bluuebunny/crossref_metadata_2025 huggingface dataset:
Limited involvement of interleukin‐6 in the pathogenesis of lethal septic shock as revealed by the effect of monoclonal antibodies against interleukin‐6 or its receptor in various murine models
Authors: Libert Claude , Vink Anne , Coulie Pierre , Brouckaert Peter , Everaerdt Bart , Van Snick Jacques , Fiers Walter
Published: March 2007

Several studies in human patients and in laboratory animals have revealed a correlation between serum interleukin (IL)‐6 levels and outcome in clinical sepsis and in related animal models, respectively. In the present study, two monoclonal antibodies were used to investigate the contribution of IL‐6 in the lethal action of tumor necrosis factor (TNF) and of lipopolysaccharide (LPS) in mice. We studied the potential protective properties of an anti‐murine (m) IL‐6 antibody and of an anti‐mIL‐6 receptor antibody. In controlled experiments, we observed that both monoclonal antibodies conferred a dose‐dependent protection to a lethal dose of mTNF. Detailed studies with the monoclonal antibodies indicate, however, that protection was no longer observed when the mTNF dose was slightly higher than the lethal dose. Likewise, the anti‐IL‐6 monoclonal antibody protected against injections of LPS at a lethal‐dose concentration, but here too failed to protect against higher doses of LPS. The anti‐IL‐6 monoclonal antibody was unable to protect against mTNF in mice sensitized by galactosamine, the corticoid receptor antagonist RU38486 or human (h) IL‐1β. Protection did not correlate with the serum concentrations of IL‐6. Finally, we demonstrate that hIL‐6 injection did not change the sensitivity of mice towards mTNF. We conclude that, although IL‐6 levels may be of value as a marker for the outcome in septic shock, this cytokine contributes only marginally in the pathogenesis leading to death. The small, but real, contribution of IL‐6 in some situations might be due to its ability to up‐regulate the level of TNF receptors.
Dissection of major histocompatibility complex (MHC) and T cell receptor contact residues in a Kb‐restricted ovalbumin peptide and an assessment of the predictive power of MHC‐binding motifs
Authors: Jameson Stephen C. , Bevan Michael J.
Published: March 2007

The effect of alanine substitution on the major histocompatibility complex (MHC) binding and T cell receptor recognition of the Kb‐restricted ovalbumin 257–264 peptide was investigated. Positions 3, 5 and 8 of the octamer were important for Kb binding, as predicted from the motifs found in Kb‐associated peptides, while mutations at positions 4, 6 and 7 affected cytotoxic T lymphocyte recognition. Substitutions at positions 1 and 2 had very minor effects on T cell recognition. In addition, we tested the capacity of sequence motifs to predict MHC binding by analysis of a series of peptides which all bear the minimal Kb motif. We found that possession of good motifs was not always sufficient to give strong MHC binding, indicating secondary effects of the residues flanking the „MHC anchor”︁ positions.
CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells
Authors: Lesley Jayne , Hyman Robert
Published: March 2007

The hyaluronic acid (HA)‐binding function of CD44 expressed on the cell surface of normal hematopoietic cells has been studied by assaying binding of fluoresceinated hyaluronic acid (FI‐HA) and adhesion to immobilized HA. As has been observed previously, normal hematopoietic cells from bone marrow and spleen do not constitutively bind HA. A CD44‐specific monoclonal antibody, IRAWB 14, which has been shown to rapidly induce HA binding in some CD44+ cell lines, was used to activate the HA‐binding function of CD44 in these normal cells. Only splenic T cells were activated by the IRAWB 14 antibody to bind FI‐HA. Upon activation, FI‐HA binding correlated with the level of CD44 expression.

Activation of HA binding allowed splenic T cells to adhere to HA immobilized on plastic and to an endothelial cell line in an HA‐dependent manner. BALB/c and AKR/J splenic T cells differ in their level of CD44 expression, and this correlated with differences in their ability to bind HA upon antibody activation. The minor subpopulation of MEL‐14− T cells were among the brightest Fl‐HA‐staining cells.

We propose, on the basis of these and other results, that there are three states of CD44 function with respect to HA binding: (a) a non‐activatable, resting state, which cannot be rapidly activated to bind HA, as seen in most hematopoietic cells; (b) an activatable state, which can be rapidly converted to HA‐binding function, in this case by the IRAWB 14 antibody, illustrated by T cells as shown here; and (c) a constitutively active state, which can bind HA without antibody activation, seen in some cell lines.
Factors modifying survival pathways of germinal center B cells. Glucocorticoids and transforming growth factor‐β, but not cyclosporin A or anti‐CD19, block surface immunoglobulin‐mediated rescue from apoptosis
Authors: Holder Michelle J. , Knox Kirstine , Gordon John
Published: March 2007

The tendency for germinal center (GC) B cells to enter apoptosis is suppressed on engaging antigen receptor with immobilized anti‐immunoglobulin; cross‐linking of surface CD40 by monoclonal antibodies provides an additional signal for rescuing GC cells from programmed death. These observations are believed to reflect events that, in vivo, would allow for the selection of centrocytes which have undergone somatic mutation on Ig V‐region genes to generate antigen receptor of high affinity. The purpose of the present study was to identify factors capable of modifying the survival pathways of GC cells. Transforming growth factor‐β, at an optimal concentration of 1 ng/ml, was found to inhibit surface immunoglobulin (sIg)‐mediated rescue of GC cells but had no influence on survival promoted through CD40. Both routes of rescue were blocked by the glucocorticoid prednisolone at pharmacological concentrations (ID50 = 10−7 M). Cyclosporin A, an antagonist of sIg‐mediated signaling in resting B cells, failed to block rescue of GC cells through either of the receptor‐activated pathways. Antibody to CD19‐which also suppresses the activation of resting B cells ‐ not only left GC cell rescue undiminished, but rather provided a modest survival signal of its own; interferon‐α behaved similarly while interferon‐γ failed to influence GC cell survival in either direction.
Environmental influence on T cell receptor α gene rearrangement and expression in vitro
Authors: Larché Mark , Rencher Samantha D. , Hurwitz Julia L.
Published: March 2007

