bluuebunny/crossref_metadata_2025 huggingface dataset:
Vaccines with Enhanced Costimulation Maintain High Avidity Memory CTL
Authors: Yang Sixun , Hodge James W. , Grosenbach Douglas W. , Schlom Jeffrey
Published: April 2014

The avidity of Ag-specific CTL is a critical determinant for clearing viral infection and eliminating tumor. Although previous studies have demonstrated that vaccines using enhanced costimulation will enhance the level and avidity of Ag-specific T cells from naive mice, there are conflicting data about the effects of vaccines using enhanced costimulation (vector or dendritic cell based) on the survival of memory T cells. In this study we have first extended previous observations that primary vaccination with a recombinant vaccinia virus (rV-) expressing a model Ag (LacZ) and a triad of T cell costimulatory molecules (B7-1, ICAM-1, and LFA-3 (designated TRICOM)) enhances the level and avidity of T cells from naive vaccinated C57BL/6 (Thy1.2) mice. Adoptive transfer of Thy1.1 memory CD8+ T cells into naive Thy1.2 C57BL/6 mice was followed by booster vaccinations with a recombinant fowlpox (rF-)-expressing LacZ (rF-LacZ) or booster vaccinations with rF-LacZ/TRICOM. Analysis of levels of β-galactosidase tetramer-positive T cells and functional assays (IFN-γ expression and lytic activity) determined that booster vaccinations with rF-LacZ/TRICOM were superior to booster vaccinations with rF-LacZ in terms of both maintenance and enhanced avidity of memory CD8+ T cells. Antitumor experiments using a self-Ag (carcinoembryonic Ag (CEA) vaccines in CEA transgenic mice bearing CEA-expressing tumors) also demonstrated that the use of booster vaccinations with vaccines bearing enhanced costimulatory capacity had superior antitumor effects. These studies thus have implications in the design of more effective vaccine strategies.
NK Cells Play a Critical Role in the Regulation of Class I-Deficient Hemopoietic Stem Cell Engraftment: Evidence for NK Tolerance Correlates with Receptor Editing
Authors: Huang Yiming , Rezzoug Francine , Xu Hong , Chilton Paula M. , Schanie Carrie L. , Fugier-Vivier Isabelle , Ildstad Suzanne T.
Published: April 2014

The role that NK cells play in the rejection of hemopoietic stem cell (HSC) and tolerance induction has remained controversial. In this study, we examined whether NK cells play a direct role in the rejection of HSC. Purified HSC from MHC class II-deficient mice engrafted readily in congenic mice, while HSC from class I-deficient donors (β2-microglobulin−/− (β2m−/−)) failed to engraft. Recipient mice lacking CD8+, CD4+, or T cells also rejected HSC from class I-deficient donors, pointing directly to NK cells as the effector in rejection of HSC. Recipients, deficient in or depleted of NK cells, engrafted readily with β2m−/− HSC. Expression of the activating Ly-49D and inhibitory Ly-49G2 receptors on recipient NK cells was significantly decreased in these β2m−/−→B6 chimeras, and the proportion of donor NK cells expressing Ly-49D was also significantly decreased. Notably, β2m−/− chimeras accepted β2m−/− HSC in second transplants, demonstrating that NK cells in the chimeras had been tolerized to β2m−/−. Taken together, our data demonstrate that NK cells play a direct role in the regulation of HSC engraftment, and down-regulation and/or deletion of specific NK subsets in mixed chimeras can contribute to the induction of NK cell tolerance in vivo. Moreover, our data show that bone marrow-derived elements significantly contribute to NK cell development and tolerance.
Expression of a Dromedary Heavy Chain-Only Antibody and B Cell Development in the Mouse
Authors: Zou Xiangang , Smith Jennifer A. , Nguyen Viet Khong , Ren Liming , Luyten Kattie , Muyldermans Serge , Brüggemann Marianne
Published: April 2014

In mature B cells of mice and most mammals, cellular release of single H chain Abs without L chains is prevented by H chain association with Ig-specific chaperons in the endoplasmic reticulum. In precursor B cells, however, surface expression of μ-H chain in the absence of surrogate and conventional L chain has been identified. Despite this, Ag-specific single H chain Ig repertoires, using μ-, γ-, ε-, or α-H chains found in conventional Abs, are not produced. Moreover, removal of H chain or, separately, L chain (κ/λ) locus core sequences by gene targeting has prevented B cell development. In contrast, H chain-only Abs are produced abundantly in Camelidae as H2 IgG without the CH1 domain. To test whether H chain Abs can be produced in mice, and to investigate how their expression affects B cell development, we introduced a rearranged dromedary γ2a H chain into the mouse germline. The dromedary transgene was expressed as a naturally occurring Ag-specific disulphide-linked homodimer, which showed that B cell development can be instigated by expression of single H chains without L chains. Lymphocyte development and B cell proliferation was accomplished despite the absence of L chain from the BCR complex. Endogenous Ig could not be detected, although V(D)J recombination and IgH/L transcription was unaltered. Furthermore, crossing the dromedary H chain mice with mice devoid of all C genes demonstrated without a doubt that a H chain-only Ab can facilitate B cell development independent of endogenous Ig expression, such as μ- or δ-H chain, at early developmental stages.
Differential Regulation of CD40-Mediated TNF Receptor-Associated Factor Degradation in B Lymphocytes
Authors: Moore Carissa R. , Bishop Gail A.
Published: April 2014

