bluuebunny/crossref_metadata_2025 huggingface dataset:
An Important Role of CD80/CD86-CTLA-4 Signaling during Photocarcinogenesis in Mice
Authors: Loser Karin , Scherer Andrea , Krummen Mathias B. W. , Varga Georg , Higuchi Tetsuya , Schwarz Thomas , Sharpe Arlene H. , Grabbe Stephan , Bluestone Jeffrey A. , Beissert Stefan
Published: April 2014

Although previous studies have shown that altered B7 costimulation plays a critical role in UV irradiation-induced regulation of immunity, the individual roles of the B7 receptors (CD28 and CTLA-4) or the B7 family members (CD80 and CD86) have not been explored. Thus, we investigated CTLA-4 signaling during photocarcinogenesis of chronically UV-B-exposed mice using an antagonistic anti-CTLA-4 Ab. Anti-CTLA-4-treated mice developed significantly fewer UV-induced tumors. Moreover, anti-CTLA-4 treatment induced long-lasting protective immunity because progressively growing UV tumors inoculated into anti-CTLA-4- and UV-treated mice that had not developed tumors were rejected. Next, we used mice deficient for CD80, CD86, or both in photocarcinogenesis studies to assess the relative contributions of these CTLA-4 ligands. Double-deficient mice showed significantly reduced UV-induced skin tumor development, whereas CD86−/− mice produced skin cancer earlier compared with CD80−/− and control mice. The growth of UV-induced tumors appears to be controlled by UV-induced suppressor T cells, because CD80−/−/CD86−/− mice had strongly reduced numbers of UV-induced CD4+CD25+ suppressor T cells. In vitro, CTLA-4 blockade inhibited the suppressor activity of UV-induced CD4+CD25+ T cells, suggesting that reduced photocarcinogenesis might be due to decreased numbers or function of suppressor T cells. Together, these data indicate that blocking CD80/86-CTLA-4 signaling induced immune protection against the development of UV-induced skin tumors. Furthermore, CD86-mediated costimulation appears to play a more critical role in the protection against photocarcinogenesis than CD80.
Identification of CD4+ T Cell-Specific Epitopes of Islet-Specific Glucose-6-Phosphatase Catalytic Subunit-Related Protein: A Novel β Cell Autoantigen in Type 1 Diabetes
Authors: Mukherjee Rinee , Wagar Danielle , Stephens Tracey A. , Lee-Chan Edwin , Singh Bhagirath
Published: April 2014

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) has been identified as a novel CD8+ T cell-specific autoantigen in NOD mice. This study was undertaken to identify MHC class II-specific CD4+ T cell epitopes of IGRP. Peptides named P1, P2, P3, P4, P5, P6, and P7 were synthesized by aligning the IGRP protein amino acid sequence with peptide-binding motifs of the NOD MHC class II (I-Ag7) molecule. Peptides P1, P2, P3, and P7 were immunogenic and induced both spontaneous and primed responses. IGRP peptides P1-, P2-, P3-, and P7-induced responses were inhibited by the addition of anti-MHC class II (I-Ag7) Ab, confirming that the response is indeed I-Ag7 restricted. Experiments using purified CD4+ and CD8+ T cells from IGRP peptide-primed mice also showed a predominant CD4+ T cell response with no significant activation of CD8+ T cells. T cells from P1-, P3-, and P7-primed mice secreted both IFN-γ and IL-10 cytokines, whereas P2-primed cells secreted only IFN-γ. Peptides P3 and P7 prevented the development of spontaneous diabetes and delayed adoptive transfer of diabetes. Peptides P1 and P2 delayed the onset of diabetes in both these models. In summary, we have identified two I-Ag7-restricted CD4+ T cell epitopes of IGRP that can modulate and prevent the development of diabetes in NOD mice. These results provide the first evidence on the role of IGRP-specific, MHC class II-restricted CD4+ T cells in disease protection and may help in the development of novel therapies for type 1 diabetes.
Frequency, Specificity, and Sites of Expansion of CD8+ T Cells during Primary Pulmonary Influenza Virus Infection
Authors: Lawrence Christopher W. , Ream Rebecca M. , Braciale Thomas J.
Published: April 2014

We have used intracellular cytokine staining and MHC class I tetramer binding in conjunction with granzyme B protease expression and in vivo BrdU uptake to characterize the primary murine CD8+ T cell response to pulmonary influenza virus infection. We have observed that the majority (>90%) of the CD8+ T cell response to the A/Japan/305/57 virus in the lung at the peak of the response (days 9–11) is directed to four epitopes (three dominant and one subdominant). Using induction of granzyme B as a surrogate to identify specific activated CD8+ T cells, we found that an unexpectedly large fraction (∼70%) of lung-infiltrating CD8+ T cells expressed granzyme B on day 6 of infection when estimates by MHC tetramer/intracellular cytokine staining yielded substantially lower frequencies (∼30%). In addition, by using intranasal administration of BrdU during infection, we obtained evidence for proliferative expansion of activated CD8+ T cells in the infected lung early (days 5–7) in the primary response. These results suggest that the frequency and number of specific CTL present in the lung early in infection may be underestimated by standard detection methods, and primary CD8+ T cell expansion may occur in both secondary lymphoid organs and the infected lung.
Protective Immunity against Disparate Tumors Is Mediated by a Nonpolymorphic MHC Class I Molecule
Authors: Chiang Eugene Y. , Stroynowski Iwona
Published: April 2014

