bluuebunny/crossref_metadata_2025 huggingface dataset:
African trypanosome infections in mice that lack the interferon‐γ receptor gene: nitric oxide‐dependent and ‐independent suppression of T‐cell proliferative responses and the development of anaemia
Authors: MABBOTT‡  , COULSON  , SMYTHIES§  , WILSON  , STERNBERG
Published: March 2003

Infection of mice with African trypanosomes leads to a severe immunosuppression, mediated by suppressor macrophages. Using ex vivo macrophage culture and in vivo cell transfer, it has been shown that nitric oxide (NO) is a potent effector product of these cells and causes both lymphocyte unresponsiveness and dyserythropoiesis. We explored the role of NO in vivo during trypanosome infection using mice with a disrupted interferon‐γ‐receptor gene, which were unable to respond with macrophage activation and NO synthesis. These mice were less effective at controlling parasitaemia than the wild types, but showed an improved splenic T‐cell responsiveness and reduced anaemia during the early stages of infection. The data indicate that, in the mouse, NO is a significant mediator of immunosuppression only in early infection. Beyond day 10 of infection, NO‐independent mechanisms are of primary significance and the control of parasitaemia and T‐cell responsiveness are not directly related.
Serum levels, ontogeny and heritability of chicken mannan‐binding lectin (MBL)
Authors: LAURSEN  , HEDEMAND  , NIELSEN  , THIEL  , KOCH  , JENSENIUS
Published: March 2003

Mannan‐binding lectin (MBL) is a serum lectin found in mammals and recently also in birds. It is thought to play an important role in the innate immune defence through binding to surface carbohydrates on micro‐organisms followed by complement activation via the MBL pathway. This results in opsonization or direct complement‐mediated killing. To gain further knowledge about the physiology and function of the protein, we developed an enzyme‐linked immunosorbent assay for chicken MBL and used this to investigate the level of MBL in different chicken strains during embryogenesis, early and adult life. The MBL concentrations in 308 chickens, representing 14 different strains, showed a non‐Gaussian, unimodal distribution profile with a mean concentration of 5·8 μg/ml (range 0·4–37·8 μg/ml). No difference between the strains could be demonstrated and no chickens were found deficient in MBL. Ontogenetic studies showed that MBL is already detectable in embryos at a gestational age of 10 days (11 days before hatching). At hatching, the level is comparable to the level found in adult chickens. This level is fairly stable during the first weeks of life, but a deficiency state develops at 4 weeks of age, whereafter the level is normalized again at 5 weeks of age. Chickens with relatively low or high MBL levels were bred with cockerels having similar MBL levels and this resulted in F1 generations with significantly different MBL levels, suggesting that the protein level is genetically influenced.
Characterization and specificity of B‐cell responses in lupus induced by Mycobacterium bovis in NOD/Lt mice
Authors: C HORSFALL  , HOWSON  , SILVEIRA  , WILLIAMS  , BAXTER
Published: March 2003

A single dose of pasteurized Mycobacterium bovis administered intravenously to prediabetic non‐obese diabetic (NOD) mice prevented the onset of type 1 diabetes but precipitated a systemic ‘autoimmune rheumatic disease’ (ARD) similar to systemic lupus erythematosus. This syndrome was characterized by haemolytic anaemia, anti‐dsDNA and anti‐Smith antigen (Sm) antinuclear autoantibodies, increased severity of sialadenitis and glomerular immune complex deposition. Here, we examine the specificity of the autoantibody responses in M. bovis‐treated NOD mice. Large amounts of antibody were detected to the Sm/ribonucleoprotein (RNP) complex, of which the 28 000 MW polypeptide appeared to be immunodominant. The IgG subclass involved in the anti‐Sm response was primarily IgG2a. Antibodies against dsDNA were also detected, but the subclass of this response was mixed, with IgG2a and IgG2b being present in equal amounts. Together, these findings argue against a role for immune deviation towards T helper type 2 (Th2) responses in pathogenesis of the disease. The anti‐dsDNA and anti‐Sm reactivities were not mediated by polyreactive antibodies since neither antigen could cross‐compete plasma antibody binding to the other in competitive enzyme‐linked immunosorbent assay. The role of polyclonal B‐cell activation was examined by measuring total γ‐globulin as well as IgG reactive with other nuclear antigens including Ro60, Ro52 and La, which although not a major component of the autoantibody responses in these mice, did show small but significant increases following immunization with M. bovis. Thus polyclonal stimulation, while likely to be occurring, was not directly responsible for production of anti‐Sm antibodies.
Involvement of c‐myc in the resistance of non‐obese diabetic mice to glucocorticoid‐induced apoptosis
Authors: MARTINS  , ÁGUAS
Published: March 2003

Non‐obese diabetic (NOD) mice spontaneously develop insulin‐dependent diabetes mellitus (IDDM) as a consequence of autoimmune aggression of β cells of the endocrine pancreas by T cells. T lymphocytes of NOD mice are resistant to apoptosis induced by glucocorticoids, or by starving or DNA‐damaging treatments, a feature that was interpreted as being linked to escape of autoreactive T cells from thymic negative selection. c‐myc is one of the gene targets of glucocorticoids (GC), its expression being down‐regulated by the activated GC–GC receptor complex. We investigated here whether expression of Myc protein, in response to dexamethasone stimulation, was the same in NOD mice and in non‐autoimmune strains, namely NON, BALB/c and C57Bl.6. We found a consistent increase in the levels of Myc protein after GC‐treatment of lymphocytes of NOD mice, a finding that was in contrast to the down‐regulation of c‐myc that we observed in lymphocytes from mice not prone to diabetes. We also report that, rather than a absolute resistance to GC‐induced cell death, NOD mice display a delayed apoptotic response to GC. We propose that the resistance of NOD mice lymphocytes to GC‐induced apoptosis is because of inhibition of the repressive action of GC‐GR complexes at the level of c‐myc transcription. This deficient action of GC–GR results in increased production of nuclear Myc protein, peculiar to NOD mice cells, following their treatment with GC.
The role of ICAM‐1/LFA‐1 and VCAM‐1/VLA‐4 interactions on T helper 2 cytokine production by lung T cells of Toxocara canis‐infected mice
Authors: TAKAMOTO  , ISOBE  , SUGANE
Published: March 2003

