bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of the tumor necrosis factor receptor p75 in mediating cytotoxicity and gene regulating activities
Authors: Medvedev Andrei E. , Sundan Anders , Espevik Terje
Published: March 2007

Using agonistic antibodies (Ab) we have examined whether the 75‐kDa chain of the tumor necrosis factor receptor (p75 TNFR) is capable of mediating cytotoxic response and gene regulation alone or in cooperation with p55 TNFR. Addition of an anti‐p75 TNFR polyclonal antiserum or anti‐p75 monoclonal antibody (mAb) plus anti‐immunoglobulin (Ig) led to cytotoxic response of human KYM‐1 rhabdomyosarcoma cells. Anti‐p75 mAb alone had no effect pointing out the importance of strong receptor stimulation for signal transduction into the cell. Simultaneous triggering of both the p55 and p75 TNFR by agonistic Ab resulted in additive cytotoxic action on KYM‐1 cells. The anti‐p75 mAb 3H5, directed to a non‐TNF binding site on the human p75 TNFR, was used to confirm further the ability of the p75 TNFR to potentiate p55 TNFR‐mediated cell death. While exhibiting no cytotoxicity by its own, 3H5 significantly augmented the cytotoxic effect of the anti‐p55 mAb htr9 towards KYM‐1 cells. Neither the anti‐p75 TNFR antiserum nor anti‐p75 mAb were cytotoxic for human U937 cells suggesting that the cytolysis resulting from p75 TNFR cross‐linking is cell specific. Noteworthy, stimulation of the p75 TNFR with mAb plus anti‐Ig or polyclonal antiserum led to a marked enhancement of the p55 TNFR‐induced U937 cell death, indicating collaboration between the two TNFR in induction of cytotoxicity also in this cell line. However, 3H5 mAb did not affect the ability of anti‐p55 mAb to lyse U937 cells. Altogether, these data demonstrate the difference between KYM‐1 and U937 cell lines with respect to the role for the p75 TNFR in mediating cytotoxicity. Both TNFR were found to mediate cytomegalovirus (CMV) promoter activation in human SW480T‐βGal cells and nuclear transcription factor χB (NF‐χB) induction in this cell line as well as in KYM‐1 cells. It was demonstrated for the first time that independent stimulation of both TNFR resulted in an additive effect on the CMV promoter activation and induction of the NF‐χB. Taken together, these results indicate that the p75 TNFR induces cytotoxicity in a cell‐specific manner and potentiates p55 TNFR‐mediated cytotoxic response and gene regulation.
An inhibitor of cytotoxic functions produced by CD8+CD57+ T lymphocytes from patients suffering from AIDS and immunosuppressed bone marrow recipients
Authors: Sadat‐Sowti Behazine , Debré Patrice , Mollet Lucile , Quint Laurent , Hadida Fabienne , Leblond Véronique , Bismuth Georges , Autran Brigitte
Published: March 2007

An inhibitor of the cytotoxic functions (ICF) mediated by human immunodeficiency virus (HIV)‐ or HLA‐specific cytotoxic T lymphocytes, natural killer and lymphokine‐activated killer (LAK) cells is secreted by CD8+CD57− T lymphocytes, a subset expanded during infection with HIV and after bone marrow transplantation. We previously showed an apparent molecular mass of 20–30 kDa for this soluble glycosylated concanavalin A‐binding inhibitor which is distinct from known cytokines. Here, we report a characterization of the mechanism of action of this CD8+CD57+ ICF. We show that the ICF‐induced inhibition of LAK cell cytolytic activity is transient, with a spontaneous recovery of cytolytic potential after 18 h. When testing interactions of ICF with a large set of cytokines we found that the ICF‐mediated inhibition of cytotoxic functions is antagonized by two cytokines: recombinant interleukin (rIL)‐4 and recombinant interferon (rIFN)‐γ. Finally, we show that ICF acts at the level of cytolytic effector cells, where it induces a significant increase of cyclic AMP (cAMP) level. In contrast, no modification of either cell surface antigen expression or of target/effector cell conjugate formation could be evidenced. Addition of rIL‐4 and rIFN‐γ reverses such an increase of cAMP levels and in parallel restores the cytolytic activity. Altogether, these data demonstrate that the glycoprotein ICF produced by CD8+CD57+ cells (1) inhibits cell‐mediated cytotoxicity by sensitizing cytolytic effector cells to the cAMP pathway, and (2) is part of a cytokine network controlling cell‐mediated cytotoxic functions.
Age‐related decrease in the proportion of germinal center B cells from mouse Peyer's patches is accompanied by an accumulation of somatic mutations in their immunoglobulin genes
Authors: González‐Fernández Africa , Gilmore David , Milstein César
Published: March 2007

Somatic hypermutation of immunoglobulin genes and the generation of memory B cells seems to take place in germinal centers, which are chronically present in Peyer's patches. Age‐associated changes in the germinal center B cell compartment of Peyer's patches and in the mutations of a χ light chain transgene were analyzed in unimmunized mice. Somatic mutations accumulate in germinal center B cells slowly and continuously to reach an apparent plateau when the animals are around 5 months old. In contrast, the proportion of germinal center B cells reaches a maximum in very young mice (about 2 months old) and decreases progressively thereafter. These results suggest that the highly mutated B cells in older mice arise by the successive accumulation of mutations in memory cells. The data also show that the optimum time for the analysis of hypermutation of transgenes in Peyer's patches is when the mice are about 5 months old.
Dysregulation of interleukin‐7 receptor may generate loss of cytotoxic T cell response in human immunodeficiency virus type 1 infection
Authors: Carini Claudio , McLane Mary Fran , Mayer Kenneth H. , Essex Max
Published: March 2007

