bluuebunny/crossref_metadata_2025_split huggingface dataset:
The glycosylphosphatidylinositol‐linked Fcγ receptor III represents the dominant receptor structure for immune complex activation of neutrophils
Authors: Hundt Matthias , Schmidt Reinhold E. 
Published: March 2007

AbstractPolymorphonuclear neutrophils (PMN) express constitutively two low‐affinity Fcγ receptors, FcγRII and FcγRIII. FcγRII is a transmembrane molecule, and FcγRIII is linked via a glycosylphosphatidylinositol (GPI) anchor to the membrane. The role of each of these receptors in activation of PMN is still unclear. We used specific cross‐linking of FcγRII via Fab fragments of IV.3 (anti‐FcγRII, CDw32) and of FcγRIII using F(ab')2 fragments of 3G8 (anti‐FcγRIII, CD16) to activate PMN. Stimulation of FcγRIII was significantly more effective in inducing a respiratory burst than cross‐linking of FcγRII. A synergistic effect was observed after simultaneous activation of FcγRIII. We could demonstrate that both FcγR mobilize calcium as intracellular signal in spite of their different membrane linkage. The kinetic of calcium mobilization after FcγR stimulation is delayed in comparison to formyl‐methionyl‐leucyl‐phenylalanine activation. In addition FcγR‐induced increase of cytoplasmic calcium is pertussis toxin insensitive. When monoclonal IgG1χ complexes were used for stimulation calcium mobilization and hydrogen peroxide (H2O2) production could also be demonstrated. Inhibition studies of this activation using monoclonal antibodies suggested that this immune complex activation was predominantly mediated via FcγRIII. Only in FcγRIII‐deficient PMN from paroxysmal nocturnal hemoglobinuria patients could a decreased H2O2 production be demonstrated to be FcγRII dependent. In normal PMN the GPI‐anchored FcγRIII structure is the predominant receptor.
Interleukin (IL)‐2 activation of p21ras in murine myeloid cells transfected with human IL‐2 receptor β chain
Authors: Izquierdo Manuel , Downward Julian , Otani Hiroki , Leonard Warren J. , Cantrell Doreen A. 
Published: March 2007

AbstractThe T cell growth factor interleukin‐2 (IL‐2) induces p21ras activation in T lymphocytes. To determine whether the IL‐2 receptor (IL‐2R) can regulate p21ras when expressed in a non‐T cell environment we have examined the ability of IL‐2 to activate p21ras in 32D murine myeloid progenitor cells transduced with human IL‐2R β chains. These cells are denoted β53 cells. 32D cells normally proliferate in response to IL‐3 but the expression of the IL‐2R β chain confers IL‐2 responsiveness to the cells. Our data show that IL‐3 is able to activate p21ras in the parental 32D cells and both IL‐2 and IL‐3 can stimulate p21ras in the IL‐2R‐expressing β53 clone of 32D. In T lymphocytes, activation of protein kinase C (PKC) with phorbol esters is sufficient to stimulate p21ras. However, in 32D and β53 cells activation of PKC with phorbol esters does not result in p21ras activation even though these cells express functional PKC. It appears, therefore, that a PKC‐mediated pathway for p21ras regulation exists in T lymphocytes but not in 32D cells. The IL‐2R can couple to p21ras independently of the concomitant presence of the PKC pathway for p21ras regulation. These data imply that multiple intracellular mechanisms may exist to regulate p21ras and that cells of different lineages may differ with regard to p21ras regulation.
T cells from atopic individuals produce IgE‐inducing activity incompletely blocked by anti‐interleukin‐4 antibody
Authors: Zhang Xiaohong , Polla Barbara , Hauser Conrad , Zubler Rudolf H. 
Published: March 2007

