bluuebunny/crossref_metadata_2025 huggingface dataset:
Intra-Refresh and Data-Partitioning for Video Streaming over IEEE 802.11e
Authors: Ali Ismail , Moiron Sandro , Fleury Martin , Ghanbari Mohammed
Published: April 2014

Intra-refresh macroblocks and data partitioning are two error-resilience tools aimed at video streaming over wireless networks. Intra-refresh macroblocks avoids the repetitive delays associated with periodic intra-coded frames, while also arresting temporal error propagation. Data-partitioning divides a compressed data stream according to the data importance, allowing packet prioritization schemes to be designed. This chapter reviews these and other error-resilience tools from the H.264 codec. As an illustration of the use of these tools, the chapter demonstrates a wireless access scheme that selectively drops packets that carry intra-refresh macroblocks. This counter-intuitive scheme actually results in better video quality than if packets containing transform coefficients were to be selectively dropped. Dropping only occurs when in the presence of wireless network congestion, as at other times the intra-coded macroblocks protect the video against random bit errors. Any packet dropping takes place under IEEE 802.11e, which is a quality-of-service addition to the IEEE 802.11 standard for wireless LANs. The chapter shows that, by this scheme, when congestion occurs, it is possible to gain up to 2 dB in video quality over assigning a stream to a single IEEE 802.11e access category. The scheme is shown to be consistently advantageous in indoor and outdoor wireless scenarios over other ways of assigning the partitioned data packets to different access categories. The chapter also contains a review of other research ideas using intra-refresh macroblocks and data-partitioning, as well as a look at the research outlook, now that the High Efficiency Video Codec (HEVC) has been released.
Performance Analysis of Biogeography Based Land Cover Feature Extractor for Building Hybrid Intelligent Models
Authors: Goel Lavika , Gupta Daya , Panchal V. K
Published: April 2014

This paper is an analytical study of the performance governing factors of the biogeography based land cover feature extraction technique which is characterized by its ability to perform differently on different natural terrain features contained in a satellite image. From the discussion, we establish the fact that the classification efficiency of BBO for a given land cover feature is inversely proportional to the degree of disorder and directly proportional to the similarity index of the digital number (DN) values of the pixels comprising that land cover feature when viewed in any of the bands of the multi-spectral satellite image. In order to verify our proposed hypotheses, we calculated the entropies and similarity indices for each of the land cover feature in two bands on two different datasets and found the same results in both the bands for each of the datasets we took, thus validating the theory. This finding is of prime importance since a prior analysis of the performance of biogeography based feature extraction technique on different types of terrain under consideration will improve the analyst's decisive capabilities for the selection of the most appropriate technique for the feature extraction task in hand. This in turn can be applied for building efficient artificially intelligent hybrid classifiers by applying the BBO technique on the extraction of those features on which it shows maximum classification efficiency as demonstrated in the paper.
LZW Chromosome Encoding in Estimation of Distribution Algorithms
Authors: Watchanupaporn Orawan , Suwannik Worasait
Published: April 2014

Estimation of distribution algorithm (EDA) can solve more complicated problems than its predecessor (Genetic Algorithm). EDA uses various methods to probabilistically model a group of highly fit individuals. Calculating the model in sophisticated EDA is very time consuming. To reduce the model building time, the authors propose compressed chromosome encoding. A chromosome is encoded using a format that can be decompressed by the Lempel-Ziv-Welch (LZW) algorithm. The authors combined LZW encoding with various EDAs and termed the class of algorithms Lempel-Ziv-Welch Estimation of Distribution Algorithms (LZWEDA). Experimental results show that LZWEDA significantly outperforms the original EDA. Finally, the authors analyze how LZW encoding transforms a fitness landscape.
An Adaptive Rayleigh-Laplacian Based MAP Estimation Technique for Despeckling SAR Images using Stationary Wavelet Transform
Authors: Das Amlan Jyoti , Talukdar Anjan Kumar , Sarma Kandarpa Kumar
Published: April 2014

Removal of speckle noise from Synthetic Aperture Radar (SAR) images is an important step before performing any image processing operations on these images. This paper presents a novel Stationary Wavelet Transform (SWT) based technique for the purpose of removing the speckle noise from the SAR returns. Maximum a posteriori probability (MAP) condition which uses a prior knowledge is used to estimate the noise free wavelet coefficients. The proposed MAP estimator is designed for this purpose which uses Rayleigh distribution for modeling the speckle noise and Laplacian distribution for modeling the statistics of the noise free wavelet coefficients. The parameters required for MAP estimator is determined by technique used for parameter estimation after SWT. Moreover an Laplacian – Gaussian based MAP estimator is also applied and the parameter estimation is done using the same method used for the proposed algorithm. For the purpose of enhancing the visual quality and to restore more edge information, a wavelet based resolution enhancement technique is also used after applying the Inverse stationary Wavelet Transform (ISWT), using interpolation technique. The experimental results show that the proposed despeckling algorithm efficiently removes speckle noise from the SAR images and restores the edge information as well.
The Epigenetic Face of Systemic Lupus Erythematosus
Authors: Ballestar Esteban , Esteller Manel , Richardson Bruce C.
Published: April 2014

