bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and cloning of a novel serine protease, RNK-Tryp-2, from the granules of a rat NK cell leukemia.
Authors: Sayers T J , Wiltrout T A , Smyth M J , Ottaway K S , Pilaro A M , Sowder R , Henderson L E , Sprenger H , Lloyd A R 
Published: December 2022

Abstract
We have biochemically purified a 27-kDa serine protease (designated RNK-Tryp-2) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) which has tryptase activity. Utilizing molecular sieve chromatography and reverse-phase HPLC, we purified RNK-Tryp-2 to homogeneity and sequenced 33 NH2-terminal amino acids. Oligonucleotide primers were used in the PCR to generate a 528-bp cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate an 884-bp RNK-Tryp-2 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 233 amino acids which does not have potential sites for N-linked glycosylation. The cDNA encodes a leader peptide of at least 25 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the N-terminus, and the His, Asp, and Ser amino acids that form the catalytic triad of serine proteases, are conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Tryp-2 is distinct protease. Southern blot analysis suggests the existence of one or more related genes. A single 1.3-kb mRNA transcript was detected by Northern blot analysis of total cellular RNA from the in vivo passaged RNK-16, rat splenocytes, lung and liver nonparenchymal cells, as well as in highly purified rat LGL and T cells. RNK-Tryp-2 is a novel serine protease that is expressed in the granules of large granular lymphocytes.
Unusual topology of an HLA-B27 allospecific T cell epitope lacking peptide specificity.
Authors: Villadangos J A , Galocha B , López de Castro J A 
Published: December 2022

Abstract
Recognition of MHC + peptide complexes by TCRs is thought to involve a large surface formed by exposed residues from the bound peptide and from the alpha-helices of the MHC protein. This interaction appears to be essentially symmetrical in the positioning of the TCR relative to the MHC molecule. In this study the topology of HLA-B27 recognition by an alloreactive TCR, 64.8P, has been analyzed with a panel of site-specific mutants that have changes at multiple positions along the peptide binding site of HLA-B27. Abrogation of transfectant target cell lysis by CTL 64.8P was obtained only with some mutations in the peptide side chain binding pockets A and B, whereas little or no effect was observed with mutations outside these pockets. CTL 64.8P efficiently lysed murine transfectant cells, including HLA-B27+ RMA-S cells. Recognition of this latter transfectant was more efficient upon increased HLA-B27 expression at 26 degrees C. The uneven distribution of mutations affecting HLA-B27 allorecognition by CTL 64.8P strongly suggests an asymmetrical topology in the interaction of this TCR with HLA-B27, in which most of the binding energy is provided by contacts with HLA-B27 and/or peptide residues located close to pockets A and B, with little contribution from other areas of the MHC or peptide molecules. Its conservation in RMA-S cells further suggests that the epitope recognized by CTL 64.8P is either peptide-independent or requires any of a set of peptides having the same amino-terminal residues.
The role of IL-2 secreted from genetically modified tumor cells in the establishment of antitumor immunity.
Authors: Bannerji R , Arroyo C D , Cordon-Cardo C , Gilboa E 
Published: December 2022

Abstract
Although the importance of CD4+ T cells for the induction of an effective CD8+ cytolytic T cell response is well documented, the mechanism by which MHC class II-negative tumor cells recruit CD4+ T help is not well understood. We have previously shown that IL-2 or IFN-gamma gene-modified CMS-5 tumor cells do not grow in syngeneic mice; however, mice which rejected the cytokine-secreting tumor cells develop a protective immune response against a challenge with parental tumor cells. Here we show that rejection of IL-2-secreting CMS-5 cells is not mediated by T cells. However, establishment of a protective immune response against CMS-5 tumor cells requires the presence of both CD4+ and CD8+ T cell subsets during the period of immunization with IL-2-secreting CMS-5 cells as well as during the effector phase. Extensive histologic analysis has failed to detect the presence of T cells at the site of immunization with either IL-2- or IFN-gamma-secreting CMS-5 cells. The main infiltrate at the site of inoculation with IL-2-secreting CMS-5 cells consisted of NK cells that appeared to play a role in their rejection. The predominant infiltrate at the site of inoculation with IFN-gamma-secreting CMS-5 cells consisted of macrophages. These observations argue against a direct role for the intact tumor cell in presenting either T helper or CTL epitopes to the immune system, and support the view that specialized APC are responsible for the in vivo priming of a T cell response against MHC class I-restricted Ag.
Differential cytokine expression in maternal blood and placenta during murine gestation.
Authors: Delassus S , Coutinho G C , Saucier C , Darche S , Kourilsky P 
Published: December 2022