In order to study the effect of costimulatory signals on T helper type 2 (Th2) cytokine production, monoclonal antibodies (mAb) against cell adhesion molecules (CAM) were added to cells in culture obtained from the lungs of Toxocara canis (Tc)‐infected mice followed by the determination of interleukin‐5 (IL‐5) and IL‐4 in the supernatants of the culture. ES‐stimulated IL‐5 production in the supernatant of total lung cells was reduced by 25% when anti‐intercellular adhesion molecule‐1 (anti‐ICAM‐1) mAb, anti‐CD11a mAb, or both anti‐ICAM‐1 and anti‐CD11a mAb together were added to the culture. The addition of anti‐CD18 mAb had no effects. Anti‐vascular cell adhesion molecule‐1 (anti‐VCAM‐1) mAb addition also reduced IL‐5 production by 60%, although the addition of anti‐very late activation antigen‐4 (anti‐VLA‐4) mAb or both anti‐VCAM‐1 and anti‐VLA‐4 mAb together were less effective. In the case of anti‐CD3 mAb stimulation, similar effects of mAb to CAM were observed. In contrast, IL‐4 production induced by anti‐CD3 mAb was reduced more markedly by the addition of either anti‐ICAM‐1 or anti‐CD11a mAb than the combination of anti‐VCAM‐1 and anti‐VLA‐4 mAb. Similar effects of mAb to CAM were observed on the production of IL‐5 and IL‐4 by CD4+ T cells purified using a fluorescence‐activated cell sorter. Coincubation with adherent cells was necessary for the significant production of IL‐5 and IL‐4 by CD4+ T cells. These results suggest that the VCAM‐1/VLA‐4 interaction is more important for IL‐5 production by CD4+ T cells in the lungs of Tc‐infected mice, and that the ICAM‐1/lymphocyte function‐associated antigen‐1 interaction is more important for the production of IL‐4.
C1 inhibitor removes the entire C1qr2s2 complex from anti‐C1Q monoclonal antibodies with low binding affinities
Authors: CHEN  , LAM  , BOACKLE
Published: March 2003

Evidence is presented for a new C1 Inhibitor (C1 INH) function. C1 INH was capable of dislodging the entire C1qr2s2 complex from C1‐activating substances that bound weakly to the globular heads of C1q. Two different mouse IgG1 monoclonal antibodies with different affinities for C1q globular heads were compared for their complement‐activating properties in the presence of normal human serum. As expected the higher affinity monoclonal antibody (Qu) was more effective in binding C1q and causing C1‐mediated C4b deposition. Unexpectedly, time responses of C1 (C1q) binding to immobilized 3C7 reached a peak then gradually decreased. However, C1q remained constantly bound to immobilized Qu. These results indicated that after C1 activation in human serum, the entire C1 complex (including C1q) was dislodged from 3C7, but not from immobilized Qu. The addition of purified C1 INH to purified C1, which had bound to immobilized 3C7, resulted in removal of C1 (C1q). Removal of the entire C1qr2s2 did not occur when C1 INH preparations were first neutralized by the addition of purified activated C1s. In summary, it is suggested that C1 INH plays a prominent role in dislodging the entire C1qr2s2 from immunoglobulin preparations which have a low binding affinity for the globular heads of C1q.
The full expression of the Ity phenotype in Ityr mice requires C3 activation by Salmonella lipopolysaccharide
Authors: NISHIKAWA  , YOSHIKAWA  , HARADA  , KITA  , KITA
Published: March 2003

Our previous study has shown that the rapid and sufficient activation of complement by Salmonella lipopolysaccharide occurs in genetically resistant (Ityr) A/J mice. To assess whether the level of complement activation by a virulent strain of Salmonella typhimurium regulates the level of murine natural resistance, we compared levels of serum complement activation by S. typhimurium and kinetics of serum‐opsonized S. typhimurium grown in macrophages using several strains of resistant (Ityr) and susceptible (Itys) mice. Itys macrophages killed intracellular S. typhimurium to the same extent as did Ityr macrophages when the pathogen was opsonized with Ityr serum. Opsonization of S. typhimurium with Itys serum reduced intracellular killing activity in Ityr macrophages to the same level as seen with Itys macrophages. Incubation of S. typhimurium with 25% Mg2+ EGTA (5 mm MgCl2–3 mm ethylene glycol‐bis (β‐aminotheyl either)‐N,N,N′,N′‐tetraacetic acid)‐chelated Ityr serum resulted in higher levels of C3 deposition onto the surface of this bacteria, C3b generation and also C3 consumption, compared with that with Mg2+ EGTA‐chelated Itys serum. Opsonization of S. typhimurium with A/J serum prior to infection increased early resistance in Itys mice. Infection with a virulent strain of S. typhimurium induced the expression of interleukin‐10 (IL‐10) mRNA at higher levels in C57BL/6 mice than in A/J mice. However, opsonization of S. typhimurium with A/J serum decreased bacterial growth in the spleen of C57BL/6 mice to the same level as observed for A/J mice in association with decreased expression levels of IL‐10 mRNA. Moreover, administration of anti‐C3 antibodies reduced the resistance of A/J mice in association with a decrease in serum levels of C3. These results indicate that the high level of complement activation via the alternative pathway in Ityr serum by a virulent strain of S. typhimurium reduces the virulence of this pathogen, which may contribute to the full expression of Ity phenotype in Ityr mice.
An antiserum raised against the recombinant cytoplasmic tail of the human CD43 glycoprotein identifies CD43 in many mammalian species
Authors: SZLANKA  , TOTH  , OCSOVSZKI  , KERESZTES
Published: March 2003

