bluuebunny/crossref_metadata_2025 huggingface dataset:
Mirror of the soul: a cortical stimulation study on recognition of facial emotions
Authors: Giussani Carlo , Pirillo David , Roux Franck-Emmanuel
Published: June 2009

Object

The capability of recognizing the expressions of facial emotions has been hypothesized to depend on a right hemispheric cortical-subcortical network. Its impairment deeply disturbs social relationships. To spare right hemispheric cortical areas involved in recognizing facial emotion, the authors used intraoperative cortical stimulation and the awake surgery technique in a consecutive series of patients. The feasibility and the interest to map them during brain mapping for neurosurgical procedures are discussed.

Methods

After a preoperative neuropsychological evaluation, 18 consecutive patients with right hemispheric lesions (5 metastases, 6 high-grade gliomas, 4 low-grade gliomas, 2 arteriovenous malformations, and 1 malignant meningioma) were tested by intraoperative cortical stimulation while performing a facial emotion recognition task along with sensorimotor and visuospatial tasks.

Results

Three hundred eighty-six cortical sites were studied. Five (1.30%) reproducible interference sites for facial emotion recognition were identified in 5 patients: 1 site in the medial segment of T1; 1 site in the posterior segment of T1; 1 site in the posterior segment of T2; and 2 sites in the supramarginal gyrus. No selective impairment was found regarding the emotion category. All facial emotion recognition sites were spared during surgery, and none of the patients experienced postoperative deficits in recognition of facial emotions.

Conclusions

The finding of interference sites in facial emotion recognition in the right posterior perisylvian area, independent to sensorimotor or visuospatial orientation processes, reinforces the theory about the role of anatomically and functionally segregated right hemisphere structures in this cognitive process. The authors advocate offering a brain mapping of facial emotion recognition to patients with right posterior perisylvian tumors.
Phase II study of ifosfamide, carboplatin, and etoposide in patients with a first recurrence of glioblastoma multiforme
Authors: Aoki Tomokazu , Mizutani Tomohiko , Nojima Kuniharu , Takagi Takehisa , Okumura Ryosuke , Yuba Yoshiaki , Ueba Tetsuya , Takahashi Jun A. , Miyatake Shin-Ichi , Nozaki Kazuhiko , Taki Waro , Matsutani Masao
Published: June 2009

Object

The prognosis of recurrent glioblastoma multiforme (GBM) remains unsatisfactory. The authors conducted a Phase II study of ifosfamide, carboplatin, and etoposide (ICE) for a first recurrence of GBM to determine whether it prolonged a patient's good-quality life.

Methods

This trial was an open-label, single-center Phase II study. Forty-two patients with a first GBM relapse after surgery followed by standard radiotherapy (60 Gy) and first-line temozolomide- or nimustine-based chemotherapy were eligible to participate. The primary end point was progression-free survival at 6 months after the ICE treatment (PFS-6), and secondary end points were response rate, toxicity, and overall survival. Chemotherapy consisted of ifosfamide (1000 mg/m2 on Days 1, 2, and 3), carboplatin (110 mg/m2 on Day 1), etoposide (100 mg/m2 on Days 1, 2, and 3), every 6 weeks.

Results

Progression-free survival at 6 months after ICE treatment was 35% (95% CI 22–50%). The median duration of PFS was 17 weeks (95% CI 10–24 weeks). The response rate was 25% (95% CI 9–34%). Adverse events were generally mild and consisted mainly of alopecia.

Conclusions

This regimen was well tolerated and has some activity and could be one of the options for patients with recurrent GBM.
Health Risk and Biological Effects of Cardiac Ionising Imaging: From Epidemiology to Genes
Authors: Foffa Ilenia , Cresci Monica , Andreassi Maria Grazia
Published: June 2009

Cardiac diagnostic or therapeutic testing is an essential tool for diagnosis and treatment of cardiovascular disease, but it also involves considerable exposure to ionizing radiation. Every exposure produces a corresponding increase in cancer risk, and risks are highest for radiation exposure during infancy and adolescence. Recent studies on chromosomal biomarkers corroborate the current radioprotection assumption showing that even modest radiation load due to cardiac catheter-based fluoroscopic procedures can damage the DNA of the cell. In this article, we review the biological and clinical risks of cardiac imaging employing ionizing radiation. We also discuss the perspectives offered by the use of molecular biomarkers in order to better assess the long-term development of health effects.
Lattice Strain Due to an Atomic Vacancy
Authors: Li Shidong , Sellers Michael  S. , Basaran Cemal , Schultz Andrew  J. , Kofke David  A.
Published: June 2009

Volumetric strain can be divided into two parts: strain due to bond distance change and strain due to vacancy sources and sinks. In this paper, efforts are focused on studying the atomic lattice strain due to a vacancy in an FCC metal lattice with molecular dynamics simulation (MDS). The result has been compared with that from a continuum mechanics method. It is shown that using a continuum mechanics approach yields constitutive results similar to the ones obtained based purely on molecular dynamics considerations.
Two New Steroidal Saponins from Allium macrostemon Bunge and Their Cytotoxity on Different Cancer Cell Lines
Authors: Chen Hai-Feng , Wang Guang-Hui , Luo Qiang , Wang Nai-Li , Yao Xin-Sheng
Published: June 2009

