bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel function of B lymphocytes from normal mice to suppress autoimmunity in (NZB × NZW)F1 mice
Authors: Ono S. , Shao D.‐Z. , Yamada S. , Yang Y. , Yamashita M. , Hamaoka T.
Published: March 2003

In systemic autoimmune‐prone (NZB × NZW)F1 (NZB/W F1) mice, B‐cell abnormalities characterized by hypergammaglobulinaemia accompanying autoantibodies have been thought to be a main cause of the disease. To examine a possible regulatory role of B cells in the disease manifestations, we injected, intravenously (i.v.), normal or autoimmune B cells into non‐irradiated NZB/W F1 mice. The injection of splenic B cells from major histocompatibility (MHC)‐matched or allogeneic normal mice caused a marked decrease in serum immunoglobulin G (IgG) levels of autoantibodies, delayed the appearance of proteinuria and prolonged life span, whereas treatment with splenic B cells from NZB/W F1 or X‐linked immunodeficient (Xid) mice failed to suppress the autoimmunity. Moreover, in vitro polyclonal antibody responses to lipopolysaccharide (LPS) of NZB/W F1‐derived B cells from the treated mice were markedly reduced. Interestingly, the treatment of NZB/W F1 mice at 16, 18 and 20 or at 20, 22 and 24 weeks of age was more effective than that at 6, 8 and 10 weeks. The treatment also inhibited the development of surface IgG+ (sIgG+) B cells and splenomegaly, prominent in aged NZB/W F1 mice. In addition, when untreated NZB/W F1 responding B cells were precultured with normal B cells in vitro for 3 days, they also diminished the autoantibody production to subsequent LPS stimulation. Hence, the present results imply a novel function of normal B cells to ameliorate autoimmune disease in NZB/W F1 mice by correcting their B‐cell abnormalities, and indicate that NZB/W F1 and Xid mice possess defects in this regulatory B‐cell function.
2B4 stimulation of YT cells induces natural killer cell cytolytic function and invasiveness
Authors: Chuang S. S. , Kim M. H. , Johnson L. A. , Albertsson P. , Kitson R. P. , Nannmark U. , Goldfarb R. H. , Mathew P. A.
Published: March 2003

2B4 is a surface molecule found on all human natural killer (NK) cells, a subset of CD8+ T cells, monocytes and basophils. It was originally identified on mouse NK cells and the subset of T cells that mediate non‐major histocompatibility complex (MHC)‐restricted killing. Recently,
9 we have cloned the human homologue of 2B4 (h2B4) and found h2B4 to also mediate non‐MHC‐restricted cytotoxicity. In this study, we examine h2B4 in regulating various functions of NK cells using a human NK cell line YT, with monoclonal antibody (mAb) C1.7, an antibody that specifically recognizes h2B4. Ligation of surface 2B4 with mAb C1.7 increases YT's ability to destroy tumour cells. In the presence of mAb C1.7, the production of interferon‐γ (IFN‐γ) by YT cells is greatly enhanced. Engagement of surface 2B4 by mAb C1.7 downregulates the expression of h2B4 at the cell surface as well as the expression of h2B4 mRNA. Also, signalling through h2B4 causes the increased expression of matrix metalloproteinase‐2, a member of the matrix degrading proteinase family. Thus, in addition to modulating cytolytic function and cytokine production of NK cells, activation through surface 2B4 may play a role in upregulating the machinery for degradation of extracellular matrices to promote invasion of the tumour by NK cells.
SHP‐1/immunoreceptor tyrosine‐based inhibition motif‐independent inhibitory signalling through murine natural killer cell receptor Ly‐49A in a transfectedB‐cell line
Authors: Motoda K. , Takata M. , Kiura K. , Nakamura I. , Harada M.
Published: March 2003

Ly‐49A is a member of the Ly‐49 family of mouse natural killer cell receptors that inhibit cytotoxicity upon recognition of their ligands, the major histocompatibility complex (MHC) class I molecules, on the target cell surface. Although Ly‐49A has an immunoreceptor tyrosine‐based inhibition motif (ITIM) in its cytoplasmic tail, relatively little is known about the mechanisms underlying its inhibitory function. We report here that antibody‐mediated co‐ligation of the B‐cell receptor (BCR) with the transfected Ly‐49A molecule results in abrogation of BCR‐induced interleukin‐2 (IL‐2) secretion and mild reduction in activation of Erk1/2 and p38 mitogen‐activated protein (MAP) kinases in the B‐cell line A20. Surprisingly, BCR‐induced calcium mobilization was unaffected by cross‐linking of BCR with Ly‐49A. Furthermore, substitution of the single tyrosine residue in ITIM with phenylalanine, did not result in a complete loss of inhibitory function, as measured by BCR‐induced IL‐2 secretion. Deletion of the N‐terminal 37 amino acid peptide, which includes the ITIM, did abrogate the inhibitory activity. Co‐immunoprecipitation experiments revealed that, upon induction of tyrosine phosphorylation, Ly‐49A recruits tyrosine phosphatase src‐homology 2 (SH2) containing tyrosine phosphatases‐1 (SHP‐1), but not inositol phosphatase src‐homology 2 (SH2) containing inositol phosphatase (SHIP), and that the tyrosine residue in the ITIM is critical for this interaction. These results suggest that transfected Ly‐49A utilizes two different inhibitory mechanisms in B‐cell signalling: ITIM‐dependent and ITIM‐independent.
Selection and characterization of MUC1‐specific CD8+ T cells from MUC1 transgenic mice immunized with dendritic‐carcinoma fusion cells
Authors: Gong J. , Apostolopoulos V. , Chen D. , Chen H. , Koido S. , Gendler S. J. , Mckenzie I. F. , Kufe D.
Published: March 2003

