bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased division of αβ TCR+ and γδ TCR+ intestinal intraepithelial lymphocytes after oral administration of cholera toxin
Authors: PENNEY L. , KILSHAW P. J. , MACDONALD T. T.
Published: November 2003

Cholera toxin (CT) or its subunits were given orally to mice and division of intestinal intraepithelial lymphocytes (IEL) in vivo measured by double immunofluorescence using 5‐bromo‐2′‐deoxyuridine (BRdU) and membrane αβ T‐cell receptors (TCR) or γδ TCR+ staining in frozen sections. Cholera toxin (10 μg) produced a two‐ to eightfold‐increase in the uptake of BRdU in αβ TCR+ IEL in the duodenum and a two‐ to fivefold increase in γδ TCR+ IEL in the ileum. Increased uptake of BRdU was also seen after a dose of 100 μg of CT, but this dose was also associated with the loss of αβ TCR+ IEL and γδ TCR+ IEL in the duodenum. CT‐A and CT‐B subunit produced increased BRdU incorporation by αβ TCR+ in the duodenum and by γδ TCR+ IEL in the ileum. Cholera toxin therefore appears to be mitogenic for IEL, probably due to an indirect mechanism.
Enhancement in antigen binding by a combination of synergy and antibody capture
Authors: Klonisch T. , Panayotou G. , Edwards P. , Jackson A. M. , Berger P. , Delves P. J. , Lund T. , Roitt I. M.
Published: November 2003

The effects of orientating pairs of synergistic monoclonal antibodies (mAb) on binding of human chorionic gonadotropin (hCG) was studied by radioimmunoassay (RIA), enzyme‐linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Antibody synergy towards hCG required two functionally intact antibodies located adjacent to each other and with different epitope specificities. We investigated whether immobilization procedures avoiding protein denaturation, increasing proper orientation and promoting higher molecular flexibility of the synergistic mAb resulted in significantly enhanced antigen binding. Synergistic mAb pairs captured through their Fc‐region by protein G or a polyclonal serum against the Fc‐part of mouse IgG could be used at 10‐fold lower coating concentrations to achieve maximal binding of the analyte as compared with the same mAb pairs coated directly onto polystyrene. The synergistic effect observed with protein A used as capture varied greatly with the subclasses of the two synergistic antibodies employed. Scatchard analysis revealed that the number of functionally synergistic antibody sites participating in the binding of hCG for one mAb pair was about 10 times higher for the protein G‐captured as compared with the directly coated synergistic pair. Biotinylated synergistic mAb pairs, coated directly or captured by streptavidin, did not display any enhanced antigen binding when tested in SPR or ELISA. With SPR, synergy was only observed when the synergistic mAb had been captured through their Fc‐region. Using protein G or a polyclonal rabbit anti‐IgG1 serum as capture reagents in SPR, synergistic triple mAb combinations against hCG were demonstrated.
Human cell‐mediated immune responses to antigenic fractions of Salmonella typhi
Authors: PÉREZ C. , CALDERÓN G. M. , XIMÉNEZ C. , MELENDRO E. I.
Published: November 2003

The proliferative responses of peripheral blood mononuclear cells of 10 subjects that had typhoid fever, and healthy volunteers without history of typhoid fever or immunization against disease, were analysed with antigen fractions from two protein extracts of Salmonella typhi. Fractions from each extract were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) electrophoresis, transferred to nitrocellulose filters by electroblotting and processed to obtain antigen‐bearing nitrocellulose particles for use in lymphocyte cultures. Although the individual proliferative responses were heterogeneous we identified two main immunogenic regions of 29–32 × 103 MW and 45–56 × 103 MW for both extracts. Even though there was no one particular antigenic fraction capable of stimulating lymphocytes from all individuals with a previous history of typhoid fever, the combination of three fractions 29–32, 41–45, 63–71 × 103 MW could be stimulatory for cells of 90% of these individuals. Also, four subjects that did not respond to unfractionated antigens gave proliferative responses to several fractions of the same extract. We have identified the main immunogenic fractions of S. typhi that might play a role during typhoid infection and postinfection immunity, and merit further purification and characterization.
B‐ and T‐cell autoantigens in pristane‐induced arthritis
Authors: BARKER R. N. , EASTERFIELD A. J. , ALLEN R. F. , WELLS A. D. , ELSON C. J. , THOMPSON S. J.
Published: November 2003

Pristane‐induced arthritis (PIA) is a murine disease resembling rheumatoid arthritis (RA) which is characterized by autoimmune responses to joint tissues. To identify the range of potential antigens targeted in PIA, proteins from arthritic or normal joint extracts were fractionated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and systematically screened for the ability to react with either serum IgG, or cultured splenic T cells, obtained from healthy or arthritic mice. Extracts from both normal and arthritic animals contained multiple proteins that were capable of reacting with murine serum IgG in immunoblotting experiments. In healthy controls, more bands were identified in extracts prepared from 30‐week‐old mice than from 8‐week‐old animals, but the widest range of proteins bound were derived from arthritic joints. Furthermore, the sera from PIA‐positive mice reacted with more bands from each of the extracts than did normal sera. Fractionated extracts prepared from healthy joints failed to stimulate the in vitro proliferation of splenic T cells from either normal or arthritic animals. When arthritic joint components were screened, T cells from healthy mice responded weakly to some fractions, but multiple fractions elicited strong proliferation by T cells from mice with PIA. A band of apparent molecular mass 60 000 was the protein most commonly bound by serum IgG from arthritic mice, and the corresponding fraction stimulated the highest responses by T cells from PIA‐positive animals. These results are consistent with the notion that the 60 000 MW mammalian heat‐shock protein is an important antigen in PIA, but that the autoimmune response diversifies with the development of arthritis to target multiple joint components.
Biosynthesis of human ficolin, an Escherichia coli‐binding protein, by monocytes: comparison with the synthesis of two macrophage‐specific proteins, C1q and the mannose receptor
Authors: LU J. , LE Y. , KON O. L. , CHAN J. , LEE S. H.
Published: November 2003