Two new steroidal saponins (1 and 2) were isolated from the dried bulbs of Allium macrostemon Bunge. Their structures were elucidated by the spectral data as 26-O-β-D-glucopyranosyl-5α-furost-25 (27)-ene-3β, 12β, 22, 26-tetraol-3-O-β-D-glucopyranosyl (1→2) [β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D-galactopyranoside (1) and 26-O-β-D-glucopyranosyl-5β-furost-20 (22)-25 (27)-dien-3β, 12β, 26-triol-3-O-β-D-glucopyranosyl (1→2)-β-D-galactopyranoside (2), respectively. Their cytotoxic activities on several cancer cell lines (MCF-7, NCI-H460, SF-268 and HepG2) were tested. 1 showed special cytotoxity on SF-268, while 2 showed cytotoxity on NCI-H460 and SF-268 cell lines, respectively.
On the Relevance of Using OpenWireless Sensor Networks in Environment Monitoring
Authors: Bagula Antoine B. , Inggs Gordon , Scott Simon , Zennaro Marco
Published: June 2009

This paper revisits the problem of the readiness for field deployments of wireless- sensor networks by assessing the relevance of using Open Hardware and Software motes for environment monitoring. We propose a new prototype wireless sensor network that finetunes SquidBee motes to improve the life-time and sensing performance of an environment monitoring system that measures temperature, humidity and luminosity. Building upon two outdoor sensing scenarios, we evaluate the performance of the newly proposed energy-aware prototype solution in terms of link quality when expressed by the Received Signal Strength, Packet Loss and the battery lifetime. The experimental results reveal the relevance of using the Open Hardware and Software motes when setting up outdoor wireless sensor networks.
Applications of Remote Sensing to Alien Invasive Plant Studies
Authors: Huang Cho-ying , Asner Gregory  P.
Published: June 2009

Biological invasions can affect ecosystems across a wide spectrum of bioclimatic conditions. Therefore, it is often important to systematically monitor the spread of species over a broad region. Remote sensing has been an important tool for large-scale ecological studies in the past three decades, but it was not commonly used to study alien invasive plants until the mid 1990s. We synthesize previous research efforts on remote sensing of invasive plants from spatial, temporal and spectral perspectives. We also highlight a recently developed state-of-the-art image fusion technique that integrates passive and active energies concurrently collected by an imaging spectrometer and a scanning-waveform light detection and ranging (LiDAR) system, respectively. This approach provides a means to detect the structure and functional properties of invasive plants of different canopy levels. Finally, we summarize regional studies of biological invasions using remote sensing, discuss the limitations of remote sensing approaches, and highlight current research needs and future directions.
A Novel Label-Free Optical Biosensor Using Synthetic Oligonucleotides from E. coli O157:H7: Elementary Sensitivity Tests
Authors: Bahşi Zehra Banu , Büyükaksoy Aligül , Ölmezcan Sinan Mert , Şimşek Fethi , Aslan Muhammed Hasan , Oral Ahmet Yavuz
Published: June 2009

SiO2-TiO2 thin films for use as fiber optic guiding layers of optical DNA biosensors were fabricated by the sol-gel dip coating technique. The chemical structure and the surface morphology of the films were characterized before immobilization. Single probe DNA strands were immobilized on the surface and the porosity of the films before the hybridization process was measured. Refractive index values of the films were measured using a Metricon 2010 prism coupler. On the surface of each film, 12 different spots were taken for measurement and calculation of the mean refractive index values with their standard deviations. The increased refractive index values after the immobilization of single DNA strands indicated that immobilization was successfully achieved. A further refractive index increase after the hybridization with target single DNA strands showed the possibility of detection of the E. coli O157:H7 EDL933 species using strands of 20-mers (5’-TAATATCGGTTGCGGAGGTG -3’) sequence.
Judgments of Critical Care Nurses About Risk for Secondary Brain Injury
Authors: McNett Molly , Doheny Margaret , Sedlak Carol A. , Ludwick Ruth
Published: June 2009

Background Interdisciplinary care for patients with traumatic brain injury focuses on treating the primary brain injury and limiting further brain damage from secondary injury. Intensive care unit nurses have an integral role in preventing secondary brain injury; however, little is known about factors that influence nurses’ judgments about risk for secondary brain injury.

Objective To investigate which physiological and situational variables influence judgments of intensive care unit nurses about patients’ risk for secondary brain injury, management solely with nursing interventions, and management by consulting another member of the health care team.

Methods A multiple segment factorial survey design was used. Vignettes reflecting the complexity of real-life scenarios were randomly generated by using different values of each independent variable. Surveys containing the vignettes were sent to nurses at 2 level I trauma centers. Multiple regression was used to determine which variables influenced judgments about secondary brain injury.

Results Judgments about risk for secondary brain injury were influenced by a patient’s oxygen saturation, intracranial pressure, cerebral perfusion pressure, mechanism of injury, and primary diagnosis, as well as by nursing shift. Judgments about interventions were influenced by a patient’s oxygen saturation, intracranial pressure, and cerebral perfusion pressure and by nursing shift. The initial judgments made by nurses were the most significant variable predictive of follow-up judgments.