AbstractWe investigated peripheral blood B and T lymphocyte functions in atopic individuals. B cells were co‐cultured with mutant EL4 thymoma cells in the presence of a standard T cell supernatant (T‐SN) with or without exogenous interleukin (IL)‐4. IgE secretion in this assay was found to be IL‐4 dependent, but not significantly different for atopic patients (n = 25) vs. normal controls (n = 25). Phytohemagglutinin plus phorbol 12‐myristate 13‐acetate (PHA+PMA)‐induced T‐SN from patients or controls was tested on normal B cells in the same assay system (in the absence of exogenous IL‐4). Compared to the controls, the IgE‐inducing activity was significantly increased for patients with asthma or allergic rhinitis (n = 12; p<0.005) but not for patients with atopic dermatitis (n = 13). The difference between the asthma or allergic rhinitis vs. the atopic dermatitis groups was significant (p>0.05). Since the assay was not inhibited by interferon (IFN)‐γ, this difference can not be attributed to IFN‐γ concentrations. Other T cell activities may be different between the patient groups or atopic T cells from the respiratory mucosa may recirculate more than those from the skin. In any case, the T cells rather than the B cells were found to be abnormal in atopic individuals.If atopic T cells were stimulated with PHA + PMA not as immediately but after a resting period of 48 h in culture medium alone, the IgE‐inducing activity, but not the total Ig‐inducing activity or the IL‐2 secretion, disappeared. In addition, a mean of 37 % of the IgE‐inducing activity (range of 13 % to 79 % for five very active T‐SN) was not inhibited by an anti‐IL‐4 antibody which neutralized exogenous IL‐4, indicating a participation of factors capable of bypassing the requirement for IL‐4 for the IgE response.
Antigen contact sites in class I major histocompatibility complex‐restricted, trinitrophenyl‐specific T cell receptors
Authors: Weltzien Hans Ulrich , Hebbelmann Sabine , Pflugfelder Ulrike , Ruh Helga , Ortmann Bodo , Martin Stefan , Iglesias Antonio 
Published: March 2007

AbstractCloned trinitrophenyl (TNP)‐specific cytotoxic T cells (CTL) were obtained from mice transgenic for the β chain of the antigen‐specific receptor (TcR) of a Kb‐restricted, TNP‐specific CTL clone (BT7.4.1). The transgene‐expressing CTL, specific for TNP/Kb were found to select for TcR α chains highly similar to that of the transgene donor clone BT7.4.1. In that way, two clones (II/7 and III/1) were identified whose TcR differed from the BT7.4.1 receptor only in their Nα‐ and Jα‐sequences, i.e. within the third complementarity‐determining regions of their α chains (CDR3α). Moreover, the TcR of clones II/7 and III/1 had both rearranged the same Jα element, thus differing from each other by only two amino acids in their Vα/Jα junctional regions. Functionally, however, clone III/1 exhibited unique cytolytic specificities for synthetic, Kb‐binding TNP‐peptides as well as for chemically TNP‐modified allogeneic (H‐2k) target cells. These findings demonstrate that (a) similar to “conventional” peptide antigens, synthetic hapten‐peptide determinants are contacted by CDR3α‐determined amino acids of the TcR and (b) in contrast to current models, CDRα also appears to influence the major histocompatibility complex restriction specificity of a given TcR.
Cell selection strategies for making antibodies from variable gene libraries: trapping the memory pool
Authors: Hawkins Robert E. , Winter Greg 
Published: March 2007

AbstractThe B cells of immunized animals can be used as a source of variable region (V) gene libraries. Such libraries offer a way of making antibodies directly in bacteria: rearranged V genes are amplified using the polymerase chain reaction, cloned and expressed as soluble fragments in bacteria, and then screened for antigen binding. Here we have used a model system to investigate antigen‐selected B cells as a source of V gene libraries. Mice were immunized with (4‐hydroxyl‐3‐nitrophenyl)acetyl (NP)‐chicken gammaglobulin, and the splenocytes harvested seven days after primary immunization. We prepared a heavy chain variable (VH) gene library from the DNA of cells selected on antigen‐coated magnetic beads, and two other libraries from the DNA or mRNA of unselected cells. The VH gene libraries were combined with the Vλ1 gene (as this light chain dominates the primary response to NP), expressed as Fv fragments in Escherichia coli and screened for binding to (4‐hydroxy‐3‐iodo‐5‐nitrophenyl)acetyl‐bovine serum albumin. The frequency of antigen‐binding clones was much greater (>50 fold) in the library from the DNA of antigen‐selected cells (17/282) or from the mRNA of unselected cells (29/282) compared to the DNA from unselected cells (0/940). Sequencing of the antigen‐binding clones revealed that they almost invariably used the V‐186.2 heavy chain, as expected from analysis of primary response hybridomas. The D segments from the mRNA library were entirely DFL16.1 (29/29), as in primary response hybridomas, whereas those from the DNA of selected cells were more diverse, using in addition to DFL16.1, other D segments (5/17) as in later response hybridomas. This suggests that the DNA library from selected cells is derived at least in part from cells destined for the memory compartment. Given the long life of memory cells, they may prove a useful source of antibody libraries in the absence of recent immunization.
Existence of mature human CD4+ T cells with genuine class I restriction
Authors: de Bueger Marleen , Bakker Astrid , Goulmy Els 
Published: March 2007