Systemic lupus erythematosus (SLE) is an archetypical systemic, autoimmune inflammatory disease characterized by the production of autoantibodies to multiple nuclear Ags. Apoptotic defects and impaired removal of apoptotic cells contribute to an overload of autoantigens that become available to initiate an autoimmune response. Besides the well-recognized genetic susceptibility to SLE, epigenetic factors are important in the onset of the disease, as even monozygotic twins are usually discordant for the disease. Changes in DNA methylation and histone modifications, the major epigenetic marks, are a hallmark in genes that undergo epigenetic deregulation in disease. In SLE, global and gene-specific DNA methylation changes have been demonstrated to occur. Moreover, histone deacetylase inhibitors reverse the skewed expression of multiple genes involved in SLE. In the present study, we discuss the implications of epigenetic alterations in the development and progression of SLE and how epigenetic drugs constitute a promising source of therapy to treat this disease.
Cutting Edge: High-Mobility Group Box 1 Preconditioning Protects against Liver Ischemia-Reperfusion Injury
Authors: Izuishi Kunihiko , Tsung Allan , Jeyabalan Geetha , Critchlow Nathan D. , Li Jianhua , Tracey Kevin J. , Demarco Richard A. , Lotze Michael T. , Fink Mitchell P. , Geller David A. , Billiar Timothy R.
Published: April 2014

High mobility group box 1 (HMGB1) is a NF released extracellularly as a late mediator of lethality in sepsis and as an early mediator of inflammation following injury. Here we demonstrate that in contrast to the proinflammatory role of HMGB1, preconditioning with HMGB1 results in protection following hepatic ischemia/reperfusion (I/R). Pretreatment of mice with HMGB1 significantly decreased liver damage after I/R. The protection observed in mice pretreated with HMGB1 was associated with a higher expression of IL-1R-associated kinase-M, a negative regulator of TLR4 signaling, compared with controls. We thus explored the possibility that HMGB1 preconditioning was mediated through TLR4 activation. HMGB1 preconditioning failed to provide protection in TLR4 mutant (C3H/HeJ) mice, but successfully reduced damage in TLR4 wild-type (C3H/HeOuj) mice. Our studies demonstrate that in contrast to the role of HMGB1 as an early mediator of inflammation and organ damage in hepatic I/R, HMGB1 preconditioning can be protective.
Cutting Edge: Impaired Transitional B Cell Production and Selection in the Nonobese Diabetic Mouse
Authors: Quinn William J. , Noorchashm Negin , Crowley Jenni E. , Reed Amy J. , Noorchashm Hooman , Naji Ali , Cancro Michael P.
Published: April 2014

Developing B cells undergo selection at multiple checkpoints to eliminate autoreactive clones. We analyzed B cell kinetics in the NOD mouse to establish whether these checkpoints are intact. Our results show that although bone marrow production is normal in NOD mice, transitional (TR) B cell production collapses at 3 wk of age, reflecting a lack of successful immature B cell migration to the periphery. This yields delayed establishment of the follicular pool and a lack of selection at the TR checkpoint, such that virtually all immature B cells that exit the bone marrow mature without further selection. These findings suggest that compromised TR B cell generation in NOD mice yields relaxed TR selection, affording autoreactive specificities access to mature pools.
Regulatory T Cells Control Uveoretinitis Induced by Pathogenic Th1 Cells Reacting to a Specific Retinal Neoantigen
Authors: Terrada Céline , Fisson Sylvain , De Kozak Yvonne , Kaddouri Mohammed , Lehoang Phuc , Klatzmann David , Salomon Benoît L. , Bodaghi Bahram
Published: April 2014

In many clinical cases, uveitis develops secondary to an infection. This could result from peripheral activation followed by ocular penetration and reactivation of T cells specific for microbial Ags expressed in the retina. To gain insights into the pathophysiology of uveitis, we developed a new mouse model based on stable retinal expression of influenza virus hemagglutinin (HA) neoantigen by adeno-associated virus-mediated gene transfer. One month thereafter, we adoptively transferred HA-specific T cells, which were activated in vitro or in vivo. Intraocular inflammation was clinically and histologically observed in all animals within 15 days. The ocular infiltrate was composed mostly of macrophages and HA-specific T cells with a proinflammatory cytokine profile. Depletion of CD4+CD25+ regulatory T cells exacerbated the disease, whereas HA-specific CD4+CD25+ T cells given i.v. controlled the disease. This novel model should allow to better study the pathophysiology and therapeutic of uveitis.
Strong TCR Signaling, TLR Ligands, and Cytokine Redundancies Ensure Robust Development of Type 1 Effector T Cells
Authors: Nembrini Chiara , Abel Brian , Kopf Manfred , Marsland Benjamin J.
Published: April 2014

