Round Bottom Research Articles

A Small Molecule Inhibitor of Endoplasmic Reticulum Oxidation 1 (ERO1) with Selectively Reversible Thiol Reactivity

Endoplasmic reticulum oxidation 1 (ERO1) is a conserved eukaryotic flavin adenine nucleotide-containing enzyme that promotes disulfide bond formation by accepting electrons from reduced protein disulfide isomerase (PDI) and passing them on to molecular oxygen. Although disulfide bond formation is an essential process, recent experiments suggest a surprisingly broad tolerance to genetic manipulations that attenuate the rate of disulfide bond formation and that a hyperoxidizing ER may place stressed cells at a disadvantage. In this study, we report on the development of a high throughput in vitro assay for mammalian ERO1alpha activity and its application to identify small molecule inhibitors. The inhibitor EN460 (IC(50), 1.9 mum) interacts selectively with the reduced, active form of ERO1alpha and prevents its reoxidation. Despite rapid and promiscuous reactivity with thiolates, EN460 exhibits selectivity for ERO1. This selectivity is explained by the rapid reversibility of the reaction of EN460 with unstructured thiols, in contrast to the formation of a stable bond with ERO1alpha followed by displacement of bound flavin adenine dinucleotide from the active site of the enzyme. Modest concentrations of EN460 and a functionally related inhibitor, QM295, promote signaling in the unfolded protein response and precondition cells against severe ER stress. Together, these observations point to the feasibility of targeting the enzymatic activity of ERO1alpha with small molecule inhibitors.

Validation of a miniaturized assay based on IFNg secretion for assessment of specific T cell immunity

A miniaturized method for detection of antigen induced secretion of IFNg by specific T cells cultured in 384 well plates has been recently reported. In order to confidently apply this assay to clinical investigations for monitoring of specific T cell immunity, an intralaboratory validation study has been undertaken. High reproducibility and linearity of reference curves was demonstrated. Consecutive replicate experiments handled by different operators using broad panels of recall antigens were reproducible when tested on individual biological samples. Kinetics of IFNg secretion with different antigens showed a plateau after 24 h culture. Similar trends were observed with secretion of TNFa, GM-CSF and IL17, suggesting that the same kinetics can be applied if other cytokines are tested with this assay. It was demonstrated that frozen-thawed cells can be tested by cell-ELISA and that when PBMC are replaced by whole blood similar reactivity profiles were observed even though cytokine concentration was lower. T cell responses were higher in round bottom than in flat bottom wells, but these plates could not be applied to cell-ELISA as clear plates are not available for scanning. In conclusion, the assay proved flexible, since plates can be frozen at different times during the process, fresh or frozen PBMC and PBMC or whole blood could be used, and robust, since reproducibility was remarkable even when different operators performed the procedures.

Culture of Bovine Embryos on a Polydimethylsiloxane (PDMS) Microwell Plate

We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.

Inhibition and Activation by CD244 Depends on CD2 and Phospholipase C-γ1

Regulation by the NK and T cell surface receptor CD244 in mice and humans depends both on engagement at the cell surface by CD48 and intracellular interactions with SAP and EAT-2. Relevance to human disease by manipulating CD244 in mouse models is complicated by rodent CD2 also binding CD48. We distinguish between contributions of mouse CD244 and CD2 on engagement of CD48 in a mouse T cell hybridoma. CD2 and CD244 both contribute positively to the immune response as mutation of proline-rich motifs or tyrosine motifs in the tails of CD2 and CD244, respectively, result in a decrease in antigen-specific interleukin-2 production. Inhibitory effects of mouse CD244 are accounted for by competition with CD2 at the cell surface for CD48. In humans CD2 and CD244 are engaged separately at the cell surface but biochemical data suggest a potential conserved intracellular link between the two receptors through FYN kinase. We identify a novel signaling mechanism for CD244 through its potential to recruit phospholipase C-gamma1 via the conserved phosphorylated tyrosine motif in the tail of the adaptor protein EAT-2, which we show is important for function.

Disease-causing Mutation in GPR54 Reveals the Importance of the Second Intracellular Loop for Class A G-protein-coupled Receptor Function

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease. When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells.

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease. When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells.