Ficolin is characterized by the presence of both collagen‐like and fibrinogen‐like sequences, and potentially has a similar overall structure as the complement protein C1q and the collectins. Previous studies have reported the presence of human ficolin mRNA predominantly in peripheral blood leucocytes. In the present study, the cellular origin of human ficolin was investigated in further detail. Preliminary studies using reverse transcriptase–polymerase chain reaction (RT–PCR) showed that ficolin mRNA was synthesized by U937 cells, a human monocyte cell line. This finding suggested that blood monocytes also normally synthesize human ficolin. Peripheral blood monocytes from adult human donors were harvested at serial time‐points (0–20 hr) after adhesion to tissue culture plates, and total RNA was isolated and assayed for ficolin mRNA by RT–PCR. Ficolin mRNA was highly expressed in monocytes throughout the first 20 hr of adhesion. In contrast, C1q and mannose receptor mRNA were not detectable during the first 8 hr of adhesion, but were highly expressed by 20 hr. Cells were harvested at longer time intervals (1, 2, 4, 6 and 8 days) to determine whether ficolin expression was temporally regulated at later stages of monocyte differentiation. Ficolin mRNA levels decreased sharply from day 1 to day 6. In contrast, the levels of both C1q and mannose receptor mRNA showed no changing trend. These results are consistent with the absence of ficolin expression in many macrophage‐rich tissues previously reported. The origin of ficolin from monocytes, together with its structural similarity to C1q and the collectins, raises the possibility that ficolin may be another plasma protein capable of binding to surface structures of micro‐organisms. Escherichia coli was therefore incubated with human serum, and bound proteins, after elution with sugars, were analysed by Western blotting using an antiserum raised against a synthetic ficolin peptide. The antiserum identified a polypeptide of approximately 42 000 MW, which is similar in size to that of ficolin as predicted from its cDNA‐derived sequence.
STEM CELL FACTOR POTENTIATES HISTAMINE SECRETION BY MULTIPLE MECHANISMS, BUT DOES NOT AFFECT tumour necrosis factor‐α RELEASE FROM RAT MAST CELLS
Authors: Lin T. J. , Bissonnette E. Y. , Hirsh A. , Befus A. D.
Published: November 2003

The effect of stem cell factor (SCF) on histamine and tumour necrosis factor‐α (TNF‐α) release from rat peritoneal mast cells (PMC) was determined and the intracellular pathways involved in the potentiation of histamine secretion were investigated. The effects of SCF (2–100 ng/ml) were examined following both short‐term (0 and 20 min) and long‐term (up to 24 hr) preincubations with SCF. Pretreatment of PMC with SCF for 0 min (concurrent) or 20 min did not induce histamine secretion directly, but significantly increased antigen (Ag)‐induced histamine secretion. SCF potentiated Ag‐induced intracellular Ca2+ increase and calcium ionophore A23187‐induced histamine secretion. Pertussis toxin (PT) inhibited SCF‐induced potentiation of IgE‐dependent histamine secretion, indicating that PT‐sensitive G‐proteins are involved in the immediate effects of SCF. In long‐term incubation experiments, SCF pretreatment for 18–24 hr significantly enhanced Ag‐induced histamine secretion, but did not affect Ag‐induced intracellular Ca2+ levels. The effects of long‐term incubation with SCF, but not the short‐term effects, were blocked by cycloheximide. Interestingly, spontaneous and Ag‐induced TNF‐α release from rat PMC were not affected by pretreatment with SCF (2–500 ng/ml) for 1 to 24 hr. Thus, through immediate and delayed mechanisms, SCF potentiates histamine release from PMC, but has no effect on TNF‐α release. The regulation of MC by SCF may be important in allergic and other inflammatory diseases.
COSTIMULATION REGULATES THE KINETICS OF interleukin‐2 RESPONSE TO BACTERIAL SUPERANTIGENS
Authors: MURAILLE E. , DEVOS S. , THIELEMANS K. , URBAIN J. , MOSER M. , LEO O.
Published: November 2003

The aim of this study was to investigate the mechanisms by which B7‐related costimulatory molecules (CD80, CD86) increase T‐cell responsiveness to extracellular ligands. As a model study, the in vitro response of purified splenic CD4+ T cells to a bacterial superantigen, SEB, was characterized. Previous analysis of this experimental model led us to conclude that expression of B7‐related molecules is strictly required in order to activate CD4+ T cells in the presence of bacterial superantigens. In the present report, we demonstrate that antigen‐presenting cell‐derived costimulatory signals regulate the kinetics of interleukin‐2 (IL‐2) production by SEB‐activated splenic CD4+ T cells. Indeed, experiments performed with purified subpopulations of antigen‐presenting cells and using B7‐transfected cell lines indicated that increased levels of CD80 and/or CD86 cell surface expression is associated with a faster kinetics of IL‐2 production in response to SEB. Accordingly, blocking of CD80 or CD86‐derived signals by specific monoclonal antibodies led to a slower kinetics of IL‐2 production in response to SEB. Thus these data demonstrate that similar strength of signal through the T‐cell receptor can lead to immune responses displaying distinct kinetics depending on the level of costimulatory ligands on APC.
Kinetics of interleukin‐6 production after experimental infection of mice with Schistosoma mansoni
Authors: KHALIL R. M. A. , HÜLTNER L. , MAILHAMMER R. , LUZ A. , MOELLER J. , MOHAMED A. A. , OMRAN S. , DÖRMER P.
Published: November 2003