Conclusions Nurses need standardized, evidence-based content for management of secondary brain injury in critically ill patients with traumatic brain injury.
Production of Both IL-27 and IFN-γ after the Treatment with a Ligand for Invariant NK T Cells Is Responsible for the Suppression of Th2 Response and Allergic Inflammation in a Mouse Experimental Asthma Model
Authors: Fujita Hiroyuki , Teng Annabelle , Nozawa Risa , Takamoto-Matsui Yukiko , Katagiri-Matsumura Haruka , Ikezawa Zenro , Ishii Yasuyuki
Published: June 2009

Using an allergen-induced airway inflammation model, we show that an injection of α-galactosylceramide (α-GalCer), a ligand for invariant NK T (iNKT) cells, induced IL-27 and that this process is essential for the attenuation of the Th2 response. After the systemic administration of α-GalCer into the mice primed with OVA in alum, Th2 cytokine production of OVA-primed CD4+ T cells in their lymph nodes, IgG1 and IgE Ab formation, and infiltration of eosinophils in bronchoalveolar lavage after the OVA challenge were suppressed. Systemic administration of rIFN-γ into OVA-primed mice could not reproduce these effects of α-GalCer. IL-27p28 was detected both in the culture supernatant of α-GalCer-stimulated spleen cells and in the serum of the α-GalCer-treated mice, but not in the iNKT cell-deficient mice. Splenic iNKT cells produced IL-27p28 in the culture supernatant upon stimulation with PMA plus ionomycin, although the transcript of IL-27p28 in the iNKT cells was constitutively expressed regardless of the stimulation. By contrast, the transcript of IL-27EBI3 was induced in the iNKT cells upon stimulation with PMA plus ionomycin in vitro and with α-GalCer treatment in vivo, suggesting that IL-27 (p28/EBI3) could be produced by iNKT cells in an activation-dependent manner. Although repeated injections of rIL-27 did not substitute for the effects of a single injection of α-GalCer, administration of rIL-27 along with rIFN-γ reproduced in vivo effects of the α-GalCer injection. These data indicate that production of both IL-27 and IFN-γ by the α-GalCer treatment is responsible for suppression of the Th2 response and allergic inflammation.
Gap Junctions at the Dendritic Cell-T Cell Interface Are Key Elements for Antigen-Dependent T Cell Activation
Authors: Elgueta Raul , Tobar Jaime A. , Shoji Kenji F. , De Calisto Jaime , Kalergis Alexis M. , Bono Maria R. , Rosemblatt Mario , Sáez Juan C.
Published: June 2009

The acquired immune response begins with Ag presentation by dendritic cells (DCs) to naive T cells in a heterocellular cell-cell contact-dependent process. Although both DCs and T cells are known to express connexin43, a gap junction protein subunit, the role of connexin43 on the initiation of T cell responses remains to be elucidated. In the present work, we report the formation of gap junctions between DCs and T cells and their role on T cell activation during Ag presentation by DCs. In cocultures of DCs and T cells, Lucifer yellow microinjected into DCs is transferred to adjacent transgenic CD4+ T cells, only if the specific antigenic peptide was present at least during the first 24 h of cocultures. This dye transfer was sensitive to gap junction blockers, such as oleamide, and small peptides containing the extracellular loop sequences of conexin. Furthermore, in this system, gap junction blockers drastically reduced T cell activation as reflected by lower proliferation, CD69 expression, and IL-2 secretion. This lower T cell activation produced by gap junction blockers was not due to a lower expression of CD80, CD86, CD40, and MHC-II on DCs. Furthermore, gap junction blocker did not affect polyclonal activation of T cell induced with anti-CD3 plus anti-CD28 Abs in the absence of DCs. These results strongly suggest that functional gap junctions assemble at the interface between DCs and T cells during Ag presentation and that they play an essential role in T cell activation.
Vav1 Regulates the Migration and Adhesion of Dendritic Cells
Authors: Spurrell David R. , Luckashenak Nancy A. , Minney Derek C. , Chaplin Anna , Penninger Joseph M. , Liwski Robert S. , Clements James L. , West Kenneth A.
Published: June 2009

Dendritic cells (DCs) are the most potent APCs for activating naive T cells, a process facilitated by the ability of immature DCs to mature and home to lymph nodes after encountering an inflammatory stimulus. Proteins involved in cytoskeletal rearrangement play an important role in regulating the adherence and motility of DCs. Vav1, a guanine nucleotide exchange factor for Rho family GTPases, mediates cytoskeletal rearrangement in hematopoietic cells following integrin ligation. We show that Vav1 is not required for the normal maturation of DCs in vitro; however, it is critical for DC binding to fibronectin and regulates the distribution but not the formation of podosomes. We also found that DC Vav1 was an important component of a signaling pathway involving focal adhesion kinase, phospholipase C-γ2, and ERK1/2 following integrin ligation. Surprisingly, Vav1−/− DCs had increased rates of migration in vivo compared with wild-type control DCs. In vitro findings show that the presence of adhesive substrates such as fibronectin resulted in inhibition of migration. However, there was less inhibition in the absence of Vav1. These findings suggest that DC migration is negatively regulated by adhesion and integrin-mediated signaling and that Vav1 has a central role in this process.
IRAK4 Kinase Activity Is Required for Th17 Differentiation and Th17-Mediated Disease
Authors: Staschke Kirk A. , Dong Sucai , Saha Joy , Zhao Jingyong , Brooks Nathan A. , Hepburn Deena L. , Xia Jinqi , Gulen Muhammet F. , Kang Zizhen , Altuntas Cengiz Z. , Tuohy Vincent K. , Gilmour Raymond , Li Xiaoxia , Na Songqing
Published: June 2009