Mice transgenic for the human MUC1 carcinoma‐associated antigen (MUC1.Tg) are tolerant to immunization with MUC1 antigen. Recent studies, however, have demonstrated that immunization of MUC1.Tg mice with fusions of MUC1‐positive tumour and dendritic cells (FC/MUC1) reverses MUC1 unresponsiveness and results in rejection of established MUC1‐positive pulmonary metastases. Here we demonstrate that lymph node cells from MUC1.Tg mice immunized with the FC/MUC1 fusion cells proliferate in response to MUC1 antigen by a mechanism dependent on the function of CD4, major histocompatibility complex (MHC) class II, B7‐1, B7‐2, CD28, CD40 and CD40 ligand. The findings demonstrate that stimulation of lymph node cells with MUC1 results in selection of MUC1‐specific CD8+ T cells. We show that the CD8+ T cells exhibit MUC1‐specific cytotoxic T lymphocyte (CTL) activity by recognition of MUC1 peptides presented in the context of MHC class I molecules Kb and Db. The MUC1‐specific CD8+ T cells also exhibit antitumour activity against MUC1‐positive metastases, but with no apparent reactivity against normal tissues. These results indicate that immunization of MUC1.Tg mice with FC/MUC1 reverses immunological unresponsiveness to MUC1 by presentation of MUC1 peptides in the presence of costimulatory signals and generates MHC‐restricted MUC1‐specific CD8+ T cells.
DNase I footprinting of the human interleukin‐5 gene promoter
Authors: Cousins D. J. , Richards D. , Kemeny D. M. , Romagnani S. , Lee T. H. , Staynov D. Z.
Published: March 2003

A characteristic feature of allergic asthma is the overexpression of the T helper type 2 (Th2) cytokines interleukin‐4 (IL‐4), IL‐5 and IL‐13 by T lymphocytes. Of these cytokines, IL‐5 is critical for the growth, survival and recruitment of eosinophils which are thought to be responsible for the tissue damage observed in asthmatic airways. The expression of human IL‐5 is primarily regulated at the transcriptional level; however, little is known about the mechanisms that control its transcription. Using nuclear extracts from allergen‐specific human T‐cell clones we have performed DNase I footprinting of the human IL‐5 promoter in order to establish sites occupied by transcription factors. We show footprints covering the conserved lymphokine element 0 [(CLE0) – 60 to – 44 base pairs (bp)] and GATA (– 73 to – 62 bp) elements, which have previously been identified to be important in the regulation of the murine IL‐5 promoter. We also describe a footprint covering a considerably extended Octamer binding site (– 249 to – 217 bp), which encompasses two hitherto unidentified CCAAT/enhancer binding protein consensus binding sites. We have also identified a previously unknown Ets binding site (– 274 to – 264 bp). These novel data on the regions of the human IL‐5 promoter that are bound by transcription factors should allow dissection of the regulatory mechanisms involved in the transcription of IL‐5 in the T‐helper lymphocytes of asthmatics.
Antiviral protection after DNA vaccination is short lived and not enhanced by CpG DNA
Authors: Oehen S. , Junt T. , López‐macías C. , Kramps T. A. N.
Published: March 2003

In this study, we investigated the potential of a DNA vaccine expressing the minimal cytotoxic T lymphocyte (CTL) epitope gp33 of the lymphocytic choriomeningitis virus glycoprotein to protect against infection of a non‐lymphoid organ and compared this to protection against a systemic infection. Furthermore, since immune stimulatory sequences have been shown to augment CTL responses, we examined the capacity of CpG DNA to enhance CTL memory. The data show that DNA vaccination with a gp33‐based gene construct induced short‐lived gp33‐specific CTL which protected against a systemic infection but not against a peripheral infection. Immune stimulatory sequences were incapable of either prolonging CTL memory or promoting protection against infection of a peripheral organ.
Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat‐shock protein 60
Authors: Macht L. M. , Elson C. J. , Kirwan J. R. , Gaston J. S. H. , Lamont A. G. , Thompson J. M. , Thompson S. J.
Published: March 2003

The hypothesis that T‐cell responses to the 60 000 MW family of heat‐shock proteins (hsp) may be related to the severity of rheumatoid arthritis (RA) was examined. Peripheral blood mononuclear cells (PBMC) from most normal individuals and both early and established RA patients proliferated in vitro in response to human hsp 60 and mycobacterial hsp 65 as well as tetanus toxoid (TT) and mycobacterial purified protein derivative (PPD). PBMC from some patients with established RA gave responses to hsp 60 that were above the normal range and/or peaked earlier than PBMC from normal individuals. The responses of PBMC from established RA to hsp 65, but not PPD or TT, were also higher than those from normal individuals, but the peak responses to all three antigens appeared delayed. Thus a selective increase in responsiveness to hsp 60 develops with disease duration in many RA patients. Six assessments of disease activity and severity were made but apart from rheumatoid factor titre, they were unrelated to the proliferative response. Similarly, disease activity and severity did not differ between those RA patients whose hsp 60 stimulated cells produced interferon‐γ and those who did not, although patients whose hsp 60‐stimulated T cells produced interleukin‐4 (IL‐4) and/or IL‐10, appeared to have less disease activity and severity than those who did not. Significant negative correlations were found between IL‐10 production by hsp 60‐stimulated cells and disease assessments. It is considered that RA is less severe in those patients whose hsp 60‐stimulated cells produce T‐helper 2 type cytokines.
Immunization onto bare skin with heat‐labile enterotoxin of Escherichia coli enhances immune responses to coadministered protein and peptide antigens and protects mice against lethal toxin challenge
Authors: Beignon A.‐S. , Briand J.‐P. , Muller S. , Partidos C. D.
Published: March 2003

