bluuebunny/crossref_metadata_2025 huggingface dataset:
Demonstration that platelet‐activating factor is capable of activating mast cells and inducing a chemotactic response
Authors: Nilsson G. , Metcalfe D. D. , Taub D. D.
Published: March 2003

Platelet‐activating factor (PAF) is generated in a variety of inflammatory conditions in which mast cells accumulate. However, little is known about the ability of PAF to influence mast cell function directly. In this study we examine the ability of PAF to activate mast cells and regulate mast cell chemotaxis. PAF was found to induce intracellular calcium mobilization and chemotactic responses in both murine and human mast cells. PAF induced transient increases in intracellular Ca2+ concentrations with a 50% effective dose of 1 n
m and induced significant migratory responses at PAF concentrations of 1 n
m to 1 µm in the human leukaemia mast cell line (HMC‐1). Using signal transduction inhibitors, both PAF‐induced calcium mobilization and migration of mast cells were shown to require activation of pertussis toxin‐sensitive G proteins. PAF‐induced calcium and chemotactic responses were cross‐desensitized by C5a. Together, these data demonstrate that PAF is capable of activating distinct signalling pathways in mast cells associated with calcium mobilization and cell migration; and that PAF may thus contribute to the regulation of mast cell responses and hyperplasia at sites of inflammation.
The effects of DNA containing CpG motif on dendritic cells
Authors: Behboudi S. , Chao D. , Klenerman P. , Austyn J.
Published: March 2003

Dendritic cells (DC) are specialized antigen‐presenting cells. DC can acquire and process antigens in the periphery before maturing and migrating to secondary lymphoid tissues where they present the antigens and deliver co‐stimulatory signals to T cells. We describe an immunostimulatory oligonucleotide containing a CpG motif that stimulated murine DC to up‐regulate co‐stimulatory molecules, induce T‐cell proliferative responses and secrete interleukin‐12 in vitro. Administration of this oligonucleotide, but not of a control oligonucleotide lacking this motif, to mice led to the disappearance of DC from the marginal zone and T‐cell areas of spleen, but not from heart or kidney. The same CpG did not cause maturation of monocyte‐derived human DC in vitro, but lipopolysaccharide‐treated monocyte‐derived DC showed enhanced functional activity and up‐regulated co‐stimulatory molecules.
Nuclear factor‐κB and caspases co‐operatively regulate the activation and apoptosis of human macrophages
Authors: Hida A. , Kawakami A. , Nakashima T. , Yamasaki S. , Sakai H. , Urayama S. , Ida H. , Nakamura H. , Migita K. , Kawabe Y. , Eguchi K.
Published: March 2003

Accumulating evidence suggests that macrophages function as major effector cells in the pathological process of various human diseases. We examined here the role of nuclear factor‐κB (NF‐κB) and caspases in the regulation of activation and apoptosis of macrophages. Activation of the human monoblastic leukaemia cell line, U937, by phorbol 12‐myristate 13‐acetate (PMA) increased the expression of CD14/CD86, and cytokine production. PMA stimulation also increased the expression of both pro‐caspase‐8 and pro‐caspase‐3 in U937, but not apoptosis or intracellular caspase‐3 activity. PMA also increased the expression of X‐chromosome‐linked inhibitor of apoptosis protein (XIAP) in U937, suggesting an inhibitory action for XIAP on the caspase cascade in PMA‐stimulated U937. Electrophoretic mobility shift assay (EMSA) showed a significant increase of nuclear NF‐κB activity in PMA‐stimulated U937. When a potent NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC), was added to U937 cell culture in the presence of PMA, apoptosis was triggered by activation of caspase‐3, which was induced by caspase‐8 activation. XIAP expression was markedly suppressed in PMA‐treated U937 in the presence of PDTC. The inhibitors of caspase‐8 and caspase‐3 mostly inhibited apoptosis of U937 treated with PMA in the presence of PDTC. Furthermore, a phenotype of U937 treated with PMA and PDTC in the presence of caspase inhibitor was almost identical to that of unstimulated U937. Our results suggest that the signalling pathways involved in the activation and apoptosis of human macrophages could be co‐operatively regulated by the use of NF‐κB and caspase inhibitors, thus enabling the control of macrophage function and number.
Co‐option of endocytic functions of cellular caveolae by pathogens
Authors: Shin J.‐S. , Abraham S. N.
Published: March 2003

It is increasingly becoming clear that various immune cells are infected by the very pathogens that they are supposed to attack. Although many mechanisms for microbial entry exist, it appears that a common route of entry shared by certain bacteria, viruses and parasites involves cellular lipid‐rich microdomains sometimes called caveolae. These cellular entities, which are characterized by their preferential accumulation of glycosylphosphatidylinositol (GPI)‐anchored molecules, cholesterol and various glycolipids, and a distinct protein (caveolin), are present in many effector cells of the immune system including neutrophils, macrophages, mast cells and dendritic cells. These structures have an innate capacity to endocytoze various ligands and traffic them to different intracellular sites and sometimes, back to the extracellular cell surface. Because caveolae do not typically fuse with lysosomes, the ligands borne by caveolar vesicles are essentially intact, which is in marked contrast to ligands endocytozed via the classical endosome–lysosome pathway. A number of microbes or their exotoxins co‐opt the unique features of caveolae to enter and traffic, without any apparent loss of viability and function, to different sites within immune and other host cells. In spite of their wide disparity in size and other structural attributes, we predict that a common feature among caveolae‐utilizing pathogens and toxins is that their cognate receptor(s) are localized within plasmalemmal caveolae of the host cell.
Isolation, cloning and functional characterization of porcine mannose‐binding lectin
Authors: Agah A. , C M. , Montalto  , Young K. , Stahl G. L.
Published: March 2003