Virus‐specific cytotoxic T lymphocytes (CTL) play a crucial role in modulating an immune response against human immunodeficiency type 1 (HIV‐1) infection. The generation of effector cytotoxic cells from CTL precursors involves intricate interactions with antigen via T cell receptors (TcR) and soluble cytokines. Interleukin (IL)‐7 can affect T cell maturation and differentiation. Here we report on a group of five HIV‐1‐positive individuals who tested negative for env‐and gag‐specific CTL activity. When exogenous recombinant human IL‐7 was added as a stimulus to the cultures, none (0/5) of the CTL‐negative individuals exhibited a CTL response. Individuals that were negative for HIV‐1‐specific CTL activity were found to lack IL‐7 receptor (IL‐7R) on CD8+ cells with a comparable reduction on CD4+ cells. Increased shedding of IL‐7R in the culture supernatant was observed. A significant reduction in receptor number was detected by binding of 125I‐labeled IL‐7 and Scatchard analysis. The lack of IL‐7R is probably not due to endogenous IL‐7, since it was not detectable in the culture supernatants of the patients studied. HIV‐1 proteins may cause down‐modulation of IL‐7R expression, either by producing an insufficient number of molecules or by rapid decay of IL‐7R on T cells. These changes may alter the cells' capability to respond to the IL‐7 growth signal, resulting in CTL failure and subsequent mishandling of the virus.
CD9 antigen is an accessory subunit of the VLA integrin complexes
Authors: Rubinstein Eric , le Naour François , Billard Martine , Prenant Michel , Boucheix Claude
Published: March 2007

The CD9 antigen is a cell surface glycoprotein of unknown function which belongs to the tetraspans family. We demonstrate here, by precipitation, Western blotting and co‐capping experiments, that this molecule is associated with a large fraction of β1 integrins in two cell lines, the pre‐B cell line NALM‐6 and the megakaryocytic cell line HEL. In HEL cells, CD9 antigen is only associated with VLA‐4. In contrast, in NALM‐6 cells, CD9 antigen is associated with both VLA‐4 and VLA‐5. On the other hand, only the β1 chain is co‐precipitated with the CD9 antigen in transfected L cells. These data show that the CD9 antigen is associated with the β1 chain rather than with a particular integrin. CD9 monoclonal antibodies (mAb) did not modify the binding of HEL and NALM‐6 cells to fibronectin, laminin or collagen. The association of CD9 antigen to VLA integrins is strengthened by the fact that both CD9 and anti‐VLA mAb induce aggregation of the two cell lines and inhibit their migration in Transwell chambers. Because the aggregating effect, but not the inhibition of migration, is observed in CEM or CD9‐transfected CEM cells, these two effects are likely to be mediated by different mechanisms.
B7/BB‐1 antigen expression on adult human microglia studied in vitro and in situ
Authors: Williams Kenneth , Ulvestad Elling , Antel Jack P.
Published: March 2007

In this study, we have examined the expression and function of B7/BB‐1 on individual glial cells, by utilizing surgically resected adult human central nervous system (CNS) tissues, tissues derived from fetal human CNS, and pathology material from cases of multiple sclerosis (MS). Immunofluorescence analysis using enriched adult human derived cultures of microglia and oligodendrocytes, and mixed microglia/astrocyte cultures, demonstrated that B7/BB‐1 was expressed on microglia. Adult human‐derived oligodendrocytes and astrocytes, and human fetal astrocytes were B7/BB‐1 negative under all culture conditions. Flow cytometry studies demonstrated a low basal level of B7/BB‐1 expression on microglia that was up‐regulated following incubation with interferon‐γ (IFN‐γ). Co‐culture of purified fresh allogeneic CD4+ T cells with microglia for 24 h resulted in clustering of T cells around microglia and microglial B7/BB‐1 expression. Preincubation of microglia with an anti BB‐1 monoclonal antibody (mAb) prior to microglia: CD4+ T cell co‐cultures resulted in partial inhibition of the ability of microglia both to present recall antigen to autologous CD4+ T cells and to present antigen to allogeneic CD4+ T cells in primary mixed lymphocyte reaction (1°MLR). The CTLA‐4 Ig fusion protein inhibited the ability of microglia to present antigen in both antigen presentation assays to an even greater extent than did the anti BB‐1 mAb. The BB‐1 antibody also inhibited the ability of microglia to stimulate previously activated T cells in a secondary 2° MLR. In sections of multiple sclerosis brain, B7/BB‐1 expression was observed on activated microglia in select parenchymal lesions, and on perivascular cells and infiltrating monocytes. B7/BB‐1 immunoreactivity was not found in normal appearing white matter from MS brain or from non‐inflammatory brain specimens. Our results indicate that the B7/BB‐1 molecule plays a functional role in the capacity of microglia to serve as CNS antigen‐presenting cells that can both initiate and perpetuate CD4+ T cell activation.
Structure of the human APO‐1 gene
Authors: Behrmann Iris , Walczak Henning , Krammer Peter H.
Published: March 2007