Abstract
To evaluate the influence of gestation on peripheral blood T cells, we measured the mRNA production of several cytokines (IL-2, IL-4, IL-10, IFN-gamma) and the p55 subunit of IL-2R at different time points in the blood of pregnant mice and in the placenta. This was made possible by the use of a new PCR technique that is precise and quantitative. Our results show that pregnancy induces profound changes in the expression of these genes in peripheral blood cells. During the first week of gestation, there is an increase in the levels of all the cytokines, followed by a state of immunodepression characterized by levels of cytokines below normal (nonpregnant mice). In the placenta low levels of IL-2 and IL-10 are detected. IFN-gamma mRNA production is higher than the blood IFN-gamma mRNA in the last week of pregnancy. However, the main difference is found for IL-4 mRNA expression where the placenta levels are 5- to 10-fold higher than the blood mRNA expression. We discuss these results in the context of the placenta as a privileged immune site, where IL-4, being the main cytokine, may play a major regulatory role.
Cefdinir (CI-983), a new oral amino-2-thiazolyl cephalosporin, inhibits human neutrophil myeloperoxidase in the extracellular medium but not the phagolysosome.
Authors: Labro M T , el Benna J , Charlier N , Abdelghaffar H , Hakim J 
Published: December 2022

Abstract
Cefdinir, a new oral 2-amino-5-thiazolyl cephalosporin, inhibited the luminol-amplified chemiluminescence (LACL) response of human neutrophils stimulated by PMA but not opsonized zymosan, in a concentration-dependent but not time-dependent manner. The LACL response to opsonized zymosan in cytochalasin B-treated neutrophils was, however, inhibited by cefdinir. Various cephalosporins, regardless of the presence of a 2-amino-5-thiazolyl moiety, did not significantly alter the neutrophil LACL response triggered by PMA and zymosan. The LACL response induced by the calcium ionophore A23187 and FMLP was also impaired by cefdinir, and this impairment was increased in cytochalasin B-treated neutrophils. Superoxide anion generation by neutrophils, measured in terms of lucigenin-amplified chemiluminescence and cytochrome c reduction, was not altered. Spontaneous and FMLP-induced neutrophil degranulation, assessed by lysozyme and beta-glucuronidase release, were not modified by cefdinir. Furthermore, cefdinir inhibited LACL generation in cell-free systems consisting of H2O2, NaI, and either horseradish peroxidase or a myeloperoxidase-containing neutrophil extract. Orthodianisidine oxidation in these two acellular systems was inhibited by cefdinir. Cefdinir did not alter neutrophil bacterial killing at concentrations that inhibited myeloperoxidase-containing neutrophil extract-dependent reactions induced by soluble stimuli. Taken together, these data strongly suggest that cefdinir directly inhibits the activity of myeloperoxidase-containing neutrophil extract released into the extracellular medium during neutrophil stimulation by soluble mediators, but has no effect on that released into the phagolysosome during phagocytosis. This unusual property of a member of the beta-lactam family could be of interest in modulating the exaggerated inflammatory process often associated with infectious diseases.
Neutrophil-activating peptides NAP-2 and IL-8 bind to the same sites on neutrophils but interact in different ways. Discrepancies in binding affinities, receptor densities, and biologic effects.
Authors: Petersen F , Flad H D , Brandt E 
Published: December 2022