Binding of mannose‐binding lectin (MBL), a C‐type lectin, and its associated serine proteases, MASP‐1 and MASP‐2, to cell surface carbohydrates activates the lectin complement pathway. As MBL plays an important role in innate immunity, it has been cloned and characterized in several species. While the pig may be used as a source of organs/tissues for xenotransplantation, little is known about its MBL, thus, we report the isolation of three monomeric forms of MBL from porcine serum. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Coomassie staining of reduced porcine MBL revealed the presence of three monomeric forms with approximate molecular masses of 30 000, 32 000 and 34 000. Protein sequencing identified these monomeric forms as one single protein, suggesting post‐translational modification. Western blot analysis demonstrated the cross‐reactivity of anti‐human MBL polyclonal antibody with porcine MBL. A full‐length porcine liver MBL cDNA was isolated and the predicted amino acid sequence exhibited 64·9% identity with human MBL and 50·2% and 56·7% identity with rat A and C MBL, respectively. Furthermore, Northern blot analysis demonstrated the presence of a single (∼1·4–1·6 kilobase pair) transcript in porcine liver. Addition of purified porcine MBL to MBL‐deficient human sera augmented N‐acetylglucosamine inhibitable C3 deposition to mannan‐coated plates in a dose‐dependent manner. Taken together, these data demonstrate that porcine and human MBL are highly conserved, sharing structural and functional characteristics.
Long‐term in vivo depletion of functional CD4+ T lymphocytes from calves requires both thymectomy and anti‐CD4 monoclonal antibody treatment
Authors: Valdez R. A. , McGuire T. C. , Brown W. C. , Davis W. C. , Knowles D. P.
Published: March 2003

In vivo depletion of lymphocyte subsets is a direct approach used for dissection of the mechanisms of protective immunity. Long‐term in vivo depletion of bovine T lymphocyte subpopulations with monoclonal antibody (mAb) treatment alone has been difficult to achieve. The objective of this study was to determine whether both thymectomy and anti‐CD4 mAb treatment would optimize long‐term in vivo depletion of functional bovine CD4+ T lymphocytes. Calves were thymectomized and treated with high doses of anti‐CD4 mAb (approximately 5 mg/kg) over 4 days followed by subsequent lower doses (approximately 0·3 mg/kg) administered twice weekly for an additional 7 weeks. Depletion of CD4+ T lymphocytes from blood, spleen and peripheral lymph nodes was significantly improved in thymectomized calves compared to thymus‐intact anti‐CD4 mAb‐treated calves. Significant differences in percentages of CD4+ T lymphocytes between thymectomized and thymus‐intact calves were sustained for the duration of the 8‐week study. Depletion of CD4+ T lymphocytes from thymectomized calves resulted in complete abrogation of lymphoproliferative responses to ovalbumin. In addition, thymectomized calves treated with anti‐CD4 mAb had significantly reduced immunoglobulin G1 and no detectable immunoglobulin G2 ovalbumin‐specific antibody responses compared to thymus‐intact anti‐CD4 mAb‐treated calves. The results of this study demonstrate that both thymectomy and treatment with anti‐CD4 mAb are required for long‐term in vivo depletion of functional bovine CD4+ T lymphocytes.
Cleavage of E2F‐1‐regulating proteins and activation of E2F‐1 during CD95‐induced death of thymocytes
Authors: Bi BaoYuan , Littlewood Nicola K. , Crispe I. Nicholas
Published: March 2003

The CD95 death receptor activates caspases that cleave a variety of intracellular substrates, including cell cycle control proteins. However, the significance of this cleavage for the induction of apoptosis is unclear. In this study, CD95‐induced cleavage of the G1/S checkpoint regulator proteins, retinoblastoma protein (pRb) and murine‐double‐minute‐2 (mdm‐2), was associated with an increased protein concentration of a key transcription factor, E2F‐1, which is regulated by both of them. Furthermore, DNA‐binding activity to E2F sites is increased. In thymocytes, CD95‐induced apoptosis was associated with increased E2F‐1 DNA‐binding activity, while thymocytes that lacked E2F‐1 were less susceptible to CD95‐induced apoptosis. We conclude that the G1/S checkpoint is an important target of CD95 signalling. CD95‐activated caspases cleave regulator proteins to increase E2F‐1 activity, and inappropriate activation of E2F‐1 is part of the mechanism of CD95‐induced apoptosis.
A carbohydrate neoepitope that is up‐regulated on human mononuclear leucocytes by neuraminidase treatment or by cellular activation
Authors: Quinn Mark T. , Swain Steve D. , Parkos Charles A. , Jutila Kathryn L. , Siemsen Daniel W. , Kurk Sandra L. , Jesaitis Algirdas J. , Jutila Mark A.
Published: March 2003

