Responses In Primary Cultures Research Articles

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

SARS-CoV-2 S protein activates NLRP3 inflammasome and deregulates coagulation factors in endothelial and immune cells

BackgroundHyperinflammation, hypercoagulation and endothelial injury are major findings in acute and post-COVID-19. The SARS-CoV-2 S protein has been detected as an isolated element in human tissues reservoirs and is the main product of mRNA COVID-19 vaccines. We investigated whether the S protein alone triggers pro-inflammatory and pro-coagulant responses in primary cultures of two cell types deeply affected by SARS-CoV-2, such are monocytes and endothelial cells.MethodsIn human umbilical vein endothelial cells (HUVEC) and monocytes, the components of NF-κB and the NLRP3 inflammasome system, as well as coagulation regulators, were assessed by qRT-PCR, Western blot, flow cytometry, or indirect immunofluorescence.ResultsS protein activated NF-κB, promoted pro-inflammatory cytokines release, and triggered the priming and activation of the NLRP3 inflammasome system resulting in mature IL-1β formation in both cell types. This was paralleled by enhanced production of coagulation factors such as von Willebrand factor (vWF), factor VIII or tissue factor, that was mediated, at least in part, by IL-1β. Additionally, S protein failed to enhance ADAMTS-13 levels to counteract the pro-coagulant activity of vWF multimers. Monocytes and HUVEC barely expressed angiotensin-converting enzyme-2. Pharmacological approaches and gene silencing showed that TLR4 receptors mediated the effects of S protein in monocytes, but not in HUVEC.ConclusionS protein behaves both as a pro-inflammatory and pro-coagulant stimulus in human monocytes and endothelial cells. Interfering with the receptors or signaling pathways evoked by the S protein may help preventing immune and vascular complications driven by such an isolated viral element.BJi2XdMkuptrjYtS_YhKHeVideo

Opposing Effects of EGF Receptor Signaling on Proliferation and Differentiation Initiated by EGF or TSH/EGF Receptor Transactivation.

Regulation of thyroid cells by thyrotropin (TSH) and epidermal growth factor (EGF) has been known but different effects of these regulators on proliferation and differentiation have been reported. We studied these responses in primary cultures of human thyroid cells to determine whether TSH receptor (TSHR) signaling may involve EGF receptor (EGFR) transactivation. We confirm that EGF stimulates proliferation and de-differentiation whereas TSH causes differentiation in the absence of other growth factors. We show that TSH/TSHR transactivates EGFR and characterize it as follows: (1) TSH-induced upregulation of thyroid-specific genes is inhibited by 2 inhibitors of EGFR kinase activity, AG1478 and erlotinib; (2) the mechanism of transactivation is independent of an extracellular EGFR ligand by showing that 2 antibodies, cetuximab and panitumumab, that completely inhibited binding of EGFR ligands to EGFR had no effect on transactivation, and by demonstrating that no EGF was detected in media conditioned by thyrocytes incubated with TSH; (3) TSH/TSHR transactivation of EGFR is different than EGFR activation by EGF by showing that EGF led to rapid phosphorylation of EGFR whereas transactivation occurred in the absence of receptor phosphorylation; (4) EGF caused downregulation of EGFR whereas transactivation had no effect on EGFR level; (5) EGF and TSH stimulation converged on the protein kinase B (AKT) pathway, because TSH, like EGF, stimulated phosphorylation of AKT that was inhibited by EGFR inhibitors; and (6) TSH-induced upregulation of thyroid genes was inhibited by the AKT inhibitor MK2206. Thus, TSH/TSHR causes EGFR transactivation that is independent of extracellular EGFR ligand and in part mediates TSH regulation of thyroid hormone biosynthetic genes.

Novel Insights Into Muscarinic and Purinergic Responses in Primary Cultures of Rat Lacrimal Gland Myoepithelial Cells.