In this study, the potential of the bare skin as a non‐invasive route for vaccination was examined. Following application of heat‐labile enterotoxin (LT) of Escherichia coli onto bare skin of BALB/c mice, strong serum anti‐LT antibody responses were observed, and mucosal immunoglobulin A (IgA) and IgG antibodies were measured in vagina washes. In addition, LT enhanced the serum and mucosal antibody and proliferative T‐cell responses to the model protein antigen β‐galactosidase (β‐gal) when coadministered onto bare skin, highlighting its potential to exert an adjuvant effect. When a peptide representing a T‐helper epitope (aa 307–319) from the haemagglutinin of influenza virus was applied onto bare skin with LT or cholera toxin (CT), it primed effectively peptide‐ and virus‐specific T cells, as measured in vitro by the interleukin‐2 (IL‐2) secretion assay. LT was shown to be as immunogenic as CT. Binding activity to GM1 gangliosides was essential for effective induction of anti‐CT serum and mucosal antibody responses. Finally, mice immunized onto bare skin with LT were protected against intraperitoneal challenge with a lethal dose of the homologous toxin. These findings give further support to a growing body of evidence on the potential of skin as a non‐invasive route for vaccine delivery. This immunization strategy might be advantageous for vaccination programmes in Third World countries, because administration by this route is simple, painless and economical.
Mechanism and therapeutic potential of DNA‐based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice
Authors: Oka Yuichiro , Fazle Akbar S. M. , Horiike Norio , Joko Kouji , Onji Morikazu
Published: March 2003

Two plasmid DNA vectors, pCAGGS(S) encoding the genes of the major envelope protein of hepatitis B virus (HBV), and pCAGGS(S + preS2) encoding the genes of the middle envelope protein were used to study the mechanism and therapeutic potential of DNA‐based immunization. Injection of these plasmids into the regenerating bilateral tibialis anterior muscle (TA) of normal C57BL/6 mice induced hepatitis B surface antigen (HBsAg)‐specific humoral and cellular immune responses. Seventy‐two hours after injection of pCAGGS(S), infiltrating cells including antigen‐presenting dendritic cells (DC) were localized around the injection site and HBsAg was expressed by both muscle cells and infiltrating cells. Spleen DC from the mice were exposed to HBsAg for up to 32 weeks after a single injection of pCAGGS(S), because these DC induced the proliferation of HBsAg‐specific memory lymphocytes in culture without exogenous HBsAg. A single injection of pCAGGS(S) or pCAGGS(S + preS2) resulted in the clearance of HBsAg in 28 out of 30 HBV‐transgenic (Tg) mice. In contrast, more than 7 monthly injections of an HBsAg‐based vaccine were required for the clearance of HBsAg in 6 out of 29 HBV‐Tg mice. Infiltrating DC at the DNA vaccine injection site may have a role in initiating HBsAg‐specific immune response, whereas the persistence of HBsAg exposed spleen DC may contribute to long‐lasting immunity. This study also suggested that DNA‐based vaccines may be a potent tool for treating chronic HBV carriers.
Extinction of expression of the genes encoding haematopoietic cell‐restricted transcription factors in T‐lymphoma × fibroblast cell hybrids
Authors: Oikawa Tsuneyuki , Yamada Toshiyuki , Kondoh Nobuo , Negishi‐Kihara Fumiko , Hitomi Yoshiaki , Suzuki Mitsuhiro , Teramoto Sayaka
Published: March 2003

We previously reported that expression of the T‐cell receptor (TCR) α and lck genes is extinguished in hybrids between mouse T‐lymphoma EL4 cells and mouse fibroblast B82 cells. In the present study, we found that the activities of the TCRα minimum enhancer and the lck promoter monitored by the luciferase or chloramphenicol acetyltransferase (CAT) assays were markedly inhibited in the hybrids. Expression of the TCF‐1, LEF‐1, GATA‐3, Ikaros, c‐myb and Fli‐1 genes, which encode the haematopoietic cell‐restricted transcription factors that appear to be responsible for the activities of the enhancer and the promoter, was fully extinguished or markedly suppressed in the hybrids. On the other hand, expression of the transcription factor genes observed in both parental cells, such as the AML1 and c‐ets‐1 genes, and that of the genes encoding ubiquitously expressed transcription factors, such as the E2A, CREB and c‐ets‐2 genes, was not significantly suppressed in the hybrids. These results suggest that the genes encoding haematopoietic cell‐restricted transcription factors are targets for negative regulation in fibroblastic background and that the repression of these genes may consequently lead to suppression of the promoter and/or enhancer activities of several T‐cell‐specific structural genes in T‐lymphoma × fibroblast cell hybrids.
Blockage of complement regulators in the conjunctiva and within the eye leads to massive inflammation and iritis
Authors: Bardenstein David S , Cheyer Christopher J , Lee Chieh , Cocuzzi Enzo , Mizuno Masashi , Okada Noriko , Medof M. Edward
Published: March 2003