APO‐1/Fas (CD95) is a type 1 transmembrane protein that belongs to the tumor necrosis factor/nerve growth factor receptor family characterized by cysteine‐rich extracellular domains. Cross‐linking of APO‐1 mediates apoptosis in a variety of cells. In the present study we report the isolation and characterization of the human APO‐1 gene spanning approximately 25 kb of human chromosome 10. The gene consists of nine exons (25 bp to > 1.44 kb) separated by eight introns (152 bp to ∼ 12 kb). The boundaries of exon 2 to 5 encoding the extracellular region do not match the boundaries of the three APO‐1 protein subdomains. Exon structure and functional protein domains correspond for exon 6 encoding the transmembrane region and for exon 9 encoding the „death domain”︁. By a polymerase chain reaction‐based approach we localized major transcriptional start sites in human spleen cells 77 and 73 nucleotides upstream of the translation initiation codon of the human APO‐1 gene. Minor initiation sites were found at positions −128, −111, −91, and −74. The 5′ flanking sequence of the human APO‐1 gene is GC rich, contains a high number of CpG dinucleotides and lacks a consensus TATA box. Consensus binding sites for the transcription factors Sp1, AP‐1, AP‐2, GAF, NF‐χB, and NF‐AT were found. The elucidation of the human APO‐1 gene structure will facilitate the study of its involvement in various diseases such as in autoimmunity.
The APO‐1/Fas (CD95) receptor is expressed in homozygous MRL/lpr mice
Authors: Mariani Sara M. , Matiba Bernd , Armandola Elena A. , Krammer Peter H.
Published: March 2007

APO‐1/Fas (CD95) is a transmembrane receptor that transduces apoptotic signals within various cells including T and B cells. The APO‐1 gene was found to be defective in lpr mice. In these mice insertion of a retrotransposon gives rise to transcription of an abnormal mRNA and only a fraction of wild‐type APO‐1 mRNA. It is not clear if lpr mice still express wild‐type APO‐1 protein. To address this question, we prepared rabbit anti‐APO‐1 antibodies (Ab) with a peptide representing the extracellular sequence corresponding to residues 5‐23 of APO‐1. The rabbit Ab reacted with thymocytes from different mouse strains and the extent of binding was correlated with the two known APO‐1 alleles. In addition, the Ab reacted with mouse cell lines expressing mouse APO‐1 mRNA but not with human cell lines. Binding of the Ab to MRL and BALB/c thymocytes was completely blocked by the immunizing peptide. Immunofluorescence analysis of MRL/lpr thymocytes showed that they still express APO‐1 protein at approximately one tenth of the wild‐type expression level on their surface. In addition, in lpr as in wild‐type mice we found a decrease of APO‐1 expression in the more mature thymic compartment. Western blot analysis of whole cell lysates from lpr and wild‐type thymocytes showed that the Ab recognized APO‐1 in both cell types. Approximately 50% of CD3+ splenocytes and 80% of in vitro activated CD3+ cells from wild‐type mice reacted with the Ab, but to a lower extent than thymocytes. The same differential reactivity was found in lpr CD3+ splenocytes. lpr T cells, however, showed a substantially lower level of APO‐1. Thus, the differential expression of APO‐1 on thymic versus peripheral lpr T cells might influence their sensitivity towards APO‐1‐mediated apoptosis.
Reduced development of CD4−8−B220+ T cells but normal autoantibody production in lpr/lpr mice lacking major histocompatibility complex class I molecules
Authors: Ohteki Toshiaki , Iwamoto Masahiro , Izui Shozo , MacDonald H. Robson
Published: March 2007

The lpr gene has recently been shown to encode a functional mutation in the Fas receptor, a molecule involved in transducing apoptotic signals. Mice homozygous for the lpr gene develop an autoimmune syndrome accompanied by massive accumulation of double‐negative (DN) CD4−8−B220+ T cell receptor‐α/β+ cells. In order to investigate the origin of these DN T cells, we derived lpr/lpr mice lacking major histocompatibility complex (MHC) class I molecules by intercrossing them with β2‐microglobulin (β2m)‐deficient mice. Interestingly, these lprβ2m–/– mice develop 13‐fold fewer DN T cells in lymph nodes as compared to lpr/lpr wild‐type (lprWT) mice. Analysis of anti‐DNA antibodies and rheumatoid factor in serum demonstrates that lprβ2m–/– mice produce comparable levels of autoantibodies to lprWT mice. Collectively our data indicate that MHC class I molecules control the development of DN T cells but not autoantibody production in Ipr/lpr mice and support the hypothesis that the majority of DN T cells may be derived from cells of the CD8 lineage.
CD4 T cells inhibit in vivo the CD8‐mediated immune response against murine colon carcinoma cells transduced with interleukin‐12 genes
Authors: Martinotti Alessia , Stoppacciaro Antonella , Vagliani Maddalena , Melani Cecilia , Spreafico Fabio , Wysocka Maria , Parmiani Giorgio , Trinchieri Giorgio , Colombo Mario P.
Published: March 2007