PurposeThe functional characteristics of receptors that regulate lacrimal gland myoepithelial cells are still somewhat unclear. To date, mainly muscarinic receptors have been of interest; however, further knowledge is needed regarding their expression and functional roles. For this purpose, primary cultures of rat lacrimal gland myoepithelial cells were established and examined functionally.MethodsRat lacrimal glands were excised, minced, and further digested, yielding mixtures of cells that were seeded in culturing flasks. After 4-6 weeks, primary monocultures of myoepithelial cells were established, verified by immunocytochemistry. The cells were stained for all muscarinic receptor subtypes (M1–M5) and examined functionally regarding intracellular [Ca2+] responses upon activation of muscarinic receptors. For methodological verification, purinergic functional responses were also studied.ResultsExpression of muscarinic receptor subtypes M2-M5 was detected, whereas expression of muscarinic M1 receptors could not be shown. Activation of muscarinic receptors by the non-selective muscarinic agonist methacholine (3 × 10−11–10−3 M) did not cause a significant increase in intracellular [Ca2+]. However, activation of purinergic receptors by the non-selective purinergic agonist ATP (10−8–10−3 M) caused a concentration-dependent increase in intracellular [Ca2+] that could be blocked by the P2 antagonists PPADS and suramin.ConclusionsPrimary cultures of rat lacrimal gland myoepithelial cells were established that displayed a heterogeneous expression of muscarinic receptors. Purinergic functional responses demonstrated a viable cell population. Upon treatment with methacholine, no significant increase in intracellular [Ca2+] could be detected, indicating that cholinergic activation of myoepithelial cells occurs via other intracellular messengers or is dependent on interaction with other cell types.

TGF-β1 Provides Neuroprotection via Inhibition of Microglial Activation in 3-Acetylpyridine-Induced Cerebellar Ataxia Model Rats.

Cerebellar ataxias (CAs) consist of a heterogeneous group of neurodegenerative diseases hallmarked by motor deficits and deterioration of the cerebellum and its associated circuitries. Neuroinflammatory responses are present in CA brain, but how neuroinflammation may contribute to CA pathogenesis remain unresolved. Here, we investigate whether transforming growth factor (TGF)-β1, which possesses anti-inflammatory and neuroprotective properties, can ameliorate the microglia-mediated neuroinflammation and thereby alleviate neurodegeneration in CA. In the current study, we administered TGF-β1 via the intracerebroventricle (ICV) in CA model rats, by intraperitoneal injection of 3-acetylpyridine (3-AP), to reveal the neuroprotective role of TGF-β1. The TGF-β1 administration after 3-AP injection ameliorated motor impairments and reduced the calbindin-positive neuron loss and apoptosis in the brain stem and cerebellum. Meanwhile, 3-AP induced microglial activation and inflammatory responses in vivo, which were determined by morphological alteration and an increase in expression of CD11b, enhancement of percentage of CD40 + and CD86 + microglial cells, upregulation of pro-inflammatory mediators, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and a downregulation of neurotrophic factor, insulin-like growth factor (IGF)-1 in the brain stem and cerebellum. TGF-β1 treatment significantly prevented all the changes caused by 3-AP. In addition, in vitro experiments, TGF-β1 directly attenuated 3-AP-induced microglial activation and inflammatory responses in primary cultures. Purkinje cell exposure to supernatants of primary microglia that had been treated with TGF-β1 reduced neuronal loss and apoptosis induced by 3-AP-treated microglial supernatants. Furthermore, the protective effect was similar to those treated with TNF-α-neutralizing antibody. These findings suggest that TGF-β1 protects against neurodegeneration in 3-AP-induced CA rats via inhibiting microglial activation and at least partly TNF-α release.

Effects of gabapentinoids on responses of primary cultures from rat dorsal root ganglia to inflammatory or somatosensory stimuli.