Experiments were designed to test whether T cell progenitors are committed to particular T cell receptor (TcR) gene rearrangement and expression patterns (prior to rearrangement) or whether such patterns are molded by the thymic microenvironment in which T cells develop. To this end, day 14 fetal thymocytes were removed from their normal environment and grown in organ culture (FTOC) for 5 to 12 days. RNA was extracted from organ‐cultured cells, processed to cDNA, and TcR α sequences were amplified by the polymerase chain reaction for cloning and sequening. By the examination of N‐region additions and V‐gene usage, and by the comparison of resultant patterns with those of early vs. adult stages of T cell differentiation in vivo, it was demonstrated that thymocytes in FTOC did not maintain early patterns of gene rearrangement. The thymocyte patterns were most dissimilar from those of normal, early ontogeny when interleukin‐4 was added to FTOC. Taken together, results demonstrated the flexibility of T cell progenitors and that environment plays a critical role in the molding of TcR α rearrangement and expression patterns.
c‐myc‐induced natural killer cell sensitivity of human melanoma cells is reversed by HLA‐B27 transfection
Authors: Peltenburg Lucy T. C. , Steegenga Wilma T. , Margaretha Krüse K. , Schrier Peter I.
Published: March 2007

In human melanoma, activation of the c‐myc oncogene results in locus‐specific down‐modulation of the HLA‐B antigen expression. Moreover, overexpression of c‐myc induces an increase of natural killer (NK) sensitivity of the tumor cells. To show that this effect on susceptibility to NK cells is mediated by the downmodulation of the HLA‐B expression rather than by the activation of the oncogene, we supertransfected a c‐myc transfectant with the gene encoding the HLA‐B27 protein. The resulting supertransfectants with HLA‐B27 surface expression were all less sensitive to NK cells than their parental cell line and showed a level of resistance equal to the original melanoma cell line with low c‐myc expression. This indicates that the induction of NK sensitivity by c‐myc activation in human melanoma cells is mediated through down‐modulation of the HLA‐B expression. These data also imply differential effects of HLA‐A and HLA‐B molecules on lysis by NK cells, because the level of NK susceptibility can apparently be defined by the level of HLA‐B, irrespective of a substantial level of HLA‐A expression present in the tumor cells.
Immunoglobulin VH‐T cell receptor Cα fusion mRNA resulting from chromosome inversion include the T cell‐associated 5′ exon ET
Authors: Fisch Paul , Foroni Letizia , Forster Alan , Lavenir Isabelle , Mengle‐Gaw Laurel , Rabbitts Terence H.
Published: March 2007

A human T cell lymphoma has been described in which an inversion of chromosome 14 results in fusion of an immunoglobulin heavy chain VH with a T cell receptor Jα segment, potentially resulting in a chimeric protein with immunoglobulin VH region recognition plus T cell receptor effector functions. Examination of the mRNA species expressed from the IgT gene in this lymphoma shows a variety of forms but all IgT mRNA include the T cell‐specific exon, ET, previously located in the distal part of the VH locus. In such mRNA species, the normal leader exon of the Ig VH segment, which encodes most of the hydrophobic signal peptide, is replaced by the short ET exon encoding mainly non‐hydrophobic residues. Two forms of this mRNA exist which lack the Ig VH leader sequence and thus potentially yield non‐membrane proteins in the T cell lymphoma.
Human Golgi β‐galactoside α‐2,6‐sialyltransferase generates a group of sialylated B lymphocyte differentiation antigens
Authors: Keppler Oliver T. , Moldenhauer Gerhard , Oppenländer Monika , Schwartz‐Albiez Reinhard , Berger Eric G. , Funderud Steinar , Pawlita Michael
Published: March 2007

The role of the human β‐galactoside α‐2,6‐sialyltransferase (hu α‐2,6‐ST) in the generation of B cell surface antigens was investigated by selecting subclones of COS cells (monkey kidney epithelial cells) constitutively expressing a transfected cDNA which encodes the hu α‐2,6‐ST (COS α‐2,6‐ST cells). Expression of hu α‐2,6‐ST in COS cells was sufficient to generate sialylated cell surface epitopes on different glycosylated antigens recognized by monoclonal antibodies to CDw75, CD76, and the unclustered monoclonal antibodies HB‐4 and EBU‐65. These epitopes were sensitive to sialidase treatment and are likely to contain terminal α‐2,6‐linked sialic acid residues. A novel antiserum raised against bacterially expessed hu α‐2,6‐ST fusion protein was used to localize the sialyltransferase in two cell lines with high expression of either endogenous (B cell line JOK‐1) or recombinant (COS α‐2,6‐ST cells) hu α‐2,6‐ST. In both cell lines, the enzyme was detected only intracellularly in the juxtanuclear region and not on the cell surface. In contrast, CDw75, formerly proposed to be identical with an α‐2,6‐ectosialyltransferase, was strongly expressed on the cell surface. The different expression patterns show that neither the CDw75 antigen nor any of the other sialylated antigens analyzed is identical with the hu α‐2,6‐ST. Furthermore, the presence of a surface‐expressed α‐2,6‐ST appears unlikely in these cell lines. We propose that CDw75, CD76, HB‐4, and EBU‐65 represent a unique group of B cell differentiation antigens the production of which requires the enzymatic activity of α‐2,6‐ST.
Interleukin‐3‐treated non‐B, non‐T cells switch activated B cells to IgG1/IgE synthesis
Authors: Ledermann Fritz , Heusser Christoph , Schlienger Claude , Le Gros Graham
Published: March 2007

