bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective Killing of Vaccinia Virus by LL-37: Implications for Eczema Vaccinatum
Authors: Howell Michael D. , Jones James F. , Kisich Kevin O. , Streib Joanne E. , Gallo Richard L. , Leung Donald Y. M.
Published: April 2014

Possible bioterrorism with smallpox has led to the resumption of smallpox (vaccinia virus) immunization. One complication, eczema vaccinatum, occurs primarily in patients with atopic dermatitis (AD). Skin lesions of patients with AD, but not psoriasis, is deficient in the cathelicidin antimicrobial peptide (LL-37) and human β-defensin-2 (HBD-2). We hypothesized that this defect may explain the susceptibility of patients with AD to eczema vaccinatum. The Wyeth vaccine strain of vaccinia virus was incubated with varying concentrations of human (LL-37) and murine (CRAMP) cathelicidins, human α-defensin (HBD-1, HBD-2), and a control peptide. Outcomes included quantification of viral PFU, vaccinia viral gene expression by quantitative real-time RT-PCR, and changes in virion structure by transmission electron microscopy. CRAMP knockout mice and control animals were inoculated by skin pricks with 2 × 105 PFU of vaccinia and examined daily for pox development. Physiologic amounts of human and murine cathelicidins (10–50 μM), but not human defensins, which had antibacterial activity, resulted in the in vitro reduction of vaccinia viral plaque formation (p < 0.0001), vaccinia mRNA expression (p < 0.001), and alteration of vaccinia virion structure. In vivo vaccinia pox formation occurred in four of six CRAMP knockout animals and in only one of 15 control mice (p < 0.01). These data support a role for cathelicidins in the inhibition of orthopox virus (vaccinia) replication both in vitro and in vivo. Susceptibility of patients with AD to eczema vaccinatum may be due to a deficiency of cathelicidin.
Chlamydia pneumoniae Multiply in Neutrophil Granulocytes and Delay Their Spontaneous Apoptosis
Authors: van Zandbergen Ger , Gieffers Jens , Kothe Henning , Rupp Jan , Bollinger Annalena , Aga Eresso , Klinger Matthias , Brade Helmut , Dalhoff Klaus , Maass Matthias , Solbach Werner , Laskay Tamás
Published: April 2014

The obligate intracellular bacterial pathogen Chlamydia pneumoniae (Cp) is responsible for a range of human diseases, including acute respiratory infection. Although experimental intratracheal infection with Cp results in a massive recruitment of neutrophil granulocytes (polymorphonuclear neutrophils (PMN)), the role of these cells in the defense against Cp is unclear. In this study the interactions of PMN with Cp were investigated. In vitro coincubation experiments showed that human granulocytes were able to internalize Chlamydia in an opsonin-independent manner. Importantly, phagocytosed Cp were not killed; the ingested bacteria survived and multiplied within PMN. Although uninfected granulocytes became apoptotic within 10 h, infected PMN survived up to 90 h. Coincubation with Cp significantly decreased the ratio of apoptotic PMN, as detected by morphological analysis, annexin V, and TUNEL staining. The observed antiapoptotic effect was associated with a markedly lower level of procaspase-3 processing and, consequently, reduced caspase-3 activity in infected PMN. LPS was found as a major, but not exclusive, component responsible for the observed antiapoptotic effect. Chlamydia LPS affected PMN apoptosis both by acting directly on the cells and by inducing the autocrine production of the antiapoptotic cytokine IL-8. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, Cp can extend the life span of neutrophil granulocytes, making them suitable host cells for survival and multiplication within the first hours/days after infection.
p47phoxDeficiency Impairs NF-κB Activation and Host Defense inPseudomonasPneumonia
Authors: Sadikot Ruxana T. , Zeng Heng , Yull Fiona E. , Li Bo , Cheng Dong-sheng , Kernodle Douglas S. , Jansen E. Duco , Contag Christopher H. , Segal Brahm H. , Holland Steven M. , Blackwell Timothy S. , Christman John W.
Published: April 2014

We examined the role of redox signaling generated by NADPH oxidase in activation of NF-κB and host defense against Pseudomonas aeruginosa pneumonia. Using mice with an NF-κB-driven luciferase reporter construct (HIV-LTR/luciferase (HLL)), we found that intratracheal administration of P. aeruginosa resulted in a dose-dependent neutrophilic influx and activation of NF-κB. To determine the effects of reactive oxygen species generated by the NADPH oxidase system on activation of NF-κB, we crossbred mice deficient in p47phox with NF-κB reporter mice (p47phox−/−HLL). These p47phox−/−HLL mice were unable to activate NF-κB to the same degree as HLL mice with intact NADPH oxidase following P. aeruginosa infection. In addition, lung TNF-α levels were significantly lower in p47phox−/−HLL mice compared with HLL mice. Bacterial clearance was impaired in p47phox−/−HLL mice. In vitro studies using bone marrow-derived macrophages showed that Toll-like receptor 4 was necessary for NF-κB activation following treatment with P. aeruginosa. Additional studies with macrophages from p47phox−/− mice confirmed that redox signaling was necessary for maximal Toll-like receptor 4-dependent NF-κB activation in this model. These data indicate that the NADPH oxidase-dependent respiratory burst stimulated by Pseudomonas infection contributes to host defense by modulating redox-dependent signaling through the NF-κB pathway.
Transcript Signatures in Experimental Asthma: Identification of STAT6-Dependent and -Independent Pathways
Authors: Zimmermann Nives , Mishra Anil , King Nina E. , Fulkerson Patricia C. , Doepker Matthew P. , Nikolaidis Nikolaos M. , Kindinger Laura E. , Moulton Elizabeth A. , Aronow Bruce J. , Rothenberg Marc E.
Published: April 2014