Engagement of CD40 on murine B cells by its ligand CD154 induces the binding of TNFR-associated factors (TRAFs) 1, 2, 3, and 6, followed by the rapid degradation of TRAFs 2 and 3. TRAF degradation occurs in response to signaling by other TNFR superfamily members, and is likely to be a normal regulatory component of signaling by this receptor family. In this study, we found that receptor-induced TRAF degradation limits TRAF2-dependent CD40 signals to murine B cells. However, TRAFs 1 and 6 are not degraded in response to CD40 engagement, despite their association with CD40. To better understand the mechanisms underlying differential TRAF degradation, mixed protein domain TRAF chimeras were analyzed in murine B cells. Chimeras containing the TRAF2 zinc (Zn) domains induced effective degradation, if attached to a TRAF domain that binds to the PXQXT motif of CD40. However, the Zn domains of TRAF3 and TRAF6 could not induce degradation in response to CD40, regardless of the TRAF domains to which they were attached. Our data indicate that TRAF2 serves as the master regulator of TRAF degradation in response to CD40 signaling, and this function is dependent upon both the TRAF Zn domains and receptor binding position.
NF-κB Couples Protein Kinase B/Akt Signaling to Distinct Survival Pathways and the Regulation of Lymphocyte Homeostasis In Vivo
Authors: Jones Russell G. , Saibil Sam D. , Pun Joyce M. , Elford Alisha R. , Bonnard Madeleine , Pellegrini Marc , Arya Sudha , Parsons Michael E. , Krawczyk Connie M. , Gerondakis Steve , Yeh Wen-Chen , Woodgett James R. , Boothby Mark R. , Ohashi Pamela S.
Published: April 2014

Protein kinase B (PKBα/Akt1) a PI3K-dependent serine-threonine kinase, promotes T cell viability in response to many stimuli and regulates homeostasis and autoimmune disease in vivo. To dissect the mechanisms by which PKB inhibits apoptosis, we have examined the pathways downstream of PKB that promote survival after cytokine withdrawal vs Fas-mediated death. Our studies show that PKB-mediated survival after cytokine withdrawal is independent of protein synthesis and the induction of NF-κB. In contrast, PKB requires de novo gene transcription by NF-κB to block apoptosis triggered by the Fas death receptor. Using gene-deficient and transgenic mouse models, we establish that NF-κB1, and not c-Rel, is the critical signaling molecule downstream of the PI3K-PTEN-PKB signaling axis that regulates lymphocyte homeostasis.
The Crystal Structure of Recombinant proDer p 1, a Major House Dust Mite Proteolytic Allergen
Authors: Meno Kåre , Thorsted Peter B. , Ipsen Henrik , Kristensen Ole , Larsen Jørgen N. , Spangfort Michael D. , Gajhede Michael , Lund Kaare
Published: April 2014

Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 Å. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.
Epigenetic Basis for the Transcriptional Hyporesponsiveness of the Human Inducible Nitric Oxide Synthase Gene in Vascular Endothelial Cells
Authors: Chan Gary C. , Fish Jason E. , Mawji Imtiaz A. , Leung Desmond D. , Rachlis Alisa C. , Marsden Philip A.
Published: April 2014

A marked difference exists in the inducibility of inducible NO synthase (iNOS) between humans and rodents. Although important cis and trans factors in the murine and human iNOS promoters have been characterized using episomal-based approaches, a compelling molecular explanation for why human iNOS is resistant to induction has not been reported. In this study we present evidence that the hyporesponsiveness of the human iNOS promoter is based in part on epigenetic silencing, specifically hypermethylation of CpG dinucleotides and histone H3 lysine 9 methylation. Using bisulfite sequencing, we demonstrated that the iNOS promoter was heavily methylated at CpG dinucleotides in a variety of primary human endothelial cells and vascular smooth muscle cells, all of which are notoriously resistant to iNOS induction. In contrast, in human cell types capable of iNOS induction (e.g., A549 pulmonary adenocarcinoma, DLD-1 colon adenocarcinoma, and primary hepatocytes), the iNOS promoter was relatively hypomethylated. Treatment of human cells, such as DLD-1, with a DNA methyltransferase inhibitor (5-azacytidine) induced global and iNOS promoter DNA hypomethylation. Importantly, 5-azacytidine enhanced the cytokine inducibility of iNOS. Using chromatin immunoprecipitation, we found that the human iNOS promoter was basally enriched with di- and trimethylation of H3 lysine 9 in endothelial cells, and this did not change with cytokine addition. This contrasted with the absence of lysine 9 methylation in inducible cell types. Importantly, chromatin immunoprecipitation demonstrated the selective presence of the methyl-CpG-binding transcriptional repressor MeCP2 at the iNOS promoter in endothelial cells. Collectively, our work defines a role for chromatin-based mechanisms in the control of human iNOS gene expression.
The Role of the MHC on Resistance to Group A Streptococci in Mice
Authors: Goldmann Oliver , Lengeling Andreas , Böse Jens , Bloecker Helmut , Geffers Robert , Chhatwal Gursharan S. , Medina Eva
Published: April 2014