The switch of activated B cells to IgE synthesis is an interleukin (IL)‐3‐dependent process. It is currently thought that specific T cells activated by antigen presented in the context of class II major histocompatibility complex are the major source of IL‐4. Recently it has been demonstrated that a splenic non‐T non‐B cell population (termed NBNT) has the capacity to produce IL‐4 following IgE and IgG receptor cross‐linkage. In this study we demonstrate that IL‐4 producing NBNT cells can induce the switch of lipopolysaccharide‐activated B cells to the synthesis of IgG1 and IgE antibodies. Furthermore, it was found that not only IgE receptor cross‐linkage but IL‐3 was able to stimulate NBNT cells to produce IL‐4 and induce the switch of B cells to IgE synthesis. NBNT cells derived from the spleen and bone marrow of SCID mice were able to produce IL‐4 on exposure to IL‐3. This suggested that the ability of IL‐3 to stimulate IL‐4 production was not dependent on prior exposure of the NBNT cells to antibody complexes in vivo. Taken together these findings represent the first observation that enough IL‐4 is produced by NBNT cells to actually influence a B cell IgG/Ig response. The findings also clearly demonstrate that B cells do not need high concentrations of IL‐4 to be directed to switch to IgG1 and IgE synthesis.
Structural and functional epitopes of the human adhesion receptor CD58 (LFA‐3)
Authors: Dengler Thomas J. , Hoffmann Jörg C. , Knolle Percy , Albert‐Wolf Marion , Roux Matthias , Wallich Reinhard , Meuer Stefan C.
Published: March 2007

CD58 (LFA‐3), a heavily glycosylated protein of 40–70 kDa, is expressed on a broad range of hematopoietic and non‐hematopoietic cells. It serves as a physiological ligand of the CD2 receptor, present on T cells and natural killer cells, and plays, thus, an important role in lymphocyte adhesion and T cell activation through CD2. Whereas several epitopes and their respective function are known for CD2, a similarly detailed characterization of CD58 is still lacking. We raised a panel of novel murine monoclonal antibodies (mAb) against recombinant human CD58 and describe here the identification of six structurally and/or functionally distinct epitopes on the CD58 molecule. All epitopes were found to be present in equal numbers on a wide range of CD58+ cells, none of them being differentially up‐regulated following cell activation or malignant transformation. Two of these epitopes represent functionally relevant sites, involved in binding of CD58 to CD2 and T cell activation via CD2. One further epitope appears to be selectively involved in CD58‐mediated activation, whereas the other three displayed no functional effects. The new mAb allow for the first time the detection of CD58 in enzyme‐linked immunosorbent assays and immunofluorescence while bound to its receptor CD2 in human serum or on freshly isolated blood cells. Finally, one mAb was found to specifically cross‐react with T11TS, the equivalent of CD58 in sheep.
Classical transplantation tolerance in the adult: the interaction between myeloablation and immunosuppression
Authors: Leong Louise Y. W. , Qin Shixin , Cobbold Stephen P. , Waldmann Herman
Published: March 2007

Allogeneic bone marrow transplantation in the neonate is an effective way of inducing permanent tolerance to donor tissue. To do the same in the immunocompetent adult requires immunosuppression to counter host‐versus‐graft alloreactivity. Conditioning with monoclonal antibodies (mAb) to CD4 and CD8 has been sufficient where donor and recipient are mismatched at only multiple “minor” histocompatibility loci, or at major histocompatibility complex (MHC) class I plus “minor” loci, but not where the mismatch involves the entire MHC. Tolerance across the MHC barrier requires extra conditioning with agents that happen to be both immunosuppressive and myeloablative, so obscuring the assessment of which effect is important. By using dimethylmyleran as a selective “space”‐creating myeloablative agent, and CD4 plus CD8 mAb as sole immunosuppressive agents, we have been able to dissect the relative requirements for immunosuppression and myeloablation. We show here that transplantation tolerance could only be achieved when both types of agent were combined together so as to guarantee sufficient donor‐type hemopoietic chimerism. We argue that the donor marrow, given sufficient space, will engraft and provide a sustained source of tolerogen overriding any host resistance that antibodies cannot control.
Enhancement of CD3‐induced activation of human intestinal intraepithelial lymphocytes by stimulation of the β7‐containing integrin defined by HML‐1 monoclonal antibody
Authors: Sarnacki Sabine , Bè Bernadette , Buc Hélène , Le Deist Françoise , Cerf‐Bensussan Nadine
Published: March 2007

Intraepithelial lymphocytes (IEL) form a large population of T cells in close contact with the intestinal lumen and differ from lymphocytes in other lymphoid compartments by their predominant CD8+ phenotype and the strong expression of the recently characterized β7‐containing integrin defined by the monoclonal antibody (mAb) HML‐1. The aim of the present in vitro study was to investigate the possible role of the integrin defined by HML‐1 in the activation of human IEL via the CD3‐T cell receptor (TcR) pathway.