Retroviral‐mediated cytokine gene transfer into tumor cells is a highly effective way of inducing tumor inhibition and immunity. We analyzed the tumorigenicity of C‐26 murine colon carcinoma cells transduced with genes encoding the two subunits of murine interleukin‐12 (IL‐12) in a polycistronic retroviral vector and selected for resistance to G418 and for IL‐12 production (30–80 pg/ml). BALB/c mice injected s.c., i.v. and intrasplenically with C‐26/IL‐12 cells from three different IL‐12‐producing clones showed delayed tumor onset as compared with mice injected with control NeoR‐transduced or parental tumor cells. Although C‐26/IL‐12 tumor‐bearing mice eventually died of lung metastasis, their survival time was twice as long as that of mice injected with control cells. In experiments with mice selectively depleted of natural killer (NK) cells before tumor cell injection, the time of tumor onset and survival of mice injected with C‐26/IL‐12 s.c. and i.v., respectively, was reduced. CD8+ T cell depletion had no effect on latency or survival, whereas removal of CD4+ T cells led to C‐26/IL‐12 tumor regression in about 40% of mice. Histological and immunocytochemical characterization of leukocytes infiltrating C‐26/IL‐12 tumors showed only slight infiltration with few T cells in non‐depleted mice but abundant infiltration by CD8+ T cells and asialo‐GM1+ NK cells in tumors of mice depleted of CD4+ T cells. The lack of CD8+ T cell infiltration is not due to a CD4‐mediated suppression of their activation because irradiated C‐26/IL‐12 cells primed for the induction of a strong cytotoxic T lymphocyte response against C‐26 parental cells and induced CD8+ effector cells that protected against C‐26/IL‐12 in a Winn assay. Rather, the results suggest that, although C‐26/IL‐12 cells injected in vivo stimulate both NK and CD8+ T cells, tumor infiltration by the latter is inhibited by CD4+ T cells.
Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport
Authors: Cerundolo Vincenzo , Kelly Adrian , Elliott Tim , Trowsdale John , Townsend Alain
Published: March 2007

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I‐restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I‐restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re‐expression of full‐length LMP7. Control experiments with LMP7, from which the putative pro‐region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.
Lipopolysaccharide‐induced interleukin‐10 in mice: role of endogenous tumor necrosis factor‐α
Authors: Barsig Johannes , Küsters Sabine , Vogt Kathrin , Volk Hans‐Dieter , Tiegs Gisa , Wendel Albrecht
Published: March 2007

Interleukin (IL)‐10 is known to protect mice against the lethal effects of lipopolysaccharides (LPS) and is considered to be an anti‐inflammatory cytokine which suppresses the production of pro‐inflammatory cytokines. We have examined the interactions of the pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) with IL‐10. Neutralization of TNF‐α in murine bone marrow‐derived macrophages resulted in a significant reduction of LPS‐inducible IL‐10 production. In mice, injection of 5 mg/kg LPS induced circulating IL‐10 with a biphasic time course exhibiting an early peak 1.5 h after challenge (synchronous with TNF‐α) and, after a nadir at 6 h, a second increase between 8 and 12 h. Treatment of mice with neutralizing anti‐mouse TNF‐α antiserum significantly increased LPS‐induced IL‐10 plasma levels between 1.5 and 6 h but diminished those at 12 h, while circulating IL‐6, interferon‐γ (IFN‐γ) and granulocyte colony‐stimulating factor (G‐CSF) concentrations were attenuated overall, without a biphasic response. Analysis of LPS‐induced IL‐10 mRNA expression in different tissues 1 h and 8 h after LPS or LPS plus anti‐TNF‐α revealed that the amount of transcripts in the liver correlated with circulating early and late IL‐10 levels. Our findings suggest that endogenous TNF‐α down‐regulates the early and up‐regulates the late LPS‐induced IL‐10 synthesis in vivo and that the liver is the major source of circulating IL‐10 after stimulation with LPS.
Expression and function of the murine CD95/FasR/APO‐1 receptor in relation to B cell ontogeny
Authors: Onel Karen B. , Tucek‐Szabo Carolyn L. , Ashany Dalit , Lacy Elizabeth , Nikolic‐Zugic Janko , Elkon Keith B.
Published: March 2007

Mice defective in Fas‐mediated apoptosis (lpr phenotype) have an intrinsic B cell abnormality that predisposes them to autoantibody production. To investigate potential roles for the Fas receptor (FasR) in B cell tolerance, FasR expression and function were evaluated at different stages of B cell development. FasR expression was very low or absent on pro‐ and pre‐B cells, but was detected in early B cell lines and was up‐regulated following IFN‐γ‐induced maturation of the pre‐B cell line 70‐Z. Whereas FasR expression was very low in resting mature sIgM+ B cells, expression was markedly increased following mitogen activation and was also elevated in two mature sIgG+ lymphoma lines. FasR expression correlated strongly with the ability of B cells to undergo Fas‐mediated apoptosis. In addition, although Fas did not appear to play a direct role in apoptosis mediated by cross‐linking of sIg with anti‐IgM, anti‐FasR and sublethal concentrations of anti‐Ig were additive in the induction of apoptosis in the early B cell line WEHI 231. These findings suggest that the Fas pathway is not involved in the elimination of pro‐ and pre‐B cells, but are compatible with an ancillary role for FasR in the elimination of early B cells and elimination of mature B cells following activation.
Characterization of Fcγ receptors on rat mucosal mast cells using a mutant FcϵRI‐deficient rat basophilic leukemia line
Authors: Boček Petr , Dráberová Lubica , Dráber Petr , Pecht Israel
Published: March 2007