Abstract
IL-8 and the neutrophil-activating peptide 2 (NAP-2) are members of the chemokine family of host defense cytokines. Although IL-8 was shown to interact with two different high affinity receptors on polymorphonuclear neutrophil granulocytes (PMN), direct demonstration of specific binding sites for NAP-2 is difficult, because the NAP-2 molecule lacks iodinable side chains. Here we present a modified labeling procedure for the chemokine that does not affect its biologic activity. The 125I-labeled NAP-2 specifically bound to PMN with two different affinities (KD = 0.65 and 22.4 nM). We observed complete cross-competition of unlabeled IL-8 with 125I-labeled-NAP-2 and of unlabeled NAP-2 with 125I-labeled IL-8, indicating the absence of monospecific binding sites for either chemokine. However, in contrast to former work by others, the total number of accessible sites was considerably lower for NAP-2 (13,000/cell) than for IL-8 (59,000/cell). In addition, PMN prepared from heparinized blood expressed significantly more receptors for NAP-2 than cells prepared from citrated blood, whereas receptor numbers for IL-8 were unchanged. Desensitization experiments suggested a regulatory role for the NAP-2 high affinity site. Short-term priming of PMN with a nonstimulatory dose of NAP-2 (or MGSA) but not with IL-8 led to drastic down-regulation of the subsequent degranulation response, challenged by higher dosages of NAP-2, MGSA, or IL-8. Reduced functional responsiveness of cells correlated with the rapid down-regulation and internalization of NAP-2 and IL-8 high affinity binding sites. Thus, our data indicate that chemokines could mediate by individual modes of interaction with common receptor's different biologic functions.
Cross-linking of ICAM-1 induces co-signaling of an oxidative burst from mononuclear leukocytes.
Authors: Rothlein R , Kishimoto T K , Mainolfi E 
Published: December 2022

Abstract
Cell adhesion molecules were first described as accessory molecules simply to bridge one cell to another. More recently, it has been realized that these molecules also transmit signals from outside of the cell to inside. We show that cross-linking of the ICAM-1 on the cell membrane with anti-ICAM-1 mAb and F(ab')2 fragments of goat anti-MIgG in the presence of suboptimal levels of the bacterial peptide FMLP results in co-stimulation of an oxidative burst from CD14 expressing PBMCs. The amplitude of the oxidative response was less than the oxidative burst induced by CD18 cross-linking, whereas the response was more prolonged. On the other hand, cross-linking by anti-L-selectin mAb plus F(ab')2 fragments of goat anti-MIgG induced a minimal oxidative burst that was not significantly greater than the response generated by anti-L-selectin mAb alone. The addition of an excess of soluble ICAM-1 to compete for the anti-ICAM-1 mAb inhibits the oxidative burst in response to ICAM-1 cross-linking but not to CD18 cross-linking. These results suggest that ICAM-1 is capable of delivering a transmembrane signal into CD14-positive PBMC.
Characterization of rabbit complement component C8. Functional evidence for the species-selective recognition of C8 alpha by homologous restriction factor (CD59).
Authors: White R V , Kaufman K M , Letson C S , Platteborze P L , Sodetz J M 
Published: December 2022

Abstract
Protection of host cells from lysis by the C5b-9 cytolytic complex is provided by a membrane-associated protein (CD59) that interacts with homologous C8 and C9 during C5b-9 assembly. This interaction restricts normal formation and function of the complex, thereby protecting cells from attack by homologous C. In this study, rabbit C8 was purified and characterized and used to investigate the role of the individual C8 subunits in homologous restriction and the basis for species selectivity by human CD59. Exchanging the disulfide-linked C8 alpha-gamma dimer in human C8 with one from rabbit was found to be sufficient to overcome restriction by human Es. Activity of the hybrid C8 and rabbit C8 toward these target cells was equivalent, thus establishing that restriction is not dependent on the species of C8 beta. Because C8 gamma was previously shown to have no role in restriction, these results suggest that within C8, structural determinant(s) recognized by CD59 reside solely on C8 alpha. Sequences determined from full-length cDNA clones for rabbit C8 alpha, C8 beta, and C8 gamma support this conclusion. All three subunits are strikingly similar to human with regard to length, m.w., N- and C-terminal residues, conserved cysteines, and overall sequence. However, when sequences are compared under high stringency, a single segment of extended sequence dissimilarity is detected between the two species of C8 alpha. Within human C8 alpha, this 37-residue segment resides near the putative membrane-interacting region and may constitute a human CD59 recognition site.
IFN-gamma, TNF, and IL-6 production by vaccinia virus immune spleen cells. An in vitro study.
Authors: Carpenter E A , Ruby J , Ramshaw I A 
Published: December 2022