The severity of infection with Streptococcus pyogenes is strongly influenced by the host’s genetics. This observation extends to the murine model of streptococcal infection, where the background of the mouse strain determines the infection outcome (BALB/c are resistant, whereas C3H/HeN are susceptible). To determine the extent to which the MHC complex (H2) contributed to diseases susceptibility, the response to S. pyogenes of congenic BALB mice from a resistant background (BALB/c), but carrying the H2k region of susceptible C3H/HeN mice (BALB/k), was examined. BALB/k were as susceptible as the H2 donor strain (C3H/HeN). Linkage analysis performed in F2 backcross ([BALB/c × C3H/HeN] × BALB/c) mice confirmed the presence of a susceptibility locus within the H2 region on proximal chromosome 17. The possibility that modulation of T cell responses to streptococcal superantigens (GAS-SAgs) by different H2 haplotypes may influence disease severity was examined. BALB/k exhibited a significantly stronger response at the level of cell proliferation and cytokine production to GAS-SAgs than did BALB/c mice. However, the fact that T cell-deficient SCID-C3H/HeN mice also exhibited a susceptible phenotype suggests a more important contribution of innate effector cells to disease susceptibility. Lower transcriptional levels of certain inflammation-related regulatory genes located on chromosome 17 were detected in macrophages from susceptible than in those from resistant mice in response to infection. These results suggest that susceptibility to S. pyogenes may be associated with an altered transcription of specific genes that may compromise the endogenous regulatory processes controlling the inflammatory cascade and favor the progression to sepsis.
Foamy Macrophages within Lung Granulomas of Mice Infected withMycobacterium tuberculosisExpress Molecules Characteristic of Dendritic Cells and Antiapoptotic Markers of the TNF Receptor-Associated Factor Family
Authors: Ordway Diane , Henao-Tamayo Marcela , Orme Ian M. , Gonzalez-Juarrero Mercedes
Published: April 2014

Highly vacuolated or foamy macrophages are a distinct characteristic of granulomas in the lungs of animals infected with Mycobacterium tuberculosis. To date these have usually been considered to represent activated macrophages derived from monocytes entering the lesions from the blood. However, we demonstrate in this study that foamy macrophages express high levels of DEC-205, a marker characteristic of dendritic cells (DCs). In addition to high expression of the DEC-205 marker, these cells were characterized as CD11b+CD11chighMHC class IIhigh, and CD40high, which are additional markers typically expressed by DCs. Up-regulation of CD40 was seen only during the early chronic stage of the lung disease, and both the expression of CD40 and MHC class II markers were down-regulated as the disease progressed into the late chronic phase. Foamy cells positive for the DEC-205 marker also expressed high levels of TNFR-associated factor-1 (TRAF-1), TRAF-2, and TRAF-3, markers associated with resistance to apoptosis. These data indicate that in addition to the central role of DCs in initiating the acquired immune response against M. tuberculosis infection, they also participate in the granulomatous response.
Activation of Innate Immunity, Inflammation, and Potentiation of DNA Vaccination through Mammalian Expression of the TLR5 Agonist Flagellin
Authors: Applequist Steven E. , Rollman Erik , Wareing Mark D. , Lidén Martin , Rozell Björn , Hinkula Jorma , Ljunggren Hans-Gustaf
Published: April 2014

Improving DNA vaccination remains a fundamental goal in vaccine research. Theoretically, this could be achieved by molecules encoded by DNA capable of activating TLRs to mimic inflammatory responses generated by infection. Therefore, we constructed an expression vector that allows mammalian cells to express the TLR5 agonist flagellin (FliC) at the cell surface. In vitro, cell lines expressing FliC stimulated production of proinflammatory cytokines and the up-regulation of costimulatory molecules on monocytes. Mice given the FliC expression vector intradermally exhibited site-specific inflammation and, in combination with vectors expressing Ags, developed dramatic increases in Ag-specific IgG as well as IgA. Surprisingly, mice also developed strong Ag-specific MHC class I-restricted cellular immunity. To determine whether vaccination using FliC vectors could elicit protective immunity to an infectious agent, mice were given dermal injections of FliC expression vector together with a vector encoding the influenza A virus nucleoprotein. This vaccination strategy elicited protective immunity to lethal influenza A virus infection. These results demonstrate that expression of DNA-encoded TLR agonists by mammalian cells greatly enhance and broaden immune responses, imposing new possibilities on DNA vaccination to infectious agents and cancer.
Efficacy of Antigen 2/Proline-Rich Antigen cDNA-Transfected Dendritic Cells in Immunization of Mice against Coccidioides posadasii
Authors: Awasthi Shanjana , Awasthi Vibhudutta , Magee D. Mitchell , Coalson Jacqueline J.
Published: April 2014

Coccidioides posadasii causes coccidioidomycosis, or Valley fever, in the endemic regions of the Southwestern United States. The susceptibility to C. posadasii infection has been attributed to a decreased Th1 cellular response. APCs, especially dendritic cells (DCs), play an important role in the activation of Th1 response. In this study, we investigated the efficacy of a DC-based vaccine against C. posadasii in a mouse model of coccidioidomycosis. We intranasally immunized C57BL6 mice with syngeneic, bone marrow-derived DCs (JAWS II cells) transfected with a cDNA encoding the protective Coccidioides-Ag2/proline-rich Ag. The immunized mice were lethally challenged with C. posadasii through either an i.p. or intranasal route. Upon necropsy after 10 days of infection, fungal burden in lung and spleen of immunized mice was significantly reduced as compared with the control animals. The lung tissue homogenates of immunized animals showed higher levels of IFN-γ. Histologically, lung tissues of immunized mice were in better condition than the control mice. To further investigate, we studied the biodistribution and trafficking of injected DCs by nuclear imaging techniques. For this purpose, the transfected DCs were radiolabeled with 111In-oxime. Scintigraphic images showed that most of the label remained in the gastrointestinal tract. A significant amount was also observed in lung, but there were negligible circulating 111In label in blood. The results suggest that the DCs have a potent immunostimulatory activity, and immunization with DCs transfected with Ag2/proline-rich Ag-cDNA induces protective immunity against C. posadasii in C57BL6 mice.
Severe CD4 T Cell-Mediated Immunopathology in Murine Schistosomiasis Is Dependent on IL-12p40 and Correlates with High Levels of IL-17
Authors: Rutitzky Laura I. , Lopes da Rosa Jessica R. , Stadecker Miguel J.
Published: April 2014