Leukosialin or CD43 is a heavily O‐glycosylated transmembrane protein expressed on all cells of the haematopoietic cell lineage with the exception of red blood cells and mature B cells. This antigen has been identified in human, mouse and rat with monoclonal antibodies. Although orthologues of many human and rodent leucocyte cell surface antigens have been described in recent years, CD43, despite its abundance on human and rodent cells, remained uncharacterized in other vertebrate species. The comparison of CD43 amino acid sequences from human, mouse and rat indicated a high level of homology in the cytoplasmic domain. A serum, (p.aCD43cp) raised against the recombinant cytoplasmic tail of the human CD43, was shown not only to recognize human CD43, but it bound to putative CD43 orthologues in many mammalian species. CD43 was found to be expressed in the same leucocyte subpopulations and circumstantial evidence suggested that CD43 is also regulated similarly during leucocyte ontogeny in all species investigated. As CD43+ cells were readily observed in fixed tissues, the p.aCD43cp serum may be used as a reliable reagent for the verification of the haematopoietic origin of infiltrations and, used together with other reagents, for the serological characterization of normal and pathological lymphoid tissues and lymphoid infiltrations in experimental work and in animal disease.
Arachidonic acid, but not its metabolites, is essential for FcγR‐stimulated intracellular killing of Staphylococcus aureus by human monocytes
Authors: ZHENG*  , ZOMERDIJK  , VAN DEN BARSELAAR  , GEERTSMA  , VAN FURTH  , NIBBERING
Published: March 2003

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcγ receptor (FcγR)‐mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcγR‐stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo‐oxygenase and lipoxygenase, markedly suppressed the FcγR‐mediated killing process. The production of O−2 by monocytes upon FcγR cross‐linking was inhibited by 4‐bromophenacyl bromide in a dose‐dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen‐dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcγR cross‐linking. Furthermore, FcγR cross‐linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcγR‐mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcγR‐stimulated intracellular killing of S. aureus by monocytes.
Human eotaxin induces eosinophil extravasation through rat mesenteric venules: role of α4 integrins and vascular cell adhesion molecule‐1
Authors: NAGAI  , LARKIN§  , HARTNELL  , LARBI  , RAZI AGHAKHANI  , WINDLEY  , DAVIES  , LOBB  , WILLIAMS  , NOURSHARGH
Published: March 2003

Eotaxin is a potent eosinophil‐specific CC‐chemokine, which has been shown to play a role in the selective induction of eosinophil accumulation in a number of allergic models of inflammation. Many aspects of the mechanism by which eotaxin induces eosinophil accumulation in vivo remain unresolved. In the present study, we investigated the direct effect of synthetic human eotaxin on leucocyte/endothelial cell interactions within rat mesenteric venules, as quantified by intravital microscopy. Topical eotaxin (30 pmol) induced rapid firm adhesion and extravasation of leucocytes within the rat mesentery, the extravasated leucocytes all being eosinophils, as determined by histological analysis. Whilst eotaxin was unable to stimulate the interaction of rat eosinophils with vascular cell adhesion molecule‐1 (VCAM‐1) under static conditions in vitro, eotaxin‐induced responses in vivo were significantly suppressed by anti‐α4 integrin and anti‐VCAM‐1 monoclonal antibodies (mAbs). The anti‐α4 integrin mAb, HP2/1 (3·5 mg/kg), inhibited the eotaxin‐induced firm adhesion and extravasation, 60 min postapplication of the chemokine, by 89% and 84%, respectively. In the same set of experiments, the anti‐VCAM‐1 mAb, 5F10 (3·5 mg/kg), inhibited leucocyte adhesion and extravasation by 61% and 63%, respectively. These results demonstrate that eotaxin‐induced migration of eosinophils through rat mesenteric venules in vivo is dependent on an α4 integrin/VCAM‐1 adhesion pathway, the significance of which may only be evident under flow conditions and/or following the ligation of other adhesion molecules expressed on eosinophils.
Immunoglobulin E suppression and cytokine modulation in mice orally tolerized to β‐lactoglobulin
Authors: PECQUET  , PFEIFER  , GAULDIE  , FRITSCHÉ
Published: March 2003