A novel rat basophilic leukemia (RBL) 2H3 subline of rat mucosal mast cells deficient in the expression of the γ chain (RBL‐γ‐) has permitted functional characterization of their low‐affinity Fcγ receptors (FcγR). A rat FcγRII analog of the mouse b2 isoform has been earlier identified and its transcript detected in RBL‐2H3 cells. We have noew isolated and sequenced the rat FcγRIIb1 isoform and observed differences between its expression in RBL‐2H3 and RBL‐γ‐. Furthermore, we demonstrate that rat mucosal mast cells express a second, low‐affinity Fcγ receptor, namely the FcγRIII. Stimulation of either cell line with IgG complexes decreased the expression of transcripts for all FcγR. Hence, ligation of FcγR on rat mucosal mast cells apparently regulate their transcription. Selective stimulation through the FcγRII or FcγRII/III systems, respectively, was accomplished by either using RBL‐γ‐ line or by saturating the FcγRI on RBL‐2H3 with monomeric IgE. RBL‐γ‐ cells, which do carry FcγRII (but lack FcγRI and FcγRIII), do not respond to IgG (and IgE) immune complexes as monitored by specific protein tyrosine phosphorylation, degranulation or cytokine secretion. This finding, together with the restoration of the functional phenotype of parental cells upon γ chain cDNA transfection into RBL‐γ‐ cells, unequivocally excludes the possible stimulation of rat mucosal mast cells by clustering of their FcγRII. FcϵRI saturation by IgE on parental RBL‐2H3 cells completely blocks their response to IgG immune complexes. Thus the FcγR on these cells do not trigger degranulation and this is not due to the absence of FcγRIII as previously suggested. Therefore, co‐clustering of FcγRII and FcγRIII on rat mucosal mast cells does not seem to stimulate them. A possible inhibitory role of FcγRII in this process is suggested and discussed.
Specific elimination of IgE production using T cell lines expressing chimeric T cell receptor genes
Authors: Lustgarten Joseph , Eshhar Zelig
Published: March 2007

B cells that are destined to secrete IgE express a membrane‐bound form of IgE (mIgE) on their cell surface. Thus, elimination of such mIgE‐positive cells should result in the suppression of IgE production, thereby alleviating the symptoms of IgE‐mediated allergy. In this study, we examined, in a model system, whether IgE‐specific effector T cells can be used specifically to eradicate IgE‐producing B cells. To this end, we endowed T cells with anti‐IgE specificity using chimeric T cell receptors (cTCR) containing the variable region domain (Fv) of the 84.1c non‐anaphylactic anti‐mouse IgE monoclonal antibody (mAb). Two configurations of chimeric receptor were used: in the first, we combined the heavy and light variable region chains of 84.1c with the constant (C) regions of the TCR α and β chains. The second construct consisted of a chimeric single‐chain receptor (scFvR), composed of a single‐chain Fv region of the 84.1c antibody and the Cβ domain of the TCR. Following transfection of the cTCR or the scFvR genes into the murine MD.45 cytotoxic T cell hybridoma or the Jurkat human T cell line, functional expression of IgE‐specific chimeric receptors was detected on the cell surface. The transfected cells secreted interleukin‐2 upon stimulation with immobilized IgE or fixed IgE‐producing hybridoma cells. Moreover, cytotoxic T cell hybridomas expressing the chimeric receptor genes specifically eliminated IgE‐secreting B cells in vitro, resulting in isotype‐specific suppression of IgE production.
Natural antibodies against the immunoglobulin F(ab′)2 fragment cause elimination of antigens recognized by the F(ab′)2 from the circulation
Authors: Yano Shinya , Kaku Seiji , Suzuki Ken‐Ichi , Terazaki Chinami , Sakayori Toshiko , Kawasaki Tomihisa , Kawamura Koji , Sugita Yuji , Hoshino Kenji , Masuho Yasuhiko
Published: March 2007

A humanized monoclonal IgG1 antibody, designated hC4G1, recognizes the fibrinogen receptor glycoprotein (GP)IIb/IIIa on platelets and inhibits platelet aggregation. When the F(ab′)2 fragment of hC4G1 (F(ab′)2 hC4G1) was administered to cynomolgus monkeys, all the monkeys showed inhibition of platelet aggregation ex vivo. Unexpectedly, a significant decrease in platelet count was observed in 5 of 18 monkeys. Antibodies against F(ab′)2 hC4G1 were detected in the plasma of these monkeys by ELISA. Antibody activity in the plasma of these monkeys was significantly correlated with the intensity of platelet decrease (r = 0.84). The natural monkey antibodies to F(ab′)2 hC4G1 were directed against the C‐terminal region of F(ab′)2 fragment common to all human and humanized IgG antibodies. Natural homo‐reactive antibodies were also detected in human plasma from 15 of 40 healthy volunteers. Specificity was closely similar to that of the monkey antibodies. Affinity‐purified human homo‐reactive antibodies enhanced phagocytosis of platelets treated with the F(ab′)2 hC4G1. Monkey plasma with high homo‐reactive antibody activity was confirmed to decrease platelet count when administered together with F(ab′)2 hC4G1 to a monkey with low antibody activity. These results suggest that F(ab′)2 of humanized and human antibodies causes elimination of the corresponding antigens from the circulation by homo‐reactive antibodies.
Exacerbated autoimmunity associated with a T helper‐1 cytokine profile shift in H‐2E‐transgenic mice
Authors: Takács Katalin , Douek Daniel C. , Altmann Daniel M.
Published: March 2007