T cell effector function is a central mechanism of adaptive immunity, and accordingly, protection of the host against pathogens. One of the primary effector molecules produced by T cells in response to such pathogens is the cytokine, IFN-γ. Although the signaling pathways associated with the production of IFN-γ are well established, disparate in vivo and in vitro results indicate that distinct pathways may become more prominent dependent upon the nature of the infection, inflammatory milieu and tissue localization. We have examined the roles and requirements of the major IFN-γ-inducing pathways in vivo and in vitro, specifically: strength of TCR signal; paracrine release of IL-12, IL-23, and IL-18; and autocrine production of IFN-γ. Our data show a dynamic interaction between these activation pathways, which allows the host a degree of flexibility and redundancy in the induction of IFN-γ. Upon strong signaling through the TCR, IL-12, IL-18, and IL-23 play negligible roles in the induction of IFN-γ, whereas autocrine IFN-γ is an important component in sustaining its own secretion. However, the absence of any one of these factors during a weaker TCR signal, results in strikingly impaired T cell IFN-γ production. Of note, TLR-activated dendritic cells (DCs) were capable of overcoming the absence of a strong TCR signal, IL-12, IL-23, or IL-18 revealing an important additional mechanism for ensuring a robust IFN-γ response. Our findings clarify the hierarchical requirements of the major IFN-γ inducing pathways and highlight the important role TLR ligand-activated DCs have to preserve them.
Suppression of Complement Regulatory Proteins (CRPs) Exacerbates Experimental Autoimmune Anterior Uveitis (EAAU)
Authors: Jha Purushottam , Sohn Jeong-Hyeon , Xu Qin , Wang Yali , Kaplan Henry J. , Bora Puran S. , Bora Nalini S.
Published: April 2014

This study was undertaken to explore the role of complement regulatory proteins (CRPs) in experimental autoimmune anterior uveitis (EAAU). We observed that the levels of CRPs, Crry and CD59, in the eyes of Lewis rats increased during EAAU and remained elevated when the disease resolved. The in vivo role of these CRPs in EAAU was explored using neutralizing mAbs, antisense oligodeoxynucleotides (AS-ODNs), and small interfering RNAs against rat Crry and CD59. Suppression of Crry in vivo at days 9, 14, or 19 by neutralizing mAb or AS-ODNs resulted in the early onset of disease, the exacerbation of intraocular inflammation, and delayed resolution. Suppression of CD59 was only effective when the Abs and ODNs were given before the onset of disease. The most profound effect on the disease was observed when a mixture of Crry and CD59 mAbs or AS-ODNs was administered. A similar effect was observed with a combination of Crry and CD59 small interfering RNA. There was no permanent histologic damage to ocular tissue after the inflammation cleared in these animals. Increased complement activation as determined by increased deposition of C3, C3 activation fragments, and membrane attack complex was observed in the eyes of Lewis rats when the function and/or expression of Crry and CD59 was suppressed. Thus, our results suggest that various ocular tissues up-regulate the expression of Crry and CD59 to avoid self-injury during autoimmune uveitis and that these CRPs play an active role in the resolution of EAAU by down-regulating complement activation in vivo.
Functional Implication of Cellular Prion Protein in Antigen-Driven Interactions between T Cells and Dendritic Cells
Authors: Ballerini Clara , Gourdain Pauline , Bachy Véronique , Blanchard Nicolas , Levavasseur Etienne , Grégoire Sylvie , Fontes Pascaline , Aucouturier Pierre , Hivroz Claire , Carnaud Claude
Published: April 2014

The cellular prion protein (PrPC) is a host-encoded, GPI-anchored cell surface protein, expressed on a wide range of tissues including neuronal and lymphoreticular cells. PrPC may undergo posttranslational conversion, giving rise to scrapie PrP, the pathogenic conformer considered as responsible for prion diseases. Despite intensive studies, the normal function of PrPC is still enigmatic. Starting from microscope observations showing an accumulation of PrPC at the sites of contact between T cells and Ag-loaded dendritic cells (DC), we have studied the contribution of PrPC in alloantigen and peptide-MHC-driven T/DC interactions. Whereas the absence of PrPC on the DC results in a reduced allogeneic T cell response, its absence on the T cell partner has no apparent effect upon this response. Therefore, PrPC seems to fulfill different functions on the two cell partners forming the synapse. In contrast, PrPC mobilization by Ab reduces the stimulatory properties of DC and the proliferative potential of responding T cells. The contrasted consequences, regarding T cell function, between PrPC deletion and PrPC coating by Abs, suggests that the prion protein acts as a signaling molecule on T cells. Furthermore, our results show that the absence of PrPC has consequences in vivo also, upon the ability of APCs to stimulate proliferative T cell responses. Thus, independent of neurological considerations, some of the evolutionary constraints that may have contributed to the conservation of the Prnp gene in mammalians, could be of immunological origin.
CARMA1 Is Critical for the Development of Allergic Airway Inflammation in a Murine Model of Asthma
Authors: Medoff Benjamin D. , Seed Brian , Jackobek Ryan , Zora Jennifer , Yang Yi , Luster Andrew D. , Xavier Ramnik
Published: April 2014

