Unstimulated Tissue Research Articles

Come together: On-chip bioelectric wound closure

There is a growing interest in bioelectric wound treatment and electrotaxis, the process by which cells detect an electric field and orient their migration along its direction, has emerged as a potential cornerstone of the endogenous wound healing response. Despite recognition of the importance of electrotaxis in wound healing, no experimental demonstration to date has shown that the actual closing of a wound can be accelerated solely by the electrotaxis response itself, and in vivo systems are too complex to resolve cell migration from other healing stages such as proliferation and inflammation. This uncertainty has led to a lack of standardization between stimulation methods, model systems, and electrode technology required for device development. In this paper, we present a ‘healing-on-chip’ approach that is a standardized, low-cost, model for investigating electrically accelerated wound healing. Our device provides a biomimetic convergent field geometry that more closely resembles actual wound fields. We validate this device by using electrical stimulation to close a 1.5 mm gap between two large (30 mm2) layers of primary skin keratinocyte to completely heal the gap twice as quickly as in an unstimulated tissue. This demonstration proves that convergent electrotaxis is both possible and can accelerate healing and offers an accessible ‘healing-on-a-chip’ platform to explore future bioelectric interfaces.

MARCKS mediates vascular contractility through regulating interactions between voltage-gated Ca2+ channels and PIP2

Phosphatidylinositol 4,5-bisphosphate (PIP2) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP2 might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP2-binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca2+ channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α1-adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP2 binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca2+ currents, which were prevented by depleting PIP2 levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP2 into the local environment where it increases voltage-gated Ca2+ channel activity.

The enteric nervous system neuropeptide, bombesin, reverses innate immune impairments during parenteral nutrition.

Lack of enteral stimulation during parenteral nutrition (PN) impairs mucosal immunity. Bombesin (BBS), a gastrin-releasing peptide analogue, reverses PN-induced defects in acquired immunity. Paneth cells produce antimicrobial peptides (AMPs) of innate immunity for release after cholinergic stimulation. Determine if BBS restores AMPs and bactericidal function during PN. Intravenously cannulated male ICR mice were randomized to Chow, PN, or PN+BBS (15 μg 3 times daily, n = 7 per group) for 5 days. Ileum was analyzed for AMPs (Protein: sPLA2 by fluorescence, lysozyme and RegIII-γ by western andcryptdin-4 by ELISA; mRNA: all by RT-PCR). Cholinergic stimulated (100 μM bethanechol) ileal specimens assessed Pseudomonas bactericidal activity. Ileum (Chow: n = 7; PN: n = 9; PN+BBS: n = 8) was assessed for Escherichia coli invasion in ex-vivo culture. PN significantly decreased most AMPs versus Chow while BBS maintained Chow levels (sPLA2: Chow: 107 + 14*, PN: 44.6 + 7.2, PN+BBS: 78.7 + 13.4* Fl/min/μL/total protein; Lysozyme: Chow: 63.9 + 11.9*, PN: 26.8 + 6.2; PN+BBS: 64.9 + 13.8* lysozyme/total protein; RegIII-γ: Chow: 51.5 + 10.0*, PN: 20.4 + 4.3, PN+BBS: 31.0 + 8.4 RegIII-γ/total protein; Cryptdin-4: Chow: 18.4 + 1.5*, PN: 12.7 + 1.6, PN+BBS: 26.1 + 2.4*† pg/mg [all *P < 0.05 vs PN and †P < 0.05 vs Chow]). Functionally, BBS prevented PN loss of bactericidal activity after cholinergic stimulation (Chow: 25.3 + 3.6*, PN: 13.0 + 3.2; PN+BBS: 27.0 + 4.7* percent bacterial killing, *P < 0.05 vs PN). BBS reduced bacterial invasion in unstimulated tissue barely missing significance (P = 0.06). The enteric nervous system (ENS) controls AMP levels in Paneth cells during PN but mucosal protection by innate immunity requires both ENS and parasympathetic stimulation.

Chronic electrical stimulation with a suprachoroidal retinal prosthesis: a preclinical safety and efficacy study.