This study was designed to confirm the tolerogenic properties of β‐lactoglobulin in a mouse model and to assess specific oral tolerance induction in humoral and cellular compartments. BALB/c mice were fed β‐lactoglobulin (BLG) or whey proteins at different ages and subsequently intraperitoneally challenged 5 days later with both BLG and a non‐specific antigen, ovalbumin (OVA). Three weeks later, oral tolerance induction was analysed in CMP‐fed, versus saline‐fed mice, by measuring specific seric and intestinal antibody responses, delayed‐type hypersensitivity (DTH), specific splenocyte proliferation, and cytokine secretion patterns. Three‐week‐old mice fed high doses of either whey proteins or BLG (respectively 3 mg/g or 5 mg/g of body weight) were found to achieve oral tolerization. At humoral and mucosal levels, anti‐BLG immunoglobulin E (IgE) were suppressed in these groups when compared with saline fed mice. With respect to cellular responses, systemic DTH and lymphocyte proliferation to BLG were also inhibited in CMP‐fed mice. Weaning time was determined to be the best period for oral tolerance induction. Kinetic analyses showed however, that a minimum of 2 weeks was required for oral tolerance detection. Finally, cytokine profiles indicated a reciprocal decrease of interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) versus an increase of IL‐10 and transforming growth factor‐β (TGF‐β) secretions in tolerized mice. Taken together, these results clearly showed that oral administration of high doses of cows’ milk proteins can induce significant hyposensitization in mice, in a specific inhibition of T helper 1 (Th1) lymphocytes with the participation of suppressor cytokines.
Syk and paxillin are differentially phosphorylated following adhesion to the plastic substrate in rat alveolar macrophages
Authors: Hirano  , Kanno
Published: March 2003

Adhesion is associated with tyrosine phosphorylation in many types of cells. Although macrophages are known to adhere and phagocytose foreign particles, the signal transduction pathway of macrophages in response to adhesion to the foreign substrate has not been fully investigated. In the present study we investigated tyrosine‐phosphorylated proteins and phosphorylation of paxillin in alveolar macrophages (AMs) following adhesion to a plastic substrate. Adhesion to a plastic dish resulted in tyrosine phosphorylation of a 68 000 MW protein, which was shown, by immunoprecipitation and immunoblotting in the present study, to be a rat Syk kinase. Treatment with erbstatin reduced both tyrosine phosphorylation of Syk and adherence of AMs, while treatment with cytochalasin B inhibited spreading of AMs but did not inhibit tyrosine phosphorylation of Syk. These results suggest that tyrosine phosphorylation of Syk plays an important role in adhesion of AMs to the plastic substrate, but not in AM spreading. Paxillin is known to be tyrosine phosphorylated following adhesion to the extracellular matrix in many types of cells. However, paxillin appeared to be serine/threonine phosphorylated rather than tyrosine phosphorylated following adhesion of AMs to the plastic substrate. Treatment with A23187 (a calcium ionophore), but not phorbol 12‐myristate 13‐acetate (PMA; a protein kinase C stimulator), induced tyrosine phosphorylation of Syk in non‐adherent AMs. Treatment with either A23187 or PMA caused electromobility changes of paxillin that were mainly a result of serine/threonine phosphorylation. These results suggest that adhesion to the plastic substrate leads to two differently regulated events in AMs: tyrosine phosphorylation of Syk and serine/threonine phosphorylation of paxillin, both of which are probably mediated by an increase in intracellular calcium.
A bone marrow‐derived stroma cell line, ST2, can support the differentiation of fetal thymocytes from the CD4−CD8−double negative to the CD4+ CD8+ double positive differentiation stage in vitro
Authors: Tong  , Kishi  , Matsuda  , Muraguchi
Published: March 2003

T‐cell precursors differentiate into mature T cells predominantly in the thymus. However, it has also been reported that T‐cell precursors mature in extrathymic organs such as the liver, bone marrow, or intestines. In order to investigate the nature of the extrathymic microenvironment that supports T‐cell maturation, we examined the effect of a bone marrow‐derived stroma cell line, ST2, on T‐cell precursors by using a reaggregate thymic organ culture (RTOC) system. We found that ST2 cells supported the differentiation of fetal thymocytes at day 14·5 of gestation from a CD4− CD8− double negative (DN) to a CD4+ CD8+ double positive (DP) differentiation stage in a manner similar to that observed in thymus. Anti‐interleukin‐7 receptor (IL‐7R) and anti‐c‐kit antibodies blocked the growth of thymocytes in RTOC with ST2 cells, but did not inhibit the generation of DP thymocytes. These data indicate that a bone marrow‐derived stroma cell, ST2, which supports B‐cell differentiation, is also able to support T‐cell development and may constitute one of the microenvironmental components for extrathymic T‐cell development.
Interaction of CTLA‐4 (CD152) with CD80 or CD86 inhibits human T‐cell activation
Authors: Vandenborre K. , Van Gool S. W. , Kasran A. , Ceuppens J. L. , Boogaerts M. A. , Vandenberghe P.
Published: March 2003

Occupancy of CTLA‐4 (cytotoxic T‐lymphocyte antigen‐4 or CD152) negatively regulates the activation of mouse T lymphocytes, as indicated by the fate of CTLA‐4‐deficient mice, by the impact of anti‐CTLA‐4 monoclonal antibodies (mAbs) on mouse T‐cell activation in vitro and by the impact of CTLA‐4 blockade on the course of experimental tumoral, autoimmune, alloimmune or infectious disease in this animal. The function of human CTLA‐4, however, remains less clear. The expression and function of human CTLA‐4 were further explored. CTLA‐4 was expressed under mitogenic conditions only, its expression being, at least partially, dependent on the secretion of interleukin‐2. Memory T cells expressed CTLA‐4 with faster kinetics than naive T cells. The functional role of human CTLA‐4 was assessed utilizing a panel of four anti‐CTLA‐4 mAbs that blocked the interaction between CTLA‐4 and its ligands. These mAbs, in immobilized form, profoundly inhibited the activation of T cells by immobilized anti‐CD3 mAb in the absence of anti‐CD28 mAb, but co‐stimulated T‐cell activation in the presence of anti‐CD28 mAb. Finally, and importantly, blockade of the interaction of CTLA‐4 with its ligands using soluble anti‐CTLA‐4 mAbs, in intact form or as Fab fragments, enhanced T‐cell activation in several polyclonal or alloantigen‐specific CD80‐ or CD80/CD86‐dependent assays, as measured by cytokine production, cellular proliferation or cytotoxic responses. It is concluded that interaction of CTLA‐4 with its functional ligands, CD80 or CD86, can down‐regulate human T‐cell responses, probably by intracellular signalling events and independent of CD28 occupancy.
Elevated apoptosis of peripheral T lymphocytes in diabetic BB rats
Authors: Jung C.‐G. , Kamiyama T. , Agui T.
Published: March 2003