C57BL/6 mice infected with the helminth Schistosoma mansoni develop small hepatic granulomas around parasite eggs, but concomitant immunization with soluble schistosome egg Ags (SEA) in CFA (SEA/CFA) causes marked exacerbation of the lesions in a Th1-dominated environment characterized by high levels of IFN-γ. We explored the cause of the severe immunopathology by using IL-12p40−/− and IL-12p35−/− mice. SEA/CFA-immunized IL-12p40−/− mice, incapable of making IL-12 or IL-23, were completely resistant to high pathology, and their SEA-stimulated lymphoid cells failed to secrete significant IFN-γ or IL-17. In contrast, SEA/CFA-immunized IL-12p35−/− mice, able to make IL-23 but not IL-12, developed severe lesions that correlated with high levels of IL-17, low IFN-γ, and an expansion of activated CD4 T cells with a CD44high/CD62Llow memory phenotype. In vivo administration of neutralizing anti-IL-17 mAb markedly inhibited hepatic granulomatous inflammation. Importantly, CBA mice, a naturally high pathology strain, also displayed elevated IL-17 levels comparable to those seen in the SEA/CFA-immunized BL/6 mice, and their lesions were similarly reduced by in vivo treatment with anti-IL-17. Our findings indicate that an IL-17-producing T cell population, likely driven by IL-23, significantly contributes to severe immunopathology in schistosomiasis.
TLRs 2 and 4 Are Not Involved in Hypersusceptibility to Acute Pseudomonas aeruginosa Lung Infections
Authors: Ramphal Reuben , Balloy Viviane , Huerre Michel , Si-Tahar Mustapha , Chignard Michel
Published: April 2014

TLRs are implicated in defense against microorganisms. Animal models have demonstrated that the susceptibility to a number of Gram-negative pathogens is linked to TLR4, and thus LPS of many Gram-negative bacteria have been implicated as virulence factors. To assess the role of this pathogen-associated molecular pattern as it is exposed on intact Pseudomonas aeruginosa, the susceptibility of mice lacking TLR4 or both TLR2 and TLR4 was examined in a model of acute Pseudomonas pneumonia. These mutant mice were not hypersusceptible to the Pseudomonas challenge and mounted an effective innate response that cleared the organism despite low levels of TNF-α and KC in the airways. Bacterial and neutrophil counts in the lung were similar in control and TLR-deficient mice at 6 and 24 h after infection. MyD88−/− mice were, however, hypersusceptible, with 100% of mice dying within 48 h with a lower dose of P. aeruginosa. Of note there were normal levels of IL-6 and G-CSF in the airways of TLR mutant mice that were absent from the MyD88−/− mice. Thus, the susceptibility of mice to P. aeruginosa acute lung infection does not go through TLR2 or TLR4, implying that Pseudomonas LPS is not the most important virulence factor in acute pneumonia caused by this organism. Furthermore, G-CSF treatment of infected MyD88−/− mice results in improved clearance and survival. Thus, the resistance to infection in TLR2/TLR4−/− mice may be linked to G-CSF and possibly IL-6 production.
Th1-Like Cytokine Induction by Heat-Killed Brucella abortus Is Dependent on Triggering of TLR9
Authors: Huang Li-Yun , Ishii Ken J. , Akira Shizuo , Aliberti Julio , Golding Basil
Published: April 2014

In this report we provide evidence, for the first time, that bacterial DNA in the context of heat-killed Brucella abortus (HKBA) engages TLR9 in dendritic cells (DC), resulting in a Th1-like cytokine response. This is based on the findings that HKBA induction of IL-12p40 is: 1) abolished in DC from TLR9−/− mice; 2) blocked by suppressive oligodeoxynucleotides; 3) simulated by bacterial DNA derived from HKBA; and 4) abrogated by DNase or methylation of the DNA from HKBA. Furthermore, the effect of HKBA can be inhibited by chloroquine, indicating that endosomal acidification is required and supporting the notion that DNA from HKBA is interacting with TLR9 at the level of the endosome, as is the case with CpG oligodeoxynucleotides. In addition to DC, HKBA can elicit IL-12p40 secretion from macrophages, in which case the effect is wholly MyD88 dependent but only partially TLR9 dependent. This probably explains why HKBA effects in vivo are only partially reduced in TLR9−/−, but absent in MyD88−/− mice. Because of their intimate interactions with T cells, the DC response is most likely to be critical for linking innate and adaptive immune responses, whereas the macrophage reaction may play a role in enhancing NK cell and bystander immune responses. In addition to IL-12p40, HKBA induces other Th1-like cytokines, namely, IFN-α and IFN-γ, in a TLR9-dependent manner. These cytokines are important in protection against viruses and bacteria, and their induction enhances HKBA as a potential carrier for vaccines.
An Immune Response Directed to Proteinase and Adhesin Functional Epitopes Protects against Porphyromonas gingivalis-Induced Periodontal Bone Loss
Authors: O’Brien-Simpson Neil M. , Pathirana Rishi D. , Paolini Rita A. , Chen Yu-Yen , Veith Paul D. , Tam Vivian , Ally Nafisa , Pike Robert N. , Reynolds Eric C.
Published: April 2014