The expression of cell‐surface antigens can delineate specific leucocyte developmental or functional stages. For example, certain membrane glycoproteins are expressed selectively on leucocyte subsets only after activation. Leucocyte activation can also induce changes in carbohydrate epitopes expressed on surface antigens. In the present studies, we report on a novel monoclonal immunoglobulin M antibody (mAb 13.22) that recognizes a unique carbohydrate epitope expressed on human leucocyte membrane proteins. Characterization of mAb 13.22 specificity by immunoblotting showed that it recognized proteins of MW ∼95 000 and 150 000, including both CD18 and CD11b. The mAb 13.22 epitope was removed by N‐glycosidase F but not by endoglycosidase H or fucosidase, demonstrating that it is an N‐linked carbohydrate antigen. Interestingly, immunoblot staining was enhanced after neuraminidase treatment, suggesting that the antibody epitope might also be partially masked by sialic acid. In resting leucocytes, the mAb 13.22 antigen was expressed strongly on neutrophils, while dull staining was present on monocytes, and no lymphocyte staining was observed. In marked contrast, treatment of leucocytes with neuraminidase resulted in exposure of a mAb 13.22 neoepitope on a subset of lymphocytes (primarily T lymphocytes and natural killer cells) as well as up‐regulated staining more than 18‐fold on monocytes. Activation of lymphocytes in culture with phytohaemagglutinin or concanavalin A also unmasked the mAb 13.22 neoepitope on ∼37% of the CD45RO+ lymphocytes. Furthermore, analysis of leucocytes collected from the synovial fluid of patients with rheumatoid arthritis showed that ∼18% of the lymphocytes present expressed the mAb 13.22 neoepitope. Taken together, our results suggest that the mAb 13.22 carbohydrate neoepitope could represent a physiologically relevant marker that is up‐regulated on leucocyte subsets during the inflammatory response.
Redox imbalance and immune functions: opposite effects of oxidized low‐density lipoproteins and N‐acetylcysteine
Authors: Viora Marina , Quaranta Maria Giovanna , Straface Elisabetta , Vari′ Rosaria , Masella Roberta , Malorni Walter
Published: March 2003

This study investigates the in vitro effects of oxidized low‐density lipoproteins (ox‐LDL), ‘physiological’ pro‐oxidants, N‐acetylcysteine (NAC), a free radical scavenger and glutathione precursor, and their combination on human peripheral blood mononuclear cell functions. We found that treatment with ox‐LDL induced a significant down‐regulation of proliferative response to mitogens, antigens and interleukin‐2. Lipid extracts from ox‐LDL were able to reproduce the same effect as the lipoprotein. On the other hand, NAC exposure induced a significant up‐regulation of proliferative responses to all the stimuli used. Moreover, we showed that natural killer (NK) cell‐mediated cytotoxic activity was significantly down‐regulated by ox‐LDL while treatment with NAC induced a significant up‐regulation of NK‐cell activity. Finally, we found that ox‐LDL and NAC exerted opposite effects on the cytokine network, interfering both at the protein secretion level and the messenger RNA synthesis level. More importantly, when NAC was used in combination with ox‐LDL the proliferative responses, NK‐cell‐mediated cytotoxic activity and cytokine production were restored to values comparable to controls. These data indicate that ox‐LDL and NAC modulate immune functions, exerting opposite effects reflecting their pro‐oxidant and antioxidant behaviours. Our results add new insights to the key role played by redox imbalance as a modulator of immune system homeostasis and suggest that an antioxidant drug such as NAC could be useful against pathologies associated with an increase in lipid peroxidation.
Rapid changes in the lymphopoietic and granulopoietic compartments of the marrow caused by stress levels of corticosterone
Authors: Laakko Tonya , Fraker Pamela
Published: March 2003

Exposure to concentrations of glucocorticoids analogous to those produced during stress, trauma and malnutrition had rapid but varying effects on the major classes of cells within the marrow. Corticosterone (CS) was given as a subdermal implant in young mice and generated 60–95 µg CS/dl of blood compared to 5–15 µg CS/dl for sham controls over a period of 36 hr. Within 24 hr CS had caused losses of 30–70% among the early pro‐B, pre‐B and immature B cells. The pre‐B cells were virtually eliminated by 36 hr and the capacity of surviving pro‐ and pre‐B cells to cycle was reduced by 70–80%. Interestingly, the earliest of B cells, the prepro‐B cells, showed considerable resistance to CS, being reduced by only 20% at 36 hr. Thus, the pattern of survival within the B‐cell compartment paralleled the expression of Bcl‐2. At the 36‐hr time‐point there were no changes in the proportion of progenitor cells, erythroid or monocytic cells, or number of nucleated cells in the marrow. By contrast, 36 hr after exposure to CS there was an increase of 30% in the proportion and absolute number of cells in the granulocytic compartment. Chronic production of CS appears to reprogramme lymphopoiesis and myelopoiesis, perhaps to preserve the first line of immune defence at the expense of the lymphoid branch. Resistance to apoptosis and modifications in the activity of the glucocorticoid receptor and cytokines produced by stromal cells are postulated as targets for CS‐driven changes.
Expression of caveolin by bovine lymphocytes and antigen‐presenting cells
Authors: Harris James , Werling Dirk , Koss Michael , Monaghan Paul , Taylor Geraldine , Howard Chris J.
Published: March 2003

Caveolin is a generic term for a family of proteins that include caveolin‐1, ‐2 and ‐3. Although the distribution of these proteins varies between cells, caveolin‐1 and ‐2 are commonly found coating membrane invaginations known as caveolae. Studies on human and murine cells suggest that caveolin/caveolae can be found in neutrophils, macrophages and mast cells, in which they are involved in the uptake of pathogens, but not in lymphocyte cell lines. Expression of caveolin‐1, ‐2 and ‐3 in bovine immune cells was investigated using confocal microscopy and Western blotting. Staining for caveolin‐1 was evident in all peripheral blood mononuclear cells (PBMC) and in CD4+, CD8+ and CD21+ lymphocytes, monocytes, macrophages and monocyte‐derived dendritic cells (DC). In addition, the caveolin‐1 antibody detected a protein with a molecular weight of approximately 22 000 in all PBMC, macrophages and DC, as well as in bovine aortic endothelial (BAE)‐1 cells and human endothelial cells by Western blotting. In macrophages and DC, caveolin co‐localized with the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) and to a lesser extent with Golgi, but not with endoplasmic reticulum. Staining was not seen on the plasma membrane in any bovine immune cells, suggesting the absence of caveolae, while in BAE‐1 cells staining was predominantly on the cell membrane. Caveolin‐2 could not be detected in any bovine cells by confocal microscopy or Western blotting, while caveolin‐3 was detected in all bovine cells by Western blotting, but not by confocal microscopy. These data provide evidence for the presence of caveolin in bovine lymphocytes and antigen‐presenting cells.
Induction of thymocyte positive selection does not convey immediate resistance to negative selection
Authors: Hare Katherine J. , Pongracz Judit , Jenkinson Eric J. , Anderson Graham
Published: March 2003

