bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential induction of Egr‐1 expression in WEHI‐231 sublines does not correlate with apoptosis
Authors: Gottschalk Alexander R. , Joseph Loren J. , Quintáns Jose
Published: March 2007

To determine whether the absence of inducible Egr‐1 expression correlates with apoptosis and growth arrest, we compared the inducible expression of two Egr family members (Egr‐1 and Egr‐2) in three sublines WEHI‐231. Expression of Egr‐2 is induced in all sublines of WEHI‐231 following surface immunoglobulin (sIg) cross‐linking, but Egr‐1 expression is induced in only two. We find that the lack of inducible Egr‐1 expression corresponded to an increase in the methylation pattern of the Egr‐1 gene. In spite of these differences in Egr‐1 expression, all the sublines demonstrate similar inhibition of [3H] thymidine incorporation following anti‐Ig treatment. Growth arrest leads to apoptosis in only two of the sublines, but apoptosis does not correlate with the absence of inducible Egr‐1 expression. Demethylation, by treatment with 5‐azacytidine, in the Egr‐1 non‐expressing subline allows for induction of Egr‐1 expression by anti‐Ig, but fails to prevent growth arrest and apoptosis. Therefore, we conclude that the lack of Egr‐1 expression is not responsible for either the apoptotic response or growth arrest induced by anti‐Ig in WEHI‐231.
Alloactivated cytotoxic T cells recognize the carboxy‐terminal domain of human immunodeficiency virus‐1 gp120 envelope glycoprotein
Authors: Clerici Magio , Shearer Gene , Hounsell Elizabeth F. , Jameson Brad , Habeshaw John , Dalgleish Angus G.
Published: March 2007

Infection with the human immunodeficiency virus (HIV) virus leads to clinical disease in humans but not in chimpanzees. Progression to disease is associated with activation of the immune system followed by loss of T helper cell function and a slow decline in CD4‐positive lymphocytes. The presence of autoreactive and cytotoxic cells in humans but not chimpanzees suggests that mechanisms other than, or in addition to, direct virus‐induced cell killing, are required for disease to develop. The observed changes are similar to those seen in chronic allogeneic disease. Here we show that a peptide from the carboxy terminus of gp120, predicted to have a structure similar to the major α‐helices of major histocompatibility complex (MHC) class I and class II, acts as a cytolytic target when presented on syngeneic cells for alloactivated cytotoxic T effector cells. These data add further evidence to the hypothesis that HIV can act as an allostimulant due to its dual properties of CD4 binding and MHC mimicry. The ability to signal nonspecifically through the T cell receptor could explain the initially paradoxical responses of proliferation, anergy and apoptosis.
Increased circulating interleukin‐6 (IL‐6) activity in endotoxin‐challenged mice pretreated with anti‐IL‐6 antibody is due to IL‐6 accumulated in antigen‐antibody complexes
Authors: Martens Erik , Dillen Chris , Put Willy , Heremans Hubertine , van Damme Jo , Billiau Alfons
Published: March 2007

Mice pretreated with monoclonal anti‐interleukin‐6 (IL‐6) antibody and then challenged with lipopolysaccharide (LPS), paradoxically develop higher levels of circulating biological IL‐6 activity, as measured by the hybridoma growth promotion assay, than mice similarly challenged but not pretreated with antibody. Here we provide evidence that this increased biological activity was entirely accounted for by the presence of increased amounts of IL‐6 protein, which could be isolated by immunoaffinity chromatography and subsequently visualized after gel electrophoresis. Chromatography on a protein G matrix and a sandwich ELISA allowed to demonstrate that all IL‐6 present in the serum was in the form of antigen‐antibody complexes. Serum samples of antibody‐treated animals which contained the highest biological activity typically contained near equimolar concentrations of IL‐6 and antibody. In vitro neutralization tests with pure antibody and IL‐6 demonstrated that, with both antibodies tested, more than 1000‐fold molar excess of antibody is needed for neutralization in the hybridoma growth assay. It is concluded that increased biological activity in serum of the anti‐IL‐6 antibody‐treated mice is due to sequestration of the endogenous IL‐6 in the form of antigen‐antibody complexes which, due to the lack of sufficient antibody excess, produce nearly full activity in the hybridoma growth assay.
Thermostability analysis of major histocompatibility complex class I molecules by temperature gradient gel electrophoresis
Authors: Tulp Abraham , Verwoerd Desirée J.
Published: March 2007