Abstract
The production of cytokines by virus immune spleen cells after in vitro restimulation was investigated. Vaccinia virus-primed spleen cells from CBA/H mice were stimulated in vitro with virus-infected UV-irradiated syngeneic cells. Both CD4+ and CD8+ T cell populations proliferated after restimulation. TNF, IL-6, and IFN-gamma were detected within 12 h of restimulation with maximal levels reached by 24 h. Adherent cells were major producers of IL-6 and TNF, whereas IFN-gamma production was dependent on CD4+ T cells and adherent cells. The IFN-gamma response was Ag specific, whereas the production of TNF and IL-6 was not. Stimulation with vaccinia virus constructs encoding IFN-gamma or TNF altered the levels of cytokines produced, in that IFN-gamma expression by stimulator cells led to increased IFN-gamma production, whereas TNF expression by stimulator cells augmented both TNF and IFN-gamma production by responder cells.
Changes in glucose transport and transporter isoforms during the activation of human peripheral blood lymphocytes by phytohemagglutinin.
Authors: Chakrabarti R , Jung C Y , Lee T P , Liu H , Mookerjee B K 
Published: December 2022

Abstract
We have explored the mechanism of stimulation of glucose transport during PHA stimulation of human peripheral blood lymphocytes (HPBT) enriched in T cells. Equilibrium exchange flux of 3-O-methyl glucose (3-O-MG) was stimulated two- and fourfold at 24 and 48 h after PHA stimulation, respectively. The increase was transient in that the flux rate returned to control (unstimulated) levels by 96 h. Immunoblotting and immunoprecipitation using specific Abs revealed that resting HPBT expresses glucose transporter isoforms GLUT-2 and GLUT-3 but not GLUT-1. After PHA stimulation, GLUT-1 expression was induced predominantly in the plasma membrane, whereas GLUT-3 expression was simultaneously down-regulated. GLUT-1 expression was detectable at 24 h, peaked at 48 h, and disappeared at 96 h. The total number of glucose transporters per cell measured as the total capacity of D-glucose displaceable cytochalasin B binding did not change significantly at any time after PHA stimulation. PHA stimulation also caused expression of high affinity IL-2R and secretion of IL-2. The IL-2 secretion was transient, which peaked at 24 h, slightly preceding the GLUT-1 expression peak and disappeared at 72 h. In PHA-activated HPBT cells synchronized at G0-G1, GLUT-1 was not expressed but was rapidly induced by exposure to IL-2. This induction did not occur in the presence of cyclosporin A, which inhibits IL-2 secretion. Based on these observations, we conclude that PHA stimulation increases glucose transport partly by inducing the expression of GLUT-1 instead of GLUT-3 and that GLUT-1 expression is induced by signals generated by IL-2 binding to its high affinity receptors.
IL-12 influences intrathymic T cell development.
Authors: Godfrey D I , Kennedy J , Gately M K , Hakimi J , Hubbard B R , Zlotnik A 
Published: December 2022

Abstract
IL-12 has been implicated in the maturation and activation of peripheral T lymphocytes and NK cells. In the present study we have investigated the potential role of IL-12 in intrathymic T cell development. Treatment of mouse fetal thymic organ culture with IL-12 caused a significant reduction in size and cell number compared with the untreated controls. Flow cytometric analysis of the thymocytes recovered from these lobes showed differential effects on individual thymocyte subsets, most but not all of which were significantly decreased. In contrast, however, we observed an increase in both the percentage and cell number of alpha beta TCR+CD4-CD8+ thymocytes. This effect could be neutralized with an anti-IL-12 antibody, demonstrating the specificity of the influence of IL-12 in the fetal thymic organ culture system. IL-12 caused proliferation of isolated thymocyte subsets, particularly CD3+CD4-CD8+ cells in the presence of IL-2 and IL-4. Additionally, IL-12 induced significant proliferation of early CD3-CD4-CD8- triple negative CD44+CD25+ pro-T cells in combination with stem cell factor. We show that both the p35 and p40 chains of IL-12 are produced in the fetal and adult mouse thymus and that thymic stromal cells are a potential source of this cytokine.
Differential activation of p21ras in CD45RA+ and CD45RO+ human T lymphocytes.
Authors: Patel H R , Renz H , Terada N , Gelfand E W 
Published: December 2022