The analysis of polygenic diseases such as asthma poses a challenging problem. In an effort to provide unbiased insight into disease pathogenesis, we took an empirical approach involving transcript expression profiling of lung tissue from mice with experimental asthma. Asthmatic responses were found to involve sequential induction of 4.7% of the tested genome; notably, there was ectopic expression of a series of genes not previously implicated in allergic or pulmonary responses. Genes were widely distributed throughout all chromosomes, but preferentially included genes involved in immunity, development, and homeostasis. When asthma was induced by two independent experimental regimens, unique gene transcript profiles were found depending upon the mode of disease induction. However, the majority of genes were common to both models representing an asthma signature genome. Analysis of STAT6-deficient mice revealed that an unexpectedly large segment of the asthma genes were STAT6 independent; this correlated with sustained inflammatory events in these mice. Notably, induction of asthma in STAT6-deficient mice resulted in gene induction not seen in wild-type mice. These results raise concern that therapeutic blockade of STAT6 in the asthmatic setting may reprogram the genetic signature, resulting in alternative lung pathology, which we indeed observed in STAT6-deficient mice. These results provide unprecedented insight into the complex steps involved in the pathogenesis of allergic airway responses; as such, these results have significant therapeutic and clinical implications.
Immune Complexes Bind Preferentially to FcγRIIA (CD32) on Apoptotic Neutrophils, Leading to Augmented Phagocytosis by Macrophages and Release of Proinflammatory Cytokines
Authors: Hart Simon P. , Alexander Karen M. , Dransfield Ian
Published: April 2014

Many human inflammatory diseases are associated with tissue deposition of immune complexes and influx of neutrophils. We show that immune complexes bind preferentially to apoptotic neutrophils via FcγRIIA (CD32) and that increased binding is associated with clustering of immune complexes on the plasma membrane of the apoptotic cell. Phagocytosis of immune complex-opsonized apoptotic neutrophils by human macrophages was substantially enhanced (4.4-fold increase compared with control apoptotic neutrophils) and stimulated macrophages to release the proinflammatory cytokines TNF-α and IL-6. Immune complexes may perturb the normal pathways for clearance of apoptotic neutrophils by augmenting their clearance at the price of proinflammatory cytokine release. This represents a novel mechanism by which immune complexes may modulate the resolution of inflammation.
Induction of C Chemokine XCL1 (Lymphotactin/Single C Motif-1α/Activation-Induced, T Cell-Derived and Chemokine-Related Cytokine) Expression by HIV-1 Tat Protein
Authors: Kim Byung Oh , Liu Ying , Zhou Betty Y. , He Johnny J.
Published: April 2014

HIV-1 Tat has been proposed as a key agent in many AIDS-related disorders, including HIV-1-associated neurological diseases. We have recently shown that Tat expression induces a significant increase in T lymphocytes in the brains of Tat transgenic mice. The CNS infiltration of T lymphocytes has been noted in AIDS patients. In the present study using this unique genetic system we attempted to understand the underlying mechanisms of Tat expression-induced infiltration of T lymphocytes by examining chemokine expression. RNase protection assay revealed that in addition to CCL2 (monocyte chemoattractant protein-1), CCL3 (macrophage inflammatory protein-1α (MIP-1α)), CCL4 (MIP-1β), CCL5 (RANTES), CXCL2 (MIP-2), and CXCL10 (inducing protein-10), XCL1 (lymphotactin/single C motif-1α/activation-induced, T cell-derived and chemokine-related cytokine) was identified to be up-regulated by Tat expression. XCL1 is a C chemokine and plays a specific and important role in tissue-specific recruitment of T lymphocytes. Thus, we further determined the relationship between Tat and XCL1 expression. Tat-induced XCL1 expression was further confirmed by XCL1-specific RT-PCR and ELISA. Combined in situ hybridization and immunohistochemical staining identified astrocytes, monocytes, and macrophages/microglia as XCL1-producing cells in vivo. Using human astrocytes, U87.MG cells, as an in vitro model, activation of XCL1 expression was positively correlated with Tat expression. Moreover, the XCL1 promoter-driven reporter gene assay showed that Tat-induced XCL1 expression occurred at the transcriptional level. Taken together, these results demonstrate that Tat directly trans-activated XCL1 expression and suggest potential roles of Tat-induced XCL1 expression in the CNS infiltration of T lymphocytes during HIV-1 infection and subsequent HIV-1-induced neurological diseases.
Metal-Protein Complex-Mediated Transport and Delivery of Ni2+ to TCR/MHC Contact Sites in Nickel-Specific Human T Cell Activation
Authors: Thierse Hermann-Josef , Moulon Corinne , Allespach Yvonne , Zimmermann Bastian , Doetze Andrea , Kuppig Stephan , Wild Doris , Herberg Friedrich , Weltzien Hans Ulrich
Published: April 2014