Current peptide-based immunotherapies for treatment of model cancers target tumor Ags bound by the classical MHC class I (class Ia) molecules. The extensive polymorphism of class Ia loci greatly limits the effectiveness of these approaches. We demonstrate in this study that the murine nonpolymorphic, nonclassical MHC class I (class Ib) molecule Q9 (Qa-2) promotes potent immune responses against multiple syngeneic tumors. We have previously shown that ectopic expression of Q9 on the surface of class Ia-negative B78H1 melanoma led to efficient CTL-mediated rejection of this tumor. In this study, we report that surface-expressed Q9 on 3LLA9F1 Lewis lung carcinoma and RMA T cell lymphoma also induces potent antitumor CTL responses. Importantly, CTL harvested from animals surviving the initial challenge with Q9-positive 3LLA9F1, RMA, or B78H1 tumors recognized and killed their cognate tumors as well as the other cancer lines. Furthermore, immunization with Q9-expressing 3LLA9F1 or RMA tumor cells established immunological memory that enhanced protection against subsequent challenge with a weakly immunogenic, Q9-bearing melanoma variant. Collectively, the generation of cross-reactive CTL capable of eliminating multiple disparate Q9-expressing tumors suggests that this nonpolymorphic MHC class I molecule serves as a restriction element for a shared tumor Ag(s) common to lung carcinoma, T cell lymphoma, and melanoma.
B Cell Receptor (BCR) Cross-Talk: IL-4 Creates an Alternate Pathway for BCR-Induced ERK Activation That Is Phosphatidylinositol 3-Kinase Independent
Authors: Guo Benchang , Rothstein Thomas L.
Published: April 2014

IL-4 has pleiotropic effects on B cells. These effects include alteration of subsequent BCR-triggered responses. To identify a molecular basis for this receptor cross-talk, we examined ERK activation and NF-κB induction. We found that treatment with IL-4, but not other cytokines, affected subsequent BCR signaling by creating a new pathway in which the need for PI3K in ERK activation was eliminated. In contrast, the need for PI3K in NF-κB induction was not altered. The new pathway for ERK required time to develop, depended on STAT6, and was blocked by inhibition of macromolecular synthesis. As in the classical pathway, BCR-induced ERK activation in the new, PI3K-independent pathway required MEK and was reflected in c-Raf. Thus, IL-4 promotes an alternate pathway through which BCR is coupled to Raf/MEK/ERK that may function to heighten the responsiveness of B cells during times of immunological stress.
Corticotropin-Releasing Hormone Contributes to the Peripheral Inflammatory Response in Experimental Autoimmune Encephalomyelitis
Authors: Benou Christina , Wang Yue , Imitola Jaime , VanVlerken Lilian , Chandras Christina , Karalis Katia P. , Khoury Samia J.
Published: April 2014

Peripheral corticotropin-releasing hormone (CRH) is thought to have proinflammatory effects. We used the model of experimental autoimmune encephalomyelitis (EAE) to study the role of CRH in an immune-mediated disease. We showed that CRH-deficient mice are resistant to EAE, with a decrease in clinical score as well as decreased cellular infiltration in the CNS. Furthermore, Ag-specific responses of primed T cells as well as anti-CD3/anti-CD28 TCR costimulation were decreased in crh−/− mice with decreased production of Th1 cytokines and increased production of Th2 cytokines. Wild-type mice treated in vivo with a CRH antagonist showed a decrease in IFN-γ production by primed T cells in vitro. This effect of CRH is independent of its ability to increase corticosterone production, because adrenalectomized wild-type mice had similar disease course and severity as control mice. We found that IκBα phosphorylation induced by TCR cross-linking was decreased in crh−/− T cells. We conclude that peripheral CRH exerts a proinflammatory effect in EAE with a selective increase in Th1-type responses. These findings have implications for the treatment of Th1-mediated diseases such as multiple sclerosis.
Phenotypic Characterization of CD3−7+ Cells in Developing Human Intestine and an Analysis of Their Ability to Differentiate into T Cells
Authors: Gunther Ute , Holloway Judith A. , Gordon John G. , Knight Andrea , Chance Victoria , Hanley Neil A. , Wilson David I. , French Ruth , Spencer Jo , Steer Howard , Anderson Graham , MacDonald Thomas T.
Published: April 2014

We have identified a large population of CD3−7+ cells in human fetal gut. Three- and four-color flow cytometry revealed a distinct surface Ag profile on this population; the majority were negative for CD4 and CD8, whereas most of the remainder expressed the CD8αα homodimer. In contrast about half of CD3+ cells expressed CD4 and half expressed CD8α. A large proportion of CD3−7+ cells expressed CD56, CD94, and CD161, and whereas CD3+ T cells also expressed CD161, they only rarely expressed CD56 or CD94. Further studies were conducted to determine whether the CD3−7+ cells have the potential to differentiate into CD3+ cells. About half of CD3−7+ cells contain intracellular CD3ε. Rearranged TCR γ-chains were detected in highly purified CD3−7+ cells as an early molecular sign of T cell commitment, and the pattern of rearrangement with V regions spliced to the most 5′ Jγ segment is reminiscent of early thymocyte differentiation. In reaggregate thymic organ cultures, CD3−7+ cells also gave rise to CD3+ T cells. Thus, we demonstrate that the CD3−7+ cells present in the human fetal gut display a distinct phenotype and are able to develop into CD3+ T cells.
Modulation of Memory CD4 T Cell Function and Survival Potential by Altering the Strength of the Recall Stimulus
Authors: Patke Deepa S. , Farber Donna L.
Published: April 2014