The open environment of the eye is continuously subject to an influx of foreign agents that can activate complement. Decay‐accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 are regulators that protect self‐cells from autologous complement activation on their surfaces. They are expressed in the eye at unusually high levels but their physiological importance in this site is unstudied. In the rat, a structural analogue termed 5I2 antigen (5I2 Ag) has actions overlapping DAF and MCP. In this investigation, we injected F(ab′)2 fragments of 5I2 mAb into the conjunctiva and aqueous humor, in the latter case with and without concomitant blockage of CD59. Massive neutrophilic infiltration of the stroma and iris resulted upon blocking 5I2 Ag activity. Frank necrosis of the iris occurred upon concomitant intraocular blockage of CD59. C3b was identified immunohistochemically, and minimal effects were seen in complement‐depleted animals and in those treated with non‐relevant antibody. The finding that blockage of 5I2 Ag function in periocular tissues and within the eye causes intense conjunctival inflammation and iritis demonstrates the importance of intrinsic complement regulators in protecting ocular tissues from spontaneous or bystander attack by autologous complement.
All‐trans‐retinoic acid and polyriboinosinic : polyribocytidylic acid in combination potentiate specific antibody production and cell‐mediated immunity
Authors: DeCicco Kathleen L. , Youngdahl John D. , Ross A. Catharine
Published: March 2003

Retinoic acid (RA), an active metabolite of vitamin A, may synergize with interferons (IFN) to evoke a heightened immune response, suggesting combination therapy as a promising treatment for various cancers. Recently, we demonstrated a strong synergism between RA and polyriboinosinic : polyribocytidylic acid (PIC), an inducer of IFN, on antibody production in immunocompromised vitamin A‐deficient animals. In the present study, we examined whether this combination could potentiate T‐cell‐dependent antibody production in non‐immunocompromised rats. Forty male Lewis rats were treated with 100 µg all‐trans‐RA, 20 µg PIC, or the combination in either an 11‐d study to evaluate antibody production, changes in lymphocyte populations, and cell proliferation, or a 21‐hr study to evaluate early changes in lymphocyte populations and gene expression. The combination of RA + PIC significantly potentiated anti‐tetanus IgG levels (P < 0·002). Similarly, this combination also increased the numbers of B cells and major histocompatibility complex (MHC) class II+ cells in spleen and lymph nodes, and natural killer (NK) cells in spleen and blood (P < 0·05). RA + PIC‐treated rats had significantly higher levels of interleukin (IL)‐10, IL‐12, and signal transducer and activator of transcription‐1 (STAT‐1) mRNA (P < 0·05), and STAT‐1 protein (P < 0·02). Treatments administered in vivo significantly modulated T‐cell proliferation to anti‐CD3/phorbol myristyl acetate + IFN‐αex vivo. These changes in antibody production, cell distribution, cytokine gene expression, and T‐cell proliferation suggest that the combination of RA + PIC stimulates humoral and cell‐mediated immunity, and deserves further testing in models of cancer chemoprevention in vivo.
CR2‐mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes
Authors: Nielsen Claus H. , Marquart Hanne V. , Prodinger Wolfgang M. , Leslie Robert G. Q.
Published: March 2003

Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane‐bound C3 convertase. We have investigated whether this might lead to the generation of a C5 convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4·4 mm MgCl2/20 mm EGTA, which abrogates the classical pathway of complement without affecting the alternative pathway. Blockade of the CR2 ligand‐binding site with the monoclonal antibody FE8 resulted in 56 ± 13% and 71 ± 9% inhibition of the C3‐fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 ± 5%) and C9 (95 ± 3%). The kinetics of C3‐fragment and MAC deposition, as well as the dependence of both processes on CR2, indicate that MAC formation is a consequence of alternative pathway activation.
Age‐dependent increase of peritoneal B‐1b B cells in SCID mice
Authors: Hinkley Kirk S. , Chiasson Rod J. , Prior Tracey K. , Riggs James E.
Published: March 2003

The impact of increasing age upon immunoglobulin production and B‐lymphocyte generation in ‘leaky’ severe combined immune‐defective (SCID) mice was examined by enzyme‐linked immunosorbent assay and flow cytometry. By 1 year of age, the mice had normal numbers of B cells in their peritoneal cavity, while their spleen had very few immunoglobulin M‐positive (IgM+) cells. The majority of B cells expressed the CD11b marker characteristic of the B‐1b subset. B‐1a (CD5+) cells were present at a lower frequency and B‐2 cells were absent. The frequency of mice producing detectable immunoglobulin increased with age, and isotype diversity within individual mice was variable. IgM production was most frequently observed followed by IgG3 and IgG2a, then IgG1, and finally IgA. The selective persistence of the B‐1 B‐cell subset in the peritoneal cavity of aging SCID mice is a natural model for the study of those genetic and environmental influences that determine lymphocyte longevity.
Purified protein derivative of tuberculin upregulates the expression of vascular endothelial growth factor in T lymphocytes in vitro
Authors: Matsuyama Wataru , Kubota Ryuji , Hashiguchi Teruto , Momi Hiroaki , Kawabata Masaharu , Nakagawa Masanori , Arimura Kimiyoshi , Osame Mitsuhiro
Published: March 2003