Nickel allergy clearly involves the activation of HLA-restricted, skin-homing, Ni-specific T cells by professional APCs. Nevertheless, knowledge concerning the molecular details of metal-protein interactions underlying the transport and delivery of metal ions to APC during the early sensitization phase and their interactions with HLA and TCRs is still fragmentary. This study investigates the role of human serum albumin (HSA), a known shuttling molecule for Ni2+ and an often-disregarded, major component of skin, in these processes. We show that Ni-saturated HSA complexes (HSA-Ni) induce and activate Ni-specific human T cells as potently as Ni salt solutions when present at equimolar concentrations classically used for in vitro T cell stimulation. However, neither HSA itself nor its Ni-binding N-terminal peptide are involved in determining the specificity of antigenic determinants. In fact, HSA could be replaced by xenogeneic albumins exhibiting sufficient affinity for Ni2+ as determined by surface plasmon resonance (Biacore technology) or atomic absorption spectroscopy. Moreover, despite rapid internalization of HSA-Ni by APC, it was not processed into HLA-associated epitopes recognizable by Ni-specific T cells. In contrast, the presence of HSA-Ni in the vicinity of transient contacts between TCR and APC-exposed HLA molecules appeared to facilitate a specific transfer of Ni2+ from HSA to high-affinity coordination sites created at the TCR/HLA-interface.
Of Mice and Not Men: Differences between Mouse and Human Immunology
Authors: Mestas Javier , Hughes Christopher C. W.
Published: April 2014

Mice are the experimental tool of choice for the majority of immunologists and the study of their immune responses has yielded tremendous insight into the workings of the human immune system. However, as 65 million years of evolution might suggest, there are significant differences. Here we outline known discrepancies in both innate and adaptive immunity, including: balance of leukocyte subsets, defensins, Toll receptors, inducible NO synthase, the NK inhibitory receptor families Ly49 and KIR, FcR, Ig subsets, the B cell (BLNK, Btk, and λ5) and T cell (ZAP70 and common γ-chain) signaling pathway components, Thy-1, γδ T cells, cytokines and cytokine receptors, Th1/Th2 differentiation, costimulatory molecule expression and function, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression. We also provide examples, such as multiple sclerosis and delayed-type hypersensitivity, where complex multicomponent processes differ. Such differences should be taken into account when using mice as preclinical models of human disease.
Cutting Edge: Activity of Human Adult Microglia in Response to CC Chemokine Ligand 21
Authors: Dijkstra Ineke M. , Hulshof Sandra , van der Valk Paul , Boddeke Hendrikus W. G. M. , Biber Knut
Published: April 2014

The approximately 50 known chemokines are classified in distinct subfamilies: CXC, CC, CX3C, and C. Although the signaling of chemokines often is promiscuous, signaling events between members of these distinct chemokine classes are hardly observed. The only known exception so far is the murine CC chemokine ligand (CCL)21 (secondary lymphoid tissue chemokine, Exodus-2, 6Ckine), which binds and activates the murine CXC chemokine receptor CXCR3. However, this exception has not been found in humans. In this study, we provide evidence that human CCL21 is a functional ligand for endogenously expressed CXCR3 in human adult microglia. In absence of CCR7 expression, CCL21 induced chemotaxis of human microglia with efficiency similar to the CXCR3 ligands CXC chemokine ligand 9 (monokine induced by IFN-γ) and CXC chemokine ligand 10 (IFN-γ-inducible protein-10). Because human CCL21 did not show any effects in CXCR3-transfected HEK293 cells, it is indicated that CXCR3 signaling depends on the cellular background in which the CXCR3 is expressed.
Cutting Edge: A New Approach to Modeling Early Lung Immunity in Murine Tuberculosis
Authors: Bhatt Kamlesh , Hickman Somia Perdow , Salgame Padmini
Published: April 2014

In this study, we report a new approach that allows dissection of distinct pathways regulating induction of early adaptive immunity in response to Mycobacterium tuberculosis (Mtb). We used traceable murine dendritic cells (DCs) and macrophage populations to chart their migratory pattern in response to Mtb, and found that only DCs receiving inflammatory stimuli from Mtb up-regulated their expression of CCR7 and migrated specifically to the draining lymph nodes (LNs). Furthermore, these Mtb-modulated DCs initiated a Th1 response only in the draining LNs. Taken together, these results demonstrate that Mtb-induced modulation of DCs is critical for their migration to regional LNs and ensuing T cell priming.
Phosphatidylinositol 3-Kinase-Dependent Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase 1/2 and NF-κB Signaling Pathways Are Required for B Cell Antigen Receptor-Mediated Cyclin D2 Induction in Mature B Cells
Authors: Piatelli Michael J. , Wardle Carrie , Blois Joseph , Doughty Cheryl , Schram Brian R. , Rothstein Thomas L. , Chiles Thomas C.
Published: April 2014

Phosphatidylinositol 3-kinase (PI-3K) has been linked to promitogenic responses in splenic B cells following B cell Ag receptor (BCR) cross-linking; however identification of the signaling intermediates that link PI-3K activity to the cell cycle remains incomplete. We show that cyclin D2 induction is blocked by the PI-3K inhibitors wortmannin and LY294002, which coincides with impaired BCR-mediated mitogen-activated protein/extracellular signal-related kinase kinase (MEK)1/2 and p42/44ERK phosphorylation on activation residues. Cyclin D2 induction is virtually absent in B lymphocytes from mice deficient in the class IA PI-3K p85α regulatory subunit. In contrast to studies with PI-3K inhibitors, which inhibit all classes of PI-3Ks, the p85α regulatory subunit is not required for BCR-induced MEK1/2 and p42/44ERK phosphorylation, suggesting the contribution of another PI-3K family members in MEK1/2 and p42/44ERK activation. However, p85α−/− splenic B cells are defective in BCR-induced IκB kinase β and IκBα phosphorylation. We demonstrate that NF-κB signaling is required for cyclin D2 induction via the BCR in normal B cells, implicating a possible link with the defective IκB kinase β and IκBα phosphorylation in p85α−/− splenic B cells and their ability to induce cyclin D2. These results indicate that MEK1/2-p42/44ERK and NF-κB pathways link PI-3K activity to Ag receptor-mediated cyclin D2 induction in splenic B cells.
A Role for IL-10-Mediated HLA-DR7-Restricted T Cell-Dependent Events in Development of the Modified Th2 Response to Cat Allergen
Authors: Reefer Amanda J. , Carneiro Raquel M. , Custis Natalie J. , Platts-Mills Thomas A. E. , Sung Sun-Sang J. , Hammer Juergen , Woodfolk Judith A.
Published: April 2014