PurposeTo assess the safety and efficacy of chronic electrical stimulation of the retina with a suprachoroidal visual prosthesis.MethodsSeven normally-sighted feline subjects were implanted for 96–143 days with a suprachoroidal electrode array and six were chronically stimulated for 70–105 days at levels that activated the visual cortex. Charge balanced, biphasic, current pulses were delivered to platinum electrodes in a monopolar stimulation mode. Retinal integrity/function and the mechanical stability of the implant were assessed monthly using electroretinography (ERG), optical coherence tomography (OCT) and fundus photography. Electrode impedances were measured weekly and electrically-evoked visual cortex potentials (eEVCPs) were measured monthly to verify that chronic stimuli were suprathreshold. At the end of the chronic stimulation period, thresholds were confirmed with multi-unit recordings from the visual cortex. Randomized, blinded histological assessments were performed by two pathologists to compare the stimulated and non-stimulated retina and adjacent tissue.ResultsAll subjects tolerated the surgical and stimulation procedure with no evidence of discomfort or unexpected adverse outcomes. After an initial post-operative settling period, electrode arrays were mechanically stable. Mean electrode impedances were stable between 11–15 kΩ during the implantation period. Visually-evoked ERGs & OCT were normal, and mean eEVCP thresholds did not substantially differ over time. In 81 of 84 electrode-adjacent tissue samples examined, there were no discernible histopathological differences between stimulated and unstimulated tissue. In the remaining three tissue samples there were minor focal fibroblastic and acute inflammatory responses.ConclusionsChronic suprathreshold electrical stimulation of the retina using a suprachoroidal electrode array evoked a minimal tissue response and no adverse clinical or histological findings. Moreover, thresholds and electrode impedance remained stable for stimulation durations of up to 15 weeks. This study has demonstrated the safety and efficacy of suprachoroidal stimulation with charge balanced stimulus currents.

Induction of Bacterial Antigen-Specific Colitis by a Simplified Human Microbiota Consortium in Gnotobiotic Interleukin-10 −/− Mice

We evaluated whether a simplified human microbiota consortium (SIHUMI) induces colitis in germfree (GF) 129S6/SvEv (129) and C57BL/6 (B6) interleukin-10-deficient (IL-10(-/-)) mice, determined mouse strain effects on colitis and the microbiota, examined the effects of inflammation on relative bacterial composition, and identified immunodominant bacterial species in "humanized" IL-10(-/-) mice. GF wild-type (WT) and IL-10(-/-) 129 and B6 mice were colonized with 7 human-derived inflammatory bowel disease (IBD)-related intestinal bacteria and maintained under gnotobiotic conditions. Quantification of bacteria in feces, ileal and colonic contents, and tissues was performed using 16S rRNA gene selective quantitative PCR. Colonic segments were scored histologically, and gamma interferon (IFN-γ), IL-12p40, and IL-17 levels were measured in supernatants of unstimulated colonic tissue explants and of mesenteric lymph node (MLN) cells stimulated by lysates of individual or aggregate bacterial strains. Relative bacterial species abundances changed over time and differed between 129 and B6 mice, WT and IL-10(-/-) mice, luminal and mucosal samples, and ileal and colonic or fecal samples. SIHUMI induced colitis in all IL-10(-/-) mice, with more aggressive colitis and MLN cell activation in 129 mice. Escherichia coli LF82 and Ruminococcus gnavus lysates induced dominant effector ex vivo MLN TH1 and TH17 responses, although the bacterial mucosal concentrations were low. In summary, this study shows that a simplified human bacterial consortium induces colitis in ex-GF 129 and B6 IL-10(-/-) mice. Relative concentrations of individual SIHUMI species are determined by host genotype, the presence of inflammation, and anatomical location. A subset of IBD-relevant human enteric bacterial species preferentially stimulates bacterial antigen-specific TH1 and TH17 immune responses in this model, independent of luminal and mucosal bacterial concentrations.