Both IL-23- and IL-1-mediated signaling pathways play important roles in Th17 cell differentiation, cytokine production, and autoimmune diseases. The IL-1R-associated kinase 4 (IRAK4) is critical for IL-1/TLR signaling. We show here that inactivation of IRAK4 kinase in mice (IRAK4 KI) results in significant resistance to experimental autoimmune encephalomyelitis due to a reduction in infiltrating inflammatory cells into the CNS and reduced Ag-specific CD4+ T cell-mediated IL-17 production. Adoptive transfer of myelin oligodendrocyte glycoprotein 35–55-specific IRAK4 KI Th17 cells failed to induce experimental autoimmune encephalomyelitis in either wild-type or IRAK4 KI recipient mice, indicating the lack of autoantigen-specific Th17 cell activities in the absence of IRAK4 kinase activity. Furthermore, the absence of IRAK4 kinase activity blocked induction of IL-23R expression, STAT3 activation by IL-23, and Th17 cytokine expression in differentiated Th17 cells. Importantly, blockade of IL-1 signaling by IL-1RA inhibited Th17 differentiation and IL-23-induced cytokine expression in differentiated Th17 cells. The results of these studies demonstrate that IL-1-mediated IRAK4 kinase activity in T cells is essential for induction of IL-23R expression, Th17 differentiation, and autoimmune disease.
Soluble TLR2 Reduces Inflammation without Compromising Bacterial Clearance by Disrupting TLR2 Triggering
Authors: Raby Anne-Catherine , Le Bouder Emmanuel , Colmont Chantal , Davies James , Richards Peter , Coles Barbara , George Christopher H. , Jones Simon A. , Brennan Paul , Topley Nicholas , Labéta Mario O.
Published: June 2009

TLR overactivation may lead to end organ damage and serious acute and chronic inflammatory conditions. TLR responses must therefore be tightly regulated to control disease outcomes. We show in this study the ability of the soluble form of TLR2 (sTLR2) to regulate proinflammatory responses, and demonstrate the mechanisms underlying sTLR2 regulatory capacity. Cells overexpressing sTLR2, or stimulated in the presence of the sTLR2 protein, are hyporesponsive to TLR2 ligands. Regulation was TLR2 specific, and affected NF-κB activation, phagocytosis, and superoxide production. Natural sTLR2-depleted serum rendered leukocytes hypersensitive to TLR2-mediated stimulation. Mice administered sTLR2 together with Gram-positive bacteria-derived components showed lower peritoneal levels of the neutrophil (PMN) chemoattractant, keratinocyte-derived chemokine; lower PMN numbers; and a reduction in late apoptotic PMN. Mononuclear cell recruitment remained unaffected, and endogenous peritoneal sTLR2 levels increased. Notably, the capacity of sTLR2 to modulate acute inflammatory parameters did not compromise the ability of mice to clear live Gram-positive bacteria-induced infection. Mechanistically, sTLR2 interfered with TLR2 mobilization to lipid rafts for signaling, acted as a decoy microbial receptor, and disrupted the interaction of TLR2 with its coreceptor, CD14, by associating with CD14. These findings establish sTLR2 as a regulator of TLR2-mediated inflammatory responses, capable of blunting immune responses without abrogating microbial recognition and may inform the design of novel therapeutics against acute and chronic inflammatory conditions.
Structural Basis for Proteolytic Specificity of the Human Apoptosis-Inducing Granzyme M
Authors: Wu Lianfeng , Wang Li , Hua Guoqiang , Liu Kan , Yang Xuan , Zhai Yujia , Bartlam Mark , Sun Fei , Fan Zusen
Published: June 2009

Granzyme M (GzmM), a unique serine protease constitutively expressed in NK cells, is important for granule-mediated cytolysis and can induce rapid caspase-dependent apoptosis of tumor cells. However, few substrates of GzmM have been reported to date, and the mechanism by which this enzyme recognizes and hydrolyzes substrates is unknown. To provide structural insights into the proteolytic specificity of human GzmM (hGzmM), crystal structures of wild-type hGzmM, the inactive D86N-GzmM mutant with bound peptide substrate, and the complexes with a catalytic product and with a tetrapeptide chloromethylketone inhibitor were solved to 1.96 Å, 2.30 Å, 2.17 Å and 2.70 Å, respectively. Structure-based mutagenesis revealed that the N terminus and catalytic triad of hGzmM are most essential for proteolytic function. In particular, D86N-GzmM was found to be an ideal inactive enzyme for functional studies. Structural comparisons indicated a large conformational change of the L3 loop upon substrate binding, and suggest this loop mediates the substrate specificity of hGzmM. Based on the complex structure of GzmM with its catalytic product, a tetrapeptide chloromethylketone inhibitor was designed and found to specifically block the catalytic activity of hGzmM.
The Presumed Hyporesponsive Behavior of Rheumatoid Arthritis T Lymphocytes Can Be Attributed to Spontaneous Ex Vivo Apoptosis rather than Defects in T Cell Receptor Signaling
Authors: Abreu Joana R. F. , Grabiec Aleksander M. , Krausz Sarah , Spijker René , Burakowski Tomasz , Maslinski Wlodzimierz , Eldering Eric , Tak Paul P. , Reedquist Kris A.
Published: June 2009