CARMA1 has been shown to be important for Ag-stimulated activation of NF-κB in lymphocytes in vitro and thus could be a novel therapeutic target in inflammatory diseases such as asthma. In the present study, we demonstrate that mice with deletion in the CARMA1 gene (CARMA1−/−) do not develop inflammation in a murine model of asthma. Compared with wild-type controls, CARMA1−/− mice did not develop airway eosinophilia, had no significant T cell recruitment into the airways, and had no evidence for T cell activation in the lung or draining lymph nodes. In addition, the CARMA1−/− mice had significantly decreased levels of IL-4, IL-5, and IL-13, did not produce IgE, and did not develop airway hyperresponsiveness or mucus cell hypertrophy. However, adoptive transfer of wild-type Th2 cells into CARMA1−/− mice restored eosinophilic airway inflammation, cytokine production, airway hyperresponsiveness, and mucus production. This is the first demonstration of an in vivo role for CARMA1 in a disease process. Furthermore, the data clearly show that CARMA1 is essential for the development of allergic airway inflammation through its role in T lymphocytes, and may provide a novel means to inhibit NF-κB for therapy in asthma.
IL-1β Breaks Tolerance through Expansion of CD25+ Effector T Cells
Authors: O’Sullivan Brendan J. , Thomas Helen E. , Pai Saparna , Santamaria Pere , Iwakura Yoichiro , Steptoe Raymond J. , Kay Thomas W. H. , Thomas Ranjeny
Published: April 2014

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn’s disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1β drives proliferation and cytokine production by CD4+CD25+FoxP3− effector/memory T cells, attenuates CD4+CD25+FoxP3+ regulatory T cell function, and allows escape of CD4+CD25− autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1β in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.
Oxygen Radicals Induce Poly(ADP-Ribose) Polymerase-Dependent Cell Death in Cytotoxic Lymphocytes
Authors: Thorén Fredrik B. , Romero Ana I. , Hellstrand Kristoffer
Published: April 2014

Cytotoxic T cells and NK cells will acquire features of apoptosis when exposed to oxygen radicals, but the molecular mechanisms underlying this phenomenon are incompletely understood. We have investigated the role of two enzyme systems responsible for execution of cell death, caspases and the poly(ADP-ribose) polymerase (PARP). We report that although human cytotoxic lymphocytes were only marginally protected by caspase inhibitors, PARP inhibitors completely protected lymphocytes from radical-induced apoptosis and restored their cytotoxic function. The radical-induced, PARP-dependent cell death was accompanied by nuclear accumulation of apoptosis-inducing factor and a characteristic pattern of large-fragment DNA degradation. It is concluded that the PARP/apoptosis-inducing factor axis is critically involved in oxygen radical-induced apoptosis in cytotoxic lymphocytes.
Quantifying and Imaging NY-ESO-1/LAGE-1-Derived Epitopes on Tumor Cells Using High Affinity T Cell Receptors
Authors: Purbhoo Marco A. , Sutton Deborah H. , Brewer Joanna E. , Mullings Rebecca E. , Hill Maxine E. , Mahon Tara M. , Karbach Julia , Jäger Elke , Cameron Brian J. , Lissin Nikolai , Vyas Paresh , Chen Ji-Li , Cerundolo Vincenzo , Jakobsen Bent K.
Published: April 2014

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157–165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10–50 NY-ESO-1/LAGE-1157–165 epitopes per cell.
Efficient Immunization and Cross-Priming by Vaccine Adjuvants Containing TLR3 or TLR9 Agonists Complexed to Cationic Liposomes
Authors: Zaks Karen , Jordan Michael , Guth Amanda , Sellins Karen , Kedl Ross , Izzo Angelo , Bosio Catharine , Dow Steven
Published: April 2014

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4+ and CD8+ T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8+ T cell responses against even low doses of protein Ags, and this effect was independent of CD4+ T cell help. Ag-specific CD8+ T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.
The Cyclic AMP-Epac1-Rap1 Pathway Is Dissociated from Regulation of Effector Functions in Monocytes but Acquires Immunoregulatory Function in Mature Macrophages
Authors: Bryn Tone , Mahic Milada , Enserink Jorrit M. , Schwede Frank , Aandahl Einar Martin , Taskén Kjetil
Published: April 2014

cAMP mediates its intracellular effects through activation of protein kinase A (PKA), nucleotide-gated ion channels, or exchange protein directly activated by cAMP (Epac). Although elevation of cAMP in lymphocytes leads to suppression of immune functions by a PKA-dependent mechanism, the effector mechanisms for cAMP regulation of immune functions in monocytes and macrophages are not fully understood. In this study, we demonstrate the presence of Epac1 in human peripheral blood monocytes and activation of Rap1 in response to cAMP. However, by using an Epac-specific cAMP analog (8-CPT-2′-O-Me-cAMP), we show that monocyte activation parameters such as synthesis and release of cytokines, stimulation of cell adhesion, chemotaxis, phagocytosis, and respiratory burst are not regulated by the Epac1-Rap1 pathway. In contrast, activation of PKA by a PKA-specific compound (6-Bnz-cAMP) or physiological cAMP-elevating stimuli like PGE2 inhibits monocyte immune functions. Furthermore, we show that the level of Epac1 increases 3-fold during differentiation of monocytes into macrophages, and in monocyte-derived macrophages cAMP inhibits FcR-mediated phagocytosis via both PKA and the Epac1-Rap1 pathway. However, LPS-induced TNF-α production is only inhibited through the PKA pathway in these cells. In conclusion, the Epac1-Rap1 pathway is present in both monocytes and macrophages, but only regulates specific immune effector functions in macrophages.
Estrogen Induces Thymic Atrophy by Eliminating Early Thymic Progenitors and Inhibiting Proliferation of β-Selected Thymocytes
Authors: Zoller Allison L. , Kersh Gilbert J.
Published: April 2014