The acquisition of functional competence represents a critical phase during intrathymic development of T cells. Thymocytes reaching this stage represent cells which have been positively selected on the basis of major histocompatibility complex reactivity, but which have also been purged of potentially autoreactive T‐cell receptor specificities by negative selection. While the developmental window in which thymocytes are subjected to positive selection is now well defined, the precise developmental timing of negative selection, in relation to positive selection events, is less clear. Moreover, the underlying mechanism allowing single‐positive thymocytes to respond to T‐cell receptor ligation by activation rather than death, remains controversial. Here we have analysed the developmental timing of negative selection in relation to positive selection, using measurement of thymocyte susceptibility to dendritic cell presentation of the superantigen staphylococcal enterotoxin B (SEB). We show that thymocytes which have received initial positive selection signals, namely CD4+ CD8+ CD69+ thymocytes, like their CD4+ CD8+ CD69− precursors, are susceptible to negative selection, indicating that induction of positive selection does not convey immediate resistance to negative selection. In contrast, newly generated CD4+ CD8− CD69+ cells are not only resistant to deletion by SEB, but respond to SEB‐mediated T‐cell receptor‐ligation by activation, indicating that the acquisition of functional competence occurs at the newly generated CD4+ CD8− CD69+ stage. Finally, by using direct retroviral infection of primary CD4+ CD8+ thymocytes, we also show that Notch‐1 activation in CD4+ CD8+ thymocytes does not correlate with, nor convey resistance to superantigen‐mediated negative selection. Thus, our data suggest that although Notch‐1 has been implicated in resistance to thymocyte apoptosis, the acquisition of resistance to negative selection occurs independently of Notch‐1 signalling.
Lung dendritic cells are primed by inhaled particulate antigens, and retain MHC class II/antigenic peptide complexes in hilar lymph nodes for a prolonged period of time
Authors: Vu Quynh , McCarthy Karin M. , McCormack Joanne M. , Schneeberger Eveline E.
Published: March 2003

Intratracheal (IT) administration of heat‐killed Listeria monocytogenes (HKL) results in an influx of macrophage and dendritic cell (DC) precursors into the lung interstitium. Low‐density, FcR+, interstitial lung cells isolated from rats instilled 24 hr before with HKL or vehicle alone, were > 90% Mar1+. After culturing with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) for 3 days, up to 24% of the loosely adherent cells were DC that stimulated allogeneic T‐cell proliferation in an mixed lymphocyte reaction (MLR) assay. After only an overnight incubation with GM‐CSF, however, the capacity of interstitial Mar1+ cells to stimulate HKL immune T‐cell proliferation without exogenous antigen was low. By contrast, when DC were isolated as major histocompatibility complex (MHC) class II+ cells from rat lungs at 1, 3, 7 and 14 days after HKL instillation and cultured overnight with GM‐CSF, their antigen presentation capacity without added exogenous antigen was robust, but declined over the 2‐week period. Interestingly, hilar lymph node DC maintained their HKL antigen‐presenting capacity for up to 2 weeks after instillation of HKL. Following IT administration of PKH‐26 labelled HKL, fluorescent or immunolabelled organisms were detected in OX62+ DC in airway epithelium, lung interstitium and hilar lymph nodes in situ and in MHC class II+ DC isolated from these sites. We conclude that newly immigrated Mar1+ lung DC precursors, while efficient in endocytosing particulate antigens, are incapable of eliciting a significant proliferative response from HKL‐sensitized T cells. By contrast, MHC class II+ DC isolated from lungs and incubated overnight with GM‐CSF induce vigorous antigen‐specific T‐cell proliferation. Antigen‐loaded lung DC in hilar lymph nodes maintain their antigen presentation capacity for up to 2 weeks.
Human monoclonal natural autoantibodies against the T‐cell receptor inhibit interleukin‐2 production in murine T cells
Authors: Robey Ian F. , Schluter Samuel F. , Akporiaye Emmanuel , Yocum David E. , Marchalonis John J.
Published: March 2003

Natural autoantibodies (NAAbs) specific for the T‐cell receptor (TCR) are present in all human sera, but individuals with rheumatoid arthritis (RA) generally produce higher titres of immunoglobulin M (IgM) isotype autoantibodies (AAbs) against Vβ TCR epitopes. To investigate possible correlations between the specificity of such AAbs and their role in immunomodulation, we generated seven B‐cell hetero‐hybridomas, secreting monoclonal IgM NAAbs, from the synovial tissue and peripheral blood of patients with RA. Here we report three anti‐TCR monoclonal autoantibodies (mAAbs) – OR2, OR5 and Syn 2H‐11 – with the ability to bind subsets of murine T cells, including the ovalbumin‐specific DO‐11.10 clone. These antibodies did not induce apoptosis in vitro, but prevented interleukin‐2 (IL‐2) production by antigen‐specific T cells. These findings suggest an immunomodulatory function for NAAbs to TCR V‐region epitopes and serve as the foundation for testing human anti‐TCR mAAbs in animal models with the eventual goal of using them as therapeutic agents in human disease.
HeLa cells cocultured with peripheral blood lymphocytes acquire an immuno‐inhibitory phenotype through up‐regulation of indoleamine 2,3‐dioxygenase activity
Authors: Logan Grant J. , Smyth Christine M. F. , Earl John W. , Zaikina Irina , Rowe Peter B. , Smythe Jason A. , Alexander Ian E.
Published: March 2003