Background Gabapentinoids are known to reduce neuropathic pain. The aim of this experimental study was to investigate whether gabapentinoids exert anti-inflammatory and/or anti-nociceptive effects at the cellular level using primary cultures of rat dorsal root ganglia (DRG). Methods Cells from rat DRG were cultured in the presence of gabapentin or pregabalin, and we tested the effects of subsequent stimulation with lipopolysaccharide (LPS) on the expression of genes (real-time polymerase chain reaction) and production of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) by specific bioassays. Using Ca2+ imaging, we further investigated in neurons the effects of gabapentinoids upon stimulation with the TRPV-1 agonist capsaicin. Results There is a small influence of gabapentinoids on the inflammatory response to LPS stimulation, namely, a significantly reduced expression of IL-6. Pregabalin and gabapentin further seem to exert a moderate inhibitory influence on capsaicin-induced Ca2+ signals in DRG neurons. Conclusions Although the single inhibitory effects of gabapentinoids on inflammatory and nociceptive responses are moderate, a combination of both effects might provide an explanation for the proposed function of these substances as an adjuvant for the reduction of neuropathic pain.

Itaconate modulates tricarboxylic acid and redox metabolism to mitigate reperfusion injury

ObjectivesCerebral ischemia/reperfusion (IR) drives oxidative stress and injurious metabolic processes that lead to redox imbalance, inflammation, and tissue damage. However, the key mediators of reperfusion injury remain unclear, and therefore, there is considerable interest in therapeutically targeting metabolism and the cellular response to oxidative stress. MethodsThe objective of this study was to investigate the molecular, metabolic, and physiological impact of itaconate treatment to mitigate reperfusion injuries in in vitro and in vivo model systems. We conducted metabolic flux and bioenergetic studies in response to exogenous itaconate treatment in cultures of primary rat cortical neurons and astrocytes. In addition, we administered itaconate to mouse models of cerebral reperfusion injury with ischemia or traumatic brain injury followed by hemorrhagic shock resuscitation. We quantitatively characterized the metabolite levels, neurological behavior, markers of redox stress, leukocyte adhesion, arterial blood flow, and arteriolar diameter in the brains of the treated/untreated mice. ResultsWe demonstrate that the “immunometabolite” itaconate slowed tricarboxylic acid (TCA) cycle metabolism and buffered redox imbalance via succinate dehydrogenase (SDH) inhibition and induction of anti-oxidative stress response in primary cultures of astrocytes and neurons. The addition of itaconate to reperfusion fluids after mouse cerebral IR injury increased glutathione levels and reduced reactive oxygen/nitrogen species (ROS/RNS) to improve neurological function. Plasma organic acids increased post-reperfusion injury, while administration of itaconate normalized these metabolites. In mouse cranial window models, itaconate significantly improved hemodynamics while reducing leukocyte adhesion. Further, itaconate supplementation increased survival in mice experiencing traumatic brain injury (TBI) and hemorrhagic shock. ConclusionsWe hypothesize that itaconate transiently inhibits SDH to gradually “awaken” mitochondrial function upon reperfusion that minimizes ROS and tissue damage. Collectively, our data indicate that itaconate acts as a mitochondrial regulator that controls redox metabolism to improve physiological outcomes associated with IR injury.

Detection of early osteogenic commitment in primary cells using Raman spectroscopy.

Major challenges in the development of novel implant surfaces for artificial joints include osteoblast heterogeneity and the lack of a simple and sensitive in vitro assay to measure early osteogenic responses. Raman spectroscopy is a label-free, non-invasive and non-destructive vibrational fingerprinting optical technique that is increasingly being applied to detect biochemical changes in cells. In this study Raman spectroscopy has been used to obtain bone cell-specific spectral signatures and to identify any changes therein during osteoblast commitment and differentiation of primary cells in culture. Murine calvarial osteoblasts (COBs) were extracted and cultured and studied by Raman spectroscopy over a 14 day culture period. Distinct osteogenic Raman spectra were identified after 3 days of culture with strong bands detected for mineral: phosphate ν3 (1030 cm-1) and B-type carbonate (1072 cm-1), DNA (782 cm-1) and collagen matrix (CH2 deformation at 1450 cm-1) and weaker phosphate bands (948 and 970 cm-1). Early changes were detected by Raman spectroscopy compared to a standard enzymatic alkaline phosphatase (ALP) assay and gene expression analyses over this period. Proliferation of COBs was confirmed by fluorescence intensity measurements using the Picogreen dsDNA reagent. Changes in ALP levels were evident only after 14 days of culture and mRNA expression levels for ALP, Col1a1 and Sclerostin remained constant during the culture period. Sirius red staining for collagen deposition also revealed little change until day 14. In contrast Raman spectroscopy revealed the presence of amorphous calcium phosphate (945-952 cm-1) and carbonated apatite (957-962 cm-1) after only 3 days in culture and octacalcium phosphate (970 cm-1) considered a transient mineral phase, was detected after 5 days of COBs culture. PCA analysis confirmed clear separation between time-points. This study highlights the potential of Raman spectroscopy to be utilised for the early and specific detection of proliferation and differentiation changes in primary cultures of bone cells.