Although high dose exposure to inhaled cat allergen (Fel d 1) can cause a form of tolerance (modified Th2 response), the T cell mechanism for this phenomenon has not been studied. T cell responses to Fel d 1 were characterized in both allergic (IgEpos) and modified Th2 (IgEnegIgGpos) responders as well as serum Ab-negative controls (IgEnegIgGneg). Fel d 1 stimulated high levels of IL-10 in PBMC cultures from all individuals, with evidence of Th2 and Th1 cytokine skewing in allergic and control subjects, respectively. Using overlapping peptides, epitopes at the N terminus of Fel d 1 chain 2 were shown to stimulate strong T cell proliferation and to preferentially induce IL-10 (peptide 2:1 (P2:1)) or IFN-γ (P2:2) regardless of the allergic status of the donor. Injection of cat extract during conventional immunotherapy stimulated expansion of IL-10- and IFN-γ-producing chain 2 epitope-specific T cells along with increased Fel d 1-specific serum IgG and IgG4 Ab. Six of 12 modified responders expressed the major HLA-DRB1 allele, *0701, and both P2:1 and P2:2 were predicted ligands for this allele. Cultures from DR7-positive modified responders produced the highest levels of IL-10 to P2:1 in addition to other major and minor epitopes within chains 1 and 2. In the presence of anti-IL-10 mAb, both T cell proliferation and IFN-γ production were enhanced in a Fel d 1- and epitope-specific manner. We conclude that IL-10-producing T cells specific for chain 2 epitopes are relevant to tolerance induction, and that DR7-restricted recognition of these epitopes favors a modified Th2 response.
The T Cells Specific for the Carboxyl-Terminal Determinants of Self (Rat) Heat-Shock Protein 65 Escape Tolerance Induction and Are Involved in Regulation of Autoimmune Arthritis
Authors: Durai Malarvizhi , Gupta Radhey S. , Moudgil Kamal D.
Published: April 2014

Immunization of Lewis rats with heat-killed Mycobacterium tuberculosis H37Ra leads to development of polyarthritis (adjuvant-induced arthritis; AA) that shares several features with human rheumatoid arthritis (RA). Immune response to the 65-kDa mycobacterial heat-shock protein (Bhsp65) is believed to be involved in induction of AA as well as in experimental modulation of this disease. However, the understanding of several critical aspects of the pathogenesis of AA in the Lewis rat has severely been hampered by the lack of information both regarding the level as well as epitope specificity of tolerance to the mammalian self (rat) homologue of Bhsp65, 65-kDa rat heat-shock protein (Rhsp65), and about the functional attributes of the T cell repertoire specific for this self protein. In this study, we established that tolerance to Rhsp65 in the Lewis rat is incomplete, and that the residual T cells primed upon challenge with this self hsp65 are disease regulating in nature. We also have defined the T cell epitopes in the C-terminal region within Rhsp65 that contribute predominantly to the immune reactivity as well as the AA-protective effect of this self protein. Furthermore, the T cells primed by peptides comprising these C-terminal determinants can be efficiently restimulated by the naturally generated epitopes from endogenous Rhsp65, suggesting that self hsp65 might also be involved in natural remission from acute AA. These novel first experimental insights into the self hsp65-directed regulatory T cell repertoire in AA would help develop better immunotherapeutic approaches for autoimmune arthritis.
Mannose Receptor Targeting of Tumor Antigen pmel17 to Human Dendritic Cells Directs Anti-Melanoma T Cell Responses via Multiple HLA Molecules
Authors: Ramakrishna Venky , Treml John F. , Vitale Laura , Connolly John E. , O’Neill Thomas , Smith Patricia A. , Jones Charles L. , He Li-Zhen , Goldstein Joel , Wallace Paul K. , Keler Tibor , Endres Michael J.
Published: April 2014

Targeting recycling endocytic receptors with specific Abs provides a means for introducing a variety of tumor-associated Ags into human dendritic cells (DCs), culminating in their efficient presentation to T cells. We have generated a human mAb (B11) against the mannose receptor that is rapidly internalized by DCs through receptor-mediated endocytosis. By genetically linking the melanoma Ag, pmel17, to Ab B11, we obtained the fully human fusion protein, B11-pmel17. Treatment of DCs with B11-pmel17 resulted in the presentation of pmel17 in the context of HLA class I and class II molecules. Thus, potent pmel17-specific T cells were cytotoxic toward gp100+ HLA-matched melanoma targets, but not HLA-mismatched melanoma or gp100− nonmelanoma tumor lines. Importantly, competitive inhibition of lysis of an otherwise susceptible melanoma cell line by cold targets pulsed with known gp100 CD8 T cell epitopes as well as a dose-dependent proliferative response to Th epitopes demonstrates that DCs can process targeted Ag for activation of cytotoxic as well as helper arms of the immune response. Thus, the specific targeting of soluble exogenous tumor Ag to the DC mannose receptor directly contributes to the generation of multiple HLA-restricted Ag-specific T cell responses.
A Type I IFN-Dependent Pathway Induced by Schistosoma mansoni Eggs in Mouse Myeloid Dendritic Cells Generates an Inflammatory Signature
Authors: Trottein François , Pavelka Norman , Vizzardelli Caterina , Angeli Veronique , Zouain Claudia S. , Pelizzola Mattia , Capozzoli Monica , Urbano Matteo , Capron Monique , Belardelli Filippo , Granucci Francesca , Ricciardi-Castagnoli Paola
Published: April 2014