The purpose of this study was to investigate the cellular source and significance of vascular endothelial growth factor (VEGF) which, as reported previously, is elevated in the sera of pulmonary tuberculous patients. We obtained peripheral blood mononuclear cells (PBMCs) from 28 patients with active pulmonary tuberculosis, from 11 healthy controls who were positive for purified protein derivative of tuberculin (PPD), and from eight healthy individuals who were negative for PPD. We incubated the PBMCs with PPD in the presence or absence of major histocompatibility (MHC) class I or class II antibody in vitro, and measured the VEGF levels of culture supernatants. We also analysed the source of cells that secrete VEGF by using flow cytometry with intracellular staining. The T lymphocytes of active tuberculous patients secreted a higher level of VEGF than those of healthy controls. This production of VEGF was inhibited by adding MHC class II antibody. The addition of MHC class I antibody, however, did not inhibit. We propose that CD4+ T lymphocytes are almost certainly the cells that produce VEGF in response to PPD. VEGF production might be associated with an antigen‐specific immune reaction via CD4+ T lymphocytes in tuberculosis.
Staphylococcal enterotoxin H contrasts closely related enterotoxins in species reactivity
Authors: Pettersson Helen , Forsberg Göran
Published: March 2003

Staphylococcus aureus enterotoxin H (SEH) belongs to the staphylococcal enterotoxin (SE) family of superantigens (SAgs). SEH has structural similarities to other SE; however, its biological properties are less well characterized. SEH binds with high affinity to human major histocompatibility complex (MHC) class II and exhibits strong mitogenic activity in human T cells, although it was found to be less potent than the related SEA. Surprisingly and in sharp contrast to related SEs, SEH did not possess superantigen activity in murine T cells and T cells from three investigated rat strains. However, SEH bound to a high extent to murine MHC class II expressing cells and when presented by these cells SEH stimulated human T cells to proliferate. Thus, SEH interacts with the murine MHC class II molecule in a functional manner. Notably, SEH had an inhibitory effect on murine SEA response, demonstrating that SEH interferes with the SEA interactions with murine cells. Despite this, murine T cells did not proliferate regardless of whether SEH was presented on human or murine MHC class II expressing cells. Consequently, SEH differs in species reactivity as compared to related SEs and lacks critical properties for T‐cell activation in mice. We propose that unlike other SEs, SEH does not interact with murine T cells since it is not recognized by murine T‐cell receptors.
Involvement of tumour necrosis factor‐α‐related apoptosis‐inducing ligand in enhanced cytotoxicity of lipopolysaccharide‐stimulated dendritic cells to activated T cells
Authors: Yu Yizhi , Liu Shuxun , Wang Wenya , Song Wengang , Zhang Minghui , Zhang Weiping , Qin Zhihai , Cao Xuetao
Published: March 2003

Dendritic cells (DC) are potent antigen‐presenting cells (APC) specialized in T‐cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone‐marrow‐derived DC activation of ovalbumin (OVA)‐specific Ia‐kb‐restricted T hybridoma cells, MF2.2D9 and OVA257–264‐specific H‐2kb‐restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand–tumour necrosis factor‐α (TNF‐α)‐related apoptosis‐inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up‐regulation of TRAIL‐expression on DC to some extent. The activation of caspase‐3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation‐induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses.
Circulating immunoglobulin A‐ and immunoglobulin G‐secreting hybridoma cells in peripheral blood preferably migrate to female genital tracts. The role of sex hormones
Authors: Wang Xiaolei , Zhao Xudong , Ben Kunlong , Cao Xiaomei , Wang Yuqi , Zhou Hongming
Published: March 2003

Antigen‐specific circulating immunoglobulin‐secreting cells (ISC) migrate to various secondary and tertiary lymphoid tissues. To understand the migration of the cells into the genital tract and its regulation by sex hormones, spleen‐derived SG2 hybridoma cells secreting immunoglobulin G2b (IgG2b) and Peyer's patch‐derived PA4 hybridoma cells secreting polymer IgA were labelled with 3H‐TdR, and intravenously injected into syngeneic mice of both sexes. Using flow cytometry, surface molecular markers of plasma cells, CD38 and CD138, and adhesion molecules, CD49d, CD162, and CD11a were found to be positive in SG2 and PA4 cells, but CD62L, α4β7 and CD44 were not expressed on these cells. The relative distribution indexes (RDIs) of the cells in genital tract and other tissues were measured. The means of RDIs of SG2 and PA4 cells in female genital tissues were 6·5 and 4·5 times as many as the means in male genital tissues, respectively. The treatment of ovariectomized mice with β‐oestradiol significantly increased the RDIs of PA4 cells in cervix and vagina, but decreased the RDIs of SG2 cells in vagina, horn of uterus, uterus and rectum (P<0·05). Progesterone treatment increased the RDIs of PA4 cells in vagina and rectum (P<0·05). The treatment with testosterone significantly increased the RDIs of SG2 and PA4 cells in epididymis and accessory sex glands (P<0·05). These results demonstrate that the female genital tract is the preferable site for the migration of circulating hybridoma cells to the male genital tract, and sex hormones play an important role in regulation of the migration of circulating ISC to genital tracts.
Irradiation up‐regulates CD80 expression through induction of tumour necrosis factor‐α and CD40 ligand expression on B lymphoma cells
Authors: Ishikawa Fumio , Nakano Hideki , Seo Akira , Okada Yayoi , Torihata Hideko , Tanaka Yuriko , Uchida Tetsuya , Miyake Hidekazu , Kakiuchi Terutaka
Published: March 2003