Mitral valve replacement and Takotsubo syndrome

Athanasiou et al. [1] should be commended for their seminal review on evidence synthesis and critical reappraisal of surgical techniques of preservation of mitral subvalvar apparatus. However, when Lillehei and colleagues in the early 1960s had proposed the chordal-sparing technique of mitral valve replacement (MVR), the entity of Takotsubo syndrome had not been described. Nearly 30 years later, Satoh et al. [2], Dote and colleagues [3] from Japan described in 1990 and 1991 this novel syndrome, characterized by transient reversible left ventricular (LV) dysfunction in the absence of coronary artery disease, with chest pain, electrocardiographic changes mimicking acute anterior myocardial infarction, but only minimal release of myocardial enzymes. Left ventriculograms revealedapeculiar shapeof theLVresembling aTakotsubo [the type of bottle with a round bottom and narrow neck — used in Japan for trapping octopus]. The syndrome is also known as acute left ventricular ballooning, transient apical ballooning, ampulla cardiomyopathy and because stress has been implicated in its pathophysiology. Human stress cardiomyopathy or broken-heart syndrome. We have earlier described the occurrence of Takotsubo syndrome in a patient after mitral valve replacement [4]. Diagnostic criteria for Takotsubo syndrome, viz. (a) new electrocardiographic abnormalities; either STelevations or T wave inversion, (b) absence of obstructive coronary artery disease, and (c) transient akinesia or dyskinesia of the left ventricle, etc. have been described [5]. Despite the characteristic, near pathognomonic pattern of LV wall motion abnormalities and the typical course of the syndrome, the clinician needs to differentiate it from three

Influence of the crucible bottom shape on the heat transport and fluid flow during the seeding process of oxide Czochralski crystal growth

AbstractFor the seeding process of oxide Czochralski crystal growth, influence of the crucible bottom shape on the heat generation, temperature and flow field of the system and the seed‐melt interface shape have been studied numerically using the finite element method. The configuration usually used in a real Czochralski crystal growth process consists of a crucible, active afterheater, induction coil with two parts, insulation, melt, gas and seed crystal. At first, the volumetric distribution of heat inside the metal crucible and afterheater inducted by the RF‐coil was calculated. Using this heat generation in the crucible wall as a source the fluid flow and temperature field of the entire system as well as the seed‐melt interface shape were determined. We have considered two cases, flat and rounded crucible bottom shape. It was observed that using a crucible with a rounded bottom has several advantages such as: (i) The position of the heat generation maximum at the crucible side wall moves upwards, compared to the flat bottom shape. (ii) The location of the temperature maximum at the crucible side wall rises and as a result the temperature gradient along the melt surface increases. (iii) The streamlines of the melt flow are parallel to the crucible bottom and have a curved shape which is similar to the rounded bottom shape. These important features lead to increasing thermal convection in the system and influence the velocity field in the melt and gas domain which help preventing some serious growth problems such as spiral growth. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Tako-Tsubo Phenomenon: Dual-Source Computed Tomography and Conventional Coronary Angiography

A 75-year-old woman was admitted to our emergency department with severe chest pain and mild dyspnea that started soon after a marital quarrel with her husband. Her 12-lead electrocardiogaphy (ECG) demonstrated an ST elevation in the precordial leads, and the plasma troponin T level was elevated to 0.29 ng/mL. An acute myocardial infarction with ST elevation was assumed, and the patient was admitted for an emergency catheter coronary angiography. Her coronary arteries were normal, but left ventriculography demonstrated a severe systolic dysfunction involving the mid and apical myocardial segments, showing apical ballooning (Supplemental Data Movie I). Thus, the diagnosis of tako-tsubo phenomenon was made. The ejection fraction was decreased to 40%. Because of a history of pulmonary embolism, the patient was referred for chest computed tomography (CT) to rule out a recurrent pulmonary embolism. Contrast-enhanced, retrospectively ECG-gated dual-source CT excluded pulmonary embolism and similarly demonstrated systolic dysfunction with apical ballooning (Supplemenal Data Movie II). After 2 days, the patient’s symptoms ceased and echocardiography showed normalized left ventricular systolic function. Tako-tsubo phenomenon is characterized by transient left ventricular systolic dysfunction of unknown etiology. Initially described in Japan [1] and named after an octopustrapping pot with a round bottom and a narrow neck, it has also been recognized more and more in Western countries [2, 3]. It predominantly occurs in postmenopausal women and frequently after emotional stress. Importantly, such a psychological stressful trigger is not observed in all individuals, and its absence does not exclude the diagnosis. A few cases have been reported in younger women and males [4]. Rarely, patients present with syncope or an out-ofhospital cardiac arrest. Symptoms clinically mimic acute myocardial infarction, with the majority of patients presenting with chest pain, dyspnea, and mild ST-segment elevation in precordial leads followed by T-wave inversion involving most leads [2]. Plasma troponin T levels are either normal or only slightly increased and thus contrast the extent of systolic dysfunction in the acute phase. Patients are usually hemodynamically stable, but clinical findings of mild to moderate congestive heart failure commonly coexist. In a minority of patients, hypotension may occur from reduced stroke volume and occasionally from dynamic left ventricular outflow tract obstruction. Cardiogenic shock may be a rare complication. Patients with tako-tsubo phenomenon generally have a good prognosis in the absence of significant underlying comorbidity. The systolic dysfunction and the regional wall-motion abnormalities are transient and full recovery usually occurs in most cases without specific therapy [2, 3]. The initial imaging for patients with tako-tsubo phenomenon is usually performed with catheter coronary angiography because of the clinical suspicion of acute myocardial infarction. The left ventriculogram shows characteristic regional wall-motion abnormalities involving the mid and apical segments (Fig. 1). There is sparing of the basal systolic function, and the wall-motion abnormality extends beyond the distribution of any one single coronary artery. Apical sparing variants have also been described [5]. Echocardiography and magnetic resonance H. Scheffel P. Stolzmann C. Karlo B. Marincek H. Alkadhi (&) Institute of Diagnostic Radiology, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland e-mail: hatem.alkadhi@usz.ch