It has been reported that interleukin‐6 (IL‐6) is expressed in cells of acute inflammatory granulomas experimentally induced in mice by eggs of Schistosoma mansoni. Moreover, in vitro IL‐6 was shown to enhance the cytotoxic activity of human platelets against larvae of S. mansoni. To elucidate further a proposed biological significance of this cytokine during the course of schistosomiasis, we studied the kinetics of IL‐6 production and concomitantly performed a histopathological analysis of the livers in BALB/c mice subcutaneously infected with S. mansoni cercariae. Over a period of 24 weeks postinfection (p.i.) we monitored serum IL‐6 levels, IL‐6 production in vitro by pokeweed mitogen (PWM)‐stimulated spleen cells as well as IL‐6 mRNA expression in livers, spleens and kidneys. We found significantly elevated IL‐6 levels in PWM‐stimulated spleen cell‐conditioned media (SCM) at weeks 6 to 20 p.i., peaking at week 10 p.i. In contrast, serum IL‐6 concentrations started to rise not before week 8 but remained significantly elevated above normal control values until week 24 p.i. The time pattern of enhanced IL‐6 mRNA expression detected in spleens and livers, but not in kidneys, as well as the rises of IL‐6 in SCM and with a delay of 2 weeks in serum samples correlated with the onset of the egg‐induced inflammatory reactions as well as the incidence and the number of the granulomas observed histopathologically in the livers of infected mice. Our data emphasize both a local and a systemic role of IL‐6 in the host immune response following infection of mice with S. mansoni.
Salivary IgG subclasses in individuals with and without homozygous IGHG gene deletions
Authors: Engström P.‐E. , Norhagen E. G. , Osipova L. , Helal A. , Wiebe V. , Brusco A. , Carbonara A. O. , Lefranc G. , Lefranc M.‐P.
Published: November 2003

In this study, the levels of salivary IgG1, IgG2, IgG3 and IgG4 from individuals with and without homozygous immunoglobulin heavy chain constant gene deletions were quantified by enzyme‐linked immunosorbent assay (ELISA). To analyse the restriction of salivary IgG subclasses, we used unstimulated whole saliva and sera collected at the same time from individuals with homozygous gene deletions, two with G1 deletion, one with G4 deletion, six with both G2 and G4 deletions and from eight individuals without IGHG gene deletions and expressing all four IgG subclasses. The median values of salivary IgG from individuals with homozygous G1, or G4, or both G2 and G4 deletions, and from individuals expressing all four subclasses were 24.2 mg/l and 23.4 mg/l, respectively. The median values of serum IgG were 13.7 g/l and 15.9 g/l, respectively. Our results show that the salivary and serum IgG levels were both within the normal range in individuals with homozygous gene deletions of either G1, or G4, or both G2 and G4.
Analysis of T‐cell hybridomas with an unusual MHC class II‐dependent ligand specificity
Authors: MENDIRATTA S. K. , SINGH N. , BAL V. , Rath S.
Published: November 2003

We have characterized two unusual T‐cell hybridomas, 1E3 and 3B8, from H‐2k mice immunized with I‐Ab‐transfected L cells (H‐2k), that are stimulated by L cells transfected with I‐Ab, I‐Ak or I‐Eb, but not by non‐transfected L cells. These hybridomas could not be stimulated by spleen cells from H‐2i3, H‐2k, H‐2b or H‐2d mice. Monoclonal anti‐I‐A antibodies did not block their responses, suggesting that mouse major histocompatibility complex (MHC) class II molecules may be peptide donors rather than restriction elements for them. The stimulation of these hybridomas by fibroblast targets was not blocked by an anti‐H‐2kk,Dk‐specific monoclonal antibody. Lipopolysaccharide (LPS)‐activated splenic and peritoneal exudate cells from H‐2k, H‐2d, H‐2i3, H‐2b as well as β2‐microglobulin‐deficient, TAP‐1‐deficient and I‐Aα‐deficient H‐2b mice stimulated these hybridomas. LPS could also activate a macrophage cell line, but not a B‐cell line, to become stimulatory for 1E3. A rat antiserum against untransfected L cells specifically and significantly blocked the response of 1E3. Thus, 1E3 may recognize a conserved murine MHC class II peptide loaded in a TAP‐1‐independent fashion on a non‐classical, monomorphic, β2‐microglobulin‐independent restriction element.
Fas‐induced programmed cell death is mediated by a Ras‐regulated O2− synthesis
Authors: GULBINS E. , BRENNER B. , SCHLOTTMANN K. , WELSCH J. , HEINLE H. , KOPPENHOEFER U. , LINDERKAMP O. , COGGESHALL K. M. , LANG F.
Published: November 2003

Fas induces apoptosis in lymphocytes via a poorly defined intracellular signalling cascade. Previously, we have demonstrated the involvement and significance of a signalling cascade from the Fas receptor via sphingomyelinases and ceramide to Ras in Fas‐induced apoptosis. Here we demonstrate rapid and transient synthesis of reactive oxygen intermediates (ROI) via activation of Ras after Fas. Genetic inhibition of Ras by transfection of transdominant inhibitory N17Ras blocked Fas‐mediated ROI synthesis and programmed cell death. Likewise, the antioxidants N‐acetyl‐cysteine and N‐t‐butyl‐phenylnitrone abolished Fas‐induced cell death, pointing to an important role for Ras‐triggered ROI synthesis in Fas‐mediated programmed cell death.
Inhibition of inducible nitric oxide synthase expression by interleukin‐4 and interleukin‐13 in human lung epithelial cells
Authors: BERKMAN N. , ROBICHAUD A. , ROBBINS R. A. , ROESEMS G. , HADDAD E. B. , BARNES P. J. , CHUNG K. F.
Published: November 2003