Endogenous N-acyl taurines regulate skin wound healing

The intracellular serine amidase, fatty acid amide hydrolase (FAAH), degrades a heterogeneous family of lipid-derived bioactive molecules that include amides of long-chain fatty acids with taurine [N-acyl-taurines (NATs)]. The physiological functions of the NATs are unknown. Here we show that genetic or pharmacological disruption of FAAH activity accelerates skin wound healing in mice and stimulates motogenesis of human keratinocytes and differentiation of human fibroblasts in primary cultures. Using untargeted and targeted lipidomics strategies, we identify two long-chain saturated NATs-N-tetracosanoyl-taurine [NAT(24:0)] and N-eicosanoyl-taurine [NAT(20:0)]-as primary substrates for FAAH in mouse skin, and show that the levels of these substances sharply decrease at the margins of a freshly inflicted wound to increase again as healing begins. Additionally, we demonstrate that local administration of synthetic NATs accelerates wound closure in mice and stimulates repair-associated responses in primary cultures of human keratinocytes and fibroblasts, through a mechanism that involves tyrosine phosphorylation of the epidermal growth factor receptor and an increase in intracellular calcium levels, under the permissive control of transient receptor potential vanilloid-1 receptors. The results point to FAAH-regulated NAT signaling as an unprecedented lipid-based mechanism of wound-healing control in mammalian skin, which might be targeted for chronic wound therapy.

Targeting γ-secretases protect against angiotensin II-induced cardiac hypertrophy.

The Notch pathway has been linked to pulmonary hypertension, but its role in systemic hypertension and, in particular in left ventricular hypertrophy (LVH), remains poorly understood. The main objective of this work was to analyse the effect of inhibiting the Notch pathway on the establishment and maintenance of angiotensin II (Ang-II)-induced arterial hypertension and LVH in adult mice with inducible genetic deletion of γ-secretase, and to test preclinically the therapeutic efficacy of γ-secretase inhibitors (GSIs). We analysed Ang-II responses in primary cultures of vascular smooth muscle cells obtained from a novel mouse model with inducible genetic deletion of the γ-secretase complex, and the effects of GSI treatment on a mouse cardiac cell line. We also investigated Ang-II-induced hypertension and LVH in our novel mouse strain lacking the γ-secretase complex and in GSI-treated wild-type mice. Moreover, we analysed vascular tissue from hypertensive patients with and without LVH. Vascular smooth muscle cells activate the Notch pathway in response to Ang-II both 'in vitro' and 'in vivo'. Genetic deletion of γ-secretase in adult mice prevented Ang-II-induced hypertension and LVH without causing major adverse effects. Treatment with GSI reduced Ang-II-induced hypertrophy of a cardiac cell line 'in vitro' and LVH in wild-type mice challenged with Ang-II. We also report elevated expression of the Notch target HES5 in vascular tissue from hypertensive patients with LVH compared with those without LVH. The Notch pathway is activated in the vasculature of mice with hypertension and LVH, and its inhibition via inducible genetic γ-secretase deletion protects against both conditions. Preliminary observations in hypertensive patients with LVH support the translational potential of these findings. Moreover, GSI treatment protects wild-type mice from Ang-II-induced LVH without affecting blood pressure. Our results unveil the potential use of GSIs in the treatment of hypertensive patients with LVH.