Previously, we reported that 100 Gy X‐ray irradiation followed by 24 hr incubation up‐regulates CD80 expression in murine B lymphoma cells, A20‐2J. In the present study, we analysed the underlying mechanisms of such up‐regulation using A20‐HL cells derived from A20‐2J cells. Irradiation of A20‐HL cells with 100 Gy enhanced CD80 expression. Incubation of untreated A20‐HL cells with those 100 Gy irradiated induced up‐regulation of CD80 expression. Irradiation of A20‐HL cells also up‐regulated the expression of tumour necrosis factor‐α (TNF‐α) and CD40 ligand (CD40L), and the amount of immunoprecipitable TNF‐α and CD40L in cell lysates. The addition of anti‐TNF‐α or anti‐CD40L monoclonal antibody (mAb) to the incubation of irradiated A20‐HL cells partially inhibited up‐regulation of CD80 expression, and the addition of both antibodies together almost completely inhibited the up‐regulation, suggesting that irradiation up‐regulated the CD80 expression through the induction of TNF‐α and CD40L expression. Irradiation also increased the accumulation of CD80, TNF‐α and CD40L mRNA. n‐tosyl‐l‐phenylalanine chloromethyl ketone (TPCK), a nuclear factor (NF)‐κB inhibitor, markedly decreased irradiation‐induced accumulation of CD80 mRNA and CD80 expression. FK506, a calcineurin inhibitor, and nifedipine, a calcium channel inhibitor, inhibited not only the expression of TNF‐α and CD40L, but also the up‐regulation of CD80 on irradiated A20‐HL cells. These results strongly suggested that irradiation induced TNF‐α and CD40L expression, which then up‐regulated CD80 mRNA and CD80 expression through activation of NF‐κB transcription factor in A20‐HL cells.
Molecular cloning and functional characterization of the pig homologue of integrin‐associated protein (IAP/CD47)
Authors: Shahein Yasser E. A. , De Andrés Damián F. , De La Lastra José M. Pérez
Published: March 2003

We report the cloning of cDNA encoding the pig homologue of human integrin‐associated protein (IAP or CD47). A pig CD47‐specific probe was generated by polymerase chain reaction (PCR) amplification of pig leucocyte cDNA, using primers based on consensus regions among the known sequences of CD47 from different species. Screening of a pig aorta smooth muscle cDNA library identified seven clones, all containing identical sequences. The clones contained an open reading frame (ORF) that encoded an 18 amino acid putative signal peptide, a 122 amino acid sequence consisting of a single extracellular immunoglobulin variable (IgV)‐like domain followed by a 147 amino acid region containing five membrane‐spanning domains and a 16 amino acid cytoplasmic tail. The amino acid sequence of the clones was 73% homologous to human IAP and therefore it was termed pig IAP or CD47. Reverse transcription–polymerase chain reaction (RT–PCR) showed that pig CD47 was expressed in a wide range of tissues and detected different alternatively spliced forms. The monoclonal antibody (mAb) BRIC 126, anti‐human CD47, was shown, by flow cytometry, to stain pig platelets as well as Chinese hamster ovary (CHO) cells transfected with the cDNA encoding pig CD47. Western blot analysis of pig erythocytes and platelets showed a molecular weight (MW) of 43 000–50 000 and of 55 000–65 000, respectively, under non‐reducing conditions. Pig CD47 was stably expressed on CHO cells and shown to bind human thrombospondin (TSP). BRIC126 antibody inhibited the binding of platelets and of CD47‐transfected cells to human TSP and to pig fibrinogen, whereas no effect was observed on control CHO cells.
Apoptosis‐inducing protein derived from hepatocyte selectively induces apoptosis in lymphocytes
Authors: Ikeda Masanobu , Yoshikawa Hideshi , Liu Jie , Nakajima Yasuo , Akahane Yoshihiro , Tasaka Kachio
Published: March 2003

The liver is where lymphocytes undergo activation‐induced cell death (AICD) at the resolution phase of an immune response, which is crucial for homeostasis of the immune system and prevention of autoimmunity. Exploring the machinery of AICD in the liver, we found that a primary culture supernatant of murine hepatocytes had an antiproliferative effect on antigen‐stimulated T clone and T lymphoma cells. Biological study showed that the antiproliferation was due to induction of apoptosis in a caspase‐dependent manner. The apoptosis‐inducing potential was sensitive to trypsin, heat (> 70°) and acid (< pH 5) treatment but could not be neutralized by anti‐tumour necrosis factor‐α, anti‐Fas ligand, or anti‐transforming growth factor‐β antibodies. Biochemical study of the isolated and purified apoptosis‐inducing component from the supernatant showed that it was a protein with a molecular mass of about 68 000–70 000. It induced apoptotic change in murine T and B cells, and to a lesser degree, in human lymphoid cells, but not in macrophages. Biochemical and biological characteristics distinguish this protein from others that have been reported to induce apoptosis of lymphocytes. The identification of an apoptosis‐inducing protein derived from murine hepatocytes, which selectively induces apoptosis in lymphocytes, suggests one possible mechanism for immune suppression in the liver.
Interleukin‐1β converting enzyme subfamily inhibitors prevent induction of CD86 molecules by butyrate through a CREB‐dependent mechanism in HL60 cells
Authors: Suzuki M. , Shinohara F. , Sato K. , Taniguchi T. , Takada H. , Rikiishi H.
Published: March 2003