The proliferative response of IEL to optimal concentrations of immobilized OKT3 was found to be similar to that of peripheral blood lymphocytes enriched in CD8+ cells. When co‐immobilized with suboptimal concentrations of OKT3, antibodies directed against CD11a, CD29 and the β7‐containing integrin defined by HML‐1 exerted a strong synergistic effect on the proliferative response and on the expression of CD25 and CD71 antigens by human IEL. These data indicate that the CD3‐TcR pathway is functional in human IEL and contrast with previous observations suggesting that the CD3‐TcR pathway was difficult to elicit in human IEL. Furthermore, the present data show that the immune response of human IEL can be modulated via interactions between integrins expressed by IEL and their respective ligands in the mucosa and suggest that IEL's activation may depend on the level of expression of integrin ligands in the epithelium, particularly of the expression of the as yet unknown ligand for the IEL‐specific integrin defined by HML‐1.
Molecular analysis of anti‐idiotypic monoclonal antibodies in the HLA‐DR antigenic system
Authors: Armandola Elena A. , Mariani Sara M. , Zwickl Markus , Hardman Norman , Ferrone Soldano
Published: March 2007

The structural organization of anti‐idiotypic (id) antibodies has been investigated mostly in haptenic systems. No information is available about the structural characteristics of anti‐id antibodies in major histocompatibility complex (MHC) antigenic systems, although these data may contribute to our understanding of the molecular basis of their functional role in the immune response. Therefore, we have determined the nucleotide and derived amino acid sequence of the VH and VL regions of the anti‐id monoclonal antibodies (mAb) F5‐444, F5‐830, F5‐963, F5‐1126, F5‐1336 and F5‐1419, which had been elicited with the syngeneic anti‐HLA‐DR1, 4, w14, w8, 9 mAb AC1.59. The six anti‐id mAb are heterogenous in their VH and VL region gene usage. This structural heterogeneity is not correlated with their target specificity and with their ability to elicit anti‐HLA‐DR antibodies. The latter characteristic is markedly influenced by a limited number of amino acid substitutions, since mAb F5‐444, which induces anti‐HLA‐DR antibodies, differs only in two residues in complementarity‐determining regions and in five residues in framework regions from mAb F5‐1126, which does not induce anti‐HLA‐DR antibodies.

The heterogeneity in VH and VL region gene usage by the six anti‐id mAb in the HLA‐DR system is at variance with the restricted VH and VL region gene usage by syngeneic anti‐id mAb in several haptenic systems. Furthermore, at variance with haptenic systems, the primary structure of the D segments of the anti‐id mAb is not correlated with their ability to induce anti‐HLA‐DR antibodies. On the other hand, the frequency of D‐D fusion events underlying the derivation of the D segments of the six anti‐id mAb in the HLA‐DR system and their average length are similar to those found in anti‐id mAb in haptenic systems. In addition, like in the latter systems, somatic mutations appear to contribute to the generation of diversity of anti‐id mAb in the HLA‐DR system.
In vivo production of interleukin‐10 by malignant cells in AIDS lymphomas
Authors: Emilie Dominique , Touitou Robert , Raphael Martine , Peuchmaur Michel , Devergnee Odile , Rea Delphine , Coumbraras Jean , Crevon Marie‐Claude , Edelman Lena , Joab Irène , Galanaud Pierre
Published: March 2007

Expression of the interleukin (IL)‐10/BCRF1 gene was studied by in situ hybridization in tissue samples from acquired immunodeficiency syndrome (AIDS) lymphomas using a BCRF1 probe which also recognizes the human IL‐10 sequence. Hybridization was detected in 8 out of 15 lymphomas. In contrast, the IL‐10/BCRF1 gene expression was detected in only 1 out of 11 lymphomas from human immunodeficiency virus (HIV)‐seronegative patients (p = 0.05). In AIDS lymphomas, the number of cells labeled with a BCRF1‐specific probe was dramatically lower than that of cells labeled with the IL‐10/BCRF1 probe. Thus, the IL‐10 rather than the BCRF1 gene was expressed. Production of IL‐10 was associated with that of IL‐10 mRNA, as shown by immunodetection of the protein in numerous cells. In contrast, BCRF1‐producing cells were rarely detected. Both in situ hybridization and immunochemical experiments indicated that malignant cells were involved in this IL‐10 synthesis. IL‐10 production in AIDS lymphomas was associated with the presence of Epstein‐Barr virus (EBV) in lymphomatous cells (p = 0.02). As IL‐10 is a potent growth factor for human B lymphocytes, these results suggest that IL‐10 may stimulate the proliferation of malignant cells in an autocrine pathway in a number of AIDS lymphomas, and that EBV and HIV may synergistically trigger its production.
Expression of interleukin (IL)‐1β, IL‐6 and their respective receptors in the normal rat brain and after injury
Authors: Yan Hong Qu , Banos Miguel Alcaros , Herregodts Patrick , Hooghe Robert , Hooghe‐Peters Elisabeth L.
Published: March 2007