Red ginseng extract blocks histamine-dependent itch by inhibition of H1R/TRPV1 pathway in sensory neurons

BackgroundKorean Red Ginseng—a steamed root of Panax ginseng Meyer—has long been used as a traditional medicine in Asian countries. Its antipruritic effect was recently found, but no molecular mechanisms were revealed. Thus, the current study focused on determining the underlying molecular mechanism of Korean Red Ginseng extract (RGE) against histamine-induced itch at the peripheral sensory neuronal level. MethodsTo examine the antipruritic effect of RGE, we performed in vivo scratching behavior test in mice, as well as in vitro calcium imaging and whole-cell patch clamp experiments to elucidate underlying molecular mechanisms. ResultsThe results of our in vivo study confirmed that RGE indeed has an antipruritic effect on histamine-induced scratching in mice. In addition, RGE showed a significant inhibitory effect on histamine-induced responses in primary cultures of mouse dorsal root ganglia, suggesting that RGE has a direct inhibitory effect on sensory neuronal level. Results of further experiments showed that RGE inhibits histamine-induced responses on cells expressing both histamine receptor subtype 1 and TRPV1 ion channel, indicating that RGE blocks the histamine receptor type 1/TRPV1 pathway in sensory neurons, which is responsible for histamine-dependent itch sensation. ConclusionThe current study found for the first time that RGE effectively blocks histamine-induced itch in peripheral sensory neurons. We believe that the current results will provide an insight on itch transmission and will be helpful in understanding how RGE exerts its antipruritic effects.

Pregnane X receptor modulates the inflammatory response in primary cultures of hepatocytes.

Bacterial sepsis is characterized by a rapid increase in the expression of inflammatory mediators to initiate the acute phase response in liver. Inflammatory mediator release is counterbalanced by the coordinated expression of anti-inflammatory molecules such as interleukin 1 receptor antagonist (IL1-Ra) through time. This study determined whether activation of pregnane X receptor (PXR, NR1I2) alters the lipopolysaccharide (LPS)-inducible gene expression program in primary cultures of hepatocytes (PCHs). Preactivation of PXR for 24 hours in PCHs isolated from wild-type mice suppressed the subsequent LPS-inducible expression of the key inflammatory mediators interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNFα) but not in PCHs isolated from Pxr-null (PXR-knockout [KO]) mice. Basal expression of key inflammatory cytokines was elevated in PCHs from PXR-KO mice. Stimulation of PCHs from PXR-KO mice with LPS alone produced enhanced levels of IL-1β when compared with wild-type mice. Experiments performed using PCHs from both humanized-PXR transgenic mice as well as human donors indicate that prolonged activation of PXR produces an increased secretion of IL1-Ra from cells through time. Our data reveal a working model that describes a pivotal role for PXR in both inhibiting as well as in resolving the inflammatory response in hepatocytes. Understanding the molecular details of how PXR is converted from a positive regulator of drug-metabolizing enzymes into a transcriptional suppressor of inflammation in liver will provide new pharmacologic strategies for modulating inflammatory-related diseases in the liver and intestine.

The viral mimetic polyinosinic:polycytidylic acid (poly I:C) induces cellular responses in primary cultures from rat brain sites with an incomplete blood–brain barrier

Primary microcultures of the organum vasculosum laminae terminalis (OVLT) and the area postrema (AP), brain sites with an incomplete blood–brain barrier, were established from topographically excised rat pup tissue, with cellular identification by marker protein-specific immunocytochemistry. Employing the ratio calcium imaging technique, we showed for the first time that polyinosinic:polycytidylic acid (poly I:C) can induce calcium signalling in single OVLT and AP cells. Poly I:C stimulation caused fast, transient rises in intracellular calcium in about 5% of neurons and astrocytes and some microglial cells. Frequently, the responses of astrocytes and microglial cells showed a shorter onset-latency compared to neurons. In addition, exposure to poly I:C led to a time dependent release of bioactive tumour necrosis factor (TNF) and interleukin-6 (IL-6) into the supernatants of OVLT and AP cultures. The demonstration of direct cellular responses of OVLT- and AP-intrinsic cells to stimulations with poly I:C is in agreement with the discovered existence of Toll-like receptor 3 (TLR3), the cognate receptor for poly I:C, in the brain.