Major histocompatibility complex (MHC) class II genes are the strongest susceptibility markers for many human autoimmune diseases. A perplexing aspect of this is that HLA alleles can confer either susceptibility or dominant protection. In nonobese diabetic (NOD) mice, the strongest known diabetes susceptibility locus is within the MHC and is presumed to be the H‐2Ag7 product. When NOD mice carry a transgenic Eαd molecule allowing expression of an H‐2E heterodimer, diabetes is prevented. We investigated whether, as in human auto‐immunity, a single class II heterodimer might protect from some autoimmune diseases while predisposing to others. NOD mice are susceptible to experimental autoimmune encephalomyelitis (EAE) induced by the proteolipoprotein (PLP) epitope 56–70. Susceptibility to EAE was analyzed in NOD mice which either have or lack transgenic H‐2E expression. We found that H‐2E expression in NOD mice has converse effects on diabetes and EAE: while diabetes is prevented, EAE is greatly exacerbated and leads to demyelination. Although PLP 56–70 could be presented both in the context of H‐2A and H‐2E, increased disease severity in H‐2E transgenic mice could not be attributed either to an enhanced T cell proliferative response to PLP or to differences in determinant spread. However, cytokine analysis of the response revealed important differences between NOD mice and their H‐2E transgenic counterparts: H‐2E expression was associated with reduced interleukin‐4 secretion and enhanced interferon‐γ (IFN‐γ) secretion by lymph node cells, while the response of central nervous system infiltrating T cells displayed a markedly enhanced IFN‐γ response. Thus, whether a particular class II molecule confers resistance or susceptibility to an autoimmune disease may depend on differential cytokine profiles elicited by particular class II/autoantigen complexes.
The presence of the HLA class. II allele DPB1*0501 in ethnic Thais correlates with an enhanced vaccine‐induced antibody response to a malaria sporozoite antigen
Authors: Stephens Henry A. F. , Brown Arthur E. , Chandanayingyong Dasnayanee , Webster H. Kyle , Sirikong Monchan , Longta Panpimon , Vangseratthana Rapeepun , Gordon Daniel M. , Lekmak Somboon , Rungruang Ekaraj
Published: March 2007

In this study, we examined the correlation between the frequency of allelic variants of the class II human leukocyte antigen (HLA) DR, DQ and DP gene loci and the quantitative humoral immune response observed in 71 Thai volunteers, subsequent to vaccination with a conjugated subunit vaccine. This vaccine was designed to induce antibodies directed against the immunodominant repeat region of the Plasmodium falciparum circumsporozoite (CS) protein. The presence of the DPB1*0501, a relatively common allele in Asian populations, was found to be associated with high vaccine‐induced CS repeat‐specific antibody responses in the volunteers. Given the increasing focus on the use of subunit vaccines in the control of infectious diseases, consideration of the influence of class II allele frequencies in ethnically diverse recipient populations may be important.
Elucidation of the protein kinase C‐dependent apoptosis pathway in distinct subsets of T lymphocytes in MRL‐lpr/lpr mice
Authors: Ohkusu Kozo , Isobe Ken‐Ichi , Hidaka Hiroyoshi , Nakashima Izumi
Published: March 2007

MRL‐lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL‐lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL‐lpr mice more extensively than in T lymphocytes from MRL‐+/+ mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H‐7, and partially prevented by PKC activator phorbol 12‐myristate 13‐acetate (PMA). High sensitivity to H‐7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose‐dependency experiments of the drug effect. Population analysis showed that both CD4+ T lymphocytes and CD8+ T lymphocytes from MRL‐lpr mice were highly sensitive to H‐7, whereas CD8+ T lymphocytes, but not CD4+ T lymphocytes, from MRL‐+/+ mice were susceptible to the reagent. Interestingly, B220+Thy‐1+CD4−CD8− T lymphocytes from MRL‐lpr mice were most sensitive to H‐7 for apoptosis induction. Correspondingly, the membrane‐translocated activated PKC‐α level in splenic T lymphocytes from MRL‐lpr was more extensively up‐regulated by PMA than in splenic T lymphocytes from MRL‐+/+. These results suggest that some signal consistently activates PKC in MRL‐lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4+CD8+ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL‐lpr mice or MRL‐+/+ mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4+ CD8+ thymocytes from the Fas‐defective MRL‐lpr mice. We conclude from these results that the PKC‐dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.
Tumor necrosis factor‐α induction of major histocompatibility complex class. II antigen expression is inhibited by interferon‐γ in a monocytic cell line
Authors: Willheim Martin , Silacci Paolo , Gessl Alois , Spittler Andreas , Szépfalusi Zsolt , Samorapoompichit Puchit , Agis Hermine , Mayr Wolfgang R. , Scheiner Otto , Förster Othmar , Mach Bernard , Boltz‐Nitulescu Georg
Published: March 2007

Regulation of major histocompatibility complex (MHC) class II antigen expression by cytokines has been suggested to play a major role in the initiation and propagation of immune and autoimmune processes. The analysis of class II gene regulation benefits greatly from the existence of mutants with defects in regulatory factors. We report the establishment of a subclone of the human monocytic cell line U937, termed C119/9, with unusual cytokine regulation of MHC class II expression. In contrast to the parental U937 cell line, only tumor necrosis factor (TNF)‐α, and not interferon (IFN)‐γ induces the expression of MHC class II antigens on C119/9 cells, and paradoxically, this induction was inhibited almost completely by IFN‐γ. The HLA‐DR induction is controlled at the transcriptional level by the first 150 bp of the class II promoter which contains all the class II consensus elements. Both HLA‐DR and ‐DQ mRNA are induced by TNF‐α treatment, and both are diminished upon co‐treatment with TNF‐α and IFN‐γ. This antagonism between TNF‐α and IFN‐γ seem to be restricted to MHC class II genes. This subline of U937 cells may be useful in further studies of MHC class II regulation.
CD40‐CD40 ligand interactions stimulate B cell antigen processing
Authors: Faassen Anne E. , Dalke David P. , Berton Michael T. , Warren Wendy D. , Pierce Susan K.
Published: March 2007

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell‐dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen‐specific T cell hybrid by B cells co‐cultured with insect cells expressing recombinant CD40L or with a CD40‐specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross‐linking the surface immunoglobulin, and processing of antigen taken up by fluid‐phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40‐induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA‐1 or CD23. CD40 ligation does not affect the biosynthesis of the class II Ek molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen‐dependent T cell‐B cell interactions.
T helper cell‐independent neutralizing B cell response against vesicular stomatitis virus: Role of antigen patterns in B cell induction?
Authors: Bachmann Martin F. , Hengartner Hans , Zinkernagel Rolf M.
Published: March 2007