Optimization of long term immunity depends on the functional persistence of memory T cells; however, there are no defined strategies for promoting memory T cell function and survival. In this study, we hypothesized that varying the strength of the recall stimulus could modulate the function and survival potential of memory CD4 T cells. We tested the ability of peptide variants of influenza hemagglutinin (HA) exhibiting strong and weak avidity for an HA-specific TCR, to modulate HA-specific memory CD4 T cells in vitro and in vivo. In vitro stimulation with a weak avidity peptide (L115) uncoupled memory CD4 T proliferation from effector cytokine production with low apoptosis, whereas stimulation with a strong avidity peptide (Y117) fully recalled memory T cell functions but triggered increased apoptosis. To determine how differential recall would affect memory T cells in vivo, we boosted BALB/c hosts of transferred, CFSE-labeled HA-specific memory CD4 T cells with native HA, Y117, and L115 variant peptides and found differences in early Ag-driven memory T cell proliferation and IL-7R expression, with subsequent changes in memory T cell yield. High avidity boosting resulted in rapid proliferation, extensive IL-7R down-regulation, and the lowest yield of HA-specific memory cells, whereas low avidity boosting triggered low in vivo proliferation, maintenance of IL-7R expression, and the highest memory T cell yield. Our results indicate that memory CD4 T cell function and survival can be modulated at the recall level, and can be optimized by low level stimulation that minimizes apoptosis and enhances responses to survival factors.
Immature Neutrophils Mediate Tumor Cell Killing via IgA but Not IgG Fc Receptors
Authors: Otten Marielle A. , Rudolph Esther , Dechant Michael , Tuk Cornelis W. , Reijmers Rogier M. , Beelen Robert H. J. , van de Winkel Jan G. J. , van Egmond Marjolein
Published: April 2014

Antitumor Abs are promising therapeutics for cancer. Currently, most Ab-based therapies focus on IgG Ab, which interact with IgG FcR (FcγR) on effector cells. In this study, we examined human and mouse neutrophil-mediated tumor cell lysis via targeting the IgA FcR, FcαRI (CD89), in more detail. FcαRI was the most effective FcR in triggering tumor cell killing, and initiated enhanced migration of neutrophils into tumor colonies. Importantly, immature neutrophils that are mobilized from the bone marrow upon G-CSF treatment efficiently triggered tumor cell lysis via FcαRI, but proved incapable of initiating tumor cell killing via FcγR. This may provide a rationale for the disappointing results observed in some earlier clinical trials in which patients were treated with G-CSF and antitumor Ab-targeting FcγR.
Targeted Delivery of the ErbB2/HER2 Tumor Antigen to Professional APCs Results in Effective Antitumor Immunity
Authors: Rohrbach Florian , Weth Robert , Kursar Mischo , Sloots Arjen , Mittrücker Hans-Willi , Wels Winfried S.
Published: April 2014

Activation of T cells by professional APCs that present peptide epitopes of tumor-associated Ags is critical for the induction of cell-mediated immunity against tumors. To facilitate targeted delivery of the ErbB2 (HER2, neu) tumor Ag to APCs in vivo, we have generated chimeric proteins that contain the extracellular domain of CTLA-4 for binding to B7 molecules on the APC surface, which is genetically fused to a human ErbB2 fragment as an antigenic determinant. Bacterially expressed CTLA-4-ErbB2 fusion protein and a similar molecule harboring in addition the translocation domain of Pseudomonas exotoxin A as an endosome escape function displayed specific binding to B7-expressing cells, followed by protein internalization and intracellular degradation. Vaccination of BALB/c mice with the fusion proteins resulted in the induction of ErbB2-specific CD8+ T cells and CTL-dependent protection from subsequent challenge with ErbB2-expressing but not ErbB2-negative murine renal carcinoma cells. In a therapeutic setting, injection of CTLA-4-ErbB2 protein vaccines caused rejection of established ErbB2-expressing tumors. Thereby, immunological memory was induced, leading to long-term systemic immunity and protection against rechallenge several months later. Our results demonstrate that these chimeric protein vaccines are effective tools for the induction of ErbB2-specific, T cell-mediated immunity.
Blocking IL-15 Prevents the Induction of Allergen-Specific T Cells and Allergic Inflammation In Vivo
Authors: Rückert René , Brandt Katja , Braun Armin , Hoymann Heinz-Gerd , Herz Udo , Budagian Vadim , Dürkop Horst , Renz Harald , Bulfone-Paus Silvia
Published: April 2014

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Rα (sIL-15Rα) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-α, IL-1β, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Rα treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Rα-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4+/CD25+ T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.
Lymphotoxin-β Receptor-Dependent Genes in Lymph Node and Follicular Dendritic Cell Transcriptomes
Authors: Huber Christoph , Thielen Caroline , Seeger Harald , Schwarz Petra , Montrasio Fabio , Wilson Mark R. , Heinen Ernst , Fu Yang-Xin , Miele Gino , Aguzzi Adriano
Published: April 2014