AbstractA human T cell receptor (TcR) α/β CD4+CD8− T cell clone (R416) is reactive with the minor histocompatibility antigen H‐Y in the context of major histocompatibility complex (MHC) class I and not class II molecules. Therewith clone R416 violates the so‐called specificity association of mature TcR α/β+ T cells. R416 displays H‐Y‐specific, HLA‐A2‐restricted proliferation as well as cytotoxicity in vitro. Its fine specificity is identical to that of a classical H‐Y‐reactive CD4−CD8+ MHC class I‐restricted CTL clone, showing that CTL expressing either CD4 or CD8 can display identical antigenic specificities. Exploiting the MHC class I restriction of this CD4+ T cell clone, it was found that interaction of CD4 with non‐TcR‐bound MHC class II molecules does not contribute to antigen specific activation of these CD4+ T cells. This coreceptor‐mismatched T cell clone was not generated in vitro but obtained by expansion of CD8‐depleted peripheral blood mononuclear cells of a female who had been immunized against H‐Y. The existence of such MHC class I‐restricted mature TcR α/β+ T cells expressing CD4 and not CD8 is relevant because it indicates that the generally accepted model for thymic selection, in which the TcR specificity alone determines CD4/CD8 expression of mature thymocytes, may not be absolute.
Biochemical analysis of the mouse mammary tumor virus long terminal repeat product. Evidence for the molecular structure of an endogenous superantigen
Authors: Knight Andrew M. , Harrison Georgina B. , Pease Richard J. , Robinson Peter J. , Julian Dyson P. 
Published: March 2007

AbstractRecent reports have shown that both exogenous and endogenous mouse mammary tumor viruses (MMTV) can encode superantigens. Transfection and transgenic studies have identified the open reading frame (ORF) present in the 3′ long terminal repeat (LTR) as encoding superantigen function. In this study, we have used an in vitro translation system in an attempt to characterize the molecular nature of the protein encoded by the 3′ ORF of Mtv‐8. Using various constructs encoding full‐length and truncated versions of the ORF product, we report that the hydrophobic region close to the amino terminus of the 36‐kDa protein can function as a transmembrane domain. Protease digestion experiments also demonstrate that the protein has a type‐II transmembrane conformation with an extra‐cytoplasmic carboxy terminus. Since this hydrophobic region is conserved between all known MMTV, we speculate that LTR ORF, including those proposed to encode the minor lymphocyte stimulatory antigens, are also capable of encoding type‐II transmembrane glycoproteins. The polymorphism between MMTV LTR ORF products, which correlates with deletion phenotypes, is predominantly in the carboxy‐terminal extracellular region, consistent with a major role in interaction with the T cell receptor.
Identification of the nonamer peptide from influenza A matrix protein and the role of pockets of HLA‐A2 in its recognition by cytotoxic T lymphocytes
Authors: Morrison Joanne , Elvin John , Latron France , Gotch Frances , Moots Robert , Strominger Jack L. , McMichael Andrew 
Published: March 2007

AbstractInfluenza matrix peptide 58–66 is shown to be the optimal nonamer for binding to HLA‐A2 and presentation to cytotoxic T lymphocytes (CTL). If titered out to 2 × 10−10 – 4 × 10−10M in CTL‐mediated lysis assays and to 3 × 10−9M in an HLA‐A2 assembly‐stabilization assay in cell lysates. The peptide was shown to make probable contacts with its carboxy terminus close to residue 116 in the floor of the cleft of HLA‐A2, close to the F pocket. The side chain of the amino‐terminal amino acid was unimportant, but its free amino and carbonyl groups in the A pocket appeared important in optimizing peptide presentation. The B pocket probably accommodates the side chain of residue 2 (isoleucine) and was shown to be critical in peptide presentation.
Human U937 cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor‐α
Authors: Bauvois Brigitte , Sancéau Josiane , Wietzerbin Juana 
Published: March 2007