Live Cell Imaging of Outward and Inward Vesiculation Induced by the Complement C5b-9 Complex

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.

Distinct Uptake Mechanisms but Similar Intracellular Processing of Two Different Toll-like Receptor Ligand-Peptide Conjugates in Dendritic Cells

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.

Apical Ballooning Syndrome

Case presentation: A 60-year-old woman presented to the emergency department 2 hours after the onset of severe retrosternal chest pain that started soon after she was told that her son had died in a car accident. A 12-lead ECG demonstrated ST-elevation in the precordial leads (Figure 1), and the plasma troponin T level was elevated at 0.07 ng/mL. A diagnosis of acute ST-elevation myocardial infarction was made, and the patient was admitted for emergency coronary angiography, which revealed normal coronary arteries. The left ventriculogram showed severe systolic dysfunction involving the mid and apical segments (Data Supplement Movie I). Figure 1. Twelve-lead ECG demonstrating ST-segment elevation in precordial leads. Physicians have long been aware of the possible association between stress and cardiovascular events. Awareness has increased of a distinct cardiac syndrome that was originally described in the Japanese population and was called Takotsubo cardiomyopathy, named after the octopus-trapping pot with a round bottom and narrow neck that resembles the left ventriculogram during systole in these patients.1,2 Other names used to describe the condition include apical ballooning syndrome (ABS), broken heart syndrome, and stress or ampulla cardiomyopathy. The precise incidence of ABS is unknown, but it may account for 1% to 2% of patients who present with an acute myocardial infarction.3 The majority of patients have a clinical presentation that is indistinguishable from an acute coronary syndrome. Most present with chest pain at rest, although some patients have dyspnea alone as their initial presenting symptom. Rarely, patients present with syncope or an out-of-hospital cardiac arrest.4 ABS appears to occur almost exclusively in postmenopausal women; however, a few cases have been reported in younger women and males.4 The patients are usually hemodynamically stable, but clinical findings of mild-to-moderate congestive heart failure …

Hypoallergens for Allergen-specific Immunotherapy by Directed Molecular Evolution of Mite Group 2 Allergens

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.

Takotsubo-Like Left Ventricular Dysfunction in an HIV-Infected Patient

Takotsubo cardiomyopathy (in Japanese language "takotsubo" is a fishing pot with a round bottom and a neck that is used for trapping octopuses) is a new syndrome, which is characterized by transient left ventricular dysfunction and by a typical left ventriculogram showing transient extensive akinesis of the apical and mid portions of the left ventricle with hypercontraction of the basal segment, from which this disease takes its name. Since 1990 sporadic cases of takotsubo cardiomyopathy were reported by Japanese authors, and only a few European reports are available. We report a case of takotsubo-like left ventricular dysfunction in an human immunodeficiency virus (HIV)-infected caucasian patient.