Nitric oxide produced by the inducible enzyme, nitric oxide synthase (iNOS), is implicated in immunological and inflammatory processes. We determined the effects of T‐helper (Th)2‐derived cytokines on the induction of iNOS from an epithelial A549 cell line and human airway epithelial cells stimulated by a mixture of interleukin‐1β (IL‐1β), interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α). Interleukin‐4 (IL‐4) and interleukin‐13 (IL‐13) but not interleukin‐10 (IL‐10) inhibited both iNOS mRNA expression and nitrite release in A549 cells. On human airway epithelial cells, IL‐4 and IL‐13 reduced iNOS mRNA expression. Dexamethasone also inhibited both iNOS expression and nitrite release. Th2 cytokines, IL‐4 and IL‐13, inhibit iNOS upregulation by Th1 cytokines, indicating an important reciprocal role of Th1 and Th2 T‐cell subsets on lung epithelial cells.
Comparison between the phenotype and function of maturing dendritic cells from spleen and lymph nodes
Authors: COATES J. P. , ROWLAND S. , HILL S. , IQBALL S. , BEDFORD P. A. , KIMBER I. , KNIGHT S. C.
Published: November 2003

We compared the capacity of mature dendritic cells (DC) from lymph nodes and maturing DC from spleens in their capacity to stimulate responses to the small hapten picryl sulphonic acid (PIC) and to the same hapten conjugated to ovalbumin (PIC–OVA) and requiring processing. Surface expression of major histocompatibility complex (MHC) class II molecules, which are up‐regulated during maturation of splenic DC, were studied as an independent marker of maturation. Freshly isolated lymph node DC had a veiled appearance and high levels of class II expression. DC separated from suspensions of spleen cells expressed the DC‐specific marker NLDC‐145, but were small, had low levels of MHC class II molecules and expressed stem cell antigen. Those DC from spleen cells cultured for 24 and 48 hr showed the development of typical veiled DC morphology and high class II expression. Lymph node DC stimulated high levels of primary T‐cell proliferation to PIC, but failed to stimulate primary responses to PIC–OVA. Splenic DC isolated immediately failed to stimulate primary responses to either antigen. More mature spleen DC stimulated responses both to PIC and PIC–OVA. Surprisingly, development of the capacity to stimulate responses to PIC preceded that of stimulating PIC–OVA responses. The capacity of the DC to process and present PIC–OVA was maintained during the culture period. The results indicate that both the form of the antigen and the source and maturity of the DC are critical in determining the responses stimulated in T lymphocytes.
Costimulatory signalling potential of murine MHC class II‐positive T‐clone cells
Authors: FROSCH S. , BONIFAS U. , ROß R. , SCHWING J. , YAGITA H. , GUO Y. , LIU Y. , RESKE‐KUNZ A. B.
Published: November 2003

Activated human and rat T cells as well as mouse T‐cell clones have been reported to synthesize and express major histocompatibility complex (MHC) class II molecules. However, the capacity of class II+ antigen (Ag) presenting T cells to induce proliferation of Ag‐specific cloned T cells has been controversial. We analysed whether the failure of some T‐cell clones to proliferate in response to Ag presented by class II+ T cells is because of a lack of costimulatory cytokine production by the antigen‐presenting cells (APC). As a model system the mouse class II+ cloned BI/O4.1 T cells were used as APC in order to activate the T cell clone KIII5. This T‐helper 1 (Th1) type, GAT (synthetic copolymer of L‐glutamic acid, L‐alanine and L‐tyrosine)‐specific clone is characterized by an efficient downregulation of interleukin‐2 receptor (IL‐2R) with time following antigenic stimulation. KIII5 cells respond to GAT‐presenting splenic antigen‐presenting cells (APC) by IL‐2 production, IL‐2R upregulation and proliferation. When BI/O4.1 T cells were used as APC, KIII5 cells produced IL‐2, but did not proliferate. Reverse transcriptase–polymerase chain reaction (RT–PCR) revealed a lack of IL‐12 production by BI/O4.1 cells. Addition of IL‐12 to a coculture of Ag‐presenting BI/O4.1 cells and KIII5 cells fully reconstituted a proliferative response. IL‐12 in synergy with IL‐2 upregulated IL‐2Rα chain expression and enhanced proliferation of KIII5 cells. Our data suggest, that class II+ T cells are not functional in inducing Ag‐mediated expansion of resting Th1 cells owing to their failure to produce IL‐12, but rather that they play a role in amplification loops during an ongoing immune response.
Lipopolysaccharide prevents apoptosis and induces responsiveness to antigen receptor cross‐linking in immature B cells
Authors: WECHSLER‐REYA R. J. , MONROE J. G.
Published: November 2003

Unlike mature B cells, immature B cells are not activated in response to antigen receptor cross‐linking. To examine the mechanisms underlying this unresponsiveness, we have studied the effects of reagents that have been shown to alter the responses of immature B cells to antigen receptor stimulation. Bacterial lipopolysaccharide (LPS) is a polyclonal B‐cell activator, and has been shown to interfere with B‐cell tolerance induction in vivo and in vitro. Here we show that LPS can also overcome the unresponsiveness of immature B cells to stimulation with anti‐receptor (anti‐μ) antibodies. LPS synergizes with anti‐μ to induce a proliferative response that exceeds the response of immature B cells to LPS alone. Moreover, pretreatment of immature cells with LPS allows them to proliferate in response to subsequent stimulation with anti‐μ antibodies. This induction of responsiveness to anti‐μ requires exposure to LPS for at least 8 hr. Although the mechanisms of induction are not fully understood, one component of the LPS effect appears to involve enhancement of immature B‐cell survival in culture. Neonatal splenic B cells undergo spontaneous apoptosis at a much higher rate than mature B cells, but we have found that LPS causes a dramatic inhibition of apoptosis, even when it is present for only the first 8 hr of culture. The ability of LPS to promote survival of immature B cells and allow them to proliferate in response to antigen receptor stimulation provides a system for investigation of the biochemical mechanisms of unresponsiveness and tolerance susceptibility.
Expression of VCAM‐1 and VLA‐4 dependent T‐lymphocyte adhesion to dermal fibroblasts stimulated with proinflammatory cytokines
Authors: GAO J.‐X. , ISSEKUTZ A. C.
Published: November 2003