Enhancing adoptively transferred T cells by stimulating with dendritic cell stimulation: exploring novel therapies (P4405)

Abstract Adoptive Cell Transfer (ACT) of tumor infiltrating lymphocytes (TILs) is a promising strategy for cancer immunotherapy. The current therapy relies on non-myeloablative lymphodepletion and high dose IL-2 therapy, which has serious side effects for patients. Optimization of the current ACT therapy, using animal models, may help reduce or eliminate these additional treatments and enhance the response rate. ACT has been evaluated in the autoimmune model RIP-GP mice, which express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) on the β-islet cells of the pancreas. Our findings showed that LCMV memory T cells stimulated with matured dendritic cells (DCs) and pulsed with peptides derived from LCMV, successfully induced diabetes. These stimulated T cells expanded well in vivo and showed strong killing activity after transfer. However, unstimulated memory tissue specific T cells rarely expanded after transfer and showed limited cytolytic activity. To evaluate this method in the tumor model, RIP-Tag2/GP double transgenic mice were used, which express the large T antigen under the control of RIP and spontaneously develop insulinomas. Islet specific T cells cocultured with DCs and LCMV-GP peptides displayed strong anti-tumor activity after transfer in vivo. Surprisingly, mice bearing insulinomas survived longer after ACT than mice treated with LCMV vaccination. Therefore, stimulating TILs before infusion has high potential for enhancing ACT therapy.

Expression profiles of Toll-like receptors 1-10 in human placenta during viral infection

Event Abstract Back to Event Expression profiles of Toll-like receptors 1-10 in human placenta during viral infection Patrycja Suski1*, Jarosław Kalinka2, Mirosława Studzińska1, Agnieszka Jabłońska1, Zbigniew J. Leśnikowski1 and Edyta Paradowska1 1 Institute of Medical Biology, Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, Poland 2 Medical University of Lodz, Department of Perinatology, First Chair of Gynecology and Obstetrics, Poland Placenta provides an important physical and immunological barrier against infectious agents and a pregnancy-specific component of the innate immune system. Toll-like receptors (TLRs) are essential for the induction of innate immunity responses in different human tissues including the maternal and fetal interface. The objective of this study was to investigate the TLR1-10 gene expressions in the human term placentas during viral infection. The explants of third-trimester chorionic villi and deciduas were exposed to infection with laboratory strains of HCMV, HSV-1 or VSV. Gene expression of TLR1-10 was assessed by quantitative real-time PCR using Power SYBR Green PCR Master Mix. The relative gene expression values were calculated using the comparative Ct method, and data were normalized against YWHAZ levels. This study clearly demonstrated that the human term placenta differentially expressed functional TLR1-10, indicating that these receptors are believed to be important for placental immune responses against viruses. We found that TLR3 mRNA levels were lower than other receptors in all examined placentas. We observed increased expression of TLR1, 9 and 10 in HCMV-infected decidua compared to unstimulated tissue. These findings demonstrated that the decidua was a critical component of the innate immunity response during HCMV infection. In contrast, infection with HSV-1 or VSV did not affect the expression of TLRs. Our results suggest that the TLRs could be important immune regulators in placenta during viral infection. Acknowledgements This work was supported by the European Regional Development Fund under the Operational Programme Innovative Economy, Grant No. POIG.01.01.02-10-107/09. Keywords: TLR expression, human placenta, hcmv, HSV-1, VSV Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Suski P, Kalinka J, Studzińska M, Jabłońska A, Leśnikowski ZJ and Paradowska E (2013). Expression profiles of Toll-like receptors 1-10 in human placenta during viral infection. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00142 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Mrs. Patrycja Suski, Institute of Medical Biology, Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, Lodz, Poland, psuski@cbm.pan.pl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Patrycja Suski Jarosław Kalinka Mirosława Studzińska Agnieszka Jabłońska Zbigniew J Leśnikowski Edyta Paradowska Google Patrycja Suski Jarosław Kalinka Mirosława Studzińska Agnieszka Jabłońska Zbigniew J Leśnikowski Edyta Paradowska Google Scholar Patrycja Suski Jarosław Kalinka Mirosława Studzińska Agnieszka Jabłońska Zbigniew J Leśnikowski Edyta Paradowska PubMed Patrycja Suski Jarosław Kalinka Mirosława Studzińska Agnieszka Jabłońska Zbigniew J Leśnikowski Edyta Paradowska Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Is p53 the Long-Sought Molecular Trigger for Cyclophilin D–Regulated Mitochondrial Permeability Transition Pore Formation and Necrosis?