To investigate the underlying mechanism for induction of CD86 molecules, we analysed the ability of the histone deacetylase (HDAC) inhibitor, sodium butyrate (NaB), to induce CD86 at the transcriptional level in HL60 cells. Our studies showed that the expression of CD86 on the cell surface was increased by 24 hr of NaB treatment, and the enhancement of CD86 mRNA expression was observed by real‐time polymerase chain reaction. When we measured NF‐κB binding activity, significant activity was induced upon NaB stimulation, which was suppressed by the addition of pyrrolidine dithiocarbamate. Butyrate also induced phosphorylated cAMP response element‐binding protein (CREB), which bound to cAMP‐responsive elements. Dibutyryl (db) ‐cAMP induced active CREB and increased the levels of CD86 by 24 hr. These observations indicated that NF‐κB and/or CREB are crucial for butyrate‐dependent activation of CD86 gene expression. We examined the inhibitory effects of various caspase inhibitors on the expression of CD86 in cells treated with NaB, because NaB also induced apoptosis with slow kinetics. Intriguingly, our results demonstrated that inhibitors of the interleukin‐1β converting enzyme subfamily (caspase‐1, ‐4, ‐5 and ‐13) blocked the butyrate‐induced increase in level of CD86. These inhibitors interfered with CD86 gene transcription in the presence of activated NF‐κB, whereas phosphorylated CREB was down‐regulated in the reactions where these inhibitors were added to inhibit CD86 gene expression. These results suggested that butyrate not only acetylates histones on the CD86 promoter through the suppression of HDAC activity, but that butyrate also regulates CREB‐mediated transcription, possibly through the caspase activities triggered by NaB.
An approach to object identification, selection and specification in object‐oriented analysis
Authors: Liang Ying , West Daune , Stowell Frank A.
Published: March 2003

When using current object‐oriented methods in the development of computer‐based information systems, problems frequently arise in the identification and specification of ‘objects’. In this paper, these problems are discussed and an approach that draws upon interpretivist systems thinking is presented that, it is argued, may alleviate some of the problems. It is proposed that a method that has been developed specifically to aid the user and analyst to appreciate a perceived problem situation might provide a useful aid in the process of identifying objects from the situation which are meaningful to the user. [An attempt has been made to use terminology that is consistent with that used in object‐oriented analysis. However, at times this terminology may be at variance with some of the interpretivist ideas put forward (e.g. the term ‘user’ is adopted, although the authors argue that the notion of ‘client’ is more appropriate to emphasize the increased involvement and responsibility of the ‘user’ in the approach advocated)]. The ability to achieve this is expected to facilitate the appropriate mapping of these meaningful objects into a technical specification through object‐oriented methods and to encourage user acceptance of the resulting computer‐based information system.
A sex‐linked Ace gene, not linked to insensitive acetylcholinesterase‐mediated insecticide resistance in Culex pipiens
Authors: Malcolm C. A. , Bourguet D. , Ascolillo A. , Rooker S. J. , Garvey C. F. , Hall L. M. C. , Pasteur N. , Raymond M.
Published: March 2003

An acetylcholinesterase (AChE) gene, Ace.x, showing 93% identity of deduced amino acid sequence to Anopheles stephensi Ace has been cloned from a Culex pipiens strain homozygous for insensitive AChE (iAChE) mediated insecticide resistance. DNA sequence of genomic DNA clones identified exons 2–5. RFLP of six clones indicated four possible alleles. Linkage analysis located Ace.x to chromosome I, less than 0.8 centimorgans from the sex locus, whereas the locus conferring resistance was 2.0 centimorgans from plum‐eye on chromosome II. Ace.1 coding for AChE1, which is associated with resistance, is therefore autosomal. We propose that Ace.x is the recently postulated Ace.2 coding for the biochemically distinct AChE2, which is not associated with resistance.
Induction of gene expression of antibacterial proteins by chitin oligomers in the silkworm, Bombyx mori
Authors: Furukawa S. , Taniai K. , Yang J. , Shono T. , Yamakawa M.
Published: March 2003

Activation of antibacterial protein genes by various chitin oligomers (from dimer to hexamer) was investigated by Northern blot analysis using cDNAs encoding cecropin B, attacin and lebocin from Bombyx mori as probes. All chitin oligomers tested were found to strongly trigger expression of cecropin B, attacin and lebocin genes. Furthermore, gene expression triggered by chitin oligomers was confirmed to occur specifically in the fat body and haemocytes. These results suggest that chitin oligomers have the same characteristics as those of lipopolysaccharide (LPS) and peptidoglycan in triggering gene expression of insect antibacterial proteins.
Mitochondrial DNA sequence divergence among greenbug (Homoptera: Aphididae) biotypes: evidence for host‐adapted races
Authors: Shufran K. A. , Burd J. D. , Anstead J. A. , Lushai G.
Published: March 2003

The full complement of known greenbug, Schizaphis graminum (Rondani), biotypes found in the USA were subjected to a molecular phylogenetic analysis based on a 1.2‐kb portion of the cytochrome oxidase I mitochondrial gene. In addition to these nine biotypes (B, C, E, F, G, H, I, J and K), a probable isolate of the enigmatic biotype A (NY), a ‘new biotype’ collected from Elymus canadensis (L.) (CWR), and an isolate from Germany (EUR) were included. Schizaphis rotundiventris (Signoret) was included as an outgroup. Genetic distances among S. graminum biotypes ranged from 0.08% to 6.17% difference in nucleotide substitutions. Neighbour‐joining, maximum parsimony and maximum likelihood analyses all produced dendrograms revealing three clades within S. graminum. Clade 1 contained the ‘agricultural’ biotypes commonly found on sorghum and wheat (C, E, K, I, plus J) and there were few substitutions among these biotypes. Clade 2 contained F, G and NY, and Clade 3 contained B, CWR and EUR, all of which are rarely found on crops. The rarest biotype, H, fell outside the above clades and may represent another Schizaphis species. S. graminum biotypes are a mixture of genotypes belonging to three clades and may have diverged as host‐adapted races on wild grasses.
Beta‐integrin of Anopheles gambiae: mRNA cloning and analysis of structure and expression
Authors: Mahairaki V. , Lycett G. , Blass C. , Louis C.
Published: March 2003