Affinity maturation and Ab class switches occur in lymphoid germinal centers (GCs), in which differentiation and maintenance depend on lymphotoxin (LT) signaling and include differentiation of follicular dendritic cells (FDCs). The events leading to FDC and GC maturation are poorly defined. Using several approaches of functional genomics, we enumerated transcripts affected in mice by suppressing LT β receptor (LTβR) signaling and/or overrepresented in FDC-enriched GC isolates. Protein expression analysis of 3 of 12 genes both enriched in FDCs and down-regulated by LTβR signaling suppression validated them as FDC markers. Functional analysis of one of these three, clusterin, suggests a role as an FDC-derived trophic factor for GC B cells. Hence, the set of genes presented in this study includes markers emanating from LTβR signaling and transcripts relevant to GC and FDC function.
Antagonism of Antiviral and Allogeneic Activity of a Human Public CTL Clonotype by a Single Altered Peptide Ligand: Implications for Allograft Rejection
Authors: Ely Lauren K. , Green Katherine J. , Beddoe Travis , Clements Craig S. , Miles John J. , Bottomley Stephen P. , Zernich Danielle , Kjer-Nielsen Lars , Purcell Anthony W. , McCluskey James , Rossjohn Jamie , Burrows Scott R.
Published: April 2014

Alloreactive T lymphocytes are central mediators of graft-versus-host disease and allograft rejection. A public CTL clonotype with specificity for the alloantigens HLA-B*4402 and B*4405 is often expanded to large numbers in healthy HLA-B*0801+ individuals, driven by cross-reactive stimulation with the common, persistent herpesvirus EBV. Since such alloreactive memory CTL expansions have the potential to influence transplantation outcome, altered peptide ligands (APLs) of the target HLA-B*0801-binding EBV peptide, FLRGRAYGL, were screened as specific antagonists for this immunodominant clonotype. One APL, FLRGRFYGL, exerted powerful antagonism of a prototypic T cell clone expressing this immunodominant TCR when costimulated with target cells presenting HLA-B*0801FLRGRAYGL. Significantly, this APL also reduced the lysis of allogeneic target cells expressing HLA-B*4402 by up to 99%. The affinities of the agonist and antagonist complexes for the public TCR, measured using solution and solid-phase assays, were 8 and 138 μM, respectively. Surprisingly, the half-life of the agonist and antagonist complexes was similar, yet the association rate for the antagonist complex was significantly slower. These observations were further supported by structural studies that suggested a large conformational hurdle was required to ligate the immunodominant TCR to the HLA-B*0801 antagonist complex. By defining an antagonist APL against an immunodominant alloreactive TCR, these findings raise the prospect of exploiting such peptides to inhibit clinical alloreactivity, particularly against clonal T cell expansions that react with alloantigens.
Mass Tag-Assisted Identification of Naturally Processed HLA Class II-Presented Meningococcal Peptides Recognized by CD4+ T Lymphocytes
Authors: Meiring Hugo D. , Kuipers Betsy , van Gaans-van den Brink Jacqueline A. M. , Poelen Martien C. M. , Timmermans Hans , Baart Gino , Brugghe Humphrey , van Schie Joost , Boog Claire J. P. , de Jong Ad P. J. M. , van Els Cécile A. C. M.
Published: April 2014

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of 14N- and 15N-labeled isoforms of the neisserial porin A serosubtype P1.5–2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities (∼30–4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4+ T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4+ T cell epitopes relevant for vaccine development.
Unusually Similar Patterns of Antibody V Segment Diversity in Distantly Related Marsupials
Authors: Baker Michelle L. , Belov Katherine , Miller Robert D.
Published: April 2014

A pattern of coevolution between the V gene segments of Ig H and L chains has been noted previously by several investigators. Species with restricted germline VH diversity tend to have limited germline VL diversity, whereas species with high levels of germline VH diversity have more diverse VL gene segments. Evidence for a limited pool of VH but diverse VL gene segments in a South American opossum, Monodelphis domestica, is consistent with this marsupial being an exception to the pattern. To determine whether M. domestica is unique or the norm for marsupials, the VH and VL of an Australian possum, Trichosurus vulpecula, were characterized. The Ig repertoire in T. vulpecula is also derived from a restricted VH pool but a diverse VL pool. The VL gene segments of T. vulpecula are highly complex and contain lineages that predate the separation of marsupials and placental mammals. Thus, neither marsupial follows a pattern of coevolution of VH and VL gene segments observed in other mammals. Rather, marsupial VH and VL complexity appears to be evolving divergently, retaining diversity in VL perhaps to compensate for limited VH diversity. There is a high degree of similarity between the VH and VL in M. domestica and T. vulpecula, with the majority of VL families being shared between both species. All marsupial VH sequences isolated so far form a common clade of closely related sequences, and in contrast to the VL genes, the VH likely underwent a major loss of diversity early in marsupial evolution.
The Secreted Peptidyl Prolyl cis,trans-Isomerase HP0175 of Helicobacter pylori Induces Apoptosis of Gastric Epithelial Cells in a TLR4- and Apoptosis Signal-Regulating Kinase 1-Dependent Manner
Authors: Basak Chaitali , Pathak Sushil Kumar , Bhattacharyya Asima , Pathak Shresh , Basu Joyoti , Kundu Manikuntala
Published: April 2014