AbstractSurface peptidase activities on the human monocytic lineage cell line U937 were characterized. Two diisopropyl phosphofluoridate (DFP)‐inhibitable serine peptidases were identified by differences in their hydrolytic activities on chromogenic peptides: one removed tripeptides from the free NH2‐terminal end of the synthetic peptide Ala‐Ala‐Phe‐p‐nitroanilide (pNA) and was not inhibited by inhibitors of metallo‐, cysteic‐, and aspartic‐proteinases, or by those of elastase‐, trypsin‐ and chymotrypsin‐like enzymes, suggesting the presence of a hitherto unidentified serine tripeptidyl endopeptidase; the other peptidase catalyzed the release of Gly‐Pro from Gly‐Pro‐pNA and was inhibited by DFP, phenylmethyl sulfonyl fluoride and diprotin A, thus resembling dipeptidyl peptidase IV (DPP IV) with respect to its substrate specificity and inhibitor profile. A group of N‐exoaminopeptidase activities specifically inhibited by bestatin, was also detected when Ala‐, Leu‐, Arg‐ and Lys‐pNA were used as substrates. The activities were surface associated and not secreted as determined by extracellular location of product and enzymatic recovery in highly purified U937 cell membranes. Peripheral monocytes and macrophages were found to virtually exhibit identical levels of these two classes of peptidase activities when compared to those detected on U937 cells. The relative contributions of these hydrolytic enzymes to the cleavage of bioactive and radioiodinated cytokines including tumor necrosis factor‐α (TNF‐α), interleukin‐1α and interferon‐γ was next examined. The results indicated that N‐aminopeptidases do not appear to participate in the catabolism of any tested cytokine. In contrast, the most interesting finding was that both serine peptidases participate in TNF‐α degradation. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native 17‐kDa molecule TNF‐α, and the concomitant release of biologically inactive fragments of ≤2 kDa. Together, these observations indicate new roles for both the DPP IV‐like enzyme and the tripeptidyl endopeptidase located at the surface of human monocytic cells, including the regulation of the extracellular TNF‐α concentration. Thus, the identification of functional ectopeptidases provides insight into their potential role in both normal and malignant monocytic function.
T cell epitope selection: dominance may be determined by both affinity for major histocompatibility complex and stoichiometry of epitope
Authors: Li Wan‐Fen , Fan Ming‐Dau , Pan Ching‐Biau , Lai Ming‐Zong 
Published: March 2007

AbstractThe majority of T cell hybridomas produced in the BALB/c mouse in response to immunization with λ, repressor cI recognize a peptide fragment comprising of residues 12 to 26 (P12–26). Some other parts of the cI (P1–14, P33–48 and P73–88) are defective in generating T cell responses in the BALB/c mouse. P73–88 may be converted into a T cell determinant if a few more amino acid residues are included (P67–88). Together with P46–67 and P80–102, most peptides derived from cI were capable of eliciting T cell responses by themselves in BALB/c mouse. The mechanisms underlying the selection of P12–26 over the other epitopes when λ repressor was used as immunogen were examined. The dominant response to P12–26 was attenuated by tolerizing with intravenous administration of P12–26. Under such treatment the T cell response to P12–26 was reduced by 80% but there was no enhancement on the responses toward other epitopes. The selection of P12–26 is, thus, unlikely to be due to a competition at the T cell level. It was also found that the dominance of P12–26 was not simply due to a higher affinity of P12–26 for major histocompatibility complex molecules. For example P12–26 binds better to I‐Ad molecule than P80–102, but co‐injection with equimole of P12–26 only slightly inhibited P80–102‐induced T cell response. Instead, it required a few molar excess of P12–26 to effectively block the association of P80–102 with I‐Ad molecules and to inhibit the T cell immunity to P80–102. Since epitopes such as P46–67, P67–88 and P80–102 were generated from λ repressor cI at a lower molar basis than that of P12–26, it is suggested that the dominance of P12–26 was probably generated by such stoichiometry difference, in addition to the higher affinity of P12–26 for I‐Ad molecules.
Selective inhibition of immune complex trapping by follicular dendritic cells with monoclonal antibodies against rat C3
Authors: van den Berg Timo K. , Döpp Ed A. , Daha Mohamed R. , Kraal Georg , Dijkstra Christine D. 
Published: March 2007