Schistosomes are helminth parasites that display a dual impact on the immune system of their hosts. Although the larval stage, also known as schistosomulum, appears to subvert the host defenses, the egg stage induces strong inflammatory reactions. Given the pivotal role of dendritic cells (DC) in initiating and regulating immune responses, we compared the distinct transcriptional programs induced in immature mouse DC by S. mansoni eggs or schistosomula. Although SLA abrogated the transcription of many genes implicated in DC functions, eggs caused myeloid DC to produce IFN-β. Autocrine/paracrine signaling through the type I IFN receptor in response to eggs was necessary for the induction of known IFN-responsive genes and enhanced the synthesis of key inflammatory products. Taken as a whole, our data provide molecular insights into the immune evasion mechanism of schistosomula and suggest an unexpected role for type I IFN in the innate response to helminth eggs.
The Contribution of the Toll-Like/IL-1 Receptor Superfamily to Innate and Adaptive Immunity to Fungal Pathogens In Vivo
Authors: Bellocchio Silvia , Montagnoli Claudia , Bozza Silvia , Gaziano Roberta , Rossi Giordano , Mambula Salamatu S. , Vecchi Annunciata , Mantovani Alberto , Levitz Stuart M. , Romani Luigina
Published: April 2014

In vitro studies have indicated the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens Candida albicans and Aspergillus fumigatus. However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling in vivo remain largely undefined. In this study we evaluate the impact of the IL-1R/TLR/myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway on the innate and adaptive Th immunities to C. albicans and A. fumigatus in vivo. It was found that 1) the MyD88-dependent pathway is required for resistance to both fungi; 2) the involvement of the MyD88 adapter may occur through signaling by distinct members of the IL-1R/TLR superfamily, including IL-1R, TLR2, TLR4, and TLR9, with the proportional role of the individual receptors varying depending on fungal species, fungal morphotypes, and route of infection; 3) individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection; and 4) MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses. Thus, the finding that the innate and adaptive immunities to C. albicans and A. fumigatus require the coordinated action of distinct members of the IL-1R/TLR superfamily acting through MyD88 makes TLR manipulation amenable to the induction of host resistance to fungi.
Enhanced CD8+ T Cell Immune Responses and Protection Elicited against Plasmodium berghei Malaria by Prime Boost Immunization Regimens Using a Novel Attenuated Fowlpox Virus
Authors: Anderson Richard J. , Hannan Carolyn M. , Gilbert Sarah C. , Laidlaw Stephen M. , Sheu Eric G. , Korten Simone , Sinden Robert , Butcher Geoffrey A. , Skinner Michael A. , Hill Adrian V. S.
Published: April 2014

Sterile immunity can be provided against the pre-erythrocytic stages of malaria by IFN-γ-secreting CD8+ T cells that recognize parasite-infected hepatocytes. In this study, we have investigated the use of attenuated fowlpox virus (FPV) strains as recombinant vaccine vectors for eliciting CD8+ T cells against Plasmodium berghei. The gene encoding the P. berghei circumsporozoite (PbCS) protein was inserted into an FPV vaccine strain licensed for use in chickens, Webster’s FPV, and the novel FPV vaccine strain FP9 by homologous recombination. The novel FP9 strain proved more potent as a vaccine for eliciting CD8+ T cell responses against the PbCS Ag. Sequential immunization with rFP9 and recombinant modified vaccinia virus Anakara (MVA) encoding the PbCS protein, administered by clinically acceptable routes, elicited potent CD8+ T cell responses against the PbCS protein. This immunization regimen elicited substantial protection against a stringent liver-stage challenge with P. berghei and was more immunogenic and protective than DNA/MVA prime/boost immunization. However, further improvement was not achieved by sequential (triple) immunization with a DNA vaccine, FP9, and MVA.
The Effect of Innate Immunity on Autoimmune Diabetes and the Expression of Toll-Like Receptors on Pancreatic Islets
Authors: Wen Li , Peng Jian , Li Zhenjun , Wong F. Susan
Published: April 2014

Viral infections have previously been implicated as a trigger of autoimmune diabetes. In this study, we compared a viral mimic with other microbial components derived from bacteria in triggering diabetes development in C57BL/6-rat insulin promoter-B7.1 mice that do not normally develop diabetes. It is striking that only the viral mimic induced the development of diabetes in our model system. Further mechanistic studies suggest that diabetes is induced, in part, by the combination of direct recognition of this virus-like stimulus by pancreatic islets through the expression of the innate immune receptor, Toll-like receptor 3. In addition, the functions of APCs are up-regulated, and this could stimulate islet Ag-reactive T cells that will attack β cells leading to autoimmune diabetes.
Acidosis Improves Uptake of Antigens and MHC Class I-Restricted Presentation by Dendritic Cells
Authors: Vermeulen Mónica , Giordano Mirta , Trevani Analía S. , Sedlik Christine , Gamberale Romina , Fernández-Calotti Paula , Salamone Gabriela , Raiden Silvina , Sanjurjo Julieta , Geffner Jorge R.
Published: April 2014