The expression of interleukin (IL)‐1β, IL‐6 and their respective receptors has been studied in the rat brain before and up to 24 h after injury. Messenger RNA transcripts of these four genes were detected by in situ hybridization (ISH) in different structures of the intact brain. The distribution was very similar for IL‐1β, IL‐6 and IL‐6 receptor (IL‐6R). The expression of IL‐1R was more widespread. Within hours after injury, an increased expression of IL‐1β, and thereafter of IL‐6 was documented. The expression of IL‐1R and IL‐6R was also increased. This expression was bilateral and not restricted to the injured area. Within 24 h, all ISH patterns had returned to normal. The molecular data were confirmed by protein data. Indeed, the distribution of IL‐6 (detected by immunocytochemistry) agreed with the ISH patterns for IL‐6. Furthermore, extracellular fluid was collected by microdialysis at the site of the lesion during 12 h and successive fractions were assayed for the presence of bioactive IL‐1 and IL‐6. Increases in IL‐1 and later in IL‐6 levels were detected. The rapid and concomitant increased expression of IL‐1β, IL‐6 and their receptors after injury stresses their possible early role in inflammatory mechanisms also in the brain, before any recruitment of inflammatory cells from remote nervous and not nervous areas.
Cloning and sequences of primate CD4 molecules: Diversity of the cellular receptor for simian immunodeficiency virus/human immunodeficiency virus
Authors: Fomsgaard Anders , Hirsch Vanessa M. , Johnson Philip R.
Published: March 2007

To study the interaction between the primate lentiviruses simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) and the CD4 receptor we have cloned and sequenced the CD4 molecule from six non‐human primate species: African green monkeys (three subspecies: sabeus, pytherethrus, aethiops), sooty mangabeys, patas monkeys, chimpanzees, rhesus macaques, and pig‐tail macaques. Molecular cDNA clones representing CD4 mRNA were generated from total RNA from peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR) amplification including reverse transcriptase in initial reactions followed by two rounds of nested amplifications. Primer sequences were selected from regions conserved among human and rodent CD4 genes. Alignments of deduced amino acid sequences revealed interesting findings. First, all of the primate CD4 molecules were about 90% identical to the human CD4 sequence except the chimpanzee (98%). Second, two macaques or two African green monkey subspecies were as distanly related as the human versus chimpanzee sequences. Third, relatedness of CD4 sequences could not be predicted on the basis of geographic origin (Asian vs. African). Finally, upon sequencing several clones from individual monkeys, a low degree of sequence variation (nucleotide substitutions, deletions, and insertions) was found within the same animal, and in case of sooty mangabeys two distict populations of CD4 molecules were present within three of four individuals. The distinguishing features involved eight amino acid changes, including a single lysine deletion relative to a primate consensus sequence in the first complementary‐determing region of V1J1. These two CD4 populations were present also at the genomic DNA level and may arrive from the two chromosomal alleles, suggesting the existence of distinct sooty mangabey subspecies. Overall, the V1J1 and to a lesser extent V2J2 were the most variable regions among the sequences examined. By construction and expression in mammalian cell lines of CD4 chimeras in which these regions of the human CD4 were replaced by those of the African green monkey and pig‐tail macaques, a higher molecular mass of the CD4 chimeras were obtained in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis suggesting that the additional N‐linked glycosylation sites present in these monkey CD4 are also used.
Selective and differential binding of interleukin (IL)‐1α, IL‐1β, IL‐2 and IL‐6 to glycosaminoglycans
Authors: Ramsden Lawrence , Rider Christopher C.
Published: March 2007

The binding of interleukin (IL)‐1α, IL‐1β, IL‐2 and IL‐6 to acidic polysaccharides was investigated by affinity chromatography of the recombinant, radioiodinated interleukins on columns of immobilized polysaccharide. Each interleukin showed selective binding retention. Overall heparin bound all four interleukins significantly, whereas chondroitin sulfate provided little retention. IL‐α and IL‐1β showed differential binding, with only the latter binding to hyaluronic acid. IL‐2 was virtually completely retained on fucoidan. Noniodinated recombinant IL‐2 bound similarly to fucoidan, and fucoidan was found to sequester IL‐2 activity in a bioassay employing IL‐2‐dependent CTLL cells. In all other cases tested, interleukin retention was partial, implying that interleukin binding sites are sparsely distributed along the polysaccharide chains. These findings suggest that during the immune response, interleukins will tend to be retained at sites of secretion by interaction with glycosaminoglycans in the extracellular matrix and on cell surfaces.
Anti‐tumor necrosis factor receptor and tumor necrosis factor agonist activity by an anti‐idiotypic antibody
Authors: Galloway Cynthia J. , Madanat Melanie S. , Sarr Timothy , Espevik Terje , Dumas Michael L. , Mitra George , Ranges Gerald E.
Published: March 2007

Tumor necrosis factor (TNF) is a cytokine which, among other properties, is a principle mediator of inflammation and septic shock. It acts upon target cells by binding to specific cell surface receptors. A10G10 is a murine monoclonal antibody which recognizes human TNF and neutralizes its activity. A rabbit polyclonal antibody directed at the antigen‐binding site of A10G10 was raised and affinity purified over an A10G10 column. The resultant anti‐idiotypic antibody recognized not only A10G10 but also both TNF receptors. It showed TNF agonist activity in two different TNF bioassays, and competed with several anti‐TNF receptor monoclonal antibodies and TNF itself for binding to cells. These results represent an example of a method for obtaining antibodies to a ligand‐specific receptor in the absence of the receptor itself.
In vivo priming of cytotoxic T lymphocyte responses in relation to in vitro up‐regulation of major histocompatibility complex class I molecules by short synthetic peptides
Authors: Zhou Xianzheng , Motal Ussama M. Abdel , Berg Louise , Jondal Mikael
Published: March 2007