Thymocytes and peripheral lymphocytes of BioBreeding (BB) diabetes‐prone (BBDP) and diabetes‐resistant (BBDR) rat were analysed by fluorescence‐activated cell sorter (FACS). The number of CD4– CD8–, CD4+ CD8–, CD4– CD8+ and CD4+ CD8+ subsets was not different between BBDP and BBDR rat thymocytes, whereas spleen and lymph nodes in BBDP rats undergo severe T‐cell lymphopenia. Notably, mature CD4– CD8+[T‐cell receptor (TCR)‐αβ+ and CD5+] cells are certainly present in the BBDP rat thymus, unlike some previous reports, suggesting that the differentiation of CD4– CD8+ from CD4+ CD8+ cells occurs normally in the BBDP rat thymus. As a cause of peripheral T‐cell lymphopenia we suspected apoptosis of recent thymic emigrants. By FACS analysis with fluorescein isothiocyanate‐labelled annexin V, elevated apoptosis was evident in BBDP rat peripheral lymphocytes. Furthermore, terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labelling (TUNEL) staining in BBDP rat splenic sections revealed that a number of TUNEL‐positive cells were observed in the T‐lymphocyte‐rich area. From these results, we postulate that an abnormally elevated apoptosis of peripheral T lymphocytes, but not impaired thymocyte differentiation, is a cause of the peripheral T‐cell lymphopenia in BBDP rats.
Deficiency of the macrophage migration inhibitory factor gene has no significant effect on endotoxaemia
Authors: Honma N. , Koseki H. , Akasaka T. , Nakayama T. , Taniguchi M. , Serizawa I. , Akahori H. , Osawa M. , Mikayama T.
Published: March 2003

By targeted disruption of the MIF gene, we have established a mouse strain deficient in macrophage (Mφ) migration inhibitory factor (MIF). Despite previous reports indicating an essential role of MIF in endotoxaemia, an injection of lipopolysaccharide (LPS) into the MIF‐deficient mice (maintained under specific pathogen‐free conditions) caused shock. No significant difference was detected between the MIF‐deficient mutant and normal mice in susceptibility to LPS for endotoxaemia or tumour necrosis factor‐α (TNF‐α) formation upon LPS injection. Peritoneal Mφ from the two strains produced TNF‐α in response to LPS with similar dose responses. Dexamethasone suppressed the LPS‐induced TNF‐α response of Mφ, but no difference was detected between the Mφ from the two strains. These results suggest that endogenous MIF has no significant effect on the LPS‐induced TNF‐α production and no effect on suppression of the response by glucocorticoids. Thus, MIF is not crucial for LPS‐induced immune responses leading to shock.
Interferon‐γ up‐regulates intercellular adhesion molecule‐1 and vascular cell adhesion molecule‐1 and recruits lymphocytes into the vagina of immune mice challenged with herpes simplex virus‐2
Authors: Parr M. B. , Parr E. L.
Published: March 2003

Lymphocyte recruitment into tissues involves interactions between adhesion molecules on vascular endothelial cells and corresponding ligands on the lymphocyte surface. In the present study we investigated the expression of four endothelial addressins in the vagina and their possible up‐regulation by interferon‐γ (IFN‐γ) in immune mice after vaginal challenge with herpes simplex virus type 2. The adhesion molecules intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) were minimally expressed in the vagina of non‐immune mice with or without vaginal challenge and in immune mice before challenge, but both were up‐regulated by IFN‐γ, directly or indirectly, in immune mice after challenge. Mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) was detected in most vaginas but was not up‐regulated by IFN‐γ in immune mice after virus challenge. E‐selectin was not detected in any vaginas. The results suggest that ICAM‐1 and VCAM‐1 may be involved in rapid, IFN‐γ‐mediated recruitment of lymphocytes to the vaginal mucosal of immune mice after local virus challenge.
Introducing endogenous antigens into the major histocompatibility complex (MHC) class II presentation pathway. Both Ii mediated inhibition and enhancement of endogenous peptide/MHC class II presentation require the same Ii domains
Authors: Frauwirth K. , Shastri N.
Published: March 2003

The invariant chain (Ii) plays a key role in regulating the antigen presentation function of major histocompatibility complex (MHC) class II molecules. Ii also influences the presentation of usually excluded endogenously synthesized proteins into the MHC class II presentation pathway. To evaluate the role of Ii in the generation of peptide–MHC class II complexes derived from endogenously synthesized proteins, we tested mutant Ii constructs in two model systems. Co‐expression of wild‐type Ii inhibits the presentation of hen‐egg lysozyme (HEL) 35–45/Ak complex, but enhances the presentation of ovalbumin (OVA) 247–265/Ak complex from endogenously synthesized HEL or OVA precursors. The differential sensitivity of these antigens to chloroquine was consistent with their being processed in distinct compartments. Nevertheless, with a panel of Ii deletion constructs we show here that both the Ii‐mediated inhibition and enhancement functions require the endosomal targeting and CLIP residues. Surprisingly, the Ii mutant lacking the endoplasmic reticulum lumenal residues 126–215, despite apparently lower expression, was at least as effective as full‐length Ii in antigen presentation assays. Thus, alternative pathways exist for processing endogenously expressed antigens, and Ii‐mediated inhibition and enhancement of peptide/MHC class II expression depend upon the same regions, with neither requiring the 89 C‐terminal, lumenal Ii residues.
The use of human CD68 transcriptional regulatory sequences to direct high‐level expression of class A scavenger receptor in macrophages in vitro and in vivo
Authors: Gough Peter J. , Gordon Siamon , Greaves David R.
Published: March 2003