Genetic associations and the clinical success of compounds targeting TCR costimulatory proteins suggest an active role for TCR signaling in the initiation and perpetuation of rheumatoid arthritis (RA). Paradoxically, T cells isolated from affected joints in RA show impaired proliferative and cytokine responses following stimulation with mitogens and recall Ags attributed in part to chronic T cell exposure to oxidative stress and inflammatory cytokines. Therefore, it is uncertain how local autoreactive TCR signaling contributes to pathology in established RA. Using single-cell analysis, we show that in contrast to results obtained in bulk culture assays, T cells from the synovial fluid of RA patients proliferate and produce cytokines (IL-2, TNF-α, and IFN-γ) as efficiently, if not more so, than T cells isolated from healthy donors and RA patient peripheral blood following TCR/CD28 stimulation. RA synovial fluid T cell hyporesponsiveness observed in bulk cultures can be attributed to spontaneous apoptosis ex vivo, which is associated with altered ratios of proapoptotic Noxa and anti-apoptotic Mcl-1 expression. The absence of RA synovial T cell proliferation and cytokine production in situ, despite the capacity of these cells to support productive TCR signaling, suggests that T cells contribute to local pathology in established RA by TCR-independent mechanisms.
Sphingosine Kinase1 Is Pivotal for FcεRI-Mediated Mast Cell Signaling and Functional Responses In Vitro and In Vivo
Authors: Pushparaj Peter N. , Manikandan Jayapal , Tay Hwee Kee , H'ng Shiau Chen , Kumar Srinivasan D. , Pfeilschifter Josef , Huwiler Andrea , Melendez Alirio J.
Published: June 2009

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcεRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcεRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcεRI stimulation including: Ca2+ signals, NFκB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1−/− and SPHK2−/− mice, which showed that SphK2 was required for FcεRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1−/− and SPHK2−/− mice and show that the calcium response and degranulation, triggered by FcεRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.
Human Placenta Expresses and Secretes NKG2D Ligands via Exosomes that Down-Modulate the Cognate Receptor Expression: Evidence for Immunosuppressive Function
Authors: Hedlund Malin , Stenqvist Ann-Christin , Nagaeva Olga , Kjellberg Lennart , Wulff Marianne , Baranov Vladimir , Mincheva-Nilsson Lucia
Published: June 2009

During mammalian pregnancy maternal-fetal tolerance involves a number of immunosuppressive factors produced by placenta. Recently, placenta-derived exosomes have emerged as new immune regulators in the maternal immune tolerance. Exosomes are membrane nanovesicles with defined morphology, which are secreted from endosomal multivesicular bodies (MVB) upon fusion with the plasma membrane. Previously, we reported that the MHC class I chain-related (MIC) proteins A and B, human ligands of the activating NK cell receptor NKG2D, are expressed by placenta, sorted to MVB of syncytiotrophoblast and probably released via MIC-bearing exosomes. In this report, we show that the second family of human NKG2D ligands, the UL-16 binding proteins (ULBP), is also expressed by placenta. Importantly, this expression was not due to placental CMV infection. Immunoelectron microscopy disclosed that ULBP1–5 are produced and retained in MVB of the syncytiotrophoblast on microvesicles/exosomes. Using human placenta explant cultures and different assays, we demonstrate that exosomes bearing NKG2D ligands are released by human placenta. Isolated placental exosomes carried ULBP1–5 and MIC on their surface and induced down-regulation of the NKG2D receptor on NK, CD8+, and γδ T cells, leading to reduction of their in vitro cytotoxicity without affecting the perforin-mediated lytic pathway. Release of placental NKG2D ligands via exosomes is an alternative mechanism for generation of bioactive soluble form of these ligands. These findings highlight a role for NKG2D ligand-bearing placental exosomes in the fetal immune escape and support the view of placenta as a unique immunosuppressive organ.
Altered Thymic Selection and Increased Autoimmunity Caused by Ectopic Expression of DRAK2 during T Cell Development
Authors: Gatzka Martina , Newton Ryan H. , Walsh Craig M.
Published: June 2009

Negative regulation of TCR signaling is an important mechanism enforcing immunological self-tolerance to prevent inappropriate activation of T cells and thus the development of autoimmune diseases. The lymphoid-restricted serine/threonine kinase death-associated protein-related apoptotic kinase-2 (DRAK2) raises the TCR activation threshold by targeting TCR-induced calcium mobilization in thymocytes and peripheral T cells and regulates positive thymic selection and peripheral T cell activation. Despite a hypersensitivity of peripheral drak2-deficient T cells, drak2-deficient mice are enigmatically resistant to induced autoimmunity in the model experimental autoimmune encephalomyelitis. To further evaluate the differential role of DRAK2 in central vs peripheral tolerance and to assess its impact on the development of autoimmune diseases, we have generated a transgenic (Tg) mouse strain ectopically expressing DRAK2 via the lck proximal promoter (1017-DRAK2 Tg mice). This transgene led to highest expression levels in double-positive thymocytes that are normally devoid of DRAK2. 1017-DRAK2 Tg mice displayed a reduction of single-positive CD4+ and CD8+ thymocytes in context with diminished negative selection in male HY TCR × 1017-DRAK2 Tg mice as well as peripheral T cell hypersensitivity, enhanced susceptibility to experimental autoimmune encephalomyelitis, and spontaneous autoimmunity. These findings suggest that alteration in thymocyte signaling thresholds impacts the sensitivity of peripheral T cell pools.
Early Life Exposure to Lipopolysaccharide Suppresses Experimental Autoimmune Encephalomyelitis by Promoting Tolerogenic Dendritic Cells and Regulatory T Cells
Authors: Ellestad Kristofor K. , Tsutsui Shigeki , Noorbakhsh Farshid , Warren Kenneth G. , Yong V. Wee , Pittman Quentin J. , Power Christopher
Published: June 2009