The mechanisms by which tumour cells escape recognition by the immune system or subvert antitumour effector responses remain poorly understood. In the course of investigating the potential of costimulatory signals in anticancer immunotherapy strategies, we have observed that HeLa cells (a human cervical carcinoma cell line) cocultured with peripheral blood lymphocytes (PBL) acquire the capacity to inhibit PBL proliferation in response to interleukin‐2 (IL‐2). This immuno‐inhibitory phenotype was further shown to result from induction of the tryptophan‐catabolizing enzyme, indoleamine 2,3‐dioxygenase (IDO), by interferon‐γ (IFN‐γ) secreted from cocultured allo‐reactive PBL. This enzyme has recently been shown to be a critically important modulator of immunological responses, most notably through the capacity to protect allogeneic concepti from alloreactive maternal lymphocytes. While the cytostatic consequences of IDO activity in tumour cells has received attention, the data presented in this report support the hypothesis that IDO activity may also act to impair antitumour immune responses.
Modulation of human monocytes by Escherichia coli heat‐labile enterotoxin B‐subunit; altered cytokine production and its functional consequences
Authors: Turcanu Victor , Hirst Timothy R. , Williams Neil A.
Published: March 2003

In murine systems, the B subunit of Escherichia coli heat‐labile enterotoxin (EtxB) is a potent immunomodulator capable of suppressing Th1‐mediated autoimmune diseases. This results from its ability to bind cell surface receptors, principally GM1‐ganglioside, and as a consequence down‐regulate the pathological T helper type 1 (Th1) response. The capacity of EtxB to alter human T‐cell responses has not been investigated. Here we show that EtxB, but not the receptor non‐binding mutant EtxB (G33D), triggers the release of interleukin (IL)‐10, IL‐6 and tumour necrosis factor‐α (TNF‐α) by human monocytes. The production of IL‐8, transforming growth factor‐β (TGF‐β) or IL‐12 was not enhanced by EtxB. Indeed, EtxB was shown to inhibit IL‐12 secretion in monocytes stimulated with interferon‐γ (IFN‐γ) and lipopolysaccharide (LPS) by an IL‐10‐independent mechanism. When EtxB‐treated monocytes were used as antigen presenting cells in an allogeneic mixed lymphocyte reaction (MLR), IL‐10 and IFN‐γ production were increased in comparison to levels seen in cultures stimulated with untreated monocytes; proliferation was unaltered. This modulation of the T‐cell response was not only evident in the primary MLR triggered by EtxB‐treated monocytes, but also upon restimulation of the responding T cells with fresh untreated monocytes; indicating that presentation by EtxB‐treated monocytes leads to altered T‐cell differentiation. Sorting experiments showed that IL‐10 secreting T cells from the MLR cultures were strong suppressors of T‐cell proliferation following their addition into a fresh primary MLR.
The ability of antigen, but not interleukin‐2, to promote n‐butyrate‐induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin‐dependent kinase inhibitors
Authors: Jackson Stephanie K. , DeLoose Annick , Gilbert Kathleen M.
Published: March 2003

The ability of the cell cycle inhibitor n‐butyrate to induce T helper 1 (Th1) cell anergy is dependent upon its ability to block the cell cycle progression of activated Th1 cells in G1. Results reported here show that although both interleukin (IL)‐2 and antigen (Ag) push Th1 cells into G1 where they are blocked by n‐butyrate, only the Ag‐activated Th1 cells demonstrate functional anergy once the n‐butyrate has been removed from the culture. Because n‐butyrate‐induced Th1 cell anergy has been linked to increased expression of the cyclin‐dependent kinase inhibitors p21Cip1 and p27Kip1, mechanistic experiments focused on the role of these inhibitors. It was found that when Th1 cells were reincubated in Ag‐stimulated secondary cultures, the Th1 cells previously exposed to Ag and n‐butyrate (anergic Th1 cells) demonstrated a cumulative increase in p21Cip1 and p27Kip1 when compared with Th1 cells previously exposed to recombinant (r)IL‐2 and n‐butyrate (non‐anergic Th1 cells). p27Kip1 in the anergic Th1 cells from the secondary cultures was associated with cyclin‐dependent kinases (cdks). In contrast, p21Cip1 in the anergic Th1 cells, although present at high levels, did not associate significantly with cdks, suggesting that p21Cip1 may target some other protein in the anergic Th1 cells. Taken together, these findings suggest that Th1 cell exposure to Ag and n‐butyrate, rather than IL‐2 and n‐butyrate, is needed to induce the cumulative increase in p21Cip1 and p27Kip1 that is associated with the proliferative unresponsiveness in anergic Th1 cells. In addition, p21Cip1 may inhibit proliferation in the anergic Th1 cells by some mechanism other than suppression of cdks that is unique to the induction of Th1 cell anergy.
Long‐term kinetics of adult human antibody repertoires
Authors: Van Dijk‐Härd Iris , Lundkvist Inger
Published: March 2003