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8+ CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.
CD40 on Adult Human Airway Epithelial Cells: Expression and Proinflammatory Effects
Authors: Cagnoni Francesca , Oddera Susanna , Giron-Michel Julien , Riccio Anna Maria , Olsson Susanna , Dellacasa Palmiro , Melioli Giovanni , Canonica G. Walter , Azzarone Bruno
Published: April 2014

CD40/CD40 ligand interaction is an important pathway for B and T cell cooperation and function; functional CD40 molecules have recently been found on nonhematopoietic cells. We detected CD40 in vivo on normal human respiratory epithelial cells and showed that its expression is increased on inflamed airway epithelium. Subsequently, we analyzed its expression and function on primary cultures of human airway epithelial cells. Our data show that CD40 is up-regulated by IFN-β and IFN-γ, its ligation increases the surface expression of CD54 and CD106 and it may stimulate the release of IL-6 and IL-8. The use of Janus kinase 3 (JAK3) and NF-κB inhibitors suggests that both basal and CD40-induced release of the two cytokines is JAK3-dependent. Using colocalization techniques, we revealed the existence of CD40/JAK3 and CD40/TNFR-associated factor 2 interplay. The extent of these interactions may be partial (2–40% of the cells) or massive (80–90% of the cells) in cultured cells. Stimulation via CD40 causes a significant increase in the number of cells expressing colocalization only in the cultures displaying low frequency of initial colocalization. Thus, airway epithelial cells, activated by CD40, may behave as effector cells of the inflammation process and should be considered priority targets for anti-inflammatory therapy. This work identifies CD40 and the correlated JAK3 signaling molecule as potential molecular targets to block the inflammatory functions of epithelial cells.
Direct In Vivo Monitoring of Acute Allergic Reactions in Human Conjunctiva
Authors: Helintö Maaret , Renkonen Risto , Tervo Timo , Vesaluoma Minna , Saaren-Seppälä Heikki , Haahtela Tari , Kirveskari Juha
Published: April 2014

Immediate allergic reactions are initiated by allergen-induced, specific IgE-mediated mast cell degranulation and involve leukocyte recruitment into the inflamed site. We compared conjunctival signs, symptoms, and in vivo leukocyte rolling and extravasation into sites of inflammation in five patients allergic to birch pollen and in 10 nonallergic controls who received a challenge to birch allergen or histamine. Both the specific allergen in allergic patients and histamine, both in patients and in healthy controls, induced symptoms and signs of an immediate allergic reaction together with leukocyte rolling within the conjunctival blood vessels. However, only allergen, not histamine, caused leukocyte extravasation into the site of inflammation in the allergic patients. Allergen also increased expression of endothelial P-selectin in conjunctival vessels and slowed the rolling of leukocytes which is required for their extravasation from blood circulation into the target tissue. Finally, i.v. heparin strongly reduced the number of slowly rolling cells during allergen- or histamine-induced reactions and this can probably hinder the leukocyte extravasation after allergen exposure. These findings suggest that slow rolling is required for leukocyte extravasation in acute allergic reactions, and it can be inhibited by heparin in vivo in therapeutically relevant conditions.
Grass Pollen Immunotherapy Induces Mucosal and Peripheral IL-10 Responses and Blocking IgG Activity
Authors: Nouri-Aria Kayhan T. , Wachholz Petra A. , Francis James N. , Jacobson Mikila R. , Walker Samantha M. , Wilcock Louisa K. , Staple Steven Q. , Aalberse Robert C. , Till Stephen J. , Durham Stephen R.
Published: April 2014

T regulatory cells and IL-10 have been implicated in the mechanism of immunotherapy in patients with systemic anaphylaxis following bee stings. We studied the role of IL-10 in the induction of clinical, cellular, and humoral tolerance during immunotherapy for local mucosal allergy in subjects with seasonal pollinosis. Local and systemic IL-10 responses and serum Ab concentrations were measured before/after a double-blind trial of grass pollen (Phleum pratense, Phl P) immunotherapy. We observed local increases in IL-10 mRNA-positive cells in the nasal mucosa after 2 years of immunotherapy, but only during the pollen season. IL-10 protein-positive cells were also increased and correlated with IL-10 mRNA+ cells. These changes were not observed in placebo-treated subjects or in healthy controls. Fifteen and 35% of IL-10 mRNA signals were colocalized to CD3+ T cells and CD68+ macrophages, respectively, whereas only 1–2% of total CD3+ cells and 4% of macrophages expressed IL-10. Following immunotherapy, peripheral T cells cultured in the presence of grass pollen extract also produced IL-10. Immunotherapy resulted in blunting of seasonal increases in serum allergen Phl p 5-specific IgE, 60- to 80-fold increases in Phl p 5-specific IgG, and 100-fold increases in Phl p 5-specific IgG4. Post-immunotherapy serum exhibited inhibitory activity, which coeluted with IgG4, and blocked IgE-facilitated binding of allergen-IgE complexes to B cells. Both the increases in IgG and the IgG “blocking” activity correlated with the patients’ overall assessment of improvement. Thus, grass pollen immunotherapy may induce allergen-specific, IL-10-dependent “protective” IgG4 responses.
Molecular and Immunological Evaluation of the Transcription Factor SOX-4 as a Lung Tumor Vaccine Antigen
Authors: Friedman Rachel S. , Bangur Chaitanya S. , Zasloff Eden J. , Fan Liqun , Wang Tongtong , Watanabe Yoshihiro , Kalos Michael
Published: April 2014