An article recently published in Cell concluded that p53 is necessary and sufficient to induce mitochondrial permeability transition pore (MPTP)–dependent necrosis through inducible p53 translocation to the matrix with cyclophilin D (CypD) binding. The results and implications are very provocative. The physiological significance of the proposed paradigm, however, is uncertain because calcium itself, which is a fundamental regulator of MPTP, is independent of p53, as shown by the authors. In addition, purified mitochondria from any unstimulated cell type or tissue, which presumably lacks p53 given the inducible mechanism proposed, have a fully functional MPTP to all the classic modes of stimulation as analyzed in vitro. Myocardial infarction produces an area of myocyte loss that is a mixture of apoptotic and necrotic cell death. Recent data from multiple laboratories have shown that necrosis can be a programmed event and that specific molecular inhibitors of this process can be potently cardioprotective. During ischemic injury, both reactive oxygen species (ROS) and calcium levels increase within cells, triggering a type of programmed necrosis through MPTP opening that is CypD-dependent.1–5 Indeed, treatment with cyclosporine A, a CypD inhibitor, as well as genetic deletion of CypD, reduces ischemia/reperfusion injury in both the brain and the heart.3–5 Thus, mitochondria are central regulators of ischemic cell death through a mechanism involving the MPTP, but until recently we have lacked an understanding of how CypD is activated and potentially induces MPTP directly. However, Moll et al6 recently published an interesting article in the journal Cell , which shows that p53 can open the MPTP leading to necrotic cell death (Figure). They propose that upon ROS stimulation, p53 translocates to the matrix of the mitochondria where it binds to CypD and somehow opens the MPTP (Figure). This function of p53 was independent of …

SiRNA-mediated knockdown ofh-caldesmon in vascular smooth muscle

Smooth muscle contraction involves phosphorylation of the regulatory myosin light chain. However, this thick-filament system of regulation cannot account for all aspects of a smooth muscle contraction. An alternate site of contractile regulation may be in the thin-filament-associated proteins, in particular caldesmon. Caldesmon has been proposed to be an inhibitory protein that acts either as a brake to stop any increase in resting or basal tone, or as a modulatory protein during contraction. The goal of this study was to use short interfering RNA technology to decrease the levels of the smooth muscle-specific isoform of caldesmon in intact vascular smooth muscle tissue to determine more carefully what role(s) caldesmon has in smooth muscle regulation. Intact strips of vascular tissue depleted of caldesmon produced significant levels of shortening velocity, indicative of cross-bridge cycling, in the unstimulated tissue and exhibited lower levels of contractile force to histamine. Our results also suggest that caldesmon does not play a role in the cooperative activation of unphosphorylated cross bridges by phosphorylated cross bridges. The velocity of shortening of the constitutively active tissue and the high basal values of myosin light chain phosphorylation suggest that h-caldesmon in vivo acts as a brake against contractions due to basally phosphorylated myosin. It is also possible that phosphorylation of h-caldesmon alone in the resting state may be a mechanism to produce increases in force without stimulation and increases in calcium. Disinhibition of h-caldesmon by phosphorylation would then allow force to be developed by activated myosin in the resting state.

Disparate Innate Immune Responses to Persistent and AcuteChlamydia pneumoniaeInfection in Chronic Obstructive Pulmonary Disease

Chlamydia pneumoniae (Cpn) infection may play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available comparing persistent and acute infection of this pathogen in the human respiratory tract. To study Cpn-induced innate immune responses in lung tissue from patients with COPD and control subjects ex vivo and in vitro. Cpn detection was done by nested polymerase chain reaction, in situ hybridization, and immunohistochemistry ex vivo in unstimulated tissue and in vitro using an acute Cpn infection model. As main endpoints for the assessment of early cellular responses, nuclear factor (NF)-kappaB activation and CXC chemokine ligand (CXCL)-8 expression were evaluated. The role of Toll-like receptors (TLRs) as recognition molecules in Cpn-induced innate responses was tested by blocking experiments. Fifteen percent of patients with COPD were chronically infected with Cpn in contrast to 0% of control subjects (p < 0.05). There were no differences in CXCL-8 and NF-kappaB expression between infected and noninfected COPD tissue ex vivo. In contrast, acute in vitro infection induced an intense innate immune response including up-regulation of TLR2. Blocking experiments demonstrated the predominant role of TLR2 in induction of the early immune response, whereas no influence on chlamydial infection rates was observed. Acute in vitro infection of human lung tissue with Cpn elicited a marked innate response via TLR2, whereas chronic chlamydial infection in patients with COPD was not associated with enhanced cellular activation. These findings suggest different roles of Cpn during acute and chronic stages of pulmonary infection.