Cytotoxic T lymphocytes (CTL) recognize target antigens as short peptides presented by major histocompatibility complex class I molecules (MHC‐I). Externally added peptides can sensitize target cells by binding directly to MHC‐I without any need for internal processing. Those which are similar in length to endogenously processed peptides are more potent in this respect than slightly longer peptides. Peptide MHC‐I interactions can also be reflected as upregulation of MHC‐I in vitro on certain cells. We have compared the capacity of Db, Kb‐ and Ld‐binding peptides, which are slightly different in length, to up‐regulate MHC‐I in vitro with their immunogenicity in vivo, in relation to generation of CTL responses. A clear correlation between these two different functions was found. We have also modified a 9‐mer Db‐binding peptide by adding cystein to the amino terminus and lysine to the amino‐ or carboxy terminus and studied the effects on MHC‐I up‐regulation and in vivo immunogenicity. Cystein and lysine contain reactive groups which are likely to influence the binding of modified peptides into the antigen‐binding groove of Db. These small modifications of the optimal 9‐mer peptide strongly influenced their functions but still there was a correlation between MHC‐I up‐regulation and CTL responses. Up‐regulation of MHC‐I in vitro may reflect a capacity of peptides to accumulate on the surface of particular antigen‐presenting cells in vivo.
Immunomodulation of streptococcal cell wall‐induced arthritis. Identification of inflammatory cells and regulatory T cell subsets by mercuric chloride and in vivo CD8 depletion
Authors: Van den Broek Maries F. , de Heer Emile , van Bruggen Mieke C. J. , de Roo Guido , Kleiverda Karin , Eulderink Frits , van den Berg Wim B.
Published: March 2007

Streptococcal cell wall (SCW)‐induced arthritis is a chronic, erosive polyarthritis which can be induced in susceptible Lewis rats by one intraperitoneal injection of a sterile, aqueous suspension of SCW. The chronic phase of the disease is dependent on T cells. Mercuric chloride is an immunomodulating agent, causing autoimmunity in BN rats, but an OX8+ cell‐mediated immunosuppression in Lewis rats. Therefore, we investigated the effect of mercuric chloride, whether or not combined with in vivo OX8 depletion, on SCW‐induced arthritis in Lewis rats. We show that (a) depletion of OX8+ cells leads to a more chronic arthritis with a more rapid onset, (b) treatment with mercuric chloride induces a rapidly developing disease which is not chronic, and (c) treatment with mercuric chloride and OX8+ cell depletion induces an arthritis with a very rapid onset and enhanced chronicity. Together with histological data this suggests an important role for OX8+ T cells in controlling both the acute and chronic phase of the disease. In addition, mercuric chloride seems to induce an early activation of T cells resulting in an enhanced onset of disease, which is controlled later by enhanced activation of OX8+ cells.
T lymphocytes mediate immunologic control of C3 gene expression
Authors: Goldman Margaret B. , Knovich Mary Ann , Goldman John N.
Published: March 2007

Immunologic control of C3 gene expression by tissue macrophages can be accomplished by treatment of spleen fragments with anti‐C3 antibody. We now demonstrate that suppression of C3 requires participation of T lymphocytes of both the CD4+ and CD8+ phenotypes. Pretreatment of splenic tissue with anti‐Thy‐1.2 monoclonal antibody blocks the ability of the anti‐C3 antibody to induce C3 suppression. Reduction in either the CD4+ or CD8+ subpopulations of T lymphocytes also abrogates C3 suppression demonstrating that both T cell subsets are required in addition to the inducing antibody. Artificially elevating intracellular levels of cAMP with cholera toxin can partially substitute for the effects mediated by T cells in this reaction. Therefore, normal expression of the C3 gene can be suppressed by a regulatory network that requires the presence of a specific inducing antibody and T lymphocytes of both the CD4+ and CD8+ subsets. This regulatory network has many similarities to regulatory networks that have been well documented in suppression of specific murine immunoglobulin allotypes.
Modulation of oral tolerance to ovalbumin by cholera toxin and its B subunit
Authors: Pierre Pascal , Denis Olivier , Bazin Hervé , Mbella Etondoh Mbongolo , Vaerman Jean‐Pierre
Published: March 2007

Oral administration to mice of ovalbumin (OVA), if given together with cholera toxin (CT) or its B subunit (CTB) prevented the hyporesponsiveness to OVA subsequently injected parenterally. Oral immunization with CT plus OVA or OVA plus CTB in fact primed the immune system, inducing a stronger response to a subsequent parenteral injection of OVA with complete Freund's adjuvant than in mice prefed only with OVA or with saline. Oral CT plus OVA also induced good serum IgG1 and IgA anti‐OVA responses, with slightly (not significant) decreased IgG2a and IgG2b responses. Our in vivo findings agree well with earlier in vitro data from others, including CT inhibition of the Th1 CD4+ T cell subset and with CT effect on B cells (induction of LPS‐stimulated IgM+ B cells to undergo increased switch differentiation to IgG1‐ and IgA‐secreting cells).
The role of natural antibodies and ABO (H) blood groups in transplantation of human lymphoid cells into mice
Authors: Huppes Wim , Paulonis John , Dijk Hans , Van Rooijen Nico , Van Bekkum Dirk W.
Published: March 2007

Recently, evidence was presented that natural antibodies (NAb) are a crucial barrier to human cellular engraftment in severely immunosuppressed normal mice (Eur. J. Immunol. 1992.22:197.). In this report we show that normal mouse serum contains low titers of NAb against human cells of blood groups type O (H) and B and high titers against human cells of blood group A. Accordingly, human peripheral blood leukocytes (PBL) of group O (H) and B donors could be grafted successfully into normal BCBA mice (H‐2h/k) following irradiation with high dose total body irradiation (TBI). PBL of blood group type A donors did not engraft in normal mice but could be transplanted without difficulty in B cell‐deficient CBA/N mice which lack NAb, after conditioning with high dose TBI. Treatment of lethally irradiated normal BCBA mice with cobra venom factor (COF), which eliminates the third factor of complement, and liposomes containing dichloromethylene diphosphonate (C12DMP), which eliminates macrophages, resulted in engraftment of human blood group type A PBL. This implies that the NAb barrier for discordant xenogeneic cell transplantation can be abrogated.