Although it has been established that high levels of estrogen can induce thymic involution, the mechanism by which this happens is not known. We have found that daily i.p. injections of the synthetic estrogen 17-β-estradiol reduce thymus cellularity by 80% over a period of 4–6 days. Although the atrophy is most strikingly observed in the CD4/CD8 double-positive (DP) thymic subset, the loss of thymocytes is not accompanied by a significant increase in thymocyte apoptosis, suggesting that direct killing of cells may not be the dominant means by which estrogens induce thymic atrophy. Instead, we find that estradiol drastically reduces the lineage-negative, Flt3+Sca-1+c-Kit+ population in the bone marrow, a population that contains thymic homing progenitors. Within the thymus, we observe that estradiol treatment results in a preferential depletion of early thymic progenitors. In addition, we find that estradiol leads to a significant reduction in the proliferation of thymocytes responding to pre-TCR signals. Reduced proliferation of DN3 and DN4 cell subsets is likely the major contributor to the reduction in DP thymocytes that is observed. The reduction in early thymic progenitors is also likely to contribute to thymic atrophy, as we show that estradiol treatment can reduce the size of Rag1-deficient thymuses, which lack pre-TCR signals and DP thymocytes.
CD4+ T Cells Can Protect APC from CTL-Mediated Elimination
Authors: Mueller Scott N. , Jones Claerwen M. , Stock Angus T. , Suter Mark , Heath William R. , Carbone Francis R.
Published: April 2014

Professional APC play a central role in generating antiviral CD8+ CTL immunity. However, the fate of such APC following interaction with these same CTL remains poorly understood. We have shown previously that prolonged Ag presentation persists in the presence of a strong CTL response following HSV infection. In this study, we examined the mechanism of survival of APC in vivo when presenting an immunodominant determinant from HSV. We show that transferred peptide-labeled dendritic cells were eliminated from draining lymph nodes in the presence of HSV-specific CTL. Maturation of dendritic cells with LPS or anti-CD40 before injection protected against CTL lysis in vivo. Furthermore, endogenous APC could be eliminated from draining lymph nodes early after HSV infection by adoptive transfer of HSV-specific CTL, yet the cotransfer of significant virus-specific CD4+ T cell help promoted prolonged Ag presentation. This suggests that Th cells may assist in prolonging class I-restricted Ag presentation, potentially enhancing CTL recruitment and allowing more efficient T cell priming.
A Membrane Form of TNF-α Presented by Exosomes Delays T Cell Activation-Induced Cell Death
Authors: Zhang Huang-Ge , Liu Cunren , Su Kaihun , Yu Shaohua , Zhang Liming , Zhang Shuangqin , Wang Jianhua , Cao Xu , Grizzle William , Kimberly Robert P.
Published: April 2014

In common with many other cell types, synovial fibroblasts produce exosomes. In this study, we show that the exosomes produced by synovial fibroblasts obtained from individuals with rheumatoid arthritis (RASF), but not exosomes produced by synovial fibroblasts obtained from individuals with osteoarthritis, contain a membrane bound form of TNF-α as demonstrated by colloidal gold immunostaining of TNF-α and confirmed by both Western blot and mass spectrometry. The RASF-derived exosomes, but not exosomes derived from fibroblasts obtained from individuals with osteoarthritis, are cytotoxic for the L929 cell, a TNF-α-sensitive cell line, and stimulate activation of NF-κB and induction of collagenase-1 in RASF. These effects are blocked by addition of soluble TNFR1 (sTNFbp), suggesting that a TNF-α-signaling pathway mediates these biological activities. sTNFbp also reduced the production of exosomes by RASF, suggesting the interruption of a positive amplification loop. Exosomes can transmit signals between cells, and RASF exosomes, effectively taken up by anti-CD3-activated T cells, activated AKT and NF-κB and rendered these activated T cells resistant to apoptosis. Neutralization of exosomal membrane TNF-α by sTNFbp partially reversed this resistance, suggesting that not only TNF-α but also additional exosomal proteins may contribute to the development of apoptosis resistance.
Signals from a Self-Antigen Induce Positive Selection in Early B Cell Ontogeny but Are Tolerogenic in Adults
Authors: Ferry Helen , Crockford Tanya L. , Leung Janson C. H. , Cornall Richard J.
Published: April 2014