In healthy humans, antibody repertoires change during ontogeny and senescence. The dynamics of antibody repertoires among adults over a longer period of time in one and the same individual has, however, not been extensively studied. In this study we analysed peripheral blood samples from five healthy adults, taken over a period of 10 weeks and once 9 years later. A competitive, quantitative polymerase chain reaction (PCR) was developed to investigate short and long‐term variations in VH gene family repertoires. Serum antibody levels to common self and non‐self antigens were determined in samples taken at the same time‐points as the cell samples to analyse possible correlations between molecular and serological expression profiles. We found a high degree of stability in the VH gene family repertoire over time as well as between individuals with a Caucasian background. A specific change in the usage of primarily the VH3 and VH5 gene families was observed in one individual at one time‐point. The deviating pattern resembled the VH gene family utilization pattern observed in naturally activated B lymphocytes. The fluctuations in VH3 and VH5 gene family expression correlated with the presence of rheumatoid factor in serum. We discuss the possible influence of polyclonal, transient stimulation of B cells on VH gene repertoires, as measured in circulating B cells.
Loxosceles spider venom induces metalloproteinase mediated cleavage of MCP/CD46 and MHCI and induces protection against C‐mediated lysis
Authors: Van Den Berg Carmen W. , De Andrade Rute M. Gonçalves , Magnoli Fabio C. , Marchbank Kevin J. , Tambourgi Denise V.
Published: March 2003

We have recently shown that sphingomyelinase D toxins from the spider Loxosceles intermedia induce Complement (C) ‐dependent haemolysis of autologous erythrocytes by the induction of cleavage of cell‐surface glycophorins through activation of a membrane‐bound metalloproteinase. The aim of this study was to investigate the effects of these toxins on C‐regulator expression and the C‐resistance of nucleated cells. Cells were incubated with Loxosceles venom/toxins and the expression of C‐regulators was assessed by flow cytometry. A reduced expression of membrane co‐factor protein (MCP) was observed, while expression of decay‐accelerating factor (DAF) and CD59 was not affected. Analysis of other cell‐surface molecules showed a reduced expression of major histocompatibility complex I (MHCI). Western blotting showed that a truncated form of MCP was released into the supernatant. Release could be prevented by inhibitors of metalloproteinases of the adamalysin family but not by inhibitors specific for matrix metalloproteinases. Cleavage of MCP was induced close to or within the membrane as demonstrated by the cleavage of transmembrane chimeras of CD59 and MCP. Although the venom/toxins induced a release of MCP, the C‐susceptibility was decreased. The mechanism of this induction of resistance may involve a change in membrane fluidity induced by the sphingomyelinase activity of the toxin/venom and/or involvement of membrane‐bound proteases. The soluble forms of MCP found in tissues and body under pathological conditions like cancer and autoimmune diseases may be released by a similar mechanism. The identity of the metalloproteinase(s) activated by the spider venom and the role in pathology of Loxoscelism remains to be established.
Maternal cell traffic bounds for immune modulation: tracking maternal H‐2 alleles in spleens of baby mice by DNA fingerprinting
Authors: Wan Wenhan , Shimizu Shoji , Ikawa Hiromichi , Sugiyama Kiyosh , Yamaguchi Nobuo
Published: March 2003

We have previously reported that the immunization of pregnant mice with T‐dependent antigens successfully induced suppression of the antigen‐specific plaque‐forming cell (PFC) response to the relevant antigens in the offspring. This suppression was not caused by the administered antigens, the antibodies produced by the pregnant mother, or lactational transfer, but was dependent on the presence of the intact maternal T cells. It was major histocompatibility complex (MHC)‐restricted manner tolerance, which continued for at least one‐sixth of the murine life. Traditionally, the placenta acts as a natural barrier, not allowing the cells to pass through. However, the results presented strongly suggested that maternal T cells pass through the placenta and subsequently induce tolerance. In this present study, we attempted to substantiate the presence of maternal cells in the fetal circulation through the use of molecular techniques. We found that a highly polymorphic microsatellite sequence within the class II Eb gene of the H‐2 complex is useful for the molecular detection of various H‐2 alleles. DNA polymorphic analysis was used for tracking maternal H‐2 alleles in the spleens of baby mice. The main procedure involved polymerase chain reaction amplification and restriction fragment length polymorphism analysis of the DNA sequence encompassing the H‐2‐specific microsatellite from the genomic DNA of baby mice. The results indicated that maternal T cells of immunized pregnant mice cross the placenta into the fetus, eventually inducing antigen‐specific immunological tolerance in the offspring.
Human autologous mixed lymphocyte reaction as an in vitro model for autoreactivity to apoptotic antigens
Authors: Amel Kashipaz Mohammad R. , Huggins Mary L. , Powell Richard J. , Todd Ian
Published: March 2003

Recent studies have indicated that cells undergoing apoptosis are the source of autoantigens which drive autoimmune responses in systemic lupus erythematosus (SLE). It has been recognized for many years that in vitro stimulation of T cells with irradiated major histocompatibility complex (MHC) class II‐bearing autologous cells results in T‐cell proliferation with immunological specificity and memory, namely the autologous mixed lymphocyte reaction (AMLR). The nature of the major stimulants in the AMLR is still unclear. We investigated whether apoptotic fragments from irradiated cells act as antigenic stimulators for AMLR or nucleohistone‐primed T cells. T‐cell proliferation in the primary AMLR was significantly suppressed by the presence of a caspase inhibitor Z‐Val‐Ala‐Asp‐CH2F (Z‐VAD.fmk), indicating that apoptotic antigens released from irradiated autologous feeder cells act as stimulators of AMLR T cells. This inhibitory effect of Z‐VAD was not caused by toxic effects, because the T‐cell response to the mitogen phytohaemagglutinin (PHA) was not inhibited by Z‐VAD. A nucleohistone preparation was shown to contain antigens that are important in the AMLR, as culture with nucleohistone (but not with thyroglobulin or hen‐egg lysozyme) primed T cells to respond with secondary kinetics in a subsequent AMLR that was also suppressed by Z‐VAD. Our data provide evidence that the AMLR constitutes a model for the evaluation of cellular and molecular mechanisms that may be relevant to the pathogenesis of SLE and similar autoimmune diseases.
Elevation of oleate‐activated phospholipase D activity during thymic atrophy
Authors: Lee Youngkyun , Song Soo‐Mee , Park Heung Soon , Kim Sungyeol , Koh Eun‐Hee , Choi Myung Sun , Choi Myung‐Un
Published: March 2003

