bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human IFN-α Protein Engineering: The Amino Acid Residues at Positions 86 and 90 Are Important for Antiproliferative Activity
Authors: Hu Renqiu , Bekisz Joseph , Schmeisser Hana , McPhie Peter , Zoon Kathryn 
Published: April 2014

AbstractHuman IFN-α is a family of structurally related proteins that exhibit a wide range of antiproliferative activities. To understand the structural basis for these different antiproliferative activities, eight recombinant human IFN-α hybrids (HY) of α21a/α2c (HY-4, HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and characterized. The data showed that the amino acid region 81–95 is important for antiproliferative activity. Site-directed mutagenesis and cassette mutagenesis studies showed that if serine (S) 86 and asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative activity was increased. We have also observed that if Y86 was replaced by isoleucine (I), the antiproliferative activity was comparable. However, if Y86 was replaced by aspartic acid (D), lysine (K), or alanine (A), the antiproliferative activity was substantially decreased. Our results indicate that Y and/or I at position 86 and Y at position 90 are very important in antiproliferative activity of human IFN-α. Circular dichroism spectra showed that the amino acid replacements at position 86 did not change the secondary structure. Thus the biological activity changes among those mutants do not appear to be due to conformational changes. The results also suggest that hydrophobic residue(s) at position 86 may be important for the interaction of the molecule with its receptor. The competitive binding data correlated with the antiproliferative activity. The N-terminal region of the molecule and the hydrophobic residues (including Y and I) on the C-helix region at positions 86 and/or 90 are important for binding and antiproliferative activities of human IFN-αs.
Kinetic Analysis of the Interactions of Complement Receptor 2 (CR2, CD21) with Its Ligands C3d, iC3b, and the EBV Glycoprotein gp350/220
Authors: Sarrias Maria Rosa , Franchini Silvia , Canziani Gabriela , Argyropoulos Emelia , Moore William T. , Sahu Arvind , Lambris John D. 
Published: April 2014

AbstractThe molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.
The Murine Cytomegalovirus pp89 Immunodominant H-2Ld Epitope Is Generated and Translocated into the Endoplasmic Reticulum as an 11-Mer Precursor Peptide
Authors: Knuehl Christine , Spee Pieter , Ruppert Thomas , Kuckelkorn Ulrike , Henklein Peter , Neefjes Jacques , Kloetzel Peter-M. 
Published: April 2014

Abstract
The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-Ld 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.
Impaired T Cell Immunity in B Cell-Deficient Mice Following Viral Central Nervous System Infection
Authors: Bergmann Cornelia C. , Ramakrishna Chandran , Kornacki Margaret , Stohlman Stephen A. 
Published: April 2014

AbstractCD8+ T cells are required to control acute viral replication in the CNS following infection with neurotropic coronavirus. By contrast, studies in B cell-deficient (μMT) mice revealed Abs as key effectors in suppressing virus recrudescence. The apparent loss of initial T cell-mediated immune control in the absence of B cells was investigated by comparing T cell populations in CNS mononuclear cells from infected μMT and wild-type mice. Following viral recrudescence in μMT mice, total CD8+ T cell numbers were similar to those of wild-type mice that had cleared infectious virus; however, virus-specific T cells were reduced at least 3-fold by class I tetramer and IFN-γ ELISPOT analysis. Although overall T cell recruitment into the CNS of μMT mice was not impaired, discrepancies in frequencies of virus-specific CD8+ T cells were most severe during acute infection. Impaired ex vivo cytolytic activity of μMT CNS mononuclear cells, concomitant with reduced frequencies, implicated IFN-γ as the primary anti viral factor early in infection. Reduced virus-specific CD8+ T cell responses in the CNS coincided with poor peripheral expansion and diminished CD4+ T cell help. Thus, in addition to the lack of Ab, limited CD8+ and CD4+ T cell responses in μMT mice contribute to the ultimate loss of control of CNS infection. Using a model of virus infection restricted to the CNS, the results provide novel evidence for a role of B cells in regulating T cell expansion and differentiation into effector cells.
Essential Role of Neutrophils in the Initiation and Progression of a Murine Model of Rheumatoid Arthritis
Authors: Wipke Brian T. , Allen Paul M. 
Published: April 2014