Positive and negative signals from self-Ags shape the B cell repertoire and the development of distinct B cell subsets, but little is known about what distinguishes these signals. To address this question, we have studied the development of anti-hen egg lysozyme MD4 Ig transgene B cells while systematically varying the level, distribution, and timing of exposure to different forms of hen egg lysozyme as a self-Ag. This process has allowed us to explore the effects of Ag independent of BCR specificity. Our findings show how the selection of autoreactive B cells is a competitive process involving immunogenic and tolerogenic forms of self-Ags. Due to a developmental switch during B cell ontogeny, autoreactive anti-hen egg lysozyme MD4 Ig transgene B cells are negatively selected by self-Ags in adult bone marrow but susceptible to positive selection by some of the same self-Ags in fetal and neonatal life. However, the persistence of B1 cells and IgM autoantibodies from early ontogeny enables autoreactive B cells from the adult bone marrow to escape negative selection. Our data suggest that this rescue may be due to the clearance or masking of self-Ag by IgM autoantibody. We discuss the implications of these findings in terms of B cell selection and the maintenance of self-tolerance during early and adult life.
IL-4 Receptor α Is an Important Modulator of IL-4 and IL-13 Receptor Binding: Implications for the Development of Therapeutic Targets
Authors: Andrews Allison-Lynn , Holloway John W. , Holgate Stephen T. , Davies Donna E.
Published: April 2014

IL-4 is a key cytokine associated with allergy and asthma. Induction of cell signaling by IL-4 involves interaction with its cognate receptors, a complex of IL-4Rα with either the common γ-chain or the IL-13R chain α1 (IL-13Rα1). We found that IL-4 bound to the extracellular domain of IL-4Rα (soluble human (sh)IL-4Rα) with high affinity and specificity. In contrast with the sequential mechanism of binding and stabilization afforded by IL-4Rα to the binding of IL-13 to IL-13Rα1, neither common γ-chain nor IL-13Rα1 contributed significantly to the stabilization of the IL-4:IL-4Rα complex. Based on the different mechanisms of binding and stabilization of the IL-4R and IL-13R complexes, we compared the effects of shIL-4Rα and an IL-4 double mutein (R121D/Y124D, IL-4R antagonist) on IL-4- and IL-13-mediated responses. Whereas IL-4R antagonist blocked responses to both cytokines, shIL-4Rα only blocked IL-4. However, shIL-4Rα stabilized and augmented IL-13-mediated STAT6 activation and eotaxin production by primary human bronchial fibroblasts at suboptimal doses of IL-13. These data demonstrate that IL-4Rα plays a key role in the binding affinity of both IL-13R and IL-4R complexes. Under certain conditions, shIL-4Rα has the potential to stabilize binding IL-13 to its receptor to augment IL-13-mediated responses. Thus, complete understanding of the binding interactions between IL-4 and IL-13 and their cognate receptors may facilitate development of novel treatments for asthma that selectively target these cytokines without unpredicted or detrimental side effects.
The HIV-Neutralizing Monoclonal Antibody 4E10 Recognizes N-Terminal Sequences on the Native Antigen
Authors: Hager-Braun Christine , Katinger Hermann , Tomer Kenneth B.
Published: April 2014

Characterization of the epitope recognized by the broadly neutralizing anti-HIV Ab 4E10 has, heretofore, focused on a linear sequence from the gp41 pretransmembrane region (PTMR). Attempts to generate neutralizing Abs based on this linear epitope sequence have been unsuccessful. We have characterized the antigenic determinants on recombinant glycosylated full-length Ags, and nonglycosylated and truncated Ags recognized by 4E10 using epitope extraction and excision assays in conjunction with MALDI mass spectrometry. The mAb recognized the peptides 34LWVTVYYGVPVWK46 and 512AVGIGAVFLGFLGAAGSTMGAASMTLTVQAR542 located at the N-terminal region of gp120 and gp41, respectively. Immunoassays verified AV(L/M)FLGFLGAA as the gp41 epitope core. Recognition of the peptide from the gp41 PTMR was detected only in constructs in which the N termini of the mature envelope proteins were missing. In this region, the epitope core is located in the sequence 672WFDITNWLWY681. We hypothesize that the hydrophobic surface of the paratope functions as a “trap” for the viral sequences, which are responsible for insertion into the host cell membrane. As the N-terminal region of gp120, the fusogenic peptide of gp41, and the PTMR of gp41 show high sequence homology among various HIV strains, this model is consistent with the broadly neutralizing capabilities of 4E10.
The Double Lysine Motif of Tapasin Is a Retrieval Signal for Retention of Unstable MHC Class I Molecules in the Endoplasmic Reticulum
Authors: Paulsson Kajsa M. , Jevon Marc , Wang James W. , Li Suling , Wang Ping
Published: April 2014

Tapasin (tpn), an essential component of the MHC class I (MHC I) loading complex, has a canonical double lysine motif acting as a retrieval signal, which mediates retrograde transport of escaped endoplasmic reticulum (ER) proteins from the Golgi back to the ER. In this study, we mutated tpn with a substitution of the double lysine motif to double alanine (GFP-tpn-aa). This mutation abolished interaction with the coatomer protein complex I coatomer and resulted in accumulation of GFP-tpn-aa in the Golgi compartment, suggesting that the double lysine is important for the retrograde transport of tpn from late secretory compartments to the ER. In association with the increased Golgi distribution, the amount of MHC I exported from the ER to the surface was increased in 721.220 cells transfected with GFP-tpn-aa. However, the expressed MHC I were less stable and had increased turnover rate. Our results suggest that tpn with intact double lysine retrieval signal regulates retrograde transport of unstable MHC I molecules from the Golgi back to the ER to control the quality of MHC I Ag presentation.
Alteration of the FcγRIIa Dimer Interface Affects Receptor Signaling but Not Ligand Binding
Authors: Powell Maree S. , Barnes Nadine C. , Bradford Tessa M. , Musgrave Ian F. , Wines Bruce D. , Cambier John C. , Hogarth P. Mark
Published: April 2014