The rising incidence of autoimmune diseases such as multiple sclerosis (MS) in developed countries might be due to a more hygienic environment, particularly during early life. To investigate this concept, we developed a model of neonatal exposure to a common pathogen-associated molecular pattern, LPS, and determined its impact on experimental autoimmune encephalomyelitis (EAE). Mice exposed to LPS at 2 wk of age showed a delayed onset and diminished severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE, induced at 12 wk, compared with vehicle-exposed animals. Spinal cord transcript levels of CD3ε and F4/80 were lower in LPS- compared with PBS-exposed EAE animals with increased IL-10 levels in the LPS-exposed group. Splenic CD11c+ cells from LPS-exposed animals exhibited reduced MHC class II and CD83 expression but increased levels of CD80 and CD86 both before and during EAE. MOG-treated APC from LPS-exposed animals stimulated less T lymphocyte proliferation but increased expansion of CD4+FoxP3+ T cells compared with APC from PBS-exposed animals. Neuropathological studies disclosed reduced myelin and axonal loss in spinal cords from LPS-exposed compared with PBS-exposed animals with EAE, and this neuroprotective effect was associated with an increased number of CD3+FoxP3+ immunoreactive cells. Analyses of human brain tissue revealed that FoxP3 expression was detected in lymphocytes, albeit reduced in MS compared with non-MS patients’ brains. These findings support the concept of early-life microbial exposure influencing the generation of neuroprotective regulatory T cells and may provide insights into new immunotherapeutic strategies for MS.
Neutrophil Elastase Represses IL-8/CXCL8 Synthesis in Human Airway Smooth Muscle Cells through Induction of NF-κB Repressing Factor
Authors: Ho Shu-Chuan , Lee Kang-Yun , Chan Yao-Fei , Kuo Lu-Wei , Ito Kazuhiro , Adcock Ian M. , Chen Bing-Chang , Sheu Joen-Rong , Lin Chien-Huang , Kuo Han-Pin
Published: June 2009

NF-κB repressing factor (NRF), a nuclear inhibitor of NF-κB, is constitutively expressed and is implicated in the basal silencing of specific NF-κB targeting genes, including IFN-β, IL-8/CXCL8, and iNOS. Little is known about the regulation of NRF and its role in response to stimuli. Airway smooth muscle (ASM) is a rich source of inflammatory mediators that may regulate the development and progression of airway inflammation. We have previously reported that NE activates NF-κB in primary human ASM (hASM), leading to induction of TGF-β1. In this study, we describe that, instead of inducing the NF-κB response gene IL-8/CXCL8, NE suppressed IL-8/CXCL8 release and mRNA expression in hASM cells. Transcriptional blockade studies using actinomycin D revealed a similar degradation rate of IL-8/CXCL8 mRNA in the presence or absence of NE, suggesting an involvement at the transcription level. Mechanistically, the NE repressive effect was mediated by inducing NRF, as shown by RT-PCR and Western blotting, which was subsequently recruited to the native IL-8/CXCL8 promoter leading to removal of RNA polymerase II from the promoter, as demonstrated by chromatin immunoprecipitation assays. Knockdown of NRF by small interfering RNA prevented NE-induced suppression of IL-8/CXCL8 expression. In contrast, NE did not induce NRF expression in A549 and Beas-2B cells, where NE only stimulates NF-κB activation and IL-8/CXCL8 induction. Forced expression of NRF in A549 cells by an NRF expression plasmid suppressed IL-8/CXCL8 expression. Hence, we describe a novel negative regulatory mechanism of NE-induced NRF, which is restricted to hASM and mediates the suppression of IL-8/CXCL8 expression.
The Coding ECP 434(G>C) Gene Polymorphism Determines the Cytotoxicity of ECP but Has Minor Effects on Fibroblast-Mediated Gel Contraction and No Effect on RNase Activity
Authors: Rubin Jenny , Zagai Ulrika , Blom Kristin , Trulson Agneta , Engström Åke , Venge Per
Published: June 2009

Eosinophil cationic protein (ECP) is a secretory protein of the eosinophil granulocyte, a cell involved in innate immunity. Functional studies have implicated ECP in numerous processes, such as tissue remodeling in allergic inflammation and cytotoxicity toward a variety of pathogens. Recent genetic studies have suggested that the ECP 434(G>C) polymorphism resulting in an arg97thr substitution would alter the function of ECP in vivo. Functional (in vitro) studies of ECP up until now have either been conducted with native preparations containing an unknown mixture of the ECP97arg and ECP97thr variants, or with recombinant proteins. Therefore, we have now for the first time extracted the native ECP97arg and ECP97thr variants from healthy blood donors and tested them functionally in vitro. Our results show that the arg97thr shift dramatically alters the cytotoxic capacity of ECP in vitro; the tested ECP97arg variants were cytotoxic toward the small-cell lung cancer cell line NCI-H69, whereas ECP97thr was noncytotoxic. RNase activity was unaffected by the arg97thr substitution. Both ECP97arg and ECP97thr stimulated fibroblast-mediated collagen gel contraction, an experimental model, which depicts wound healing, in a dose-dependent manner. In conclusion, our results demonstrate that the ECP 434(G>C) gene polymorphism affects the functional properties of native ECP, but also that there is a dissociation between different biological activities; the arg97thr substitution impairs the cytotoxic potential of ECP but less the gel contraction and not at all the RNase activity.
LL-37 Complexation with Glycosaminoglycans in Cystic Fibrosis Lungs Inhibits Antimicrobial Activity, Which Can Be Restored by Hypertonic Saline
Authors: Bergsson Gudmundur , Reeves Emer P. , McNally Paul , Chotirmall Sanjay H. , Greene Catherine M. , Greally Peter , Murphy Philip , O'Neill Shane J. , McElvaney Noel G.
Published: June 2009