Apoptosis contributes to the pathology of gastric epithelial cell damage that characterizes Helicobacter pylori infection. The secreted peptidyl prolyl cis, trans-isomerase of H. pylori, HP0175 executed apoptosis of the gastric epithelial cell line AGS in a dose- and time-dependent manner. The effect of HP0175 was confirmed by generating an isogenic mutant of H. pylori disrupted in the HP0175 gene. The apoptosis-inducing ability of this mutant was impaired compared with that of the wild type. The effect of HP0175 was mediated through TLR4. Preincubation of the gastric epithelial cell line AGS with anti-TLR4 mAb inhibited apoptosis induced by HP0175. Downstream of TLR4, apoptosis signal-regulating kinase 1 activated MAPK p38, leading to the caspase 8-dependent cleavage of Bid, its translocation to the mitochondria, mitochondrial pore formation, cytochrome c release, and activation of caspases 9 and 3. We show for the first time that a secreted bacterial Ag with peptidyl prolyl cis,trans-isomerase activity signals through TLR4, and that this Ag executes gastric epithelial cell apoptosis through a signaling pathway in which TLR4 and apoptosis signal-regulating kinase 1 are central players.
Molecular and Functional Characterization of NKG2D, NKp80, and NKG2C Triggering NK Cell Receptors in Rhesus and Cynomolgus Macaques: Monitoring of NK Cell Function during Simian HIV Infection
Authors: Biassoni Roberto , Fogli Manuela , Cantoni Claudia , Costa Paola , Conte Romana , Koopman Gerrit , Cafaro Aurelio , Ensoli Barbara , Moretta Alessandro , Moretta Lorenzo , De Maria Andrea
Published: April 2014

An involvement of innate immunity and of NK cells during the priming of adaptive immune responses has been recently suggested in normal and disease conditions such as HIV infection and acute myelogenous leukemia. The analysis of NK cell-triggering receptor expression has been so far restricted to only NKp46 and NKp30 in Macaca fascicularis. In this study, we extended the molecular and functional characterization to the various NK cell-triggering receptors using PBMC and to the in vitro-derived NK cell populations by cytofluorometry and by cytolytic activity assays. In addition, RT-PCR strategy, cDNA cloning/sequencing, and transient transfections were used to identify and characterize NKp80, NKG2D, CD94/NKG2C, and CD94/NKG2A in M. fascicularis and Macaca mulatta as well as in the signal transducing polypeptide DNAX-activating protein DAP-10. Both M. fascicularis and M. mulatta NK cells express NKp80, NKG2D, and NKG2C molecules, which displayed a high degree of sequence homology with their human counterpart. Analysis of NK cells in simian HIV-infected M. fascicularis revealed reduced surface expression of selected NK cell-triggering receptors associated with a decreased NK cell function only in some animals. Overall surface density of NK cell-triggering receptors on peripheral blood cells and their triggering function on NK cell populations derived in vitro was not decreased compared with uninfected animals. Thus, triggering NK cell receptor monitoring on macaque NK cells is possible and could provide a valuable tool for assessing NK cell function during experimental infections and for exploring possible differences in immune correlates of protection in humans compared with cynomolgus and rhesus macaques undergoing different vaccination strategies.
Novel Fibrogenic Pathways Are Activated in Response to Endothelial Apoptosis: Implications in the Pathophysiology of Systemic Sclerosis
Authors: Laplante Patrick , Raymond Marc-André , Gagnon Gabrielle , Vigneault Normand , Sasseville A. Marie-Josée , Langelier Yves , Bernard Monique , Raymond Yves , Hébert Marie-Josée
Published: April 2014

Apoptosis of endothelial cells (EC) is appreciated as a primary pathogenic event in systemic sclerosis. Yet, how apoptosis of EC leads to fibrosis remains to be determined. We report that apoptosis of EC triggers the release of novel fibrogenic mediators. Medium conditioned by apoptotic EC (SSC) was found to inhibit apoptosis of fibroblasts, whereas medium conditioned by EC in which apoptosis was blocked (with either pan-caspase inhibition or Bcl-xL overexpression) did not. PI3K was activated in fibroblasts exposed to SSC. This was associated with downstream repression of Bim-EL and long-term up-regulation of Bcl-xL protein levels. RNA interference for Bim-EL in fibroblasts blocked apoptosis. SSC also induced PI3K-dependent myofibroblast differentiation with expression of α-smooth muscle actin, formation of stress fibers, and production of collagen I. A C-terminal fragment of the domain V of perlecan was identified as one of the fibrogenic mediators present in SSC. A synthetic peptide containing an EGF motif present on the perlecan fragment and chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, induced PI3K-dependent resistance to apoptosis in fibroblasts and myofibroblast differentiation. Human fibroblasts derived from sclerodermic skin lesions were more sensitive to the antiapoptotic activities of the synthetic peptide and chondroitin 4-sulfate than fibroblasts derived from normal controls. Hence, we propose that a chronic increase in endothelial apoptosis and/or increased sensitivity of fibroblasts to mediators produced by apoptotic EC could form the basis of a fibrotic response characterized by sustained induction of an antiapoptotic phenotype in fibroblasts and persistent myofibroblast differentiation.
Gene Therapy to Manipulate Effector T Cell Trafficking to Tumors for Immunotherapy
Authors: Gough Michael , Crittenden Marka , Thanarajasingam Uma , Sanchez-Perez Luis , Thompson Jill , Jevremovic Dragan , Vile Richard
Published: April 2014