Abstract
CD45RA+ and CD45RO+ human T cells differ in their functional responses to different forms of mitogenic stimulation. However, little is known about the intracellular signaling events related to the functional differences in these two subsets. In the present study, we examined the ability of different Abs to the TCR/CD3 complex to activate the p21ras proto-oncogene product in CD45RA+ and CD45RO+ T cells. Stimulation with soluble OKT3 anti-CD3 mAb induced activation of p21ras in CD45RA+ cells but not in CD45RO+ cells, although soluble anti-CD3 was able to induce both activation of CD3-associated kinases and IL-2 responsiveness in CD45RO+ cells. Similar results were obtained by using UCHT1, a different anti-CD3 Ab. In contrast, cross-linked anti-CD3 or the combination of phorbol ester and ionomycin were found to activate p21ras equally in both T cell subsets. Our results demonstrate a selective inability of CD45RO+ human T cells to activate p21ras in response to soluble anti-CD3 mAb.
Immunologic and molecular characterization of Amb p V allergens from Ambrosia psilostachya (western Ragweed) pollen.
Authors: Ghosh B , Rafnar T , Perry M P , Bassolino-Klimas D , Metzler W J , Klapper D G , Marsh D G 
Published: December 2022

Abstract
We have purified and characterized the Amb p V allergen (A1 variant) from western ragweed (Ambrosia psilostachya) pollen. This allergen was found to be highly cross-reactive with the Amb a VA1 allergen from short ragweed (A. artemisiifolia) pollen in a competitive double-Ab radioimmunoassay (DARIA) and the two allergens showed concordant allergenic potency in histamine-release experiments. We cloned and sequenced several Amb p V genes from western ragweed pollen and flowers by direct PCR of genomic DNA. The amino acid sequences deduced from the nucleotide sequences indicated the presence of multiple forms of Amb p V that could be broadly classified into two groups: Amb p VA and Amb p VB variants. The sequences of the Amb p VA variants are highly homologous to Amb a V (about 90% identity) and very similar to the protein sequence that we obtained. The Amb p VB variants share approximately 65% amino acid homology with Amb a V and have five to seven cysteine residues as compared with the eight found in Amb a V and Amb t V. Two cysteine residues that form disulfide bonds in other Amb Vs (positions 19 and 43 in Amb a V) are replaced by serine and alanine in the Amb p VB1 and Amb p VB2 variants. We have generated model structures of Amb p VA1, VA2, VA3, and VB1 variants from the nuclear magnetic resonance-derived structure of Amb a VA1 by homology modeling. Comparison of antigenic epitopes predicted for the structures of Amb p V variants and Amb a VA1 explains the observed cross-reactivity of the two ragweed proteins and suggests the epitopes likely to be involved in Ab recognition.
Exploration of requirements for peptide binding to HLA DRB1*0101 and DRB1*0401.
Authors: Hill C M , Liu A , Marshall K W , Mayer J , Jorgensen B , Yuan B , Cubbon R M , Nichols E A , Wicker L S , Rothbard J B 
Published: December 2022

Abstract
The individual amino acid contacts responsible for peptide binding to DRB1*0101 and/or DRB1*0401 were defined using a quantitative binding assay. The differential contribution of each amino acid in two well studied T cell determinants, HA307-319 and RMBP 90-102, was delineated by comparing the IC50 values of analogues of varying length. This analysis confirmed the importance of a hydrophobic amino acid located near the amino-terminus for binding to both alleles and revealed that the contribution of the carboxyl-terminal amino acids differed between DRB1*0101 and DRB1*0401. Taking advantage of previous experiments demonstrating that all of the residues could be replaced with alanine, with the exception of the key hydrophobic amino acid, simplified analogues composed of polyalanines were used to prove 1) optimal binding depended on the position of the hydrophobic side chain relative to the amino- and carboxyl-termini; 2) aromatic amino acids were superior to aliphatic side chains at this position; and 3) a significant amount of free energy of binding arises from hydrogen bonding between the class II binding site and the amide bonds of the ligand. The role of each carbonyl and amide nitrogen was measured by assaying analogues containing reduced peptide bonds or N-methyl amino acids. Serine, but not glycine, could be used as a framework amino acid for peptide ligands, indicating that the beneficial aspects of these simplified structures was the combination of retaining the correct orientation of the peptide bonds, the restriction of the conformational freedom by limiting the possible phi/psi angles of the peptide, and avoidance of deleterious side-chain contacts. Collectively, these data were consistent with the peptide binding in a nonrepeating conformation with the vast majority of the free energy of binding arising from hydrogen bonds with the peptide backbone and a single, key hydrophobic side chain interacting in a conserved pocket in both DRB1*0101 and DRB1*0401.