Macrophages (Mφ) play a key role in innate and acquired immunity. The study of Mφ biology has been hampered by the absence of suitable gene regulatory sequences for the overexpression of heterologous genes in Mφ. The human CD68 gene encodes a glycoprotein that is expressed in monocytes and Mφ, and therefore represents an attractive candidate gene for the generation of a Mφ‐specific gene‐targeting vector. A transgene expression cassette that combines 2·9 kb of CD68 5′ flanking sequence with the 83‐bp first intron (IVS‐1) of the CD68 gene, directed high‐level, long‐lasting expression of class A human scavenger receptor (hSR‐A) isoforms in the murine Mφ cell line, RAW‐264. By using this CD68 expression cassette to generate Mφ cell lines that overexpress a soluble secreted form of the extracellular portion of type I human SR‐A, we were able to purify significant quantities of this protein and show its ability to inhibit SR‐A‐mediated endocytosis. Analysis of two independent lines of transgenic mice that expressed type III human SR‐A under the control of the CD68 gene sequences revealed transgene mRNA expression in elicited Mφ populations and in mouse tissues in a pattern that was consistent with Mφ‐specific gene targeting. These data show that CD68 transcriptional regulatory sequences can be used to direct high‐level transgene expression in Mφin vitro and in vivo.
The genetic and immunopathological processes underlying collagen‐induced arthritis
Authors: Luross JEFF A. , Williams NEIL A.
Published: March 2003

Animal models of rheumatoid arthritis (RA) have provided substantial insights into basic pathogenic mechanisms of chronic inflammatory arthritis and autoimmune disease in general. Of the variety of models reported, collagen‐induced arthritis (CIA) has been the most characterized in terms of both its pathogenesis and its underlying immunological basis. Collagen‐induced arthritis has also been the model of choice in terms of testing potential new therapeutic agents for the treatment of human RA. Nevertheless, the complex nature of the balance between T‐cell cytokines and the chronic inflammatory processes is only recently becoming clear. This review focuses on these developments, highlighting their implications for our understanding of RA and for the use of CIA as a suitable animal model.
γδ T cells are critical for the induction of anterior chamber‐associated immune deviation
Authors: Xu Yijun , Kapp Judith A.
Published: March 2003

Anterior chamber‐associated immune deviation (ACAID) is a systemic form of tolerance that is elicited by introducing antigens into the anterior chamber of the eye. ACAID is characterized by deficiencies in delayed‐type hypersensitivity and complement‐fixing antibodies upon subsequent challenge with antigen. The mechanisms responsible for the generation of this form of tolerance are not yet completely clear. Here we asked whether γδ T cells, which are critical in the induction of oral tolerance and nasal tolerance, play a role in ACAID. The percentage of splenic γδ T cells was higher in mice that received antigen via the anterior chamber compared to untreated mice. In addition, CD44 was up‐regulated on some splenic γδ and αβ T cells after the intraocular injection of antigen. Moreover, administration of antigen into the anterior chamber did not induce ACAID in the C57BL/6 mice pretreated with anti‐mouse δ‐chain monoclonal antibody or in the γδ T‐cell‐receptor‐deficient (δ−/−) mice. γδ T cells from wild‐type mice reconstituted ACAID when transferred into the δ−/− mice before injection of antigen, verifying that the deficiency in δ−/− mice results from the lack of γδ T cells rather than from an inadvertent change caused by deletion of the δ‐chain. These findings indicate that γδ T cells play a very important role in ocular tolerance.
Tissue distribution of products of the mouse decay‐accelerating factor (DAF) genes. Exploitation of a Daf1 knock‐out mouse and site‐specific monoclonal antibodies
Authors: Lin Feng , Fukuoka Yoshihiro , Spicer Andrew , Ohta Rieko , Okada Noriko , Harris Claire L. , Emancipator Steven N. , Medof M. Edward
Published: March 2003

Decay‐accelerating factor (DAF) is a membrane regulator of C3 activation that protects self cells from autologous complement attack. In humans, DAF is uniformly expressed as a glycosylphosphatidylinositol (GPI)‐anchored molecule. In mice, both GPI‐anchored and transmembrane‐anchored DAF proteins are produced, each of which can be derived from two different genes (Daf1 and Daf2). In this report, we describe a Daf1 gene knock‐out mouse arising as the first product of a strategy for targeting one or both Daf genes. As part of the work, we characterize recently described monoclonal antibodies against murine DAF protein using deletion mutants synthesized in yeast, and then employ the monoclonal antibodies in conjunction with wild‐type and the Daf1 knock‐out mice to determine the tissue distribution of the mouse Daf1 and Daf2 gene products. To enhance the immunohistochemical detection of murine DAF protein, we utilized the sensitive tyramide fluorescence method. In wild‐type mice, we found strong DAF labelling of glomeruli, airway and gut epithelium, the spleen, vascular endothelium throughout all tissues, and seminiferous tubules of the testis. In Daf1 knock‐out mice, DAF labelling was ablated in most tissues, but strong labelling of the testis and splenic dendritic cells remained. In both sites, reverse transcription‐polymerase chain reaction analyses identified both GPI and transmembrane forms of Daf2 gene‐derived protein. The results have relevance for studies of in vivo murine DAF function and of murine DAF structure.
Herpes simplex virus 1 infected neuronal and skin cells differ in their susceptibility to complement attack
Authors: Rautemaa Riina , Helander Tuula , Meri Seppo
Published: March 2003