In chronic inflammatory conditions, mononuclear cells infiltrate the involved connective tissue and may persist, perhaps because of binding to adhesion molecules on connective tissue cells, as well as extracellular matrix. Here we investigated whether vascular cell adhesion molecule‐1 (VCAM‐1) may be induced on human dermal fibroblasts by proinflammatory cytokines. Expression of VCAM‐1 was determined by Northern blotting, cell enzyme‐linked immunosorbent assay (ELISA) and flow cytometry. Only trace amounts of VCAM‐1 mRNA or protein were constitutively expressed on dermal fibroblasts but both were rapidly (within 4 hr) upregulated by tumour necrosis factor‐α (TNF‐α) and interleukin‐1α (IL‐1α) and somewhat slower (20 hr) by interferon‐γ (IFN‐γ). The combination of TNF‐α and IFN‐γ had at least an additive effect. The adhesion function of the expressed VCAM‐1 was examined by studying the adhesion of the Jurkat T lymphocytes to fibroblasts. Adhesion of Jurkat cells to dermal fibroblasts was markedly increased by stimulation of fibroblasts with TNF‐α and IFN‐γ (22% of cells adhered versus 9% on unstimulated fibroblasts). The increased adhesion was inhibited to that on unstimulated fibroblasts by monoclonal antibody (mAb) to domain 1 of VCAM‐1, but not by mAb to domain 4. MAb to very late antigen‐4 (VLA‐4), the integrin counter‐receptor on lymphocytes for VCAM‐1, completely inhibited the increase in Jurkat cell adhesion to activated fibroblasts and partly also inhibited basal adhesion to unstimulated fibroblasts. These results suggest that VCAM‐1 expression by dermal fibroblasts is inducible at the mRNA, protein and functional levels by proinflammatory cytokines. The VLA‐4/VCAM‐1 pathway maybe involved in adhesive interactions between T lymphocytes and activated fibroblasts in the skin during chronic dermal inflammatory conditions.
Opsonic human antibodies from an endemic population specific for a conserved epitope on the M protein of group A streptococci
Authors: BRANDT E. R. , HAYMAN W. A. , CURRIE B. , CARAPETIS J. , WOOD Y. , JACKSON D. C. , COOPER J. , MELROSE W. D. , SAUL A. J. , GOOD M. F.
Published: November 2003

This study demonstrates the presence of epitope‐specific opsonic human antibodies in a population living in an area endemic for group A streptococci (GAS) infection. Antibodies recognizing a conserved C‐terminal region epitope (p145, sequence in single letter amino acids: LRRDLDASREAKKQVEKALE) of the M protein of GAS were isolated from human patients by affinity chromatography and were shown to be of the immunoglobulin G1 (IgG1) and IgG3 subclasses. These antibodies could reduce the number of colonies of serotype 5 GAS in an in vitro opsonization assay by 71–92%, compared with an equal amount of IgG from control adult donors living in non‐endemic areas and without antibodies to p145. Addition of the peptide, p145, completely inhibited this opsonization. Indirect immunofluorescence showed that p145‐specific antibodies were capable of binding to the surface of M5 GAS whereas control IgG did not. Using chimeric peptides, which contain overlapping segments of p145, each 12 amino acids in length, inserted into a known helical peptide derived from the DNA binding protein of yeast, GCN4, we have been able to further define two minimal regions within p145, referred to as pJ2 and pJ7. These peptides, pJ2 and pJ7, were able to inhibit opsonization by p145 specific antibodies. Finally, we have observed an association between the age‐related development of immunity to GAS and the acquisition of antibodies to the conserved epitope, p145, raising the possibility of using this epitope as a target in a prophylactic vaccine administered during early childhood.
Complement regulatory protein expression by a human oligodendrocyte cell line: cytokine regulation and comparison with astrocytes
Authors: GASQUE P. , MORGAN B. P.
Published: November 2003

Rat oligodendrocytes spontaneously activate complement (C) and lack the C inhibitor CD59. As a consequence, rat oligodendrocytes are susceptible to lysis by autologous C in vitro. Expression of C inhibitors on human oligodendrocytes in vitro and other human glia has yet to be well characterized. We have previously shown expression at the mRNA level of the membrane inhibitors CD59, decay‐accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46) in human astrocytes. We here examine the expression of membrane and secreted C inhibitors by the oligodendrocyte cell line, HOG. HOG cells abundantly expressed CD59, assessed at protein and mRNA level, and expressed DAF and MCP, albeit at a lower level. Expression of all three inhibitors was enhanced by incubation with interferon‐γ or with phorbol ester (PMA). Complement receptor type 1 (CR1; CD35) was neither expressed constitutively nor induced by cytokines. HOG also constitutively secreted C1‐inhibitor, S‐protein and clusterin. Factor H was secreted only after stimulation with cytokines. C4b binding protein was expressed at a very low level and was detected only at the mRNA level by reverse transcriptase–polymerase chain reaction (RT–PCR). For comparison, astrocyte expression of CD59, DAF, MCP and CR1 was confirmed at the mRNA and protein levels. HOG did not activate C spontaneously, as judged by the lack of deposition of C fragments, and were not lysed by C even after inhibition of CD59 and DAF using specific monoclonal antibodies.
Molecular characterization of the V regions of four IgE antibodies specific for trichosanthin
Authors: WANG Y. , YEH M.
Published: November 2003