STIM1 and Orai1 mediate thrombin-induced Ca2+ influx in rat cortical astrocytes

In astrocytes, thrombin leads to cytoplasmic Ca2+ elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca2+ increase generated by an interplay between ER-Ca2+ release and store-operated Ca2+ entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca2+ depletion and binds Orai1 to activate Ca2+ influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca2+ response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca2+ influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca2+ influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca2+ response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca2+ release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca2+ influx.

Crucial role for IPS-1 signaling in mediating anti-viral responses and clearance of Respiratory Syncytial Virus (170.4)

Abstract Upon replication of Respiratory Syncytial Virus (RSV), double-stranded viral RNA can be detected in the endosomes via Toll-like receptor 3 (TLR3) signaling or by the cytoplasmic helicases retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated gene (Mda5). The latter interact downstream with IFNβ promoter stimulator (IPS-1). We previously demonstrated that TLR3 deficiency worsens RSV-induced lung pathology, yet leaves viral clearance intact. The present study examined the importance of IPS-1 signaling for anti-viral responses in primary cultures of alveolar epithelial cells (AECs), in dendritic cells (DCs), and macrophages (mΦ), as well as in vivo using IPS-1 knockout (KO) mice. In AECs, RSV induced IFNβ, IL6, CXCL1, CCL2 and CCL5 in an IPS-1 dependent manner. In DCs, the expression of IFNβ and CCL2 also required IPS-1. Conversely, RSV-induced cytokine and chemokine expression was enhanced in IPS-1 KO mΦ, while T-cells of infected IPS-1 KO mice showed a predominant Th1 profile with increased IFNγ. Plaque assays revealed higher RSV titers in IPS-1 deficient mice, accompanied by more severe granulocytic infiltration. Our data suggest a significant contribution of the IPS-1 pathway to the RSV-induced type I interferon response and inflammatory cytokine/chemokine production. Future experiments, using bone marrow chimeras, will help to elucidate the role of IPS-1 in both the structural and hematopoietic mediated response to viral infection.

Riluzole and gabapentinoids activate glutamate transporters to facilitate glutamate-induced glutamate release from cultured astrocytes

We have recently demonstrated that the glutamate transporter activator riluzole paradoxically enhanced glutamate-induced glutamate release from cultured astrocytes. We further showed that both riluzole and the α2δ subunit ligand gabapentin activated descending inhibition in rats by increasing glutamate receptor signaling in the locus coeruleus and hypothesized that these drugs share common mechanisms to enhance glutamate release from astrocytes. In the present study, we examined the effects of riluzole and gabapentin on glutamate uptake and release and glutamate-induced Ca2+ responses in primary cultures of astrocytes. Riluzole and gabapentin facilitated glutamate-induced glutamate release from astrocytes and significantly increased glutamate uptake, the latter being completely blocked by the non-selective glutamate transporter blocker DL-threo-β-benzyloxyaspartic acid (DL-TBOA). Riluzole and gabapentin also enhanced the glutamate-induced increase in intracellular Ca2+ concentrations. Some α2δ subunit ligands, pregabalin and L-isoleucine, enhanced the glutamate-induced Ca2+ response, whereas another, 3-exo-aminobicyclo[2.2.1]heptane-2-exo-carboxylic acid (ABHCA), did not. The enhancement of glutamate-induced intracellular Ca2+ response by riluzole and gabapentin was blocked by the DL-TBOA and an inhibitor of Na+/Ca2+ exchange, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943). Gabapentin's enhancement of Ca2+ increase was specific to glutamate stimulation, as it was not mimicked with stimulation by ATP. These results suggest that riluzole and gabapentin enhance Na+-glutamate co-transport through glutamate transporters, induce subsequent Ca2+ influx via the reverse mode of Na+/Ca2+ exchange, and thereby facilitate Ca2+-dependent glutamate release by glutamate in astrocytes. The present study also demonstrates a novel target of gabapentinoid action in astrocytes other than α2δ subunits in neurons.