A method utilizing directly labeled probes and flow cytometry is described for the quantitation in mouse serum of NAb, reacting with human cells. Using sera of H‐2b/k mice we show that murine NAb react with human stem cells, granulocytes, lymphocytes and monocytes of blood group A and only weakly with similiar cells from blood group O (H) and B donors. Sera of H‐2b, H‐2d and H‐2k mice of different ages and microflora possess NAb against human erythrocytes of blood group type A and occasionally demonstrate weak titers against erythrocytes of blood groups B and O (H) and the Rhesus factor.
Cytokine regulation of the interleukin‐1 receptor antagonist protein in U937 cells
Authors: Berger Ann E. , Carter Donald B. , Hankey Susan O. , McEwan Robert N.
Published: March 2007

A naturally occurring receptor‐level antagonist of interleukin‐1 (IRAPorIL‐1 ra) has recently been cloned. To determine what stimuli might regulate this inhibitor, cytokines were tested for their effects on the steady‐state level of IRAP mRNA in phorbol ester‐differentiated U937 cells. The cytokines tested fell into one of three groups: (a) inducers: granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), IL‐4, (b) weak inducers (<2‐fold stimulation): [IL‐lα, IL‐Iγ, and transforming growth factor‐β (TGF‐γ)] and (c) cytokines with no effect: (IL‐2, platelet‐derived growth factor, acidic fibroblast grouth factor, basic fibroblast growth factor, epidermal growth factor, granulocyte colony‐stimulating factor, IL‐3, IL‐5, IL‐6, interferon‐y, multi‐colony stimulating factor, tumor necrosis factor ‐a and IRAP itself. One hundred U/ml of either GM‐CSF or IL‐4 was the dose inducing peak IRAP mRNA expression; that peak expression occurred 12 h after addition of cytokine. GM‐CSF induced a 34 ±15‐fold increase in IRAP mRNA, and IL‐4 induced a 15± 6‐fold increase. In the same RNA samples, GM‐CSF increased IL‐ip mRNA 5.9 ± 1.7‐fold, but IL‐4 decreased IL‐Iγ mRNA to half that of control levels (0.45 ± 0.17). Thus, a single stimulus (IL‐4) decreased the expression of an agonist (IL‐1) while it increased the expression of an antagonist (IRAP). When U937 cells were treated with both IL‐4 and GM‐CSF, the level of IRAP mRNA induced was additive, suggesting that the cytokines acted differently to increase IRAP mRNA levels. The level of IL‐1 mRNA in cells treated with both IL‐4 and GM‐CSF was intermediate. Dexamethasone and cycloheximide inhibited all mRNA increases and did not reverse IL‐4‐induced decreases in IL‐1 mRNA. These studies have identified two cytokines which induce IRAP in the monocytic cells studied, and have partially characterized the differential regulation of IL‐1 and its antagonist, IRAP.
CD3 antigen‐mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets
Authors: Robinson Anne T. , Miller Nigel , Alexander Denis R.
Published: March 2007

T lymphocytes may be separated into subsets according to their expression of CD45 isoforms. The CD45R0+ T cell subset has been reported to proliferate in response to recall antigen and to mitogenic mAb to a much greater extent than the CD45RA+ subset. This difference could be due to more efficient coupling of the T cell antigen receptor complex to mitogenic signaling pathways. To investigate this possibility, CD3 antigen‐induced calcium signals, diacylglycerol (DAG) production and protein kinase C (PKC) activation levels were compared in CD45RA+ and CD45R0+ human T lymphocyte subsets derived from peripheral blood. The mean CD3‐induced rise in intracellular calcium was 80% greater in CD45R0+ than in CD45RA+ cells. Basal DAG levels in CD45R0+ cells were found to be, on average, 60% higher than in CD45RA+ cells (p = 0.002), but the CD3‐induced production of DAG over background was not different in the two subsets (p = 0.4). Basal PKC activity, and CD3‐induced PKC activation levels over background, were found to be 50% and 140% higher, respectively, in CD45R0+ cells than in CD45RA+ cells (p = 0.015 and 0.023). The CD45R0+ subset contained a higher proportion of cells expressing activation markers, such as CD25, CD71 and major histocompatibility complex class II, when compared to the CD45RA+ subset. Our results suggest that the elevated basal DAG levels observed in the CD45R0+ subset may reflect the recent activation of these cells. Both the higher basal DAG and CD3‐induced elevation in intracellular calcium observed in the CD45R0+ cells may contribute to the greater PKC activation signals triggered by CD3 mAb in this subset. These findings elucidate the greater response of CD45R0+ T cells to mitogenic stimuli compared to CD45RA+ cells.
Recognition of shared melanoma antigen by HLA‐A2‐restricted cytolytic T cell clones derived from human tumor‐infiltrating lymphocytes
Authors: Viret Christophe , Davodeau Francois , Guilloux Yannick , Bignon Jean‐Denis , Semana Gilbert , Breathnach Richard , Jotereau Francine
Published: March 2007