AbstractNeutrophils are prominent participants in the joint inflammation of human rheumatoid arthritis (RA) patients, but the extent of their role in the inductive phase of joint inflammation is unknown. In the K/B×N mouse RA model, transfer of autoreactive Ig from the K/B×N mouse into mice induces a rapid and profound joint-specific inflammatory response reminiscent of human RA. We observed that after K/B×N serum transfer, the earliest clinical signs of inflammation in the ankle joint correlated with the presence of neutrophils in the synovial regions of recipient mouse ankle joints. In this study, we investigated the role of neutrophils in the early inflammatory response to transferred arthritogenic serum from the K/B×N transgenic mouse. Mice were treated with a neutrophil-depleting mAb before and following transfer of arthritogenic serum and scored for clinical indications of inflammation and severity of swelling in ankle joints and front paws. In the absence of neutrophils, mice were completely resistant to the inflammatory effects of K/B×N serum. Importantly, depletion of neutrophils in diseased recipient mice up to 5 days after serum transfer reversed the inflammatory reaction in the joints. Transfer of serum into mice deficient in the generation of nitrogen or oxygen radicals (inducible NO synthase 2 or gp91phox genes, respectively) gave normal inflammatory responses, indicating that neither pathway is essential for disease induction. These studies have identified a critical role for neutrophils in initiating and maintaining inflammatory processes in the joint.
Decreased Expression of Toll-Like Receptor-4 and MD-2 Correlates with Intestinal Epithelial Cell Protection Against Dysregulated Proinflammatory Gene Expression in Response to Bacterial Lipopolysaccharide
Authors: Abreu Maria T. , Vora Puja , Faure Emmanuelle , Thomas Lisa S. , Arnold Elizabeth T. , Arditi Moshe 
Published: April 2014

AbstractThe lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS. Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types. Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC). The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS. Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-κB reporter gene in response to purified, protein-free LPS. No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS. IEC responded vigorously to IL-1β in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling. IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2. To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines. Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-κB and IL-8 reporter genes in response to LPS. We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4. The remainder of the intracellular LPS signaling pathway is functionally intact.
The Cytoplasmic Domain of L-Selectin Participates in Regulating L-Selectin Endoproteolysis
Authors: Matala Erik , Alexander Shelia R. , Kishimoto Takashi K. , Walcheck Bruce 
Published: April 2014

Abstract
Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca2+-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.
The Essential Role of Lipopolysaccharide-Binding Protein in Protection of Mice Against a Peritoneal Salmonella Infection Involves the Rapid Induction of an Inflammatory Response
Authors: Heinrich Jan-Michael , Bernheiden Martin , Minigo Gabriela , Yang Kang Kang , Schütt Christine , Männel Daniela N. , Jack Robert S. 
Published: April 2014

Abstract
Acute and chronic hyperinflammation are of major clinical concern, and many treatment strategies are therefore directed to inactivating parts of the inflammatory system. However, survival depends on responding quickly to pathogen attack, and since the adaptive immune system requires several days to adequately react, we rely initially on a range of innate defenses, many of which operate by activating parts of the inflammatory network. For example, LPS-binding protein (LBP) can transfer the LPS of Gram-negative bacteria to CD14 on the surface of macrophages, and this initiates an inflammatory reaction. However, the importance of this chain of events in infection is unclear. First, the innate system is redundant, and bacteria have many components that may serve as targets for it. Second, LBP can transfer LPS to other acceptors that do not induce inflammation. In this study, we show that innate defense against a lethal peritoneal infection with Salmonella requires a direct proinflammatory involvement of LBP, and that this is a major nonredundant function of LBP in this infection model. This emphasizes that blocking the LBP-initiated inflammatory cascade disables an essential defense pathway. Any anti-inflammatory protection that may be achieved must be balanced against the risks inherent in blinding the innate system to the presence of Gram-negative pathogens.
Evidence of IL-18 as a Novel Angiogenic Mediator
Authors: Park Christy C. , Morel Jacques C. M. , Amin M. Asif , Connors Matthew A. , Harlow Lisa A. , Koch Alisa E. 
Published: April 2014