Anti-inflammatory pathways and alcoholic liver disease: Role of an adiponectin/interleukin-10/heme oxygenase-1 pathway

The development of alcoholic liver disease (ALD) is a complex process involving both the parenchymal and non-parenchymal cells in the liver. Enhanced inflammation in the liver during ethanol exposure is an important contributor to injury. Kupffer cells, the resident macrophages in liver, are particularly critical to the onset of ethanol-induced liver injury. Chronic ethanol exposure sensitizes Kupffer cells to activation by lipopolysaccharide via Toll-like receptor 4. This sensitization enhances production of inflammatory mediators, such as tumor necrosis factor-alpha and reactive oxygen species, that contribute to hepatocyte dysfunction, necrosis, apoptosis, and fibrosis. Impaired resolution of the inflammatory process probably also contributes to ALD. The resolution of inflammation is an active, highly coordinated response that can potentially be manipulated via therapeutic interventions to treat chronic inflammatory diseases. Recent studies have identified an adiponectin/interleukin-10/heme oxygenase-1 (HO-1) pathway that is profoundly effective in dampening the enhanced activation of innate immune responses in primary cultures of Kupffer cells, as well as in an in vivo mouse model of chronic ethanol feeding. Importantly, induction of HO-1 also reduces ethanol-induced hepatocellular apoptosis in this in vivo model. Based on these data, we hypothesize that the development of therapeutic agents to regulate HO-1 and its downstream targets could be useful in enhancing the resolution of inflammation during ALD and preventing progression of early stages of liver injury.

Brain distribution of carboxy terminus of Hsc70-interacting protein (CHIP) and its nuclear translocation in cultured cortical neurons following heat stress or oxygen–glucose deprivation

Carboxy terminus of Hsc70-interacting protein (CHIP) is thought to be a cytoprotective protein with protein quality control roles in neurodegenerative diseases and myocardial ischemia. This study describes the localization of CHIP expression in normal rodent brain and the early CHIP response in primary cultures of cortical neurons following ischemic stress models: heat stress (HS) and oxygen-glucose deprivation (OGD). CHIP was highly expressed throughout the brain, predominantly in neurons. The staining pattern was primarily cytoplasmic, although small amounts were seen in the nucleus. More intense nuclear staining was observed in primary cultured neurons which increased with stress. Nuclear accumulation of CHIP occurred within 5-10 min of HS and decreased to baseline levels or lower by 30-60 min. Decrease in nuclear CHIP at 30-60 min of HS was associated with a sharp increase in delayed cell death. While no changes in cytoplasmic CHIP were observed immediately following OGD, nuclear levels of CHIP increased slightly in response to OGD durations of 30 to 240 min. OGD-induced increases in nuclear CHIP decreased slowly during post-ischemic recovery. Nuclear CHIP decreased earlier in recovery following 120 min of OGD (4 h) than 30 min of OGD (12 h). Significant cell death first appeared between 12 and 24 h after OGD, again suggesting that delayed cell death follows closely behind the disappearance of nuclear CHIP. The ability of CHIP to translocate to and accumulate in the nucleus may be a limiting variable that determines how effectively cells respond to external stressors to facilitate cell survival. Using primary neuronal cell cultures, we were able to demonstrate rapid translocation of CHIP to the nucleus within minutes of heat stress and oxygen-glucose deprivation. An inverse relationship between nuclear CHIP and delayed cell death at 24 h suggests that the decrease in nuclear CHIP following extreme stress is linked to delayed cell death. Our findings of acute changes in subcellular localization of CHIP in response to cellular stress suggest that cellular changes that occur shortly after exposure to stress ultimately impact on the capacity and capability of a cell to recover and survive.