We have isolated an mRNA encoding a β integrin subunit of the malaria mosquito Anopheles gambiae. Our analysis predicts a protein that is very similar to βPS, the fruitfly orthologue. The gene is expressed during all developmental stages and it is found in all body parts, including the midgut. Finally, the expression of the gene does not seem to be modulated during blood meals, except for a substantial increase 48 h posthaematophagy, when digestion is nearly complete.
Effect in vitro of Tifell (formocresol‐eugenol) on macrophage adhesion
Authors: Segura  , Jiménez‐Rubio  , Guerrero  , Calvo  , Feito
Published: March 2003

The purpose of this study was to investigate the effect in vitro on macrophage adhesion of Tifell (formocresol‐eugenol), used as an intracanal medicament and in therapeutic pulpotomies in primary teeth, Macrophages were obtained from Wistar rats. As a test of macrophage phagocytic function the adherence capacity of macrophages to a plastic surface was determined. Assays were carried out in Eppendorf tubes after 15 min of incubation at 37°C in a humidified atmosphere of 5% carbon dioxide. The adherence index (AI) was calculated. Results showed that Tifell decreased significantly (P < 0.05) the AI of macrophages. Half maximal inhibition of AI was obtained at 1:334.5 Tifell dilution (AI = 27.75; P < 0.05). Taking into account that substrate adherence is the first step in the phagocytic action of macrophages and in antigen presentation, Tifell could inhibit macrophage function and modulate immune and inflammatory responses in dental pulp and periapical tissues.
Evaluating the sizes of Lightspeed® instruments
Authors: Schrader  , Sener  , Barbakow
Published: March 2003

The diameters of six different sized Lightspeed cutting heads were measured using a modified optical micrometer. Six specimens of instrument sizes 20, 22.5, 27.7, 30, 32.5 and 45 were examined. Except for one cutting head of size 30, all the recorded diameters were oversized compared to the expected diameter and the permitted tolerance (+0.005 mm). Perfectly machined instruments would increase by 12.5%, 9.09% and 8.3% from instrument sizes 20–22.5, 27.5–30 and 30–32.5, respectively. Under the conditions of this study, the corresponding increases were 7.1%, 6.75% and 8.54%. Instrument size 20 had the widest range and is the smallest instrument in the Lightspeed set. The largest differences between the recorded and expected means were registered for the size 45 group. An earlier study using a different measuring method reported that Lightspeed instruments were mostly undersized.
Crown cementation and pulpal health
Authors: Lam C. W. , Wilson P. R.
Published: March 2003

Literature review A direct result of the liquid continuum within the pulpo‐dentine complex is the effect of restorative dentistry on the health of the dentalpulp. Better understanding of the role of the complex in relation to restorative dentistry enables strategies to be devised in preserving pulp vitality. A review of the literature produced good laboratory evidence to support the prophylactic sealing of crown preparations with dentine bonding agents.
Leakage of five root canal sealers
Authors: Miletić I. , Anić I. , Pezelj‐Ribarić S. , Jukić S.
Published: March 2003

Aim The aim of this study was to examine the apical sealing ability of five root canal sealers using a fluid transport model.

Methodology A comparison of root canal sealers AH26, AH Plus, Diaket, Apexit, and Ketac‐Endo were tested on 60 single‐rooted teeth. The coronal part of each tooth was removed at the amelo‐cemento junction, and also 3 mm of the root tip. Root canals were instrumented using the ‘step‐back’ technique with Gates Glidden drills and irrigated with 2.5% NaOCl. The specimens were divided into five groups of 10 samples each and filled with test materials and gutta‐percha points by the cold lateral condensation technique. Ten teeth were used as a control group, out of which five served as negative and five as positive controls. The leakage was measured by the movement of an air bubble in a capillary glass tube connected to the experimental root section.

Results The differences in leakage amongst Ketac‐Endo (0.318μL; SD 0.084), AH26 (0.319μL; SD 0.075), AH Plus (0.330L; SD 0.085) Apexit (0.360μL; SD 0.127) and Diaket (0.387μL; SD 0.140) were not statistically significant (P 0.05).

Conclusions Under the conditions of this study, all five sealers produced a satisfactory seal.
A comparison of thermocouple and infrared thermographic analysis of temperature rise on the root surface during the continuous wave of condensation technique
Authors: Mc Cullagh J. J. P. , Setchell D. J. , Gulabivala K. , Hussey D. L. , Biagioni P. , Lamey P.‐J. , Bailey G.
Published: March 2003

Aim

This study was designed to use two methods of temperature measurement to analyse and quantify the in vitro root surface temperature changes during the initial stage of the continuous wave technique of obturation of 17 single‐rooted premolar teeth with standard canal preparations.

Methodology

A model was designed to allow simultaneous temperature measurement with both thermocouples and an infrared thermal imaging system. Two thermocouples were placed on the root surface, one coronally and the other near the root apex. A series of thermal images were recorded by an infrared thermal imaging camera during the downpack procedure.

Results

The mean temperature rises on the root surface, as measured by the two thermocouples, averaged 13.9 °C over the period of study, whilst the infrared thermal imaging system measured an average rise of 28.4 °C at the same sites. Temperatures at the more apical point were higher than those measured coronally. After the first wave of condensation, the second activation of the plugger in the canal prior to its removal always resulted in a secondary rise in temperature. The thermal imaging system detected areas of greater temperature change distant from the two selected thermocouple sites.

Conclusions

The continuous wave technique of obturation may result in high temperatures on the external root surface. Infrared thermography is a useful device for mapping patterns of temperature change over a large area.