Activation of the Lipid Droplet Controls the Rate of Lipolysis of Triglycerides in the Insect Fat Body. A role for Lsdp1 (Lipid storage droplet protein 1)

The hydrolysis of triglyceride (TG) stored in the lipid droplets (LD) of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation of cAMP-dependent kinase cascade (PKA). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the LD was investigated. The activity of purified TG-lipase determined using in vivo TG-radiolabeled LD was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against LD isolated from AKH-stimulated fat bodies than unstimulated tissue. Investigation of the phosphorylation state of LD- associated proteins showed that in vivo stimulation of lipolysis promotes a rapid phosphorylation of a 42-44kDa protein. This protein was identified by mass spectrometry as Lsdp1. In vivo phosphorylation of this protein reached a peak ~10min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with LD isolated 10min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase, and LD, without additional cellular components. In vitro phosphorylation of LD catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the LD. Lsdp1 is the main targetof PKA, suggesting that this protein is a major player in theactivation of lipolysis in insects. Funding provided by OAES and NIH.

Fragile X Mental Retardation Protein Shifts between Polyribosomes and Stress Granules after Neuronal Injury by Arsenite Stress orIn VivoHippocampal Electrode Insertion

Fragile X mental retardation protein (FMRP), the lack of which causes fragile X syndrome, is an RNA-binding protein encoded by the FMR1 gene. FMRP accompanies mRNAs from the nucleus to dendritic regions and is thought to regulate their translation at synapses. It has been shown that FMRP moves into nontranslating stress granules (SGs) during heat stress of cultured fibroblasts (Mazroui et al., 2002). We used a novel method to isolate SGs from neurons by virtue of their TIA-1 (T-cell intracellular antigen 1) protein component, and found that FMRP moved out of polyribosomes and into SGs subsequent to oxidative stress. We then examined FMRP changes in subcellular localization resulting from mechanically induced neuronal injury by placement of electrodes into the dentate gyrus and the perforant path of the hippocampus in vivo. During the first 10 min after electrode insertion into one hippocampus, FMRP shifted into SGs and away from polyribosomes, in both hippocampi. Although the injury discharge subsided beyond 10 s, FMRP levels in polyribosomes and stress granules did not return to basal levels until 30 min after electrode penetration. Our findings suggest that procedures for in vivo induction of long-term potentiation or long-term depression should incorporate a 30 min rest period after electrode insertion, and indicate that the contralateral hippocampus cannot be considered an unstimulated control tissue.

Activation of the Lipid Droplet Controls the Rate of Lipolysis of Triglycerides in the Insect Fat Body

The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA)). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the lipid droplet, was investigated in fat body adipocytes. The activity of purified TG-lipase determined using in vivo TG-radiolabeled lipid droplets was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against lipid droplets isolated from hormone-stimulated fat bodies than against lipid droplets isolated from unstimulated tissue. In vivo stimulation of lipolysis promotes a rapid phosphorylation of a lipid droplet protein with an apparent mass of 42-44 kDa. This protein was identified as "Lipid Storage Droplet Protein 1" (Lsdp1). In vivo phosphorylation of this protein reached a peak approximately 10 min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with lipid droplets isolated 10 min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase and lipid droplets. In vitro phosphorylation of lipid droplets catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase, demonstrating a prominent role PKA and protein phosphorylation on the activation of the lipid droplets. AKH-induced changes in the properties of the substrate do not promote a tight association of the lipase with the lipid droplets. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the lipid droplet. This activation is achieved by PKA-mediated phosphorylation of the lipid droplet. Lsdp1 is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects.