AbstractThe role of complement component C3 in the trapping of immune complexes by follicular dendritic cells (FDC) was studied in the rat, by means of the C3‐specific monoclonal antibody ED11. Immunocytochemistry revealed the presence of C3 on FDC, where it co‐localized with trapped peroxidase anti‐peroxidase complexes. Furthermore, C3 was detected on reticular cells occupying the T cell areas of peripheral lymphoid organs, which are not involved in the handling of immune complexes. The in vivo administration of anti‐C3 abolished the trapping of immune complexes in splenic follicles, but was unable to release preexisting complexes from the FDC. Trapping of immune complexes was also prevented by treatment of rats with cobra venom factor (CoVF). While CoVF caused massive depletion of C3 from serum, ED11 treatment had no such effect. The effect of anti‐C3 appeared at least in part to be due to an inhibition of complement activation by immune complexes. We also analyzed earlier stages of the trapping process, with respect to their C3 dependence. Upon systemic injection immune complexes are initially observed in the marginal zone. Administration of anti‐C3 reduced this localization, indicating a role for C3 in the entry of immune complexes into the spleen. Our results confirm experiments in CoVF‐treated animals and extend the evidence for a role of C3 in the follicular trapping process using anti‐C3 in vivo. The mechanism of immune complex trapping and the role of complement therein is discussed.
The glucocorticoid antagonist RU38486 mimics interleukin‐1 in its sensitization to the lethal and interleukin‐6‐inducing properties of tumor necrosis factor
Authors: Brouckaert Peter , Everaerdt Bart , Fiers Walter 
Published: March 2007

AbstractAlthough tumor necrosis factor (TNF) is undoubtedly a major mediator of the antitumor and shock‐inducing activities of lipopolysaccharide (LPS), the outcome of a challenge with TNF is highly dependent on the presence or absence of other substances or conditions. We have previously shown that to obtain lethality in mice after TNF administration both TNF receptor (TNF‐R) types have to be triggered. This is illustrated by the fact that recombinant human (rh) TNF, which is a selective murine (m) TNF‐R55 agonist, is not lethal, whereas mTNF, which binds both mTNF‐R55 and mTNF‐R75, is lethal in mice. Triggering of TNF‐R75 is, however, no longer needed when sensitizers such as galactosamine or low doses of LPS or interleukin (IL)‐1 are also present. Here, we report that this selective species specificity of TNF is also reflected in patterns of induced IL‐6: both rmTNF and rhTNF could induce considerable IL‐6 peak levels in the plasma (up to 10 ng/ml) 2 to 3 h after TNF administration. However, only rmTNF was capable of inducing the same pattern of sustained IL‐6 levels previously observed after lethal LPS doses, while rhTNF only caused induction of transient IL‐6 levels, as found after nonlethal LPS doses. We also observed that the sensitizer IL‐1 could complement rhTNF to induce such a sustained IL‐6 induction. Since we were interested in sensitizers with a defined mechanism of action, we further investigated the effects of the glucocorticoid and progesterone inhibitor RU38486 on the lethal and IL‐6‐inducing properties of TNF. We observed that RU38486 closely mimicked IL‐1: both had similar effects on IL‐6 induction and sensitized mice to the lethal effects of TNF with comparable efficiency and kinetics. Using a monoclonal anti‐IL‐1R antibody, we finally observed that the effects of RU38486 were most probably not mediated by IL‐1. These observations suggest that a glucocorticoid‐antagonistic activity might be a key factor in the pathways leading to septic shock and that such activity could be a key target for the pharmacological manipulation of sepsis.
Peripheral tolerance to an islet cell‐specific hemagglutinin transgene affects both CD4+ and CD8+ T cells
Authors: Lo David , Freedman Jessica , Hesse Sabine , Palmiter Richard D. , Brinster Ralph L. , Sherman Linda A. 
Published: March 2007

AbstractTo study the basis for immunological tolerance of peripheral tissue‐specific antigens, a transgenic mouse line was established that expresses the influenza hemagglutinin (HA) on pancreatic islet β cells (Ins‐HA transgenic mice). When followed up to 14 months of age, Ins‐HA transgenic mice did not develop spontaneous autoimmune disease. Upon immunization with HA‐expressing viruses, high titers of HA‐specific circulating antibody were detected; however, T cell responses by both the T helper and T cytolytic compartment were markedly reduced as compared with transgene‐negative littermates, and no evidence could be found for islet infiltrates. Adoptive transfer of histocompatible lymphocytes from transgene‐negative mice plus virus into irradiated Ins‐HA hosts resulted in islet inflammation dominated by CD4+ T cells, indicating that the HA antigen was accessible to activated T cells. These results suggest that T cells can be rendered tolerant of antigens expressed outside the thymus.