The aggregation of cell surface FcRs by immune complexes induces a number of important Ab-dependent effector functions. However, despite numerous studies that examine receptor function, very little is known about the molecular organization of these receptors within the cell. In this study, protein complementation, mutagenesis, and ligand binding analyses demonstrate that human FcγRIIa is present as a noncovalent dimer form. Protein complementation studies found that FcγRIIa molecules are closely associated. Mutagenesis of the dimer interface, as identified by crystallographic analyses, did not affect ligand binding yet caused significant alteration to the magnitude and kinetics of receptor phosphorylation. The data suggest that the ligand binding and the dimer interface are distinct regions within the receptor, and noncovalent dimerization of FcγRIIa may be an essential feature of the FcγRIIa signaling cascade.
Level of Expression of IL-13Rα2 Impacts Receptor Distribution and IL-13 Signaling
Authors: Daines Michael O. , Tabata Yasuhiro , Walker Bradley A. , Chen Weiguo , Warrier Manoj R. , Basu Saswata , Hershey Gurjit K. Khurana
Published: April 2014

IL-13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including IL-4Rα, IL-13Rα1, and IL-13Rα2. IL-4Rα and IL-13Rα1 form a heterodimeric signaling receptor for IL-13. In contrast, IL-13Rα2 binds IL-13 with high affinity but does not signal. IL-13Rα2 exists on the cell surface, intracellularly, and in soluble form, but no information is available regarding the relative distributions of IL-13Rα2 among these compartments, whether the compartments communicate, and how the relative expression levels impact IL-13 responses. Herein, we investigated the distribution of IL-13Rα2 in transfected and primary cells, and we evaluated how the total level of IL-13Rα2 expression impacted its distribution. Our results demonstrate that the distribution of IL-13Rα2 is independent of the overall level of expression. The majority of the IL-13Rα2 protein existed in intracellular pools. Surface IL-13Rα2 was continually released into the medium in a soluble form, yet surface expression remained constant supporting receptor trafficking to the cell surface. IL-13Rα2 inhibited IL-13 signaling proportionally to its level of expression, and this inhibition could be overcome with high concentrations of IL-13.
PanR1, a Dominant Negative Missense Allele of the Gene Encoding TNF-α (Tnf), Does Not Impair Lymphoid Development
Authors: Rutschmann Sophie , Hoebe Kasper , Zalevsky Jonathan , Du Xin , Mann Navjiwan , Dahiyat Bassil I. , Steed Paul , Beutler Bruce
Published: April 2014

A dominant hypomorphic allele of Tnf, PanR1, was identified in a population of G1 mice born to N-ethyl-N-nitrosourea-mutagenized sires. Macrophages from homozygotes produced no detectable TNF bioactivity, although normal quantities of immunoreactive TNF were secreted. The phenotype was confined to a critical region on mouse chromosome 17, and then ascribed to a C→A transversion at position 3480 of the Tnf gene, corresponding to the amino acid substitution P138T. As a result of subunit exchange, the protein exerts a dominant-negative effect on normal TNF trimers, interfering with the trimer/receptor interaction. Homozygotes are highly susceptible to infection by Listeria monocytogenes, confirming the essential role of TNF in innate immune defense. However, PanR1 mutant mice show normal architecture of the spleen and Peyer’s patches, suggesting that TNF is not essential for the formation of these lymphoid structures.
Peyer’s Patches Are Required for the Induction of Rapid Th1 Responses in the Gut and Mesenteric Lymph Nodes during an Enteric Infection
Authors: Kwa Sue-fen , Beverley Peter , Smith Adrian L.
Published: April 2014

The Peyer’s patches (PP) and mesenteric lymph nodes (MLN) are structural components of the gut-associated lymphoid tissues and contribute to the induction of immune responses toward infection in the gastrointestinal tract. These secondary lymphoid organs provide structural organization for efficient cellular interactions and the initiation of primary adaptive immune responses against infection. Immunity against primary infection with the enteric apicomplexan parasite, Eimeria vermiformis, depends on the rapid induction of local Th1 responses. Lymphotoxin (LT)-deficient mice which have various defects in secondary lymphoid organs were infected with E. vermiformis. The relative susceptibility of LTα−/−, LTβ−/−, LTα+/−β+/− mice and bone marrow chimeras, indicated that rapid protective Th1 responses required both PP and MLN. Moreover, the timing of Th1 induction in both MLN and gut was dependent on the presence of PP suggesting a level of cooperation between immune responses induced in these distinct lymphoid structures. The delay in Th1 induction was attributable to the delayed arrival of a broad range of dendritic cell subsets in the MLN and a substantial reduction of CD8α−CD11bhigh B220− dendritic cells in PP-deficient mice.
Group B Streptococcus Induces Macrophage Apoptosis by Calpain Activation
Authors: Fettucciari Katia , Fetriconi Ilaria , Mannucci Roberta , Nicoletti Ildo , Bartoli Andrea , Coaccioli Stefano , Marconi Pierfrancesco
Published: April 2014