Long‐term potentiation in the rat dentate gyrus is associated with enhanced Arc/Arg3.1 protein expression in spines, dendrites and glia

Electron microscopic immunocytochemical methods were used to determine the localization, subcellular distribution and expression of activity-regulated cytoskeletal protein (Arc/Arg3.1) in dentate gyrus after unilateral induction of long-term potentiation (LTP) in the perforant pathway of anaesthetized rats. At 2 h post-induction, immunoreaction product was visible in the dentate gyrus in both the granule cell and molecular layers. Arc expression was higher in the potentiated than the unstimulated contralateral hemisphere. Single-section electron microscopy analysis in unstimulated tissue and in tissue prepared 2 and 4 h after LTP induction showed Arc immunoreactivity (Arc-IR) in dendrites, dendritic spines and glia. Arc-IR was associated with synaptic and non-synaptic plasma membrane apposed to axon terminals and with cytoplasmic organelles, including the cytoskeleton. Arc-IR was also present in neuronal perikarya and there was occasional labelling of nuclei and axons. At 2 h post-LTP induction, there were significant increases in Arc-IR within the granule cell and molecular layers of the dentate gyrus and particularly within the middle molecular layer relative to the inner and outer molecular layers. This increase in Arc expression 2 h after LTP induction was blocked by the N-methyl-D-aspartate receptor antagonist (RS)-3-2-carboxypiperazin-4-yl-propyl-1-phosphonic acid. In animals killed 4 h after LTP induction, Arc expression had declined and differences between the potentiated and unpotentiated hemispheres were no longer significant. Our data provide ultrastructural evidence for a transient LTP-associated increase in the expression of Arc protein in the middle molecular layer of the dentate gyrus, with preferential targeting to dendrites, dendritic spines and glia.

Plasticity of synaptic ribbons of the rat pineal gland in vitro — Minor effects of electrical stimulation

Synaptic ribbons (SRs) of mammalian pinealocytes exhibit day/night changes in number and size, changes that are apparently regulated by the suprachiasmatic nucleus via postganglionic sympathetic nerve fibres. Since the neural control of SR changes is far from clear and as pinealocytes produce action potentials, we undertook to investigate whether electrical stimulation affects SR changes. Isolated rat pineal glands removed during the daytime were kept in vitro for 0, 30, 60, 90 or 120 min, with or without continuous electrical stimulation (1 mA, 1 Hz), followed by the quantification of SR profiles (SRPs) by transmission electron microscopy. SRs were categorised as to whether they lay less than 100 nm away from the pinealocyte plasmalemma (SRPs(near)) or more distant from it (SRPs(dist)) and the lengths of the profiles were measured. Cultured pineal organs showed a significant numerical depression of SRPs(near), irrespective of whether the organs had been electrically stimulated or not. SRPs(near) length revealed a significant increase at 60 min in unstimulated control tissue and at 30 min in electrically stimulated glands. SRPs(dist) length decreased significantly at 30 min in control glands and after 60 min in electrically stimulated glands. Thus, action potentials inside the pineal gland appear to be minor factors regulating SR numbers. In future pineal studies, SRPs(near) and SRPs(dist) should be considered separately as they differ in plasticity.

T-cell proliferation induced by local application of LPS in the nasal mucosa of nonatopic children