There is an abundance of antimicrobial peptides in cystic fibrosis (CF) lungs. Despite this, individuals with CF are susceptible to microbial colonization and infection. In this study, we investigated the antimicrobial response within the CF lung, focusing on the human cathelicidin LL-37. We demonstrate the presence of the LL-37 precursor, human cathelicidin precursor protein designated 18-kDa cationic antimicrobial protein, in the CF lung along with evidence that it is processed to active LL-37 by proteinase-3. We demonstrate that despite supranormal levels of LL-37, the lung fluid from CF patients exhibits no demonstrable antimicrobial activity. Furthermore Pseudomonas killing by physiological concentrations of exogenous LL-37 is inhibited by CF bronchoalveolar lavage (BAL) fluid due to proteolytic degradation of LL-37 by neutrophil elastase and cathepsin D. The endogenous LL-37 in CF BAL fluid is protected from this proteolysis by interactions with glycosaminoglycans, but while this protects LL-37 from proteolysis it results in inactivation of LL-37 antimicrobial activity. By digesting glycosaminoglycans in CF BAL fluid, endogenous LL-37 is liberated and the antimicrobial properties of CF BAL fluid restored. High sodium concentrations also liberate LL-37 in CF BAL fluid in vitro. This is also seen in vivo in CF sputum where LL-37 is complexed to glycosaminoglycans but is liberated following nebulized hypertonic saline resulting in increased antimicrobial effect. These data suggest glycosaminoglycan–LL-37 complexes to be potential therapeutic targets. Factors that disrupt glycosaminoglycan–LL-37 aggregates promote the antimicrobial effects of LL-37 with the caveat that concomitant administration of antiproteases may be needed to protect the now liberated LL-37 from proteolytic cleavage.
Modulating the Expression of IFN Regulatory Factor 8 Alters the Protumorigenic Behavior of CD11b+Gr-1+ Myeloid Cells
Authors: Stewart Trina J. , Liewehr David J. , Steinberg Seth M. , Greeneltch Kristy M. , Abrams Scott I.
Published: June 2009

CD11b+Gr-1+-expressing cells, termed myeloid-derived suppressor cells, can mediate immunosuppression and tumor progression. However, the intrinsic molecular events that drive their protumorigenic behavior remain to be elucidated. Although CD11b+Gr-1+ cells exist at low frequencies in normal mice, it also remains unresolved whether they are biologically distinct from those of tumor-bearing hosts. These objectives were investigated using CD11b+Gr-1+ cells from both implantable (4T1) and autochthonous (mouse mammary tumor virus-polyomavirus middle T Ag (MMTV-PyMT)) mouse models of mammary carcinoma. Limited variation was observed in the expression of markers associated with immunoregulation between CD11b+Gr-1+ cells of both tumor models, as well as with their respective controls (Cnt). Despite limited differences in phenotype, tumor-induced CD11b+Gr-1+ cells were found to produce a more immunosuppressive cytokine profile than that observed by Cnt CD11b+Gr-1+ cells. Furthermore, when admixed with tumor cells, CD11b+Gr-1+ cells from tumor-bearing mice significantly enhanced neoplastic growth compared with counterpart cells from Cnt mice. However, the protumorigenic behavior of these tumor-induced CD11b+Gr-1+ cells was significantly diminished when the expression of IFN regulatory factor 8, a key myeloid-associated transcription factor, was enhanced. The loss of this protumorigenic effect occurred independently of the host immune system and correlated with a CD11b+Gr-1+ cytokine/chemokine production pattern that resembled cells from nontumor-bearing Cnt mice. Overall, our data indicate that 1) tumor-induced CD11b+Gr-1+ cells from both cancer models were phenotypically similar, but biologically distinct from their nontumor-bearing counterparts and 2) modulation of IFN regulatory factor 8 levels in tumor-induced CD11b+Gr-1+ cells can significantly abrogate their protumorigenic behavior, which may have important implications for cancer therapy.
Differential Regulation of P2X7 Receptor Activation by Extracellular Nicotinamide Adenine Dinucleotide and Ecto-ADP-Ribosyltransferases in Murine Macrophages and T Cells
Authors: Hong Shiyuan , Schwarz Nicole , Brass Anette , Seman Michel , Haag Friedrich , Koch-Nolte Friedrich , Schilling William P. , Dubyak George R.
Published: June 2009