Evaluation of a carp primary hepatocyte culture system for screening chemicals for oestrogenic activity

The presence of endocrine disrupting chemicals (EDCs) in the environment has driven the development of screening and testing assays to both identify chemicals with hormonal activity and evaluate their potential to cause adverse effects. As the number of animals used for research and regulatory purposes rises, and set against a desire to reduce animal testing, there is increased emphasis on the development and application of in vitro techniques to evaluate chemical risks to the environment. Induction of vitellogenin (VTG) in isolated fish liver cells has been used successfully to identify a wide range of EDCs, including both natural and synthetic oestrogens and a variety of other xenoestrogens. However, the vitellogenic response reported for hepatocytes in culture has been shown to vary widely, making comparisons between studies difficult. The work presented in this paper explored the variability of the vitellogenic response in primary cultures of common carp ( Cyprinus carpio) hepatocytes following exposure to the model oestrogenic compound, 17β-oestradiol (E2). As expected, variability in the vitellogenic response was observed, both in terms of the sensitivity and magnitude of VTG induction, for hepatocytes isolated from different fish. An apparent difference was observed in the response of isolated hepatocytes based on the sex of the donor fish; maximum levels of E2-stimulated VTG synthesis in hepatocytes derived from females appeared higher (1962 ng mL −1 ± 487 [ n = 9] compared with 1194 ng mL −1 ± 223 for hepatocytes from males [ n = 9]) and EC 50 values lower (1.61 ± 0.4 μM E2 for females and 2.12 ± 0.2 μM E2 for males). However, these differences were not statistically significant, likely in part due to the variation observed in the vitellogenic response. In particular, hepatocytes derived from female fish showed more variation than their male counterparts (the co-efficient of variation for females was 77% compared to 28% for males). Despite the variation observed in the vitellogenic response between different cultures, data from the different donor fish could be compared by standardising responses relative to the maximum VTG induction in each culture following exposure to E2. Adopting this approach in the future will allow for data from different hepatocyte cultures and from donor fish of different sexes, age and stage of maturity to be compared with greater consistency. Measurement of vtg mRNA expression was relatively more sensitive to the oestrogenic effects of E2 exposure than measurement of VTG protein (the LOEC at the transcriptome level was 10-fold lower [0.01 μM E2] than at the protein level [0.1 μM E2]) and changes in vtg mRNA expression showed less variation between individual hepatocyte isolations. Measurement of vtg mRNA in the hepatocyte culture system therefore may offer the most sensitive and consistent option for the screening of chemicals with oestrogenic activity in fish primary hepatocyte cultures.

Use of an in vitro chemoresponse assay to define a subset of colorectal carcinomas responsive to cetuximab

e15127 Background: Cetuximab (Erbitux) is a chimeric monoclonal antibody that binds to the extracellular domain of the epidermal growth factor receptor. This interaction interferes with ligand binding and activation of the receptor blocking the downstream signaling of EGFR and impairing cell growth and proliferation. Cetuximab also has been shown to mediate antibody dependent cellular cytotoxicity. Thus, a singular approach to drug mechanism likely would not capture the true effects of cetuximab. Cetuximab is FDA-approved to treat head and neck cancer and colorectal carcinomas and is being evaluated for use in non-small cell lung and endometrial cancer. Due to the low population response rate of cetuximab, an integrated biomarker that can identify patients with an increased likelihood for response would be of great clinical utility. The current study describes an in vitro chemoresponse assay to predict response of primary cultures of human colorectal tumor specimens to cetuximab. Methods: The chemoresponse assay (ChemoFx) was developed using four different immortalized carcinoma cell lines (NCI-H292, NCI-H522, NCI-H1666, Calu3). The chemoresponse assay was also performed on 54 first passage primary cultures of human colorectal tumor specimens. Cell lines and specimens were treated with a 10 dose concentration range of cetuximab for 72 hours, stained with a nuclear dye, and remaining post-treatment live cells were counted. Resulting dose-response curves were analyzed. Results: Two of the examined cell lines showed response to cetuximab treatment; EC50 values for NCI-H292 and NCI-H1666 were 825nM and 13nM, respectively. NCI-H522 and Calu3 were deemed non-responsive to cetuximab. Dose-response curves of the 54 primary colorectal cultures revealed that 8% of the cultures tested were responsive to cetuximab, 22% had an intermediate response, and 70% were deemed non-responsive. These results are consistent with a reported clinical response rate of 11% for cetuximab in colorectal carcinoma patients. Conclusions: Initial results demonstrate that in vitro chemoresponse testing may be useful in predicting patient response to cetuximab. This could increase the efficacy of the current chemotherapy decision-making process for oncologists and their patients. No significant financial relationships to disclose.