytosis, a process believed to be disrupted during the early stages of LOAD. The CD2-associated protein (CD2AP) gene is strongly associated with LOAD risk (p=5.2x10-11) and plays a role in receptor-mediated endocytosis. We hypothesised that CD2AP contributes to amyloid precursor protein (APP) metabolism and subsequent amyloid –beta (Aβ) generation through its regulation of clathrin-mediated endocytosis. To test this, we initially characterised the effects of CD2AP knockdown on APP metabolism and Aβ production in a human neuronal cell line. A recent study found that another GWAS-identified risk gene PICALM, regulates trafficking of Aβ across the endothelial cells which form the blood brain barrier (BBB). We hypothesise that CD2AP acts in a similar way to PICALM and that knocking down CD2AP diminishes Aβ trafficking across the BBB, decreasing the amount of Aβ cleared from the brain. To investigate this, we are characterising the effects of CD2AP knockdown in an in vitro model of the BBB. Endogenous CD2AP expression was knocked down in H4 and hMEC/D3 cells using siRNA. Knockdown was quantified by western blot and Image J analysis. Levels of APP, Aβ, and APP metabolites were quantified using ELISAs. The rate of receptor-mediated endocytosis was measured by a transferrin uptake assay. Co-localisation of CD2AP with APP and endosomal markers was assessed using immunocytochemistry. Transcytosis in hMEC/D3 cells will be assessed using Transwell assays. We observed a significant 30.27±0.07 % increase in Aβ40 expression (p=0.0046). Expression levels of APP and APP processing metabolites was not significantly altered. Results from our experiments in progress will be made available at AAIC 2016. Knockdown of CD2AP expression resulted in a significant increase in extracellular amyloid-B (Aβ) levels, which did not appear to be directly related to changes in APP processing. This study is ongoing and novel data will be presented at AAIC 2016. Overall, our study provides valuable insights into how CD2AP contributes to LOAD susceptibility. Effect of CD2AP siRNA treatment of H4 cells on extracellular Aβ40 protein levels. Results are expressed as a percentage of oligofectamine only treated cells. Four biological replicates were analysed for each assay. Statistical significance of CD2AP siRNA vs. control siRNA treatment was determined by two-tailed student's t-test., **p = 0.0046; *= 0.0223 Error bars = SEM.

Effect of pomegranate powder on the heat inactivation of Escherichia coli O104:H4 in ground chicken

Health concerns have led to a search for natural plant-based antimicrobials. Ellagic acid has been shown to have antimicrobial activity against foodborne pathogens. The objective of this study was to assess the effect of a high-ellagic acid commercial pomegranate on the heat resistance of Escherichia coli O104:H4 in ground chicken. A full 24 factorial design was used, consisting of temperature treatment with four levels (55.0, 57.5, 60.0, and 62.5 °C) and pomegranate with four levels (0.0, 1.0, 2.0, and 3.0 wt/wt. % containing 70% ellagic acid). Experiments were conducted twice, providing a total of 32 survival curves. A three-parameter Weibull primary model was used to describe survival kinetics. Secondary models were then developed to estimate the shape parameter β (i.e., curvature representing susceptibility of cells to stress), scale parameter γ (i.e., time to reach the first decimal reduction) and the 5.0-log lethality time t5.0 (i.e., time to reach a 5.0-log reduction), all as polynomial functions of temperature and pomegranate powder concentration. The positive effect of pomegranate concentration on both β and γ demonstrated that the phenolic-rich pomegranate powder causes E. coli O104:H4 cells to become more susceptible to heat, increasing the steepness and concavity of the isothermal survival curves. It was estimated that the 5.0-log reduction time would reach a minimum at a pomegranate powder concentration of 1%, producing a 50% decrease in lethality time, in comparison to that without added pomegranate powder. Nonetheless, a mixed-effect omnibus regression further confirmed that the greatest difference in the thermal resistance of E. coli O104:H4 happened between tests with and without pomegranate powder. In fact, adding more than 1.0% pomegranate powder, at a constant temperature, resulted only in a marginal decrease in thermal resistance. Meat processors can use the model to design lethality treatments in order to achieve specific reductions of E. coli O104:H4 in ground chicken.

Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4

The Shiga toxigenic Escherichia coli O104:H4 isolated during the 2011 European outbreak expresses Shiga toxin 2a and possess virulence genes associated with the enteroaggregative E. coli (EAEC) pathotype. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga-toxin adsorption, but it is not clear whether the AAF/I fimbriae are involved in the colonization and biofilm formation on food and environmental matrices such as the surface of fresh produce. We deleted the gene encoding for the AAF/I fimbriae main subunit (AggA) from an outbreak associated E. coli O104:H4 strain, and evaluated the role of AAF/I fimbriae in the adherence and colonization of E. coli O104:H4 to spinach and abiotic surfaces. The deletion of aggA did not affect the adherence of E. coli O104:H4 to these surfaces. However, it severely diminished the colonization and biofilm formation of E. coli O104:H4 on these surfaces. Strong aggregation and biofilm formation on spinach and abiotic surfaces were observed with the wild type strain but not the isogenic aggA deletion mutant, suggesting that AAF/I fimbriae play a crucial role in persistence of O104:H4 cells outside of the intestines of host species, such as on the surface of fresh produce.