Various phospholipases are thought to be associated with the in vitro apoptosis of thymocytes. In the present study, the in vivo phospholipase D (PLD) activity of rat thymus was studied after whole‐body X‐irradiation or injection of dexamethasone (DEX). Using exogenous [14C]dipalmitoyl phosphatidylcholine (PC) as the substrate, an elevation of oleate‐activated PLD activity was observed during thymic atrophy. The activity increases were sevenfold at 48 hr after 5‐Gy irradiation and fourfold at 72 hr after injection of 5 mg/kg DEX. The elevation of PLD activity appeared to parallel extensive thymus shrinkage. An increased level of thymic phosphatidic acid (PA), the presumed physiological product of PLD action on PC, was also detected. By comparing the acyl chains of PA with those of other phospholipids, PA appeared to originate from PC. To assess the role of PLD during thymic atrophy, thymocytes and stromal cells were isolated. Although thymocytes themselves exhibited significant PLD activation, the major elevation in PLD activity (greater than fourfold) was found in isolated stromal cells. PLD was also activated during in vitro phagocytosis of apoptotic thymocytes by the macrophage‐like cell line P388D1. This in vitro phagocytosis was significantly inhibited by PLD action blockers, such as 2,3‐diphosphoglycerate and 1‐butanol. These observations strongly suggest that the alteration of oleate‐activated PLD activity is part of an in vivo event in the progression of thymic atrophy, including phagocytic clearance of apoptotic thymocytes.
Phagocytosis of bacille Calmette–Guérin‐infected necrotic macrophages induces a maturation phenotype and evokes antigen‐presentation functions in dendritic cells
Authors: Goldmann Oliver , Rohde Manfred , Medina Eva
Published: March 2003

The interaction of pathogens with dendritic cells (DCs) seems to play a critical role in the initiation of the immune response. Tissue damage and induction of an inflammatory reaction are events frequently associated with the progression of the infection. Although DCs are very efficient at phagocytosing pathogens, the capacity of these cells to uptake microbes from a necrotic environment has not yet been proven. Here we have investigated the ability of murine bone marrow‐derived DCs to maturate and acquire antigen‐presentation functions when cocultured with bacille Calmette–Guérin (BCG)‐infected necrotic macrophages. Immature DCs exhibited a prominent capacity to ingest necrotic material as demonstrated by flow cytometry analysis and confocal microscopy. Furthermore, after exposure to BCG‐infected necrotic macrophages, DCs underwent phenotypic changes, including the up‐regulation of maturation specific markers (major histocompatibility complex class II, CD40, CD80, and CD86) and the capacity to stimulate antigen‐specific CD4+ T cells with higher efficiency than when they were directly infected with a similar number of bacteria. Antigen presentation following phagocytosis of BCG‐infected necrotic macrophages was demonstrated by their ability to stimulate in vitro proliferation and interferon‐γ production of antigen‐specific CD4+ T cells. These results suggest that the functional changes occurring in DCs after interaction with a pathogen can be favoured when the encounter takes place in a necrotic environment and it may constitute an important mechanism for the amplification of class II‐restricted immune responses induced during infection.
Antigen‐specific dose‐dependent system for the study of an inheritable and reversible phenotype in mouse CD4+ T cells
Authors: Firpo Eduardo J. , Kong Raymond K. , Zhou Qinghong , Rudensky Alexander Y. , Roberts James M. , Franza B. Robert
Published: March 2003

The transgenic T‐cell receptor in mouse TEa CD4+ lymphocytes recognizes an endogenous peptide, Eα52‐68, presented in the context of the major histocompatibility complex class II molecule I‐Ab. In response to an optimal peptide concentration TEa cells enter the cell cycle and proliferate. However, a single exposure to high doses of the specific peptide diminished cell expansion upon subsequent restimulation. This hyporesponsive, or anergic, phenotype can still be detected after multiple restimulations indicating that the hyporesponsiveness persists despite cell division and it was inherited by daughter cells. Furthermore, we demonstrated that this hypoproliferative response is associated with high p27Kip1 and cyclin E protein levels, and reduced intracellular interleukin‐2 (IL‐2) expression. Addition of exogenous IL‐2 was required to reset p27Kip1 levels in the progeny derived from hyporesponsive TEa cells. Thus, we have established antigen dose‐dependent induction of a reversible, inheritable (i.e. epigenetic) phenotype and we have identified at least three components of the network of interactions: p27Kip1 cyclin E, and IL‐2 expression.
Mycobacterium bovis bacillus Calmette–Guérin‐infected dendritic cells potently activate autologous T cells via a B7 and interleukin‐12‐dependent mechanism
Authors: Cheadle Eleanor J. , Selby Peter J. , Jackson Andrew M.
Published: March 2003