Three melanoma‐specific cytotoxic T lymphocytes (CTL) clones were derived from the tumor‐infiltrating lymphocyte (TIL) of human melanoma M17, and were used to study the expression of immunogenic melanoma peptides on allogeneic tumors. Antibody inhibition studies showed that two of these TIL clones were restricted by an HLA‐A2 molecule which was identified as A2.1 by gene sequencing. The third CTL clone was not restricted by HLA‐A2, but by a B or C HLA antigen. HLA‐A2‐restricted CTL clones M17‐1 and M17‐2 lysed 5 and 12 out of 15 HLA‐A2+ allogeneic melanomas, respectively. Since they did not lyse autologous Epstein‐Barr virus B cells, HLA‐A2.1‐transfected P815 cells,13 HLA‐A2+ non‐melanoma tumor cell lines and 10 HLA‐A2− melanomas, these clones appeared specific for melanoma‐restricted epitopes presented by the HLA‐A2.1 molecule. We then tried to determine why a few HLA‐A2+ melanomas were refractory to TIL lysis. By using a combination of flow cytometry analysis, partial cloning and sequencing of their HLA‐A2 genes, we show that failure to lyse did not result from low expression or polymoprhism of the HLA‐A2 molecule, or from deficient expression of the adhesion molecules ICAM‐1 and LFA‐3 by these melanomas. Taken together, our data confirm at the clonal level the existence of shared melanoma antigens recognized by TIL in the HLA‐A2.1 context. They further show that individual peptides derived from these antigens are expressed by a large majority of HLA‐A2+ melanomas. Identification of such peptides appears crucial for the future of vaccination therapies.
A role for immunoglobulin D: interference with tolerance induction
Authors: Carsetti Rita , Köhler Georges , Lamers Marinus C.
Published: March 2007

We have studied the induction and maintenance of tolerance in the B lymphocyte compartment. Neonatal and adult transgenic mice which expressed either surface IgM (sIgM) or sIgM and sIgD anti‐2,4,6‐trinitrophenyl (TNP) were treated with soluble mono‐ and multivalent forms of TNP‐modified carriers. We compared the B cell compartment of mice treated with antigen and of littermates injected with phosphate‐buffered saline. Antigen‐mediated cross‐linking of membrane‐bound IgM (sIgM) caused deletion of B cells both in neonatal and adult mice with μ and χ transgenes. Deletion was the result of apoptosis. In mice that carried an additional δ transgene sIgD interfered with tolerance induction. The stage in which the cells were sensitive to deletion was characterized as a transitional stage between immature (sIgMdull, heat‐stable antigenbright, B220dull, sIgD−) and more mature (IgMbright, heat‐stable antigendull, B220bright, slgD−) B cells. Surviving cells were functional as measured by receptor‐mediated changes in the intracellular free Ca2+ concentration. We propose that when the immature B cells have reached the final stages of maturation IgM always transmits negative signals in the absence of T cell help. When B cells need to be screened against self reactivity IgM is the only antigen receptor expressed. The presence of sIgD protects resting B cells from deletion and allows them to initiate an effective immune response.
Macrophage colony‐stimulating factor gene transfer into tumor cells induces macrophage infiltration but not tumor suppression
Authors: Dorsch Marion , Hock Hanno , Kunzendorf Ulrich , Diamantstein Tibor , Blankenstein Thomas
Published: March 2007

In order to analyze the effect of a high local concentration of macrophage colony‐stimulating factor (M‐CSF; CSF‐1) on tumor growth, the plasmacytoma cell line J558L was transfected with the human M‐CSF gene and injected into syngeneic BALB/c mice. In contrast to the parental tumors, M‐CSF transfectants were heavily infiltrated by macrophages as evidenced by immunohistochemistry with antibodies to Mac‐1 and Mac‐3 and by isolation of the macrophages from the tumor. Nevertheless, tumor growth was only slightly affected by M‐CSF and M‐CSF‐producing cells grew as tumor in all cases. The growth retardation of M‐CSF‐producing cells varied depending on the experiment and seemed to be due to an indirect effect because the growth rate of the cells in vitro had not changed upon gene transfer. Attempts to activate the tumor‐infiltrating macrophages for tumor suppression by systemic application of interferon‐γ and/or lipopolysaccharide were not successful. Altogether, our results suggest that M‐CSF is a potent chemoattractant for macrophages in vivo but alone is not sufficient to activate these macrophages for tumoricidal activity.
CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny
Authors: Fung‐Leung Wai‐Ping , Wallace Valerie A. , Gray Dawn , Sha William C. , Pircher Hanspeter , Teh Hung‐Sia , Loh Dennis Y. , Mak Tak W.
Published: March 2007

During thymic development, immature thymocytes expressing major histocompatibility complex (MHC) class I‐restricted T cell receptors (TcR) differentiate into CD8+ T cells with cytolytic functions. To evaluate the role of CD8 in positive and negative selection during thymic ontogeny, mice rendered CD8‐null by gene targeting were bred with three lines of transgenic mice expressing unique MHC class I‐restricted TcR. In all three instances CD8 was required for positive selection of MHC class I‐restricted transgenic T cells. The efficiency of positive selection decreased in accordance with a reduced level of CD8 expression on thymocytes. Surprisingly, there was a differential requirement for CD8 expression in negative selection of MHC class I‐restricted thymocytes, depending on the antigen specificity of TcR. These observations show that CD8 is essential for positive selection but is differentially required for negative selection of MHC class I‐restricted T cells. Thus thymic selection, at least for negative selection, can occur in the absence of the CD8 accessory molecule.