Herpes simplex virus type 1 (HSV‐1) infection in neurons is lifelong and generally asymptomatic. Reactivation of this latent infection results in skin blistering whereas the respective peripheral neurons are rarely affected. Why the neuronal cells are spared while the skin cells are sacrificed is not well understood. In the present study our aim was to study whether neuronal and skin cells differ in their ability to control complement attack during HSV‐1 infection. Human embryonal skin (HES) cells and neuronal Paju cells were infected by HSV‐1 in vitro. Both types of infected cells activated complement but were initially resistant to membrane attack complex (MAC) deposition. During the first hours of infection the expression of the endogenous complement regulators decay accelerating factor (DAF) and CD59 increased on both HES and Paju cells. By 12 hr the infected HES cells had lost their ability to control complement attack. The expression of DAF and CD59 decreased and the cells became targets for MAC attack. In contrast, complement regulator expression on the Paju cells did not decrease below the initial level and complement C5b‐9 deposition was found only on 10% of the Paju cells at 12 hr. The results suggest that HSV‐infected neuronal cells are better than skin cells in protecting themselves against complement attack. This may contribute to the persistence of a latent HSV‐1 infection in neuronal cells for prolonged periods.
Stimulation of naïve T‐cell adhesion and immunological synapse formation by chemokine‐dependent and ‐independent mechanisms
Authors: Bromley Shannon K. , Dustin Michael L.
Published: March 2003

Chemokines adsorbed to the cell surface play an important role in the initial interactions of T cells with endothelial cells, and may also have a role in T‐cell interactions with dendritic cells. Therefore, we examined the effect of surface‐adsorbed chemokines on the interaction of naïve murine splenic T cells with supported bilayers containing intercellular adhesion molecule (ICAM)‐1, or with bone marrow‐derived cultured dendritic cells in the presence and absence of relevant MHC–peptide complexes. Naïve T cells formed immunological synapses, defined as a ring of lymphocyte function associated (LFA)‐1–ICAM‐1 interactions surrounding a central cluster of MHC–peptide complexes, on supported planar bilayers containing ICAM‐1 and relevant MHC–peptide complexes. Chemokines stimulated an increase in the percentage of naïve cells that adhered to ICAM‐1, but did not increase the average number of LFA‐1–ICAM‐1 interactions in the contact area. In contrast, relevant MHC–peptide complexes resulted in a small increase in the proportion of interacting T cells, but stimulated an 8‐fold increase in the number of LFA‐1–ICAM‐1 interactions in each contact formed. Naïve T cells displayed a significant basal adhesion to bone marrow dendritic cells that was further increased when relevant chemokines were adsorbed to the dendritic cell surface. However, basal and antigen‐stimulated T‐cell adhesion to dendritic cells was not sensitive to pertussis toxin. Thus, there are chemokine‐independent mechanisms that initiate adhesion between T cells and dendritic cells.
Role of stromal‐cell derived factor‐1 in the development of autoimmune diseases in non‐obese diabetic mice
Authors: Matin Khairul , Salam M. Abdus , Akhter Joynab , Hanada Nobuhiro , Senpuku Hidenobu
Published: March 2003

The chemokine stromal‐cell derived factor‐1 (SDF‐1) controls maturation, trafficking, and homing of certain subsets, lymphoid cells including immunogenic B and T cells, as a ligand of the CXCR4 chemokine receptor. Insulin‐dependent diabetes mellitus (IDDM) and Sjögren's syndrome (SS), both highly regulated autoimmune diseases, develop spontaneously in non‐obese diabetic (NOD) mice. To investigate the role of SDF‐1 in the development of autoimmune diseases, we injected groups of NOD female mice with antibodies to SDF‐1 (anti‐SDF‐1), which resulted in a 30% reduction of diabetes up to 30 weeks of age, delayed average diabetes onset by 10 weeks, and suppressed insulitis. Autoimmune sialoadenitis was evident in anti‐SDF‐1‐injected mice (SDF‐1‐Ig group) at the same level as in all groups of mice, whether injected with non‐specific antibodies or not. In addition, in the SDF‐1‐Ig group, a greater number of immunoglobulin M (IgM)− IgD− B220low CD38+ CD43+ CD23− progenitor B cells and IgM+ IgD+ B220high CD43− CD38+ CD24+ CD23+ mature B cells remained in the bone marrow, whereas infiltration of mature IgM+ B cells was less extensive in peripheral tissues. Our results suggested that anti‐SDF‐1 antibodies injection was effective in inhibiting diabetes and insulitis without affecting autoimmune sialoadenitis or SS in NOD mice. SDF‐1 may be an essential chemokine for trafficking and migration of autoreactive B cells in the development of diabetes.
Cytotoxic T lymphocyte‐associated antigen‐4 inhibits integrin‐mediated stimulation
Authors: Gatta Lucia , Calviello Gabriella , Di Nicuolo Fiorella , Pace Luigia , Ubaldi Vanessa , Doria Gino , Pioli Claudio
Published: March 2003