Porphyromonas gingivalis, a pathogen associated with periodontitis, bound to fibrinogen, fibronectin, hemoglobin, and collagen type V with a similar profile to that of its major virulence factor, the cell surface RgpA-Kgp proteinase-adhesin complex. Using peptide-specific, purified Abs in competitive inhibition ELISAs and epitope mapping assays, we have identified potential adhesin binding motifs (ABMs) of the RgpA-Kgp complex responsible for binding to host proteins. The RgpA-Kgp complex and synthetic ABM and proteinase active site peptides conjugated to diphtheria toxoid, when used as vaccines, protected against P. gingivalis-induced periodontal bone loss in the murine periodontitis model. The most efficacious peptide and protein vaccines were found to induce a high-titer IgG1 Ab response. Furthermore, mice protected in the lesion and periodontitis models had a predominant P. gingivalis-specific IL-4 response, whereas mice with disease had a predominant IFN-γ response. The peptide-specific Abs directed to the ABM2 sequence (EGLATATTFEEDGVA) protected against periodontal bone loss and inhibited binding of the RgpA-Kgp complex to fibrinogen, fibronectin, and collagen type V. Furthermore, the peptide-specific Abs directed to the ABM3 sequence (GTPNPNPNPNPNPNPGT) protected against periodontal bone loss and inhibited binding to hemoglobin. However, the most protective Abs were those directed to the active sites of the RgpA and Kgp proteinases. The results suggest that when the RgpA-Kgp complex, or functional binding motif or active site peptides are used as a vaccine, they induce a Th2 response that blocks function of the RgpA-Kgp complex and protects against periodontal bone loss.
Pyocyanin and Its Precursor Phenazine-1-Carboxylic Acid Increase IL-8 and Intercellular Adhesion Molecule-1 Expression in Human Airway Epithelial Cells by Oxidant-Dependent Mechanisms
Authors: Look Dwight C. , Stoll Lynn L. , Romig Sara A. , Humlicek Alicia , Britigan Bradley E. , Denning Gerene M.
Published: April 2014

Pseudomonas aeruginosa secretes numerous factors that alter host cell function and may contribute to disease pathogenesis. Among recognized virulence factors is the redox-active phenazine pyocyanin. We have recently demonstrated that the precursor for pyocyanin, phenazine-1-carboxylic acid (PCA), increases oxidant formation and alters gene expression in human airway epithelial cells. We report in this work that PCA and pyocyanin increase expression of ICAM-1 both in vivo and in vitro. Moreover, phenazines enhanced cytokine-dependent increases in IL-8 and ICAM-1. Antioxidant intervention studies indicated both similarities and differences between PCA and pyocyanin. The thiol antioxidant N-acetyl cysteine, extracellular catalase, and inducible NO synthase inhibitors inhibited ICAM-1 and IL-8 increases in response to both phenazines. However, pyocyanin was significantly more sensitive to N-acetylcysteine inhibition. Interestingly, hydroxyl radical scavengers inhibited the response to pyocyanin, but not to PCA. These studies suggest that P. aeruginosa phenazines coordinately up-regulate chemokines (IL-8) and adhesion molecules (ICAM-1) by mechanisms that are, at least in part, oxidant dependent. However, results indicate that the mechanisms by which PCA and pyocyanin exert their effects are not identical, and not all antioxidant interventions are equally effective in inhibiting phenazine-mediated proinflammatory effects.
Viral IL-6 Blocks Neutrophil Infiltration during Acute Inflammation
Authors: Fielding Ceri A. , McLoughlin Rachel M. , Colmont Chantal S. , Kovaleva Marina , Harris Dean A. , Rose-John Stefan , Topley Nicholas , Jones Simon A.
Published: April 2014

Pathologies arising as a consequence of human herpesvirus-8 (HHV8) infections are closely associated with the autocrine activity of a HHV8 encoded IL-6 (vIL-6), which promotes proliferation of infected cells and their resistance to apoptosis. In this present report, studies show that vIL-6 may also be important in influencing the host’s immunological response to secondary infections. Using peritoneal inflammation as a model of acute bacterial infection, vIL-6 was found to specifically block neutrophil recruitment in vivo through regulation of inflammatory chemokine expression. This response was substantiated in vitro where activation of STAT3 in human peritoneal mesothelial cells by vIL-6 was associated with enhanced CCL2 release. Although vIL-6 did not effect CXCL8 production, IL-1β-induced secretion of this neutrophil-activating chemokine was significantly suppressed by vIL-6. These data suggest that vIL-6 has the capacity to suppress innate immune responses and thereby influence the outcome of opportunistic infections in HHV8-associated disease.
Fas-Mediated Acute Lung Injury Requires Fas Expression on Nonmyeloid Cells of the Lung
Authors: Matute-Bello Gustavo , Lee Janet S. , Liles W. Conrad , Frevert Charles W. , Mongovin Steven , Wong Venus , Ballman Kimberly , Sutlief Steven , Martin Thomas R.
Published: April 2014