The developmental transcription factor SOX-4 has been shown to be highly and differentially overexpressed in primary small cell lung carcinomas (SCLC). To examine the potential of SOX-4 for broad use as a lung cancer vaccine, we have evaluated the expression of SOX-4 in a panel of primary adenocarcinoma, squamous, and large cell tumor samples as well as in a panel of established small cell and non-small cell lung carcinoma tumor cell lines. SOX-4 mRNA is shown to be overexpressed in a substantial fraction of each of these lung tumor types. To examine the immunological potential of SOX-4, we have evaluated the presence of SOX-4-specific CD4 and CD8 T cells in PBMC of healthy donors and the presence of SOX4-specific Abs in sera from SCLC patients. We demonstrate the presence of both CD4 and CD8 T cells that recognize naturally processed epitopes derived from SOX-4 as well as the presence of SOX-4-specific Abs in sera from SCLC patients, but not in sera from healthy donors. The lung tumor-specific overexpression and demonstration of a comprehensive Ag-specific immune response specific for SOX-4 support the use of this molecule in the development of whole gene-, peptide-, or protein-based vaccination strategies against lung cancer. Furthermore, the identification of naturally processed T cell and Ab epitopes from SOX-4 provides valuable tools for the development of peptide-based vaccination strategies against lung cancer as well as to monitor SOX-4-specific responses in vaccinated patients.
Textbook Germinal Centers?
Authors: Manser Tim
Published: April 2014

Models for the development and function of germinal centers (GCs) have been so widely discussed in the original literature that they now appear in immunology textbooks. Unfortunately, many of the tenets of these models have not yet been subjected to adequate experimental scrutiny. Indeed, recent studies have called several of their principal assumptions into question. In addition, the term germinal center has been applied to a diverse assortment of focal processes of B cell proliferation and differentiation. This variability might be explained by alterations in the progression of a single textbook GC process. Alternatively, distinct developmental pathways may create unique classes of GCs with specialized functions.
Suppression of CD4+ T Lymphocyte Effector Functions by CD4+CD25+ Cells In Vivo
Authors: Martin Bruno , Banz Alice , Bienvenu Boris , Cordier Corinne , Dautigny Nicole , Bécourt Chantal , Lucas Bruno
Published: April 2014

CD4+CD25+ regulatory T cells have been extensively studied during the last decade, but how these cells exert their regulatory function on pathogenic effector T cells remains to be elucidated. Naive CD4+ T cells transferred into T cell-deficient mice strongly expand and rapidly induce inflammatory bowel disease (IBD). Onset of this inflammatory disorder depends on IFN-γ production by expanding CD4+ T cells. Coinjection of CD4+CD25+ regulatory T cells protects recipient mice from IBD. In this study, we show that CD4+CD25+ regulatory T cells do not affect the initial activation/proliferation of injected naive T cells as well as their differentiation into Th1 effectors. Moreover, naive T cells injected together with CD4+CD25+ regulatory T cells into lymphopenic hosts are still able to respond to stimuli in vitro when regulatory T cells are removed. In these conditions, they produce as much IFN-γ as before injection or when injected alone. Finally, when purified, they are able to induce IBD upon reinjection into lymphopenic hosts. Thus, prevention of IBD by CD4+CD25+ regulatory T cells is not due to deletion of pathogenic T cells, induction of a non reactive state (anergy) among pathogenic effector T cells, or preferential induction of Th2 effectors rather than Th1 effectors; rather, it results from suppression of T lymphocyte effector functions, leading to regulated responses to self.
Prolonged Antigen Presentation, APC-, and CD8+ T Cell Turnover during Mycobacterial Infection: Comparison withListeria monocytogenes
Authors: van Faassen Henk , Dudani Renu , Krishnan Lakshmi , Sad Subash
Published: April 2014

We expressed the CTL epitope of OVA (OVA257–264) in an acute (Listeria monocytogenes (LM)-OVA) and a chronic intracellular pathogen (Mycobacterium bovis (BCG)-OVA), to evaluate the kinetics of Ag presentation. LM-OVA proliferated rapidly in vivo, resulting in profound LM-OVA expansion within the first 24 h of infection, culminating in the generation of a potent CD8+ T cell response, which peaked on day 7 but underwent a rapid attrition subsequently. In contrast, BCG-OVA exhibited reduced growth in vivo, resulting in a delayed CD8+ T cell response that increased progressively with time. Relative to LM-OVA, BCG-OVA induced persistently increased numbers of apoptotic (annexin V+) CD8+ T cells. Ag presentation in vivo was evaluated by transferring Thy1.2+ carboxyfluorescein-labeled OT1 transgenic CD8+ T cells into infected Thy1.1+ congeneic recipient mice. LM-OVA induced rapid Ag presentation that was profound in magnitude, with most of the transferred cells getting activated within 4 days and resulting in a massive accumulation of activated donor CD8+ T cells. In contrast, Ag presentation induced by BCG-OVA was delayed, weaker in magnitude, which peaked around the second week of infection and declined to a low level subsequently. Increasing the dose of BCG-OVA while enhancing the magnitude of Ag presentation did not change the kinetics. Furthermore, a higher dose of BCG-OVA also accelerated the attrition of OVA257–264-specific CD8+ T cells. Relative to LM-OVA, the dendritic cells in BCG-OVA-infected mice were apoptotic for prolonged periods, suggesting that the rapid death of APCs may limit the magnitude of Ag presentation during chronic stages of mycobacterial infection.
The Cell-Cell Adhesion Molecule Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 1 Inhibits IL-2 Production and Proliferation in Human T Cells by Association with Src Homology Protein-1 and Down-Regulates IL-2 Receptor
Authors: Chen Charng-Jui , Shively John E.
Published: April 2014