Inhibition of Intrinsic Thrombin Generation

Background: The contact phase of coagulation is of physiologic/pathophysiologic importance, whenever unphysiologic polynegative substances such as cell fragments (microparticles) get in contact with blood. There are several clinically used inhibitors of intrinsic thrombin generation. Here the inhibitory concentrations 50% (IC50) of these anticoagulants are measured by the highly specific thrombin generation assay INCA. Methods: Unfrozen pooled normal citrated plasma in polystyrole tubes was supplemented at 23°C in duplicate with 0–2 IU/ml low molecular weight heparin (dalteparin), 0–2 IU/ml unfractionated heparin, 0–500 KIU/ml aprotinin, or 0–40 mM arginine. 50 μl plasma or 1 IU/ml thrombin standard were pipetted into a polystyrole microtiter plate with flat bottom. 5 μl SiO2/CaCl2 - reagent (INCA activator) were added and after 0–30 min incubation at 37°C 100 μl 2.5 M arginine, pH 8.6, were added; arginine inhibits hemostasis activation and depolymerizes generated fibrin within 20 min at 23°C. The in the physiologic 37°C incubation phase generated thrombin was then chromogenically detected. The intra-assay CV values were < 5%. Results and Discussion: The approximate IC50 were 0.01 IU/ml dalteparin, 0.02 IU/ml heparin, 25 KIU/ml aprotinin, and 12 mM arginine. The efficiency of any anticoagulant on intrinsic thrombin generation should be measured for each individual patient. Abbreviations: IIa, thrombin; ΔA, increase in absorbance; APTT, activated partial thromboplastin time; CRT, coagulation reaction time (at 37°C in water-bath); F-wells, polystyrole microtiter plates with flat bottom; IC50, inhibitory concentration 50%; INCA, intrinsic coagulation activity assay; IU, international units; KIU, kallikrein inhibiting unis; LMWH, low molecular weight heparin; mA, milli-absorbance units; PSL, pathromtin SL®; RT, room temperature (23°C); U-wells, polystyrole microtiter plates with round bottom.

Ferrous Ions and Reactive Oxygen Species Increase Antigen-binding and Anti-inflammatory Activities of Immunoglobulin G

Polyspecific antibodies represent a first line of defense against infection and regulate inflammation, properties hypothesized to rely on their ability to interact with multiple antigens. We demonstrated that IgG exposure to pro-oxidative ferrous ions or to reactive oxygen species enhances paratope flexibility and hydrophobicity, leading to expansion of the spectrum of recognized antigens, regulation of cell proliferation, and protection in experimental sepsis. We propose that ferrous ions, released from transferrin and ferritin at sites of inflammation, synergize with reactive oxygen species to modify the immunoglobulins present in the surrounding microenvironment, thus quenching pro-inflammatory signals, while facilitating neutralization of pathogens.

Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

Studies involving Toll-like receptor 3 (TLR3)-deficient mice suggest that this receptor binds double-stranded RNA. In the present study, we analyzed ligand/receptor interactions and receptor-proximal events leading to TLR3 activation. The mutagenesis approach showed that certain cysteine residues and glycosylation in TLR3 amino-terminal leucine-rich repeats were necessary for ligand-induced signaling. Furthermore, inactive mutants had a dominant negative effect, suggesting that the signaling module is a multimer. We constructed a chimeric molecule fusing the amino-terminal ectodomain of TLR3 to the transmembrane and carboxyl terminal domains of CD32a containing an immunoreceptor tyrosine-based motif. Expression of TLR3-CD32 in HEK293T cells and the myeloid cell line U937 resulted in surface localization of the receptor, whereas the nonrecombinant molecule was intracellularly localized. The synthetic double-stranded RNAs poly(I-C) and poly(A-U) induced calcium mobilization in a TLR3-CD32 stably transfected U937 clone but not in control cells transfected with other constructs. An anti-TLR3 antibody also induced Ca(2+) flux but only when cross-linked by a secondary anti-immunoglobulin antibody, confirming that multimerization by the ligand is a requirement for signaling. The inhibitors of lysosome maturation, bafilomycin and chloroquine, inhibited the poly(I-C)-induced biological response in immune cells, showing that TLR3 interacted with its ligand in acidic subcellular compartments. Furthermore, TLR3-CD32 activation with poly(I-C) was only observed within a narrow pH window (pH 5.7-6.7), whereas anti-TLR3-mediated Ca(2+) flux was pH-insensitive. The importance of an acidic pH for TLR3-ligand interaction becomes critical when using oligomeric poly(I-C) (15-40-mers). These observations demonstrate that engagement of TLR3 by poly(I-C) at an acidic pH, probably in early phagolysosomes or endosomes, induces receptor aggregation leading to signaling.