Fas (CD95) is a membrane surface receptor, which, in the lungs, is expressed in macrophages, neutrophils, and epithelial cells. In mice, Fas activation leads to a form of lung injury characterized by increased alveolar permeability. We investigated whether Fas-mediated lung injury occurs primarily as a result of Fas activation in myeloid cells (such as macrophages) or in nonmyeloid cells (such as epithelial cells). Chimeric mice lacking Fas in either myeloid or nonmyeloid cells were generated by transplanting marrow cells from lpr mice (which lack Fas) into lethally irradiated C57BL/6 mice (MyFas− group) or vice versa (MyFas+ group). Additional mice transplanted with marrow cells from their same strain served as controls (Fas+ ctr and Fas− ctr groups). Sixty days after transplantation, the mice received intratracheal instillations of the Fas-activating mAb Jo2 (n = 10/group), or an isotype control Ab (n = 10/group), and were euthanized 24-h later. Only animals expressing Fas in nonmyeloid cells (Fas+ ctr and MyFas−) showed significant increases in lung neutrophil content and in alveolar permeability. These same mice showed tissue evidence of lung injury and caspase-3 activation in cells of the alveolar walls. Despite differences in the neutrophilic response and lung injury, there was no statistical difference in the lung cytokine concentrations (KC and MIP-2) among groups. We conclude that Fas-mediated lung injury requires expression of Fas on nonmyeloid cells of the lungs. These findings suggest that the alveolar epithelium is the primary target of Fas-mediated acute lung injury, and demonstrate that apoptotic processes may be associated with neutrophilic inflammation.
Vitamin D3 Confers Protection from Autoimmune Encephalomyelitis Only in Female Mice
Authors: Spach Karen M. , Hayes Colleen E.
Published: April 2014

The prevalence of multiple sclerosis (MS) increases significantly with decreasing UV B light exposure, possibly reflecting a protective effect of vitamin D3. Consistent with this theory, previous research has shown a strong protective effect 1,25-dihydroxyvitamin D3 in experimental autoimmune encephalomyelitis (EAE), an MS model. However, it is not known whether the hormone precursor, vitamin D3, has protective effects in EAE. To address this question, B10.PL mice were fed a diet with or without vitamin D3, immunized with myelin basic protein, and studied for signs of EAE and for metabolites and transcripts of the vitamin D3 endocrine system. The intact, vitamin D3-fed female mice had significantly less clinical, histopathological, and immunological signs of EAE than ovariectomized females or intact or castrated males. Correlating with reduced EAE, the intact, vitamin D3-fed female mice had significantly more 1,25-dihydroxyvitamin D3 and fewer CYP24A1 transcripts, encoding the 1,25-dihydroxyvitamin D3-inactivating enzyme, in the spinal cord than the other groups of mice. Thus, there was an unexpected synergy between vitamin D3 and ovarian tissue with regard to EAE inhibition. We hypothesize that an ovarian hormone inhibited CYP24A1 gene expression in the spinal cord, so the locally-produced 1,25-dihydroxyvitamin D3 accumulated and resolved the inflammation before severe EAE developed. If humans have a similar gender difference in vitamin D3 metabolism in the CNS, then sunlight deprivation would increase the MS risk more significantly in women than in men, which may contribute to the unexplained higher MS incidence in women than in men.
Human Male Genital Tract Secretions: Both Mucosal and Systemic Immune Compartments Contribute to the Humoral Immunity
Authors: Moldoveanu Zina , Huang Wen-Qiang , Kulhavy Rose , Pate Mitchell S. , Mestecky Jiri
Published: April 2014

In contrast to numerous studies of female genital tract secretions, the molecular properties of Abs and the magnitude of humoral responses in human male genital tract secretions to naturally occurring Ags and to mucosal and systemic immunizations have not been extensively investigated. Therefore, seminal plasma (SP) collected from healthy individuals was analyzed with respect to Ig levels, their isotypes, molecular forms of IgA, and for the presence of Abs to naturally occurring Ags, or induced by systemic or mucosal immunizations with viral and bacterial vaccines. The results indicated that in SP, IgG and not IgA, is the dominant Ig isotype, and that IgM is present at low levels. IgA is represented by secretory IgA, polymeric IgA, and monomeric IgA. In contrast to the female genital tract secretions in which IgA2 occurs in slight excess, the distribution of IgA subclasses in SP resembles that in plasma with a pronounced preponderance of IgA1. The IgG subclass profiles in SP are also similar to those in serum. Thus, SP is an external secretion that shares common features with both typical external secretions and plasma. Specifically, SP contains naturally occurring secretory IgA Abs to environmental Ags of microbial origin and to an orally administered bacterial vaccine, and plasma-derived IgG Abs to systemically injected vaccines. Therefore, both mucosal and systemic immunization with various types of Ags can induce humoral responses in SP. These findings should be considered in immunization strategies to induce humoral responses against sexually transmitted infections, including HIV-1.
Characterization of Latent Membrane Protein 2 Specificity in CTL Lines from Patients with EBV-Positive Nasopharyngeal Carcinoma and Lymphoma
Authors: Straathof Karin C. , Leen Ann M. , Buza Elizabeth L. , Taylor Graham , Huls M. Helen , Heslop Helen E. , Rooney Cliona M. , Bollard Catherine M.
Published: April 2014