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mφ) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mφ apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, α-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mφ apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mφ apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.
Functional Significance of Factor H Binding toNeisseria meningitidis
Authors: Schneider Muriel C. , Exley Rachel M. , Chan Hannah , Feavers Ian , Kang Yu-Hoi , Sim Robert B. , Tang Christoph M.
Published: April 2014

Neisseria meningitidis is an important cause of septicemia and meningitis. To cause disease, the bacterium must successfully survive in the bloodstream where it has to avoid being killed by host innate immune mechanisms, particularly the complement system. A number of pathogenic microbes bind factor H (fH), the negative regulator of the alternative pathway of complement activation, to promote their survival in vivo. In this study, we show that N. meningitidis binds fH to its surface. Binding to serogroups A, B, and C N. meningitidis strains was detected by FACS and Far Western blot analysis, and occurred in the absence of other serum factors such as C3b. Unlike Neisseria gonorrhoeae, binding of fH to N. meningitidis was independent of sialic acid on the bacterium, either as a component of its LPS or its capsule. Characterization of the major fH binding partner demonstrated that it is a 33-kDa protein; examination of insertion mutants showed that porins A and B, outer membrane porins expressed by N. meningitidis, do not contribute significantly to fH binding. We examined the physiological consequences of fH bound to the bacterial surface. We found that fH retains its activity as a cofactor of factor I when bound to the bacterium and contributes to the ability of N. meningitidis to avoid complement-mediated killing in the presence of human serum. Therefore, the recruitment of fH provides another mechanism by which this important human pathogen evades host innate immunity.
C-Reactive Protein Increases Cytokine Responses toStreptococcus pneumoniaethrough Interactions with Fcγ Receptors
Authors: Mold Carolyn , Du Clos Terry W.
Published: April 2014

Streptococcus pneumoniae is the most common organism responsible for community acquired pneumonia and meningitis. In pneumococcal pneumonia, a strong local inflammatory cytokine response reduces the frequency of bacteremia and increases survival. The initiation of this cytokine response by innate recognition of bacterial cell wall components through TLR has been described, but the role of soluble innate mediators has received limited attention. C-reactive protein (CRP) is an acute phase protein that binds phosphocholine residues on S. pneumoniae cell walls. CRP interacts with phagocytic cells through FcγRI and FcγRII and activates the classical complement pathway. CRP is protective in mouse pneumococcal bacteremia by increasing complement-dependent clearance and killing of bacteria. We studied the cytokine response of PBMC stimulated with CRP-opsonized S. pneumoniae to determine the effect of CRP interaction with FcγR. CRP dramatically increased the production of TNF-α and IL-1β in response to S. pneumoniae. These increases were blocked by phosphocholine, which inhibits CRP binding to S. pneumoniae, by inhibitors of FcγR signaling, and by mAb to FcγRI and FcγRII. A mutated rCRP with decreased FcγR binding had a decreased ability to stimulate TNF-α release, compared with wild-type CRP. Individuals who were homozygous for the R-131 allele of FcγRIIA, which has a higher affinity for CRP, showed higher responses to CRP-opsonized bacteria than did individuals homozygous for the H-131 allele, further implicating this receptor. The results indicate that CRP recognition of S. pneumoniae and binding to FcγR may enhance the early protective cytokine response to infection.
Regulation of Complement Activation by C-Reactive Protein: Targeting of the Inhibitory Activity of C4b-Binding Protein
Authors: Sjöberg Andreas P. , Trouw Leendert A. , McGrath Fabian D. G. , Hack C. Erik , Blom Anna M.
Published: April 2014

C-reactive protein (CRP) is the major acute phase protein in humans. It has been shown that CRP interacts with factor H, an inhibitor of the alternative pathway of complement, and now we demonstrate binding of CRP to the fluid-phase inhibitor of the classical pathway, C4b-binding protein (C4BP). C4BP bound to directly immobilized recombinant CRP as well as CRP attached to phosphorylcholine. The binding was sensitive to ionic strength and was enhanced in the presence of calcium. C4BP lacking β-chain and protein S, which is a form of C4BP increasing upon inflammation, bound CRP with higher affinity than the C4BP-protein S complex. The binding could not be blocked with mAbs directed against peripheral parts of the α-chains of C4BP while the isolated central core of C4BP obtained by partial proteolytic digestion bound CRP, indicating that the binding site for CRP is localized in the central core of the C4BP molecule. Furthermore, we found complexes in serum from a patient with an elevated CRP level and trace amounts of CRP were also identified in a plasma-derived C4BP preparation. We were also able to detect C4BP-CRP complexes in solution and established that C4BP retains full complement regulatory activity in the presence of CRP. In addition, we found that C4BP can compete with C1q for binding to immobilized CRP and that it inhibits complement activation locally. We hypothesize that CRP limits excessive complement activation on targets via its interactions with both factor H and C4BP.