Recognition of the Apical Ballooning Syndrome in the United States

Acute dilated cardiomyopathy is an uncommon but often life-threatening cardiovascular event. Despite varied clinical presentations that may include cardiogenic shock, ventricular tachyarrhythmias, and chest pain mimicking acute myocardial infarction, a growing list of potentially reversible pathogeneses should be considered in the initial differential diagnosis. These may include cardiotoxins, such as excessive alcohol, cocaine, or antiretroviral agents; nutritional deficiencies (eg, thiamine, selenium, carnitine); endocrine disturbances (hyperthyroidism, hypothyroidism, pheochromocytoma); viral myocarditis; Lyme carditis; hypersensitivity reactions; infiltrative processes, such as hemochromatosis; peripartum disease; and tachycardia-induced cardiomyopathy.1 Recently, a newly described left ventricular apical ballooning syndrome has been added to the growing list of diagnostic possibilities.2 See p 472 The apical ballooning syndrome is characterized by the abrupt onset of angina-like chest pain, ECG changes that typically demonstrate ST-segment elevation, diffuse T-wave inversions and abnormal QS-wave development, discrete wall motion abnormalities involving the lower anterior wall and apex on echocardiography or left ventriculography, and limited myocardial enzyme release relative to the extent of ventricular akinesia. The clinical presentation mimics acute myocardial infarction but always occurs in patients without evidence for hemodynamically significant coronary arterial stenoses by angiography. It was first described in the Japanese literature in 1991 by Dote and colleagues,2 who proposed the term “tako-tsubo” (Japanese for octopus trap) cardiomyopathy on the basis of the peculiar appearance of a rounded bottom and narrow neck on the end-systolic left ventriculogram. The clinical features and outcomes of the syndrome have been well characterized in Asian populations in studies by Kurisu et al3 (n=30 patients) and Abe et al4 (n=17 patients) and in an important multicenter observational study of 88 patients by Tsuchihashi et al.5 The “tako-tsubo” syndrome has been reported to account for 1% of admissions for suspected acute myocardial infarction in Japan during the past decade.5 A …

Une pratique funéraire originale du Néolithique moyen bourguignon : des vestiges de crémation à Lyon (Rhône)

Un secteur du quartier de Vaise (Lyon, Rhone) a fait l'objet d'une fouille preventive au 51-53 quai Paul Sedallian. En rive droite de la Saone, a environ six kilometres au nord de la confluence actuelle de cette riviere avec le fleuve Rhone, l'occupation attribuee au Neolithique moyen bourguignon (NMB) se caracterise par un sol anthropique SL303, une fosse FS229 et l'ensemble E246. SL303, observe sur une faible extension (43 m2), pourrait faire partie de l'installation caracterisee par la fosse FS229 et l'ensemble E246, qui livrent des residus de cremation. Un os humain brule provient de SL303. Les os ont ete brules a l'etat frais et sont en majorite ceux d'un adulte, seuls quelques elements appartiennent a un enfant. L'occupation NMB est difficile a caracteriser, cependant FS229 et E246 se rapportent a une activite funeraire et peu d'elements plaident en faveur d'un habitat. Parmi le corpus ceramique, coupes et ecuelles representent l'essentiel des recipients identifies (dix sur onze), une seule jarre a ete reconnue. L'echantillon semble incomplet ou biaise. Soit l'apparente selection de ce mobilier est a mettre en relation avec l'etat de conservation du site (fort impact destructeur des amenagements industriels), soit elle depend de la nature de l'installation; les recipients traduiraient dans ce cas un choix en relation avec les gestes funeraires. En outre, la repartition des types par ensemble archeologique permet d'opposer la serie des recipients issus du sol SL303 a celle des excavations (E246 et FS229). La difference entre les formes representees dans chaque contexte est sans doute fonctionnelle et conduit a interpreter le sol SL303 comme un niveau de circulation, alors que E246 et FS229 se rapportent a des depots de vestiges de cremation. D'un point de vue archeologique, les faits E246, FS229 et SL303 sont lies. La typologie de la ceramique, ainsi que leur contenu (os humains brules), assurent la synchronie de E246 et de FS229. Le sol SL303, proche, mais sans relation stratigraphique, appartient a la meme phase chronoculturelle. Pour affirmer le lien archeologique entre les vestiges, un datage par le Centre de datation radiocarbone de Lyon sur os humains brules a ete realise. Le resultat (Lyon-2164 (Oxa) : 5080 +/- 30 BP), en adequation avec la typologie ceramique NMB, assure l'attribution chronoculturelle et l'association des recipients avec les os humains brules. Les vestiges de Sedallian illustrent la seule cremation certifiee pour le NMB. Le corpus ceramique et la date sont en concordance pour attribuer cette occupation a une phase ancienne de ce groupe culturel, en position meridionale par rapport a la zone eponyme de la Franche-Comte et de la Bourgogne.