Viral proteins expressed by EBV-associated tumors provide target Ags for immunotherapy. Adoptive T cell therapy has proven effective for posttransplant EBV-associated lymphoma in which all EBV latent Ags are expressed (type III latency). Application of immunotherapeutic strategies to tumors such as nasopharyngeal carcinoma and Hodgkin’s lymphoma that have a restricted pattern of EBV Ag expression (type II latency) is under investigation. Potential EBV Ag targets for T cell therapy expressed by these tumors include latent membrane proteins (LMP) 1 and 2. A broad panel of epitopes must be identified from these target Ags to optimize vaccination strategies and facilitate monitoring of tumor-specific T cell populations after immunotherapeutic interventions. To date, LMP2 epitopes have been identified for only a limited number of HLA alleles. Using a peptide library spanning the entire LMP2 sequence, 25 CTL lines from patients with EBV-positive malignancies expressing type II latency were screened for the presence of LMP2-specific T cell populations. In 21 of 25 lines, T cell responses against one to five LMP2 epitopes were identified. These included responses to previously described epitopes as well as to newly identified HLA-A*0206-, A*0204/17-, A29-, A68-, B*1402-, B27-, B*3501-, B53-, and HLA-DR-restricted epitopes. Seven of the nine newly identified epitopes were antigenically conserved among virus isolates from nasopharyngeal carcinoma tumors. These new LMP2 epitopes broaden the diversity of HLA alleles with available epitopes, and, in particular, those epitopes conserved between EBV strains provide valuable tools for immunotherapy and immune monitoring.
Cutting Edge: Direct Suppression of B Cells by CD4+CD25+ Regulatory T Cells
Authors: Lim Hyung W. , Hillsamer Peter , Banham Allison H. , Kim Chang H.
Published: April 2014

Regulatory T cells (Tregs) can potentially migrate to the B cell areas of secondary lymphoid tissues and suppress T cell-dependent B cell Ig response. T cell-dependent Ig response requires B cell stimulation by Th cells. It has been unknown whether Tregs can directly suppress B cells or whether they must suppress Th cells to suppress B cell response. We report here that Foxp3+ Tregs are found in T-B area borders and within germinal centers of human lymphoid tissues and can directly suppress B cell Ig response. Although Tregs can effectively suppress T cells, they can also directly suppress B cell response without the need to first suppress Th cells. The direct suppression of B cell Ig production by Tregs is accompanied by inhibition of Ig class switch recombination.
Cutting Edge: Activation of the Aryl Hydrocarbon Receptor by 2,3,7,8-Tetrachlorodibenzo-p-dioxin Generates a Population of CD4+CD25+ Cells with Characteristics of Regulatory T Cells
Authors: Funatake Castle J. , Marshall Nikki B. , Steppan Linda B. , Mourich Dan V. , Kerkvliet Nancy I.
Published: April 2014

Activation of the aryl hydrocarbon receptor (AhR) by its most potent ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to immune suppression in mice. Although the underlying mechanisms responsible for AhR-mediated immune suppression are not known, previous studies have shown that activation of the AhR must occur within the first 3 days of an immune response and that CD4+ T cells are primary targets. Using the B6-into-B6D2F1 model of an acute graft-vs-host response, we show that activation of AhR in donor T cells leads to the generation of a subpopulation of CD4+ T cells that expresses high levels of CD25, along with CD62Llow, CTLA-4, and glucocorticoid-induced TNFR. These donor-derived CD4+CD25+ cells also display functional characteristics of regulatory T cells in vitro. These findings suggest a novel role for AhR in the induction of regulatory T cells and provide a new perspective on the mechanisms that underlie the profound immune suppression induced by exposure to TCDD.
Absence of Leukotriene B4 Receptor 1 Confers Resistance to Airway Hyperresponsiveness and Th2-Type Immune Responses
Authors: Terawaki Kan , Yokomizo Takehiko , Nagase Takahide , Toda Akiko , Taniguchi Masahiko , Hashizume Kohei , Yagi Takeshi , Shimizu Takao
Published: April 2014

Bronchial asthma is an increasingly common disorder that remains poorly understood and difficult to manage. The disease is characterized by airway hyperresponsiveness, chronic inflammation, and mucus overproduction. Based on the finding that leukotriene B4 receptor 1 (BLT1) is expressed highly in Th2 lymphocytes, we analyzed the roles of BLT1 using an OVA-induced bronchial asthma model. BLT1-null mice did not develop airway hyperresponsiveness, eosinophilic inflammation, and hyperplasia of goblet cells. Attenuated symptoms were accompanied by reduced IgE production, and accumulation of IL-5 and IL-13 in bronchoalveolar lavage fluid, suggesting attenuated Th2-type immune response in BLT1-null mice. Peribronchial lymph node cells of sensitized BLT1-null mice showed much attenuated proliferation and production of Th2 cytokines upon re-stimulation with Ag in vitro. Thus, LTB4-BLT1 axis is required for the development of Th2-type immune response, and blockade of LTB4 functions through BLT1 would be novel and useful in the effort to ameliorate bronchial asthma and related Th2-biased immune disorders.
The Adaptor Protein 3BP2 Binds Human CD244 and Links this Receptor to Vav Signaling, ERK Activation, and NK Cell Killing
Authors: Saborit-Villarroya Ifigènia , Del Valle Juana M. , Romero Xavier , Esplugues Enric , Lauzurica Pilar , Engel Pablo , Martín Margarita
Published: April 2014

Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-γ secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-γ release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-γ release involve distinct NK pathways.
Maintenance of Immune Tolerance Depends on Normal Tissue Homeostasis
Authors: Boonman Zita F. H. M. , van Mierlo Geertje J. D. , Fransen Marieke F. , de Keizer Rob J. W. , Jager Martine J. , Melief Cornelis J. M. , Toes René E. M.
Published: April 2014