The neutralizing antibody response against vesicular stomatitis virus (VSV) was studied to analyze conditions necessary for induction of mature B cells. The glycoprotein of VSV (VSV‐G) is expressed as repetitive epitopes that are in a rigid densely packed paracristalline form in the virus envelope; in contrast, VSV‐G is present in a mobile randomly ordered form on infected cells and in micelles. We found that the rigid, paracristalline form of VSV‐G spaced about 5–10 mm on VSV virons induced potent primary and secondary neutralizing B cell responses which were independent of T helper cells, whereas the more randomly distributed, mobile forms of VSV‐G induced primary and secondary B cell responses that were more tightly controlled by T helper cells. These data suggest that (i) cross‐linking is a critical signal for B cell activation and antibody production, and that this signal alone does not necessarily anergize or delete mature B cells, (ii) the more regularly and rigidly ordered in a distance of 5–10 nm repetitive identical antigenic determinants are, the less are primary and secondary B cell responses controlled by T cells. We therefore propose that B cells take antigen organization as a marker for foreigness.
Default development of cloned human naive CD4 T cells into interleukin‐4‐ and interleukin‐5‐producing effector cells
Authors: Yang Liang‐Peng , Byun Dae‐Gyoo , Demeure Christian E. , Vezzio Nadia , Delespesse Guy
Published: March 2007

It was recently demonstrated that naive human and mouse CD4 T cells release low but sufficient levels of interleukin (IL)‐4 at priming to support their development into IL‐4 producers. To determine whether this IL‐4 is produced by a minor subset of cells, freshly isolated human naive CD4 T cells were directly cloned by limiting dilution in the absence of exogenous IL‐4. More than 95% of neonatal and 60% of adult naive T cells seeded in single‐cell cultures could be expanded upon stimulation with anti‐CD3 mAb immobilized on CD32‐B7.1 L cell transfectants in the presence of IL‐2. All 171 clones derived from four neonates and two adults produced IL‐4 and IL‐5 at generally high levels. Like the allergen‐specific human Th2 clones described in the literature, these T cell clones produced little or no interferon γ upon stimulation via their T cell receptor/CD3 complex, whereas they released high levels of this cytokine when activated with phorbol 12‐myristate 13‐acetate + ionomycin. Cells cloned and expanded in the presence of anti‐IL4 + anti‐IL‐4R mAb produced much lower levels of IL‐4 and IL‐5. It is concluded that almost every single naive human CD4 T cell primed and expanded in the absence of exogenous IL‐4 releases sufficient autocrine IL‐4 to support its clonal expansion into high IL‐4/IL‐5 producers.
IL‐18 time‐dependently modulates Th1/Th2 cytokine production by ligand‐activated NKT cells
Authors: Uchida Takefumi , Kinoshita Manabu , Fukasawa Masashi , Habu Yoshiko , Shinomiya Nariyoshi , Seki Shuhji
Published: March 2007

While IL‐18 synergizes with IL‐12 to induce a Th1 immune response, it also promotes a Th2 response. Here we investigate the modulatory role of IL‐18 on the Th1/Th2 cytokine response. The injection of α‐galactosylceramide (α‐GalCer), a ligand for NKT cells, elevated mouse serum levels of both IFN‐γ and IL‐4. When the mice were treated 2 h before α‐GalCer challenge with IL‐18, IFN‐γ production but not IL‐4 production was remarkably up‐regulated. In contrast, pretreatment with IL‐18 6 h before the challenge enhanced IL‐4 production. However, this IL‐18‐enhanced IL‐4 production was not elicited in mice injected with anti‐CD3 Ab. Liver mononuclear cells (MNC) produced a similar cytokine production pattern when MNC from mice treated with IL‐18 either 2 h or 6 h before challenge were stimulated with α‐GalCer in vitro. Expression of SOCS1 and SOCS3 was notably up‐regulated in the liver MNC from mice pretreated 6 h before with IL‐18; in particular, SOCS3 expression was confined to the liver NKT cells. Inhibition of SOCS3 by RNA interference up‐regulated the phosphorylation of STAT3 and suppressed in vitro IL‐4 production by IL‐18‐primed liver MNC stimulated with α‐GalCer, but it did not affect IFN‐γ production. These results suggest that IL‐18 time‐dependently modulates Th1/Th2 cytokine production in ligand‐activated NKT cells by regulating/inducing SOCS3 expression.
Arginase activity mediates reversible T cell hyporesponsiveness in human pregnancy
Authors: Kropf Pascale , Baud David , Marshall Sara E. , Munder Markus , Mosley Angelina , Fuentes José M. , Bangham Charles R. M. , Taylor Graham P. , Herath Shanti , Choi Beak‐San , Soler Germán , Teoh Tg , Modolell Manuel , Müller Ingrid
Published: March 2007