Abstract
Angiogenesis, or new blood vessel growth, is a key process in the development of synovial inflammation in rheumatoid arthritis (RA). Integral to this pathologic proliferation are proinflammatory cytokines. We hypothesized a role for IL-18 as an angiogenic mediator in RA. We examined the effect of human IL-18 on human microvascular endothelial cell (HMVEC) migration. IL-18 induced HMVEC migration at 1 nM (p &lt; 0.05). RA synovial fluids potently induced endothelial cell migration, but IL-18 immunodepletion resulted in a 68 ± 5% decrease in HMVEC migration (p &lt; 0.05). IL-18 appears to act on HMVECs via αvβ3 integrin. To test whether IL-18 induced endothelial cell tube formation in vitro, we quantitated the degree of tube formation on Matrigel matrix. IL-18, 1 or 10 nM, resulted in a 77% or 87% increase in tube formation compared with control (p &lt; 0.05). To determine whether IL-18 may be angiogenic in vivo, we implanted IL-18 in Matrigel plugs in mice, and IL-18 at 1 and 10 nM induced angiogenesis (p &lt; 0.05). The angiogenesis observed appears to be independent of the contribution of local TNF-α, as evidenced by adding neutralizing anti-TNF-α Ab to the Matrigel plugs. In an alternative in vivo model, sponges embedded with IL-18 or control were implanted into mice. IL-18 (10 nM) induced a 4-fold increase in angiogenesis vs the control (p &lt; 0.05). These findings support a novel function for IL-18 as an angiogenic factor in RA and may elucidate a potential therapeutic target for angiogenesis-directed diseases.
Inhibition of H2 Histamine Receptor-Mediated Cation Channel Opening by Protein Kinase C in Human Promyelocytic Cells
Authors: Suh Byung-Chang , Lee Hyun , Jun Dong-Jae , Chun Jang-Soo , Lee Jong-Hee , Kim Kyong-Tai 
Published: April 2014

Abstract
Histamine, through H2 receptors, triggers a prominent rise in intracellular free Ca2+ concentration ([Ca2+]i) in addition to an elevation of cAMP level in HL-60 promyelocytes. Here we show that the histamine-induced [Ca2+]i rise was due to influx of Ca2+ from the extracellular space, probably through nonselective cation channels, as incubation of the cells with SKF 96365 abolished the histamine-induced [Ca2+]i rise, Na+ influx, and membrane depolarization. The Ca2+ influx was specifically inhibited by pretreatment of the cells with PMA or extracellular ATP with 50% inhibitory concentrations of 0.12 ± 0.03 nM and 185 ± 17 μM, respectively. Western blot analysis of protein kinase C (PKC) isoforms revealed that PMA (≤1 nM) and ATP (300 μM) caused selective translocation of PKC-δ to the particulate/membrane fraction. Costimulation of the cells with histamine and SKF 96365 partially reduced histamine-induced granulocytic differentiation, which was evaluated by looking at the extent of fMet-Leu-Phe-induced [Ca2+]i rise and superoxide generation. In conclusion, nonselective cation channels are opened by stimulation of the H2 receptor, and the channels are at least in part involved in the induction of histamine-mediated differentiation processes. Both effects of histamine were selectively inhibited probably by the δ isoform of PKC in HL-60 cells.
The Generation of Both T Killer and Th Cell Clones Specific for the Tumor-Associated Antigen HER2 Using Retrovirally Transduced Dendritic Cells
Authors: Meyer zum Büschenfelde Christian , Metzger Jochen , Hermann Christine , Nicklisch Nicole , Peschel Christian , Bernhard Helga 
Published: April 2014