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X7 purinergic receptor (P2X7R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X7R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X7R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X7R function in murine macrophages. Coexpression of the cloned murine P2X7R with ART2.1 or ART2.2 in HEK293 cells verified that P2X7R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X7R. Rather, NAD potentiated ATP-dependent P2X7R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X7R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X7R channel opening in T cells, this P2X7R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X7R signaling in murine macrophages and also suggest that the cellular context in which P2X7R signaling occurs differs between myeloid and lymphoid leukocytes.
CD11c+CD8α+ Dendritic Cells Promote Protective Immunity to Respiratory Infection withBordetella pertussis
Authors: Dunne Pádraic J. , Moran Barry , Cummins Robert C. , Mills Kingston H. G.
Published: June 2009

CD11c+CD8α+ and CD103+ dendritic cells (DC) have been shown to promote regulatory T cell responses and mediate tolerance in the gastrointestinal tract. These cells have also been identified in the lung, but their role in immunity to respiratory tract infection is not clear. In this study, we have used a murine model of infection with Bordetella pertussis to examine the function of DC subtypes in protective immunity in the lungs. We found a dramatic increase in the numbers of CD11c+CD8α+ DC in the cervical lymph nodes within 4 h of challenge with B. pertussis and these DC could acquire particulate Ag from the upper respiratory tract. CD11c+CD8α+ DC also infiltrated the lung with a peak 7 days after B. pertussis challenge. The infiltrating CD11c+CD8α+ DC expressed MHC, costimulatory and activation markers indicative of mature DC. The CD11c+CD8α+ DC in the cervical lymph nodes expressed IL-4 and IL-10 and lower levels of IFN-γ, but in the lungs expressed predominantly IFN-γ. Depletion of CD8α+ cells early in infection attenuated Th1 responses in the lungs and significantly reduced bacterial clearance. Conversely, transfer of FLT3 ligand (FL)-expanded CD11c+CD8α+ DC enhanced bacterial clearance, whereas GM-CSF-expanded conventional DC had no effect. The numbers of CD11c+CD8α+CD103+ cells were also increased during the early phase of infection. Blocking CD103 function caused a significant delay in bacterial clearance and a reduction in cellular infiltration into the lungs. These findings demonstrate that CD11c+CD8α+ and CD11c+CD103+DC play a protective role in mediating immunity to B. pertussis infection in the respiratory tract.
The Transmembrane E3 Ligase GRAIL Ubiquitinates and Degrades CD83 on CD4 T Cells
Authors: Su Leon L. , Iwai Hideyuki , Lin Jack T. , Fathman C. Garrison
Published: June 2009

Ubiquitination of eukaryotic proteins regulates a broad range of cellular processes, including T cell activation and tolerance. We have previously demonstrated that GRAIL (gene related to anergy in lymphocytes), a transmembrane RING finger ubiquitin E3 ligase, initially described as induced during the induction of CD4 T cell anergy, is also expressed in resting CD4 T cells. In this study, we show that GRAIL can down-modulate the expression of CD83 (previously described as a cell surface marker for mature dendritic cells) on CD4 T cells. GRAIL-mediated down-modulation of CD83 is dependent on an intact GRAIL extracellular protease-associated domain and an enzymatically active cytosolic RING domain, and proceeds via the ubiquitin-dependent 26S proteosome pathway. Ubiquitin modification of lysine residues K168 and K183, but not K192, in the cytoplasmic domain of CD83 was shown to be necessary for GRAIL-mediated degradation of CD83. Reduced CD83 surface expression levels were seen both on anergized CD4 T cells and following GRAIL expression by retroviral transduction, whereas GRAIL knock-down by RNA interference in CD4 T cells resulted in elevated CD83 levels. Furthermore, CD83 expression on CD4 T cells contributes to T cell activation as a costimulatory molecule. This study supports the novel mechanism of ubiquitination by GRAIL, identifies CD83 as a substrate of GRAIL, and ascribes a role for CD83 in CD4 T cell activation.
Immunization with the DNA-Encoding N-Terminal Domain of Proteophosphoglycan of Leishmania donovani Generates Th1-Type Immunoprotective Response against Experimental Visceral Leishmaniasis
Authors: Samant Mukesh , Gupta Reema , Kumari Shraddha , Misra Pragya , Khare Prashant , Kushawaha Pramod Kumar , Sahasrabuddhe Amogh Anant , Dube Anuradha
Published: June 2009

Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) which exist as secretory as well as surface-bound forms in both promastigotes and amastigotes. The structure and function of PPGs have been reported to be species and stage specific as in the case of Leishmania major and Leishmania mexicana; there has been no such information available for Leishmania donovani. We have recently demonstrated that PPG is differentially expressed in sodium stibogluconate-sensitive and -resistant clinical isolates of L. donovani. To further elucidate the structure and function of the ppg gene of L. donovani, a partial sequence of its N-terminal domain of 1.6 kb containing the majority of antigenic determinants, was successfully cloned and expressed in prokaryotic as well as mammalian cells. We further evaluated the DNA-encoding N-terminal domain of the ppg gene as a vaccine in golden hamsters (Mesocricetus auratus) against the L. donovani challenge. The prophylactic efficacy to the tune of ∼80% was observed in vaccinated hamsters and all of them could survive beyond 6 mo after challenge. The efficacy was supported by a surge in inducible NO synthase, IFN-γ, TNF-α, and IL-12 mRNA levels along with extreme down-regulation of TGF-β, IL-4, and IL-10. A rise in the level of Leishmania-specific IgG2 was also observed which was indicative of enhanced cellular immune response. The results suggest the N-terminal domain of L. donovani ppg as a potential DNA vaccine against visceral leishmaniasis.