Background: Recently, there has been a great deal of interest in the role of bacterial products in altering the allergic immune response in early life. Objective: We sought to develop a model of bacterial inflammation in human subjects to characterize the ex vivo response to LPS in the nasal mucosa of young nonatopic children. Methods: Using the explant technique, we cultured nasal mucosa in the presence or absence of increasing concentrations of LPS (0.001-1 μg LPS/mL) for 24 hours. Immunocytochemistry and in situ hybridization were used to phenotype the cells and cytokines in the explant, and immunofluorescence was used to study proliferation of T cells in explants cultured with LPS and 5-bromo-2′-deoxyuridine (BrdU). Results: CD68, CD3, tryptase, elastase, IFN- γ IL-12, and IL-10 immunoreactivity was detected in unstimulated tissue. LPS stimulation caused a dose-dependent increase in the number of CD3+ (n = 7, P <.05), CD68+ (n = 7, P < .01), elastase-positive (n = 7, P < .01), tryptase-positive (n = 7, P < .05), IL-2 –positive (n = 6, P < .05), IL-12 –positive (n = 6, P < .05), and IFN- γ–positive (n = 6, P < .05) cells compared with media alone. LPS had no effect on major basic protein, IL-4, IL-5, or IL-13 immunoreactivity. LPS (0.1 μg/mL) caused an increase in the number of BrdU-positive cells as early as 2 hours after incubation (n = 8, P < .001). The maximal BrdU immunoreactivity was seen 24 hours after incubation with LPS, at which time approximately 70% of BrdU-positive cells were CD3+ T cells. Conclusions: Our results suggest that LPS is capable of inducing local inflammation, TH1 cytokine immunoreactivity, and proliferation of CD3+ activated T cells in the absence of de novo cell recruitment. This model holds promise for future studies investigating the importance of bacterial products in regulating local immune function in young atopic children. (J Allergy Clin Immunol 2002;110:771-6.)

NO-induced relaxation of labouring and non-labouring human myometrium is not mediated by cyclic GMP.

1. In myometrial strips from near-term non-labouring human uterus, addition of oxytocin (OT) evoked dose-dependent (10 - 3000 nM) phasic contractions that were antagonized by atosiban (1 microM) and relaxed by addition of the nitric oxide donor S-nitroso L-cysteine (Cys-NO). In near-term labouring myometrium, however, addition of OT was ineffective at raising additional tone. 2. In both labouring and non-labouring tissue, Cys-NO mediated relaxation of spontaneous or OT-induced contractions (IC(50)=1 microM) was unaffected by prior addition of the guanylyl cyclase (GC) inhibitors ODQ (1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one; 1 microM), or methylene blue (MB; 10 microM). 3. Elevation of intracellular cyclic GMP accompanying 30 microM Cys-NO addition in non-labouring tissue (7.5 fold) or in labouring tissues (2.5 fold) was completely blocked in tissues that had been pre-treated with ODQ or MB. 4. Charybdotoxin (ChTx), iberiotoxin (IbTx) and kaliotoxin (KalTx) all shifted the Cys-NO inhibition curve to the right and reduced the degree of relaxation produced by maximal Cys-NO treatment (100 microM in non-labouring tissue; in labouring tissue, KalTx prevented Cys-NO mediated relaxation in both stimulated and unstimulated tissue. 5. Addition of the NO-donor S-nitroso N-acetyl penicillamine (SNAP) produced a dose-dependent relaxation of pregnant myometrium while 3-morpholinosyndonimine (SIN-1) did not. The failure of SIN-1 to relax OT-induced contractions was not due to a failure of the donor to stimulate myometrial GC. 6. We demonstrate that despite the ability of NO to stimulate myometrial GC in pregnant uterine muscle, relaxations are independent of cyclic GMP action. Effects of K(+)-channel inhibitors suggests that NO-induced relaxation in human uterine smooth muscle may be subserved by direct or indirect activation of one or more calcium-activated K(+)-channels.

Role of inducible nitric oxide synthase in the regulation of leucocyte recruitment

Constitutively produced nitric oxide released by endothelial cells has been shown to act as an endogenous agent which inhibits the rolling and adhesion of leucocytes in the microcirculation. However, during various types of inflammation, expression of the inducible form of nitric oxide synthase (iNOS) can dramatically increase the amount of nitric oxide present in tissues. Furthermore, as iNOS can be expressed by a wide variety of cell types, the distribution of nitric oxide is likely to be altered relative to that in unstimulated tissue. Under these conditions, it is less well understood whether iNOS-derived nitric oxide retains the anti-adhesive capabilities of constitutively produced nitric oxide. This review summarizes work done to examine this issue. Three main approaches have been used. In vitro studies have examined the role of iNOS in adhesive interactions between stimulated endothelial cells and leucocytes, providing evidence of an anti-adhesive effect of iNOS. In addition, the role of iNOS has been examined in vivo in animal models of inflammation using pharmacological iNOS inhibitors. These experiments were extended by the advent of the iNOS-deficient (iNOS(-/-)) mouse. Intravital microscopy studies of these mice have indicated that, under conditions of low-dose endotoxaemia, iNOS-derived nitric oxide can inhibit leucocyte rolling and adhesion. The potential mechanisms for these effects are discussed. In contrast, several other studies have observed either no effect or an enhancing effect of iNOS on inflammatory leucocyte recruitment. Taken together, these studies suggest that the importance of iNOS in modulating leucocyte recruitment can vary according to the type of inflammatory response.