Complex regulation of T cell functions during pregnancy is required to ensure materno‐fetal tolerance. Here we reveal a novel pathway for the temporary suppression of maternal T cell responses in uncomplicated human pregnancies. Our results show that arginase activity is significantly increased in the peripheral blood of pregnant women and remarkably high arginase activities are expressed in term placentae. High enzymatic activity results in high turnover of its substrate L‐arginine and concomitant reduction of this amino acid in the microenvironment. Amino acid deprivation is emerging as a regulatory pathway of lymphocyte responses and we assessed the consequences of this enhanced arginase activity on T cell responses. Arginase‐mediated L‐arginine depletion induces down‐regulation of CD3ζ, the main signalling chain of the TCR, and functional T cell hyporesponsiveness. Importantly, this arginase‐mediated T cell suppression was reversible, as inhibition of arginase activity or addition of exogenous L‐arginine restored CD3ζ chain expression and T cell proliferation. Thus, L‐arginine metabolism constitutes a novel physiological mechanism contributing to the temporary suppression of the maternal immune response during human pregnancy.
Can Four‐Membered Heterophosphete Structures Exist? The Contribution of Phosphorus d Orbitals to Antiaromaticity
Authors: Mucsi Zoltán , Kötvélyesi Tamás , Viskolcz Béla , Csizmadia Imre G. , Novák Tibor , Keglevich György
Published: February 2007

The large instability of heterophosphete compounds, composed of a four‐membered unsaturated ring with a pentavalent phosphorus atom and a heteroatom, has been identified and characterised by first‐principle molecular orbital computations, at several levels of theory. It has been shown that strong electron‐withdrawing groups bound to the phosphorus atom are able to stabilize the system. The extent of antiaromaticity of these compounds is examined and quantified by a linear aromatic and antiaromatic scale.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)
Electroactive Nanostructured Membranes (ENM): Synthesis and Electrochemical Properties of Redox Mediator‐Modified Gold Nanoparticles Using a Dendrimer Layer‐by‐Layer Approach
Authors: Crespilho Frank N. , Ghica M. Emilia , Zucolotto Valtencir , Nart Francisco C. , Oliveira Osvaldo N. , Brett Christopher M. A.
Published: February 2007

The layer‐by‐layer (LbL) self‐assembly process has become an important tool in the fabrication of nanostructured devices for electrochemical applications, especially in those cases where control at the molecular level is required. In this paper we present a system based on electroactive nanostructured membranes (ENM) with ITO‐PVS/PAMAM‐Au LbL electrodes, in which a redox mediator (Me) is electrodeposited around the Au nanoparticles to form an ITO‐PVS/PAMAM‐Au@Me system. The redox mediators used were Co, Fe, Ni and Cu hexacyanoferrates. The 3‐bilayer ITO‐PVS/PAMAM‐Au@Me system was characterized electrochemically by cyclic voltammetry and electrochemical impedance spectroscopy. All hexacyanoferrate modified electrodes showed electrocatalytic activity towards hydrogen peroxide, thus demonstrating that this new approach can be used in biosensors and nanodevices, where a redox mediator is required.
Biosensor Based on Self‐Assembling Glucose Oxidase and Dendrimer‐Encapsulated Pt Nanoparticles on Carbon Nanotubes for Glucose Detection
Authors: Xu Lihuan , Zhu Yihua , Tang Longhua , Yang Xiaoling , Li Chunzhong
Published: February 2007

A novel amperometric glucose biosensor based on layer‐by‐layer (LbL) electrostatic adsorption of glucose oxidase (GOx) and dendrimer‐encapsulated Pt nanoparticles (Pt‐DENs) on multiwalled carbon nanotubes (CNTs) was described. Anionic GOx was immobilized on the negatively charged CNTs surface by alternatively assembling a cationic Pt‐DENs layer and an anionic GOx layer. Transmission electron microscopy images and ζ‐potentials proved the formation of layer‐by‐layer nanostructures on carboxyl‐functionalized CNTs. LbL technique provided a favorable microenvironment to keep the bioactivity of GOx and prevent enzyme molecule leakage. The excellent electrocatalytic activity of CNTs and Pt‐DENs toward H2O2 and special three‐dimensional structure of the enzyme electrode resulted in good characteristics such as a low detection limit of 2.5 μM, a wide linear range of 5 μM–0.65 mM, a short response time (within 5 s), and high sensitivity (30.64 μA mM−1 cm−2) and stability (80% remains after 30 days).
Prediction of elastic displacement response spectra in Europe and the Middle East
Authors: Akkar Sinan , Bommer Julian J.
Published: February 2007

Empirical equations are presented for the prediction of displacement response ordinates for damping ratios of 2, 5, 10, 20 and 30% of critical and for response periods up to 4s, using 532 accelerograms from the strong‐motion databank from Europe and the Middle East. The records were all re‐processed and only employed for regressions at periods within the usable range, defined as a fraction of the filter cut‐off and depending on the instrument type (digital or analogue), earthquake magnitude and site class. The equations can be applied to predict the geometric mean displacement and pseudo‐acceleration spectra for earthquakes with moment magnitudes (M) between 5 and 7.6, and for distances up to 100km. The equations also include style‐of‐faulting and site class as explanatory variables. The predictions obtained from these new equations suggest that earlier European equations for spectral displacements underestimate the ordinates at longer periods as a result of severe filtering and the use of the spectral ordinates at periods too close to the filter cut‐off. The results also confirm that the period defining the start of the constant displacement plateau in the Eurocode 8 (EC8) spectrum is excessively short at 2s. The results not only show that the scaling factor defined in EC8 for estimating the spectral ordinates at damping ratios different from 5% of critical are a good general approximation, but also that this scaling varies with magnitude and distance (reflecting the influence of duration) and also displays a mild dependence on response period. Copyright © 2007 John Wiley & Sons, Ltd.
The influence of evaluators on state medicaid policies: Florida and South Carolina's experience
Authors: Bigos Yvonne , Johnson Jennifer , Hendlin Rae , Harkreader Steve , Truitt Andrea
Published: February 2007

This chapter describes conditions and strategies that played a role in affecting actions taken by the Florida legislature and the South Carolina General Assembly in their respective Medicaid programs.