Ags expressed at immune privileged sites and other peripheral tissues are able to induce T cell tolerance. In this study, we analyzed whether tolerance toward an intraocular tumor expressing a highly immunogenic CTL epitope is maintained, broken, or reverted into immunity in the event the anatomical integrity of the eye is lost. Inoculation of tumor cells into the anterior chamber of the eye of naive B6 mice leads to progressive intraocular tumor growth, an abortive form of CTL activation in the tumor-draining submandibular lymph node, and systemic tolerance as evidenced by the inability of these mice to reject an otherwise benign tumor cell inoculum. Loss of anatomical integrity of the eye as a consequence of phthisis resulted in loss of systemic tolerance and the emergence of effective antitumor immunity against an otherwise lethal tumor challenge. Phthisis was accompanied by dendritic cell maturation and preceded the induction of systemic tumor-specific CTL immunity. Our data show that normal tissue homeostasis and anatomical integrity is required for the maintenance of ocular tolerance and prevention of CTL-mediated immunity. These data also indicate that tissue injury in the absence of viral or microbial infection can act as a switch for the induction of CTL immunity.
CD4+CD25+ Regulatory T Cells Inhibit the Maturation but Not the Initiation of an Autoantibody Response
Authors: Fields Michele L. , Hondowicz Brian D. , Metzgar Michele H. , Nish Simone A. , Wharton Gina N. , Picca Cristina C. , Caton Andrew J. , Erikson Jan
Published: April 2014

To investigate the mechanism by which T regulatory (Treg) cells may control the early onset of autoimmunity, we have used an adoptive transfer model to track Treg, Th, and anti-chromatin B cell interactions in vivo. We show that anti-chromatin B cells secrete Abs by day 8 in vivo upon provision of undeviated, Th1- or Th2-type CD4+ T cell help, but this secretion is blocked by the coinjection of CD4+CD25+ Treg cells. Although Treg cells do not interfere with the initial follicular entry or activation of Th or B cells at day 3, ICOS levels on Th cells are decreased. Furthermore, Treg cells must be administered during the initial phases of the Ab response to exert full suppression of autoantibody production. These studies indicate that CD25+ Treg cells act to inhibit the maturation, rather than the initiation, of autoantibody responses.
Dendritic Cell-Specific Intercellular Adhesion Molecule 3-Grabbing Nonintegrin/CD209 Is Abundant on Macrophages in the Normal Human Lymph Node and Is Not Required for Dendritic Cell Stimulation of the Mixed Leukocyte Reaction
Authors: Granelli-Piperno Angela , Pritsker Alla , Pack Maggi , Shimeliovich Irina , Arrighi Jean-Francois , Park Chae Gyu , Trumpfheller Christine , Piguet Vincent , Moran Thomas M. , Steinman Ralph M.
Published: April 2014

The C-type lectin dendritic cell-specific ICAM 3-grabbing nonintegrin (DC-SIGN)/CD209 efficiently binds several pathogens, including HIV-1. DC-SIGN is expressed on monocyte-derived DCs in culture, and importantly, it is able to sequester HIV-1 within cells and facilitate transmission of virus to CD4+ T cells. To investigate DC-SIGN function, we have generated new mAbs. We report in this study that these and prior anti-DC-SIGN mAbs primarily label macrophages in the medullary sinuses of noninflamed human lymph node. In contrast, expression is not detected on most DCs in the T cell area, except for occasional cells. We also noted that IL-4 alone can induce expression of DC-SIGN in CD14+ monocytes and circulating blood DCs. However, blockade of DC-SIGN with Abs and DC-SIGN small interfering RNA did not result in a major reduction in the capacity of these DCs to transfer HIV to T cells, confirming significant DC-SIGN-independent mechanisms. The blocking approaches did reduce HIV-1 transmission by DC-SIGN-transfected cells by >90%. DC-SIGN blockade also did not reduce the ability of DCs to stimulate T cell proliferation in the MLR. These results indicate that DC-SIGN has the potential to contribute to macrophage function in normal human lymph node, and that DCs do not require DC-SIGN to transmit HIV or to initiate T cell responses.
Immunostimulatory CpG Oligonucleotides Abrogate Allergic Susceptibility in a Murine Model of Maternal Asthma Transmission
Authors: Fedulov Alexey , Silverman Eric , Xiang Yuhong , Leme Adriana , Kobzik Lester
Published: April 2014

We tested the potential of CpG oligodeoxynucleotides (ODN) to reverse the increased susceptibility to allergic airways disease in neonatal mice in a model of maternal transmission of asthma risk. Offspring of OVA-sensitized and challenged BALB/c mother mice were subjected to an intentionally suboptimal sensitization protocol that has minimal effects on normal mice, but results in airway hyperresponsiveness (AHR) and airway inflammation (AI) in babies of asthmatic mother mice. We evaluated pulmonary function and AI in CpG- or control ODN-treated offspring. CpG treatment of neonates on day 4 of life prevents the AHR otherwise seen in this model (enhanced pause at 100 mg/ml methacholine: CpG, 0.9 ± 0.1; ODN control, 3.8 ± 0.6; n = 62; p < 0.005). It also prevented the development of AI, as evident in decreased bronchoalveolar lavage eosinophilia (CpG, 1.2 ± 0.3%; ODN, 31.4 ± 4.1%; n = 56; p < 0.005), diminished the severity of AI on histopathology, and resulted in lower IL-5 levels in bronchoalveolar lavage fluid. The effect of CpG persisted for at least 4–6 wk and was allergen independent. Treatment with CpG just before OVA aerosol challenge also prevented allergic responses. The data support the potential for immunomodulatory therapy with CpG in early life to reduce susceptibility to asthma.