We report here the cDNA cloning and variable region nucleic acid sequences of the heavy and the light chains of four monoclonal IgE and one monoclonal IgG1 antibodies against trichosanthin (TCS), all of which recognized a similar or related antigenic determinant. Sequence analysis revealed that the VH regions of the four IgE antibodies use different V gene segments from the 7183, 36–60 and J558 families. The DH genes varied extensively in nucleotide sequence and length, with D segments DSP2, DFL16 and DQ52 being used with equal frequency. All JH gene segments except JH1 were present in the ε transcripts. In contrast, there appeared to be restricted L‐chain utilization. All four L‐chain V genes cloned belonged to the Vκ21 family and a bias in the use of Jκ1 gene was also observed. The VL sequences of the anti‐TCS monoclonal antibodies showed much less diversity than the VH sequences. We propose that the L‐chain rather than H‐chain may play a more significant role in specific binding with allergenic determinant on TCS.
α2‐macroglobulin stimulation of protein tyrosine phosphorylation in macrophages via the mannose receptor for Fcγ receptor‐mediated phagocytosis activation
Authors: MURAI M. , ARAMAKI Y. , TSUCHIYA S.
Published: November 2003

Macrophage phagocytic activity has previously been shown to be increased by binding of modified α2‐macroglobulin (α2M) with mannose residues at termini of sugar chains to mannose receptors on macrophages, with a subsequent increase in the number of Fcγ receptors at the cell surface. In the present study, an examination was made of the association of protein tyrosine kinase with the increase in number of Fcγ receptors following binding of modified α2M to mannose receptors. The phagocytosis of IgG‐opsonized sheep red blood cells through the action of the Fcγ receptor by modified α2M was inhibited by mannose and herbimycin A and slightly so by genistein. The mannose receptor would thus appear to be associated with tyrosine kinase activity. By Western blotting, tyrosine phosphorylated proteins with molecular weights of 32 000, 34 000, 36 000, 65 000, 85 000 and 110 000 appeared or increased upon treating macrophages with modified α2M. The degree of tyrosine phosphorylated proteins was the same for control macrophages following incubation in the presence of mannose and herbimycin A. Genistein treatment affected only tyrosine phosphorylated proteins of 65 000 and 110 000. The binding of modified α2M to mannose receptors was demonstrated by the inducement of tyrosine kinase activation that was sensitive to herbimycin A, followed by an increase in Fcγ receptors and consequently greater phagocytosis.
Saponin adjuvant primes for a dominant interleukin‐10 production to ovalbumin and to Trypanosoma cruzi antigen
Authors: TADOKORO C. E. , MACEDO M. S. , ABRAHAMSOHN I. A.
Published: November 2003

The adjuvant activity of saponin for T‐cell responses was evaluated and compared with that of complete Freund’s adjuvant (CFA) in two antigen systems: a lysate of the protozoa Trypanosoma cruzi and ovalbumin (OA). Strong delayed‐type hypersensitivity and T‐cell proliferate responses, comparable with those stimulated by CFA, were observed for both antigens following immunization with saponin as adjuvant. Upon in vitro secondary antigen stimulation, high interleukin‐10 (IL‐10) and low interferon‐γ (IFN‐γ) levels were observed in lymph node (LN) cell cultures from saponin‐immunized mice in contrast with the high IFN‐γ and decreased IL‐10 production by LN cells from CFA‐immunized mice. Production of IL‐10 and IFN‐γ in these conditions was CD4‐activation dependent. Concanavalin A (Con A)‐stimulated interleukin‐4 (IL‐4) production was higher in saponin‐immunized mice than in CFA‐immunized mice. IL‐10 produced by LN cells from saponin‐immunized mice suppressed IFN‐γ production and Con A‐induced proliferation. Taken together, these data are consistent with in vivo stimulation of both T‐helper (Th)1 and Th2‐type cells by immunization with saponin; in vitro a Th2‐type cytokine response with high IL‐10 production predominates, indicating preferential priming towards a Th2‐type response. Immunization with CFA induced a Th1‐type cytokine response. To our knowledge, this is the first report in which an adjuvant is shown to prime for a dominant IL‐10 production.
The influence of IL‐7 on V(D)J recombination
Authors: DO& uml;BBELING U.
Published: November 2003

The influence of interleukin‐7 (IL‐7) on V(D)J recombination was investigated directly in the V(D)J recombination competent pre‐B‐cell line 38B9. The addition of IL‐7 to the medium reduced the V(D)J recombination rate by 52–64%. This reduction was insensitive to the addition of cyclosporin A, indicating that the repression by IL‐7 is not mediated by phosphatase 2B. The repression mechanism of IL‐7 did not synergize with those of the protein kinase C activator 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and the intracellular Ca2+ mobilizer thapsigargin. The action of IL‐7 was blocked by the addition of the protein kinase A stimulator caffeine and the synthetic glucocorticoid dexamethasone. IL‐7 did not change the m‐RNA levels of the V(D)J recombination activating genes RAG‐1 and RAG‐2, therefore IL‐7 must exert its influence on V(D)J recombination either by post‐transcriptional regulation of the RAG genes or by the regulation of other genes that are involved in V(D)J recombination. Although IL‐7 may be necessary for the induction of and maintenance of V(D)J recombination during some stages of lymphocyte precursor development, it reduces the V(D)J recombination activity in pre‐B cells.
MUTZ‐3, a monocytic model cell line for interleukin‐4 and lipopolysaccharide studies
Authors: QUENTMEIER H. , DUSCHL A. , HU Z.‐B. , SCHNARR B. , ZABORSKI M. , DREXLER H. G.
Published: November 2003