Strategies that generate tumor Ag-specific effector cells do not necessarily cure established tumors. We hypothesized that the relative efficiency with which tumor-specific effector cells reach the tumor is critical for therapy. We demonstrate in this study that activated T cells respond to the chemokine CCL3, both in vitro and in vivo, and we further demonstrate that expression of CCL3 within tumors increases the effector T cell infiltrate in those tumors. Importantly, we show that adenoviral gene transfer to cause expression of CCL3 within B16ova tumors in vivo increases the efficacy of adoptive transfer of tumor-specific effector OT1 T cells. We additionally demonstrate that such therapies result in endogenous immune responses to tumor Ags that are capable of protecting animals against subsequent tumor challenge. Strategies that modify the “visibility” of tumors have the potential to significantly enhance the efficacy of both vaccine and adoptive transfer therapies currently in development.
Manipulation of Allergen-Induced Airway Remodeling by Treatment with Anti-TGF-β Antibody: Effect on the Smad Signaling Pathway
Authors: McMillan Sarah J. , Xanthou Georgina , Lloyd Clare M.
Published: April 2014

Airway inflammation and remodeling are important pathophysiologic features of chronic asthma. Previously, we have developed a mouse model of prolonged allergen challenge which exhibits many characteristics of chronic asthma such as goblet cell hyperplasia and subepithelial collagen deposition, in association with an increase in lung expression of the profibrotic mediator, TGF-β. The aim of this study was to determine the effects of blockade of TGF-β on the development of airway inflammation and remodeling using our murine model of prolonged allergen challenge. Importantly anti-TGF-β Ab was administered therapeutically, with dosing starting after the onset of established eosinophilic airway inflammation. Therapeutic treatment of mice with anti-TGF-β Ab significantly reduced peribronchiolar extracellular matrix deposition, airway smooth muscle cell proliferation, and mucus production in the lung without affecting established airway inflammation and Th2 cytokine production. Thus, our data suggest that it might be possible to uncouple airway inflammation and remodeling during prolonged allergen challenge. In addition, anti-TGF-β Ab treatment was shown to regulate active TGF-β signaling in situ with a reduction in the expression of phospho-Smad 2 and the concomitant up-regulation of Smad 7 in lung sections. Therefore, this is the first report to suggest that anti-TGF-β Ab treatment prevents the progression of airway remodeling following allergen challenge even when given in a therapeutic mode. Moreover, the molecular mechanism behind this effect may involve regulation of active TGF-β signaling.
NF-κB Mediates the Stimulation of Cytokine and Chemokine Expression by Human Articular Chondrocytes in Response to Fibronectin Fragments
Authors: Pulai Judit I. , Chen Hong , Im Hee-Jeong , Kumar Sanjay , Hanning Charles , Hegde Priti S. , Loeser Richard F.
Published: April 2014

Fibronectin fragments (FN-f) that bind to the α5β1 integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β). Constitutive and FN-f-inducible expression of GRO-α and GRO-γ were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1β expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-κB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.
Resistin, an Adipokine with Potent Proinflammatory Properties
Authors: Bokarewa Maria , Nagaev Ivan , Dahlberg Leif , Smith Ulf , Tarkowski Andrej
Published: April 2014

The adipokine resistin is suggested to be an important link between obesity and insulin resistance. In the present study, we assessed the impact of resistin as inflammatogenic cytokine in the setting of arthritis. In vitro experiments on human PBMC were performed to assess cytokine response and transcription pathways of resistin-induced inflammation. Proinflammatory properties of resistin were evaluated in animal model by intra-articular injection of resistin followed by histological evaluation of the joint. Levels of resistin were assessed by ELISA in 74 paired blood and synovial fluid samples of patients with rheumatoid arthritis. Results were compared with the control group comprised blood samples from 34 healthy individuals and 21 synovial fluids from patients with noninflammatory joint diseases. We now show that resistin displays potent proinflammatory properties by 1) strongly up-regulating IL-6 and TNF-α, 2) responding to TNF-α challenge, 3) enhancing its own activity by a positive feedback, and finally 4) inducing arthritis when injected into healthy mouse joints. Proinflammatory properties of resistin were abrogated by NF-κB inhibitor indicating the importance of NF-κB signaling pathway for resistin-induced inflammation. Resistin is also shown to specifically accumulate in the inflamed joints of patients with rheumatoid arthritis and its levels correlate with other markers of inflammation. Our results indicate that resistin is a new and important member of the cytokine family with potent regulatory functions. Importantly, the identified properties of resistin make it a novel and interesting therapeutic target in chronic inflammatory diseases such as rheumatoid arthritis.
Neuropeptide Release from Dental Pulp Cells by RgpB via Proteinase-Activated Receptor-2 Signaling
Authors: Tancharoen Salunya , Sarker Krishna Pada , Imamura Takahisa , Biswas Kamal Krishna , Matsushita Kenji , Tatsuyama Shoko , Travis James , Potempa Jan , Torii Mitsuo , Maruyama Ikuro
Published: April 2014