Mycobacteria are potent adjuvants, can survive intracellularly and have been safely used for many years as vaccines against tuberculosis and leprosy. They are thus important potential vectors for recombinant vaccines. Many of their adjuvant properties are mediated following phagocytosis by dendritic cells (DC), which are in turn critical for priming naïve T cells. Although the maturation of DC in response to mycobacteria, such as Mycobacterium bovis bacillus Calmette–Guérin (BCG), is well described the subsequent responses of autologous T cells to mycobacterium‐infected DC remains uncharacterized. In our experiments DC infected with BCG expressed more co‐stimulatory molecules than tumour‐necrosis factor‐α (TNF‐α) ‐treated DC and stimulated more potent mixed leucocyte reactions. When autologous T cells were co‐cultured with BCG‐exposed DC they became highly activated, as determined by display of CD25, CD54 and CD71 on both CD4+ and CD8+ cells. In contrast, the response of T cells to TNF‐α‐matured DC was significantly less. Cytokine production from T cells cultured with BCG‐exposed DC was enhanced with elevated secretion of interleukin‐2 (IL‐2), IL‐10 and interferon‐γ (IFN‐γ) and was produced by both CD4+ and CD8+ lymphocytes as determined by intracellular staining. In particular, IFN‐γ secretion was increased from 50 pg/ml to 25 000 pg/ml and IL‐10 secretion increased from 20 pg/ml to 300 pg/ml in BCG‐exposed DC co‐cultures. Blocking antibodies to B7.1 and B7.2 or IL‐12 significantly reduced the secretion of IFN‐γ and reductions were also seen in the expression of CD25 and CD71 by CD4+ cells. These data demonstrate that mycobacterially infected DC are particularly potent activators of autologous T cells compared to TNF‐α‐exposed DC and that the resultant T cells are functionally superior.
Activation of macrophages and interference with CD4+ T‐cell stimulation by Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium
Authors: Zur Lage Susanne , Goethe Ralph , Darji Ayub , Valentin‐Weigand Peter , Weiss Siegfried
Published: March 2003

Mycobacterium avium subspecies paratuberculosis (M. ptb) and M. avium subspecies avium (M. avium) are closely related but exhibit significant differences in their interaction with the host immune system. The macrophage line, J774, was infected with M. ptb and M. avium and analysed for cytokine production and stimulatory capacity towards antigen‐specific CD4+ T cells. Under all conditions J774 cells were activated to produce proinflammatory cytokines. No influence on the expression of major histocompatibility complex (MHC) class II, intracellular adhesion molecule‐1 (ICAM‐1), B7.1, B7.2 or CD40 was found. However, the antigen‐specific stimulatory capacity of J774 cells for a CD4+ T‐cell line was significantly inhibited after infection with M. ptb, but not with M. avium. When a T‐cell hybridoma expressing a T‐cell receptor identical to that of the T‐cell line was used, this inhibition was not observed, suggesting that costimulation which is essential for the CD4+ T‐cell line is influenced by the pathogenic bacterium M. ptb.
Tumour‐induced polarization of tumour vaccine‐draining lymph node T cells to a type 1 cytokine profile predicts inherent strong immunogenicity of the tumour and correlates with therapeutic efficacy in adoptive transfer studies
Authors: Winter Hauke , Hu Hong‐Ming , Poehlein Christian H. , Huntzicker Erik , Osterholzer John J. , Bashy Jaffar , Lashley David , Lowe Bruce , Yamada Jane , Alvord Gregory , Urba Walter J. , Fox Bernard A.
Published: March 2003

Previously we have shown that vaccination with the poorly immunogenic B16BL6‐D5 melanoma (D5) elicits a dominant type 2 (T2) cytokine response that fails to protect the host from a subsequent tumour challenge. Here we investigated whether the inherent immunogenicity of a tumour can be correlated with its ability to bias the anti‐tumour cytokine response towards either a type 1 (T1) or a T2 profile. The immune response to six tumours of different inherent immunogenicity was assayed. By isolating l‐selectinlow T cells from tumour vaccine draining lymph nodes (TVDLN), it was possible to detect tumour‐specific cytokine responses from both immunogenic, poorly immunogenic and non‐immunogenic tumours. Immunogenic tumours (MCA‐304, MCA‐309, MPR‐4) induced a predominant tumour‐specific T1 cytokine response. In contrast, weakly (MCA‐310, MPR‐3) and poorly/non‐immunogenic tumours (MPR‐5, D5) sensitized T cells with a predominant tumour‐specific T2 cytokine response. A significant correlation (P < 0·025) between immunogenicity and the ratio of tumour‐specific interferon‐γ : interleukin‐4 (IL‐4) secretion by TVDLN T cells was identified. We then documented that non‐therapeutic T cells primed by the poorly immunogenic D5, recognized ‘tumour‐rejection’ antigens and that reprogramming their cytokine response, by in vitro culture with IL‐12 and anti‐IL‐4, to a T1 profile uncovered therapeutic efficacy. In contrast, TVDLN T cells primed by a therapeutic vaccine lose therapeutic efficacy when cultured with IL‐4. These results provide insights into the development of a protective anti‐tumour immune response and strengthen the hypothesis that a T1 cytokine response is critical for T‐cell‐mediated tumour regression.
End‐user computing and information systems methodologies
Authors: Taylor M. J. , Moynihan E. P. , Wood‐Harper A. T.
Published: March 2003

End‐user computing is a growing area within the information technology (IT) industry. The number and size of end‐user‐developed applications is steadily increasing, yet little attention is paid to the way in which such applications are developed, and their impact upon organizations. In this paper we examine the relationship between information systems methodologies and the end‐user‐computing environment. In particular, the potential benefits and disadvantages of the use of information systems methodologies for end‐user‐computing, and strategies by which such usage can be accomplished are discussed. This paper is partly the result of a research programme involving case studies in 34 UK organizations aimed at improving business systems.