The human monocytic cell lines MUTZ‐3 and MONO‐MAC‐6 express the lipopolysaccharide (LPS) receptor CD14. Paralleling the situation in peripheral blood monocytes (PBMo), recombinant human interleukin‐4 (IL‐4) down‐regulated the expression of CD14 on the cell surface of MUTZ‐3, but not that of MONO‐MAC‐6 cells. In addition, preincubation with IL‐4 prevented the LPS‐induced up‐regulation of IL‐1β mRNA levels in MUTZ‐3, but not in MONO‐MAC‐6 cells. We examined whether the differential responsiveness of the cell lines was due to the missing expression of the IL‐4 receptor (IL‐4R) α or γc chain in MONO‐MAC‐6 cells. Flow cytometric and immunoprecipitation analysis revealed expression of both IL‐4R chains in both cell lines. In addition, short‐term stimulation with IL‐4 induced tyrosine‐phosphorylation of the γc chain. As both cell lines also expressed signal transducer and activator of transcription 6 (STAT 6), our data suggested that the differential reaction patterns of MUTZ‐3 and MONO‐MAC‐6 cells were not due to a generally defective IL‐4R complex. Interestingly, long‐term (48 hr) treatment with LPS rendered MONO‐MAC‐6 cells sensitive to IL‐4. LPS up‐regulated expression of monocyte‐specific esterase (MSE) mRNA as well as CD14 protein in MONO‐MAC‐6 cells; both effects were inhibited by IL‐4. This stimulation was not paralleled by an increase of IL‐4R mRNA or protein expression supporting the above hypothesis of a constitutively present and active IL‐4R. We discuss possible causes for the differential reaction patterns of MUTZ‐3 and MONO‐MAC‐6 cells to IL‐4.
Cytokine production of neutrophils is limited to interleukin‐8
Authors: ALTSTAEDT J. , KIRCHNER H. , RINK L.
Published: November 2003

Since granulocytes are one of the first cell types at sites of inflammation, investigation of their capacity to produce cytokines has concentrated on interleukin‐1β (IL‐1β), IL‐6, IL‐8 and tumour necrosis factor‐α (TNF‐α). However, the results are subject to controversy. Incapability to produce cytokines as well as a broad panel of cytokines induced by isolation procedures are reported. The purpose of this study was to investigate the capacity of non‐prestimulated neutrophils to produce the above‐mentioned cytokines in response to stimulation with lipopolysaccharide or zymosan. In reverse transcription–polymerase chain reactions, we found IL‐8 mRNA directly after isolation in unstimulated cells, whereas mRNA for IL‐1β, IL‐6 and TNF‐α only appeared after stimulation. By means of flow cytometry we ruled out the possibility of prestimulation of the neutrophils during isolation, proving that IL‐8 mRNA is produced constitutively by neutrophils. In enzyme‐linked immunosorbent assays we found that, compared with controls, only IL‐8 was released at significantly higher levels after 24 hr of stimulation, giving a further indication that neutrophils have an immunoregulatory influence driven by IL‐8. We can confirm neither a constitutive nor a post‐stimulatory release of IL‐1β, IL‐6, or TNF‐α by neutrophils, as had been reported by others. These observations may be due to prestimulation, handling and culturing of the granulocytes, or to monocyte contamination. Collectively, our results show that granulocytes have a preformed capacity to produce the cytokine IL‐8 and that the production of proinflammatory cytokines by neutrophils is limited to IL‐8.
Lipids from Mycobacterium leprae cell wall are endowed with an anti‐inflammatory property and inhibit macrophage function in vivo
Authors: MOURA A. C. N. , MARIANO M.
Published: November 2003

In general, the majority of bacteria are pro‐inflammatory when injected in experimental animals. However, Mycobacterium leprae has no inflammatory effect when injected into mouse footpad, but using the delipidated mycobacteria we observed a mild significant increase in footpad oedema. Other mycobacteria, Mycobacterium bovis‐BCG or M. tuberculosis induce a strong paw oedema. Furthermore, M. leprae reduced locally the BCG‐induced inflammatory reaction in mouse footpad, whereas delipidated M. leprae did not influence this reaction. Both M. leprae and M. leprae cell wall lipids blocked immune phagocytosis in vivo by inflammatory macrophages (from an induced focus). In contrast delipidated M. leprae stimulated the phagocytosis reaction. Neither intact M. leprae, delipidated M. leprae, nor its lipids had any toxic effect on macrophages or on cell migration. Although M. leprae did not interfere on cell influx and cell type in an induced‐inflammatory site, this mycobacterium led to the appearance of a distinct cell population in vivo. The hypothesis is that M. leprae would transform macrophages in epithelioid cells, suggested by morphology analysis of cells by fluorescence‐activated cell sorter and observed under optic microscopy.
DIFFERENTIAL EXPRESSION OF FUNCTION‐RELATED ANTIGENS ON NEWBORN AND ADULT MONOCYTE SUBPOPULATIONS
Authors: MURPHY F. J. , REEN D. J.
Published: November 2003