Dental pulp inflammation often results from dissemination of periodontitis caused mostly by Porphyromonas gingivalis infection. Calcitonin gene-related peptide and substance P are proinflammatory neuropeptides that increase in inflamed pulp tissue. To study an involvement of the periodontitis pathogen and neuropeptides in pulp inflammation, we investigated human dental pulp cell neuropeptide release by arginine-specific cysteine protease (RgpB), a cysteine proteinase of P. gingivalis, and participating signaling pathways. RgpB induced neuropeptide release from cultured human pulp cells (HPCs) in a proteolytic activity-dependent manner at a range of 12.5–200 nM. HPCs expressed both mRNA and the products of calcitonin gene-related peptide, substance P, and proteinase-activated receptor-2 (PAR-2) that were also found in dental pulp fibroblast-like cells. The PAR-2 agonists, SLIGKV and trypsin, also induced neuropeptide release from HPCs, and HPC PAR-2 gene knockout by transfection of PAR-2 antisense oligonucleotides inhibited significantly the RgpB-elicited neuropeptide release. These results indicated that RgpB-induced neuropeptide release was dependent on PAR-2 activation. The kinase inhibitor profile on the RgpB-neuropeptide release from HPC revealed a new PAR-2 signaling pathway that was mediated by p38 MAPK and activated transcription factor-2 activation, in addition to the PAR-2-p44/42 p38MAPK and -AP-1 pathway. This new RgpB activity suggests a possible link between periodontitis and pulp inflammation, which may be modulated by neuropeptides released in the lesion.
Characterization of the Anti-Tissue Transglutaminase Antibody Response in Nonobese Diabetic Mice
Authors: Sblattero Daniele , Maurano Francesco , Mazzarella Giuseppe , Rossi Mauro , Auricchio Salvatore , Florian Fiorella , Ziberna Fabiana , Tommasini Alberto , Not Tarcisio , Ventura Alessandro , Bradbury Andrew , Marzari Roberto , Troncone Riccardo
Published: April 2014

Type 1 diabetes mellitus is an autoimmune disorder characterized by destruction of insulin-producing pancreatic β cells by T lymphocytes. In nonobese diabetic (NOD) mice, a role has been hypothesized for dietary gluten proteins in the onset of diabetes, and because gluten dependence is the major feature of celiac disease, together with production of Abs to the autoantigen tissue transglutaminase (tTG), we looked for the presence of anti-tTG Abs in the serum of NOD mice and, to establish their origin, analyzed the Ab repertoire of NOD mice using phage display Ab libraries. We found significant levels of serum anti-tTG Abs and were able to isolate single-chain Ab fragments to mouse tTG mainly from the Ab libraries made from intestinal lymphocytes and to a lesser extent from splenocytes. Data from NOD mice on a gluten-free diet suggest that the anti-tTG response is not gluten-dependent. The intestinal Ab response to tTG is a feature of NOD mice, but the underlying mechanisms remain obscure.
Gene Expression Profiling of the Effect of High-Dose Intravenous Ig in Patients with Kawasaki Disease
Authors: Abe Jun , Jibiki Toshiaki , Noma Seiji , Nakajima Tosiharu , Saito Hirohisa , Terai Masaru
Published: April 2014

Kawasaki disease (KD) is an acute vasculitis of infants and young children, preferentially affecting the coronary arteries. Intravenous infusion of high dose Ig (IVIG) effectively reduces systemic inflammation and prevents coronary artery lesions in KD. To investigate the mechanisms underlying the therapeutic effects of IVIG, we examined gene expression profiles of PBMC and purified monocytes obtained from acute patients before and after IVIG therapy. The results suggest that IVIG suppresses activated monocytes and macrophages by altering various functional aspects of the genes of KD patients. Among the 18 commonly decreased transcripts in both PBMC and purified monocytes, we selected six genes, FCGR1A, FCGR3A, CCR2, ADM, S100A9, and S100A12, and confirmed the microarray results by real-time RT-PCR. Moreover, the expressions of FcγRI and FcγRIII on monocytes were reduced after IVIG. Plasma S100A8/A9 heterocomplex, but not S100A9, levels were elevated in patients with acute KD compared with those in febrile controls. Furthermore, S100A8/A9 was rapidly down-regulated in response to IVIG therapy. Persistent elevation of S100A8/A9 after IVIG was found in patients who later developed coronary aneurysms. These results indicate that the effects of IVIG in KD may be mediated by suppression of an array of immune activation genes in monocytes, including those activating FcγRs and the S100A8/A9 heterocomplex.
Arginine-Rich Anti-Vascular Endothelial Growth Factor (Anti-VEGF) Hexapeptide Inhibits Collagen-Induced Arthritis and VEGF-Stimulated Productions of TNF-α and IL-6 by Human Monocytes
Authors: Yoo Seung-Ah , Bae Dong-Goo , Ryoo Jae-Woong , Kim Hae-Rim , Park Gyeong-Sin , Cho Chul-Soo , Chae Chi-Bom , Kim Wan-Uk
Published: April 2014

Vascular endothelial growth factor (VEGF) has been suggested to play a critical role in the pathogenesis of rheumatoid arthritis (RA). We previously identified a novel RRKRRR hexapeptide that blocked the interaction between VEGF and its receptor through the screening of peptide libraries. In this study, we investigated whether anti-VEGF peptide RRKRRR (dRK6) could suppress collagen-induced arthritis (CIA) and regulate the activation of mononuclear cells of RA patients. A s.c. injection of dRK6 resulted in a dose-dependent decrease in the severity and incidence of CIA and suppressed synovial infiltration of inflammatory cells in DBA/1 mice. In these mice, the T cell responses to type II collagen (CII) in lymph node cells and circulating IgG Abs to CII were also dose-dependently inhibited by the peptides. In addition, VEGF directly increased the production of TNF-α and IL-6 from human PBMC. Synovial fluid mononuclear cells of RA patients showed a greater response to VEGF stimulation than the PBMC of healthy controls. The major cell types responding to VEGF were monocytes. Moreover, anti-VEGF dRK6 inhibited the VEGF-induced production of TNF-α and IL-6 from synovial fluid mononuclear cells of RA patients and decreased serum IL-6 levels in CIA mice. In summary, we observed first that dRK6 suppressed the ongoing paw inflammation in mice and blocked the VEGF-induced production of proinflammatory cytokines. These data suggest that dRK6 may be an effective strategy in the treatment of RA, and could be applied to modulate various chronic VEGF-dependent inflammatory diseases.
Plasmodium vivax Hospitalizations in a Monoendemic Malaria Region: Severe Vivax Malaria?
Authors: Quispe Antonio M. , Pozo Edwar , Guerrero Edith , Durand Salomón , Baldeviano G. Christian , Edgel Kimberly A. , Graf Paul C. F. , Lescano Andres G.
Published: April 2014