Role of inducible nitric oxide synthase in the regulation of leucocyte recruitment

Constitutively produced nitric oxide released by endothelial cells has been shown to act as an endogenous agent which inhibits the rolling and adhesion of leucocytes in the microcirculation. However, during various types of inflammation, expression of the inducible form of nitric oxide synthase (iNOS) can dramatically increase the amount of nitric oxide present in tissues. Furthermore, as iNOS can be expressed by a wide variety of cell types, the distribution of nitric oxide is likely to be altered relative to that in unstimulated tissue. Under these conditions, it is less well understood whether iNOS-derived nitric oxide retains the anti-adhesive capabilities of constitutively produced nitric oxide. This review summarizes work done to examine this issue. Three main approaches have been used. In vitro studies have examined the role of iNOS in adhesive interactions between stimulated endothelial cells and leucocytes, providing evidence of an anti-adhesive effect of iNOS. In addition, the role of iNOS has been examined in vivo in animal models of inflammation using pharmacological iNOS inhibitors. These experiments were extended by the advent of the iNOS-deficient (iNOS-/-) mouse. Intravital microscopy studies of these mice have indicated that, under conditions of low-dose endotoxaemia, iNOS-derived nitric oxide can inhibit leucocyte rolling and adhesion. The potential mechanisms for these effects are discussed. In contrast, several other studies have observed either no effect or an enhancing effect of iNOS on inflammatory leucocyte recruitment. Taken together, these studies suggest that the importance of iNOS in modulating leucocyte recruitment can vary according to the type of inflammatory response.

Oxidized-Ldl Enhances Coronary Vasoconstriction by Increasing the Activity of α and ε Protein Kinase C Isoforms

31 Oxidized low density lipoprotein (ox-LDL) plays a critical role in the development of atherosclerotic coronary vasospasm; however, the cellular mechanisms involved are not fully understood. We tested the hypothesis that ox-LDL enhances coronary vasoconstriction by increasing the activity of specific protein kinase C (PKC) isoforms in coronary smooth muscle. Active stress was measured in de-endothelialized porcine coronary artery strips, [Ca 2+ ] i was monitored in single coronary smooth muscle cells loaded with fura-2, and the whole tissue, cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies using Western blot analysis. Ox-LDL (100 μg/ml) caused a slow but significant increase in active stress to 1.2±0.2 x10 3 N/m 2 , that was completely inhibited by the PKC inhibitors staurosporine and calphostin C, with no significant increase in [Ca 2+ ] i . Ox-LDL enhanced coronary contraction to increasing concentrations of 5-hydroxytryptamine (5-HT) and extracellular KCl with no additional increases in [Ca 2+ ] i . Direct activation of PKC by phorbol 12,13-dibutyrate (PDBu, 0.1 μM) caused a contraction similar in magnitude and time course to ox-LDL induced contraction. PDBu also enhanced 5-HT and KCl-induced contraction with no additional increases in [Ca 2+ ] i . Both ox-LDL and PDBu caused an increase in PKC activity in the particulate fraction and a decrease in the cytosolic fraction and increased the particulate/cytosolic PKC activity ratio. Western blot analysis revealed α-, δ-, ε- and ζ-PKC isoforms. In unstimulated tissue, α- and ε-PKC were mainly cytosolic, δ-PKC was mainly in the particulate fraction, while ζ-PKC was equally distributed in the cytosolic and particulate fractions. Ox-LDL and PDBu caused translocation of α- and ε-PKC from the cytosolic to particulate fraction, with minimal effect on the distribution of δ-PKC and ζ-PKC. Native LDL did not significantly affect coronary contraction, [Ca 2+ ] i or PKC activity. These results suggest that ox-LDL enhances coronary vasoconstriction via activation and translocation of α- and ε-PKC isoforms in coronary smooth muscle.