The cell adhesion molecule, carcinoembryonic Ag-related cellular adhesion molecule 1, shown by others to both activate and inhibit T cell proliferation, exhibits a reciprocal relationship to IL-2R expression over the time course of activation of PBMCs, and upon Ab ligation, inhibits both the production of IL-2 and cell proliferation. Carcinoembryonic Ag-related cellular adhesion molecule 1 associates with CD3 and is found in lipid rafts of PBMCs, is phosphorylated on the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of the -4L isoform, and associates with Src homology protein-1, providing an explanation for its inhibitory activity. When the ITIM-containing -4L and non-ITIM-containing -4S isoforms are transfected into Jurkat cells that produce, but do not depend on IL-2 for growth, both IL-2 production and cell proliferation are differentially inhibited, demonstrating that the two isoforms signal via different pathways. When the two isoforms are transfected into Kit-225 cells that depend on IL-2 for growth, IL-2Rβ and γ, but not α subunits are down-regulated, and the -4L, but not the -4S isoform inhibits cell proliferation by 6-fold in an IL-2 dose-response study.
A Keratinocyte-Responsive γδ TCR Is Necessary for Dendritic Epidermal T Cell Activation by Damaged Keratinocytes and Maintenance in the Epidermis
Authors: Jameson Julie M. , Cauvi Gabrielle , Witherden Deborah A. , Havran Wendy L.
Published: April 2014

A unique population of T lymphocytes, designated dendritic epidermal T cells (DETC), homes to the murine epidermis during fetal development. DETC express a canonical γδ TCR, Vγ3/Vδ1, which recognizes Ag expressed on damaged, stressed, or transformed keratinocytes. Recently, DETC were shown to play a key role in the complex process of wound repair. To examine the role of the DETC TCR in DETC localization to the epidermis, maintenance in the skin, and activation in vivo, we analyzed DETC in the TCRδ−/− mouse. Unlike previous reports in which the TCRδ−/− skin was found to be devoid of any DETC, we discovered that TCRδ−/− mice have αβ TCR-expressing DETC with a polyclonal Vβ chain repertoire. The αβ DETC are not retained over the life of the animal, suggesting that the γδ TCR is critical for the maintenance of DETC in the skin. Although the αβ DETC can be activated in response to direct stimulation, they do not respond to keratinocyte damage. Our results suggest that a keratinocyte-responsive TCR is necessary for DETC activation in response to keratinocyte damage and for DETC maintenance in the epidermis.
Inhibition of NFAT Specifically in T Cells Prevents Allergic Pulmonary Inflammation
Authors: Diehl Sean , Krahl Troy , Rinaldi Lisa , Norton Ryan , Irvin Charles G. , Rincón Mercedes
Published: April 2014

NFAT is a family of transcription factors important in the regulation of cytokine genes and is widely expressed in different lymphoid and nonlymphoid tissues. Consequently, the role of NFAT in CD4+ T cells during an in vivo immune response is not completely clear. In this study, we use transgenic mice expressing a dominant negative NFAT mutant exclusively in T cells to address the role of NFAT in T cells during a Th2 immune response in a model of allergic airway inflammation. We have observed that inhibition of NFAT in T cells results in a reduction of Ag-specific Th2 Ab levels and IL-4 production by CD4+ T cells. The accumulation of eosinophils in the bronchoalveolar lavage is delayed in dominant negative NFAT-transgenic mice. These mice are also more resistant to the development of lung pathology in response to allergen exposure. We, therefore, conclude that activation of NFAT in CD4+ T cells is required for the development of a Th2 immune response in vivo and allergic airway inflammation.
Aberrant Toll Receptor Expression and Endotoxin Hypersensitivity in Mice Lacking a Functional TGF-β1 Signaling Pathway
Authors: McCartney-Francis Nancy , Jin Wenwen , Wahl Sharon M.
Published: April 2014

TGF-β1 plays a central role in maintaining normal immune function and deficiency of this potent immunosuppressive molecule is linked to uncontrolled inflammation, cachexia, and multiorgan failure as seen in the TGF-β1 null mouse. Infiltration of inflammatory cells into vital organs of the null mouse is accompanied by increased gene expression of inflammatory cytokines, including TNF-α and IL-1β, as well as inducible NO synthase, each regulated by NF-κB. Treatment with the proteasome inhibitor MG132 to prevent NF-κB activation dramatically reduced NO production and expression of inflammatory cytokines. This inflammatory phenotype with NF-κB activation in the TGF-β1 null mouse, in the absence of any identifiable pathogen, suggested activation of innate immune responses. Because Toll-like receptors (TLR) are essential in the activation of innate immunity, we examined inflamed tissue from TGF-β1 null and wild-type mice for expression of TLR4, the receptor that interacts with bacterial cell wall LPS to initiate an NF-κB-dependent signaling pathway, leading to gene transcription of inflammatory mediators. Increased TLR4 mRNA expression observed in TGF-β1 null mice as well as in mice lacking the TGF-β transcription factor Smad3 was associated with LPS hyperresponsiveness leading to increased expression of inflammatory cytokines and NO and endotoxemia. Furthermore, mice lacking both TGF-β1 and a functional TLR4 were resistant to endotoxin shock. Constitutive and/or environmental activation of TLR4 and downstream elements, in the absence of TGF-β suppression, may impact on innate and adaptive immunity and contribute to massive uncontrolled inflammation.