The negative role exerted by cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4) in the regulation of T‐cell activity, as induced by T‐cell receptor (TCR)/CD3 and CD28 costimulation, has been widely described. In the present work we investigated the role of CTLA‐4 in the control of cell activation, as induced by costimulation of the adhesion molecule lymphocyte function‐associated antigen‐1 (LFA‐1) in murine CD4+ T cells. Results show that CTLA‐4 engagement inhibits interleukin‐2 (IL‐2) production, not only when induced by CD3/CD28 costimulation, but also when CD4+ T cells are costimulated by anti‐CD3 and anti‐LFA‐1 monoclonal antibodies (mAbs). LFA‐1 has been described to induce Ca2+ mobilization also in the absence of TCR engagement. Moreover, we found that CTLA‐4 engagement negatively affects Ca2+ mobilization and NF‐AT activation, as induced by LFA‐1 engagement alone. PLCγ1 phosphorylation was also dampened within minutes after CTLA‐4 engagement. Altogether these data indicate that through the control of signals induced by different receptors, CTLA‐4 could be a global attenuator of T‐cell activation.
Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs
Authors: Mayer Balázs , Zolnai Anna , Frenyó László V. , Jancsik Veronika , Szentirmay Zoltán , Hammarström Lennart , Kacskovics Imre
Published: March 2003

Maternal immunity is mediated exclusively by colostral immunoglobulins in ruminants. As the neonatal Fc receptor (FcRn) is suggested to be involved in the transport of immunoglobulin G (IgG) in the mammary gland, we cloned this receptor from sheep and analysed its expression in the mammary gland around the time of parturition and also in the small intestine from the newborn lamb. FcRn heavy‐chain mRNA was detected (by using in situ hybridization) exclusively in the acinar and ductal epithelial cells in mammary gland biopsies both before and after parturition. Immunohistochemistry revealed that the cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition. A remarkable difference was observed in the pattern after lambing, where the apical side of the cells was strongly stained. The presence of the FcRn in the acinar and ductal epithelial cells of the mammary gland, and the obvious change in distribution before and after parturition, indicate that the FcRn plays an important role in the transport of IgG during colostrum formation in ruminants. Immunohistochemical analysis detected a strong apical and a weak basal FcRn signal in the duodenal crypt cells of a neonatal lamb, which have been previously demonstrated to secrete IgG1 in newborn ruminants. The FcRn was not detected in the duodenal enterocytes, which absorb intact IgG from the colostrum in a non‐specific manner. These data suggest that FcRn is involved in IgG1 secretion in ruminant epithelial cells.
Insulin‐like growth factor‐1 activates Akt and Jun N‐terminal kinases (JNKs) in promoting the survival of T lymphocytes
Authors: Walsh Patrick T. , Smith Loraine M. , O'Connor Rosemary
Published: March 2003

Insulin‐like growth factor 1 receptor (IGF‐1R) expression is augmented on T cells upon ligation of CD28, and this promotes IGF‐1‐mediated protection from Fas‐induced cell death for up to 6 days. To determine the mechanism of action of IGF‐1R in T‐cell expansion, we investigated the signalling pathways activated by IGF‐1 in T cells and in Jurkat cells. We found that IGF‐1 transiently induces Akt, jun N‐terminal kinases (JNK), and c‐Jun phosphorylation in activated T cells, with JNK and c‐Jun phosphorylation occurring faster than Akt phosphorylation. To mimic IGF‐1R expression levels in CD28‐stimulated Jurkat cells these cells were stably transfected to over‐express the IGF‐1R. Jurkat/IGF‐1R cells exhibited enhanced constitutive Akt phosphorylation compared with mock‐transfected controls, but IGF‐1 induced transient phosphorylation of MKK4, JNKs, and c‐Jun. Inhibition of PI‐3 kinase activity and Akt phosphorylation with LY294002 totally suppressed IGF‐1‐mediated protection from Fas killing in activated T cells, but only partially suppressed IGF‐1‐mediated protection in Jurkat/IGF‐1R cells. However, either dicumarol in T cells or a dominant negative JNK1 (APF) in Jurkat/IGF‐1R cells greatly suppressed IGF‐1‐mediated protection from Fas killing. Together, these data demonstrate that IGF‐1‐mediated activation of JNKs and PI‐3 kinase contributes to normal T‐cell survival, whereas the JNK pathway may be more important in Jurkat leukaemia cells.
IT outsourcing in insurance services: risk, creative contracting and business advantage
Authors: Willcocks Leslie P. , Lacity Mary C.
Published: March 2003

Abstract. Risks in IT outsourcing have received little systematic academic attention, despite growing evidence of wide disappointment and difficulty as well as some successes in outsourcing IT. This paper explores the 1993–98 case history of Polaris, a software intermediary in the European insurance sector. Despite the considerable risks inherent in outsourcing business‐critical technology, Polaris was found to be achieving business advantage from such a strategy. This paper details 10 major risk areas in IT outsourcing distilled from previous research. It focuses on the sourcing, contractual and management choices made at Polaris. It assesses, against prior research findings, the degree to which these choices mitigated short‐ and long‐term risk, and whether learning points flow from the analysis for other IT outsourcing arrangements.
Behavioural effects of attitudes toward constraint in CASE: the impact of development task and project phase
Authors: Day Donald L.
Published: March 2003

This paper examines the effects of systems development task and phase upon individuals’ attitudes and behaviour while using computer‐aided systems engineering (CASE) tools. A previous empirical study of developers in several countries is revisited. Findings indicate that the mediating effects of task and phase upon the relationship among constraints, attitudes and behaviour are weak. They thereby support earlier research, which claims significance between attitudes and behaviour, by eliminating the potentially mediating effects of task and phase. Findings also indicate that CASE tool users associate implementation, integration and maintenance in terms of tasks and constraints, suggesting a reassessment of traditional life cycle models.