AbstractInduction of antitumor immunity involves the presence of both CD8+ CTLs and CD4+ Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34+ hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8+ CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4+ Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4+ T cells were cloned that released IFN-γ upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8+ and CD4+ T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.
Apoptosis and Altered Dendritic Cell Homeostasis in Lupus Nephritis Are Limited by Anti-CD154 Treatment
Authors: Kalled Susan L. , Cutler Anne H. , Burkly Linda C. 
Published: April 2014

AbstractAutoimmunity results from a failure in central and/or peripheral tolerance; however, the events that initiate and maintain this dysfunction remain unclear. To better understand the mediators involved in autoimmunity, we investigated the cellular mechanisms maintaining disease in the (SWR × NZB)F1 (SNF1) mouse model of systemic lupus erythematosus. Previously, we have shown that autoimmunity in this model is dependent on CD40-CD154 interactions. Herein, our studies reveal that the severity of disease in SNF1 mice correlates with a marked increase in the frequency of apoptotic splenocytes, including a higher proportion of apoptotic dendritic cells (DC) in vivo. In addition, we demonstrate a significant disease-related increase in the absolute number of splenic CD11chigh DC. The increased DC number appears to be attributable to DC proliferation and enhanced migration to the spleen, most likely induced by elevated splenic expression of secondary lymphoid chemokine. Importantly, these imbalances in apoptosis, secondary lymphoid chemokine expression, and DC homeostasis were reduced or normalized by anti-CD154 treatment. Thus, our data demonstrate CD154-dependent regulation of apoptosis and DC homeostasis in mice with lupus-like autoimmune disease. We suggest that these mechanisms comprise an autostimulatory loop, maintaining the cascade of autoimmunity by DC presentation of self-Ags derived from apoptotic cells and CD154-mediated costimulation.
IL-13 and IFN-γ: Interactions in Lung Inflammation
Authors: Ford Jean G. , Rennick Donna , Donaldson Debra D. , Venkayya Rajeev , McArthur Cliff , Hansell Elisabeth , Kurup Viswanath P. , Warnock Martha , Grünig Gabriele 
Published: April 2014

AbstractChronic inflammatory diseases of the lungs, such as asthma, are frequently associated with mixed (Th2 and Th1) T cell responses. We examined the impact of critical Th1 and Th2 cytokines, IFN-γ and IL-13, on the responses in the lungs. In a mouse model of airway inflammation induced by mixed T cell responses, the number of Th1 (IFN-γ-positive) cells was found to be negatively correlated with airway hyperreactivity. In these mice, blockade of IL-13 partially inhibited airway hyperreactivity and goblet cell hyperplasia but not inflammation. In contrast, in mice that responded with a polarized Th2 response to the same Ag, blockade of IL-13 inhibited airway hyperreactivity, goblet cell hyperplasia, and airway inflammation. These results indicated that the presence of IFN-γ would modulate the effects of IL-13 in the lungs. To test this hypothesis, wild-type mice were given recombinant cytokines intranasally. IFN-γ inhibited IL-13-induced goblet cell hyperplasia and airway eosinophilia. At the same time, IFN-γ and IL-13 potentiated each other’s effects. In the airways of mice given IL-13 and IFN-γ, levels of IL-6 were increased as well as numbers of NK cells and of CD11c-positive cells expressing MHC class II and high levels of CD86. In conclusion, IFN-γ has double-sided effects (inhibiting some, potentiating others) on IL-13-induced changes in the lungs. This may be the reason for the ambiguous role of Th1 responses on Th2 response-induced lung injury.