Severe malaria caused by Plasmodium vivax is no longer considered rare. To describe its clinical features, we performed a retrospective case control study in the subregion of Luciano Castillo Colonna, Piura, Peru, an area with nearly exclusive vivax malaria transmission. Severe cases and the subset of critically ill cases were compared with a random set of uncomplicated malaria cases (1:4). Between 2008 and 2009, 6,502 malaria cases were reported, including 106 hospitalized cases, 81 of which fit the World Health Organization definition for severe malaria. Of these 81 individuals, 28 individuals were critically ill (0.4%, 95% confidence interval = 0.2–0.6%) with severe anemia (57%), shock (25%), lung injury (21%), acute renal failure (14%), or cerebral malaria (11%). Two potentially malaria-related deaths occurred. Compared with uncomplicated cases, individuals critically ill were older (38 versus 26 years old, P < 0.001), but similar in other regards. Severe vivax malaria monoinfection with critical illness is more common than previously thought.
Efficacy of Three Different Regimens of Primaquine for the Prevention of Relapses of Plasmodium vivax Malaria in the Amazon Basin of Peru
Authors: Durand Salomón , Cabezas Cesar , Lescano Andres G. , Galvez Mariela , Gutierrez Sonia , Arrospide Nancy , Alvarez Carlos , Santolalla Meddly L. , Bacon David J. , Graf Paul C. F.
Published: April 2014

We evaluated the efficacy of three primaquine (PQ) regimes to prevent relapses with Plasmodium vivax through an open-label randomized trial in Loreto, Peru. Vivax monoinfections were treated with chloroquine for 3 days and PQ in three different regimes: 0.5 mg/kg per day for 5 days (150 mg total), 0.5 mg/kg per day for 7 days (210 mg total), or 0.25 mg/kg per day for 14 days (210 mg total). Biweekly fever assessments and bimonthly thick smears were taken for 210 days. Recurrences after 35 days were considered relapses. One hundred eighty cases were enrolled in each group; 90% of cases completed follow-up. There were no group-related differences in age, sex, or parasitemia. Relapse rates were similar in the 7- and 14-day regimes (16/156 = 10.3% and 22/162 = 13.6%, P = 0.361) and higher in the 5-day group (48/169 = 28.4%, P < 0.001 and P = 0.001, respectively). The 7-day PQ regimen used in Peru is as efficacious as the recommended 14-day regimen and superior to 5 treatment days.
Maripa Hantavirus in French Guiana: Phylogenetic Position and Predicted Spatial Distribution of Rodent Hosts
Authors: de Thoisy Benoît , Matheus Séverine , Catzeflis François , Clément Luc , Barrioz Sébastien , Guidez Amandine , Donato Damien , Cornu Jean-François , Brunaux Olivier , Guitet Stéphane , Lacoste Vincent , Lavergne Anne
Published: April 2014

A molecular screening of wild-caught rodents was conducted in French Guiana, South America to identify hosts of the hantavirus Maripa described in 2008 in a hantavirus pulmonary syndrome (HPS) case. Over a 9-year period, 418 echimyids and murids were captured. Viral RNA was detected in two sigmodontine rodents, Oligoryzomys fulvescens and Zygodontomys brevicauda, trapped close to the house of a second HPS case that occurred in 2009 and an O. fulvescens close to the fourth HPS case identified in 2013. Sequences from the rodents had 96% and 97% nucleotide identity (fragment of S and M segments, respectively) with the sequence of the first human HPS case. Phylogenetic reconstructions based on the complete sequence of the S segment show that Maripa virus is closely related to Rio Mamore hantavirus. Using environmental descriptors of trapping sites, including vegetation, landscape units, rain, and human disturbance, a maximal entropy-based species distribution model allowed for identification of areas of higher predicted occurrence of the two rodents, where emergence risks of Maripa virus are expected to be higher.
A New Molecular Surveillance System for Leishmaniasis
Authors: Pandey Kishor , Pandey Basu Dev , Mallik Arun Kumar , Acharya Jyoti , Kato Kentaro , Kaneko Osamu , Ferreira Pedro Eduardo
Published: April 2014

Presently, global efforts are being made to control and eradicate the deadliest tropical diseases through the improvement of adequate interventions. A critical point for programs to succeed is the prompt and accurate diagnosis in endemic regions. Rapid diagnostic tests (RDTs) are being massively deployed and used to improve diagnosis in tropical countries. In the present report, we evaluated the hypothesis of, after use for diagnosis, the reuse of the Leishmania RDT kit as a DNA source, which can be used downstream as a molecular surveillance and/or quality control tool. As a proof of principle, a polymerase chain reaction-based method was used to detect Leishmania spp. minicircle kinetoplast DNA from leishmaniasis RDT kits. Our results show that Leishmania spp. DNA can be extracted from used RDTs and may constitute an important, reliable, and affordable tool to assist in future leishmaniasis molecular surveillance methods.