Umbilical cord blood and adult peripheral blood monocytes were separated into two subpopulations, based on the intensity of CD14 expression, and the coexpression of various antigens associated with monocyte function was examined. The majority of cord and adult monocytes expressed CD14 at a high density (CD14bright) while approximately 15% of monocytes expressed this antigen at a lower level (CD14dim). Three times as many CD14dim monocytes expressed CD16 (FcγRIII) as did CD14bright monocytes in both cord and adult preparations, while its level of expression (mean fluorescence intensity) was significantly reduced on cord CD14dim monocytes relative to adult CD14dim monocytes. The percentage of cord blood CD14dim monocytes expressing human leucocyte antigen‐DR (major histocompatibility complex class II antigen) was significantly reduced relative to adult CD14dim cells; however, the level of expression of this antigen was greater on both cord and adult CD14dim monocytes compared with CD14bright cells. Cord CD14bright cells expressed CD36 (OKM5) to a greater extent than did their adult counterparts. The level of expression of CD62L was reduced on cord CD14dim cells compared with adult CD14dim cells. CD11b (CR3) was expressed both on a higher percentage of cells and also with a greater intensity on both cord and adult CD14bright monocytes compared with their CD14dim counterpart. These results show that significant differences exist between cord and adult monocyte subpopulations with regard to expression of various antigens associated with specific effector function.
Cross‐linking of CD26 by antibody induces tyrosine phosphorylation and activation of mitogen‐activated protein kinase
Authors: HEGEN M. , KAMEOKA J. , DONG R.‐P. , SCHLOSSMAN S. F. , MORIMOTO C.
Published: November 2003

CD26, a T‐cell activation antigen that has dipeptidyl peptidase IV activity in its extracellular domain and has also been shown to play an important role in T‐cell activation. The earliest biochemical events seen in stimulated T lymphocytes activated through the engagement of the T‐cell receptor (TCR) is the tyrosine phosphorylation of a panel of cellular proteins. In this study we demonstrate that antibody‐induced cross‐linking of CD26 in CD26‐transfected Jurkat cells induced tyrosine phosphorylation of several intracellular proteins with a similar pattern to that seen after TCR/CD3 stimulation. Herbimycin A, an inhibitor of the src family protein tyrosine kinases dramatically inhibited this CD26‐mediated effect on tyrosine phosphorylation. Major tyrosine phosphorylated proteins were identified by immunoblotting, and included p56lck, p59fyn, zeta associated protein‐tyrosine kinase of 70 000 MW (ZAP‐70), mitogen‐activated protein (MAP) kinase, c‐Cbl, and phospholipase Cγ. CD26‐induced tyrosine phosphorylation of MAP kinase correlated with increased MAP kinase activity. In addition, CD26 was costimulatory to CD3 signal transduction since co‐cross‐linking of CD26 and CD3 antigens induced prolonged and increased tyrosine phosphorylation in comparison with CD3 activation alone. We therefore conclude that CD26 is a true costimulatory entity that can up‐regulate the signal transducing properties of the TCR.
The beneficial effects of treatment with tamoxifen and anti‐oestradiol antibody on experimental systemic lupus erythematosus are associated with cytokine modulations
Authors: DAYAN M. , ZINGER H. , KALUSH F. , MOR G. , AMIR‐ZALTZMAN Y. , KOHEN F. , STHOEGER‡ Z. , MOZES E.
Published: November 2003

In an attempt to elucidate the role of oestrogens in systemic lupus erythematosus (SLE) we investigated the effects of treatment with an oestrogen antagonist – tamoxifen and a monoclonal anti‐oestradiol (anti‐E2) antibody on mice in which experimental systemic lupus erythematosus (SLE) was induced by a human monoclonal anti‐DNA antibody bearing the 16/6 idiotype (16/6 Id). Thus, groups of BALB/c female mice were immunized with the 16/6 Id and 3 weeks following the booster injection, when antibody titres were elevated in the injected mice, treatment protocols with anti‐oestradiol or tamoxifen were initiated. Control groups that were not immunized with the 16/6 Id but were similarly treated with the above agents were included in the study. The treatment with the above agents had no effect on the total autoantibody titres; however, a decrease in the immunoglobulin G (IgG)2a/IgG1 ratio of the anti‐DNA antibodies was determined in the 16/6 Id immunized and treated mice. Further, both the anti‐oestradiol and tamoxifen had beneficial effects on the clinical manifestations (white blood cell counts, levels of protein in the urine and immune complex deposits in the kidneys) of the 16/6 Id immunized and treated mice. We have previously observed a significant elevation in interleukin‐1 (IL‐1) and tumour necrosis factor‐α (TNF‐α) secretion in mice with experimental SLE and a reduction in IL‐2, IL‐4 and interferon‐γ (INF‐γ) levels as compared with the levels detected in healthy controls. Treatment with either the anti‐oestradiol antibody or with tamoxifen restored the levels of all the above cytokines to the normal levels observed in the control mice.These findings suggest that cytokine modulation may be the basis for the therapeutic effects of both anti‐oestrogens in experimental SLE.
CD44‐stimulated dendrite formation (‘spreading’) in activated B cells
Authors: SANTOS‐ARGUMEDO L. , KINCADE P. W. , PARTIDA‐SÁNCHEZ S. , PARKHOUSE R. M. E.
Published: November 2003

A rat monoclonal antibody (mAb) (NIM‐R8), insolubilized by binding to plastic plates, induced a rapid and extensive formation of dendrite processes (‘spreading’) in B lymphocytes activated by anti‐IgM and interleukin‐4 (IL‐4) or anti‐CD38 and IL‐4. In contrast, resting B cells were unable to spread similarly on the NIM‐R8‐coated plates. The NIM‐R8 antibody recognized a 90 000 MW surface glycoprotein (gp90) present on both B and T lymphocytes. The expression of this molecule was greatly increased after polyclonal (lipopolysaccarhide, anti‐IgM plus IL‐4 or concanavalin A) activation. The NIM‐R8 mAb with or without IL‐2 or IL‐4 was unable to induce proliferation of splenic lymphocytes. Following the demonstration that the NIM‐R8 mAb recognizes the murine equivalent of human CD44, the induction of spreading of activated B lymphocytes was studied using a panel of mAb recognizing different epitopes of murine CD44. All of these different mAb induced similar spreading of activated B cells. The ligand‐inducible spreading of activated B lymphocytes may be an important mechanism for providing an increased cell‐surface area for cell–cell or cell–matrix interactions, and thus may be an important factor controlling the response of activated lymphocytes.