Empty major histocompatibility complex (MHC) class I molecules present on the surface of RMA‐S (26°C) cells were loaded with the iodinated peptides APGNYPAL, FAPGNYPAL (SEV‐9) and RGYVYQGL (VSV‐8), respectively. The thermostability of these peptide‐loaded MHC class I molecules was assessed using temperature gradient native polyacrylamide gel electrophoresis. A linear temperature gradient perpendicular to the direction of electrophoresis yielded a graphical representation of the melting of MHC class I molecules. The class I signal disappeared when the peptide melted out of the groove, and gave rise to a second signal due to released peptide. APGNYPAL‐loaded class I molecules melted at 11°C with considerable release even at 0°C. VSV‐8‐loaded class I molecules melted first at 36°C, whereas SEV‐9‐loaded molecules melted at about 22°C. A discrimination between the binding of SEV‐9 to Kb and Db molecules was seen in the melting patterns. Results are discussed in correlation with known crystallographic structures of class I molecules containing peptides in the binding groove.
Helper activity of CD4+ αβ T cells is required for the avian γδ T cell response
Authors: Arstila T. Petteri , Toivanen Paavo , Lassila Olli
Published: March 2007

We have studied the in vitro activation of chicken γδ T cells. Both splenic αβ and γδ T cells obtained from complete Freund's adjuvant‐primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4+ cells or αβ T cell receptor (TcR)‐positive cells were removed, both the proliferation and the blast formation of γδ T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)‐activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4‐ or αβ TcR‐depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the γδ T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti‐γδ TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti‐γδ TcR monoclonal antibody, a strong proliferation of γδ T cells was observed. In contrast, both Vβ1‐ and Vβ2‐expressing αβ T cells proliferated in vitro in response to stimulation with the relevant anti‐TcR monoclonal antibody alone. Depletion of either Vβ1+ or Vβ2+ T cell subset alone had no negative effect on the proliferation or blast formation of γδ T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4+ αβ T cells (both Vβl‐ and Vβ2‐expressing) play a role in the activation and response of chicken γδ T cells.
The novel subset of CD14+/CD16+ blood monocytes exhibits features of tissue macrophages
Authors: Ziegler‐Heitbrock H. W. Löms , Fingerle Günter , Ströbel Marion , Schraut Winfried , Stelter Felix , Schütt Christine , Passlick Bernward , Pforte Almuth
Published: March 2007

The CD14+/CD16+ cells account for about 10 % of all blood monocytes. They are characterized by a low level expression of the CD14 molecule and a high level expression of the CD16 (FcγR III) molecule. Polymerase chain reaction analysis of mRNA prevalence in CD14+/CD16+ cells (compared to the regular CD14+ + blood monocytes) demonstrates low levels of CD14 transcripts and high levels of CD16 transcripts, suggestive of a transcriptional control for both of these proteins. Analysis of additional cell surface molecules in three‐color immunofluorescence reveals that CD14+/CD16+ cells express the FcγR II in all, and FcγR I and ICAM‐1 in some donors. Furthermore, class II antigens are expressed at fourfold higher levels, while both, CD11b and CD33 cell surface proteins, are decreased by a factor of two. Transcript levels were reduced in CD14+/CD16+ cells for all three cell surface molecules. Since these phenotypic markers of the CD14+/CD16+ blood monocytes are reminiscent of tissue macrophages, we performed a comparative analysis with alveolar macrophages (AM). These cells are similar to the CD14+/CD16+ monocytes in that they show low levels of CD14 and strong expression of CD16. Furthermore, similar to the CD14+/CD16+ cells, the AM also exhibit higher levels of class II and lower levels of CD11b and CD33 when compared to the regular CD14++ blood monocytes.

In vitro induction of maturation of blood monocytes by 5 day culture of peripheral blood mononuclear cells in 10 % human serum will result in decreased CD14 and increased CD16 cell surface expression on the monocyte derived macrophages. At the same time, these cells acquire increased levels of class II and decreased levels of CD11b and CD33. Taken together, these data show that CD14+/CD16+ monocytes, while still in circulation, have acquired features in common with mature tissue macrophages.
CD23‐mediated homotypic cell adhesion: The role of proteolysis
Authors: Moulder Kevin , Barton Aisling , Weston Beverley
Published: March 2007

CD23 is a multifunctional molecule expressed by cells of lymphoid, myeloid and hematopoietic lineages. As a cell surface molecule CD23 acts both as a low‐affinity receptor for IgE (FcεRII) and as a cell adhesion molecule. CD23 can undergo autoproteolysis to release soluble 37–25‐kDa CD23 (s‐CD23) molecules with a range of cytokine activities. Here we show a causal link between the two apparently disparate functions of autoproteolysis and cell adhesion. The Epstein‐Barr virus‐transformed B cell line RPMI‐8866 formed macroscopic cell clusters solely via CD23. Cell adhesion was inhibited by mAb to CD23 and by IgE. Cell adhesion was also dependent on serum as cells grown in serum‐free media failed to form clusters. In serum‐free conditions cell adhesion could be induced by the addition of not only 10 % FCS but also s‐CD23. As s‐CD23 is reported to possess proteolytic activity we screened a range of proteases to determine whether they also could induce cell adhesion in serum‐free medium. It was found that chymotrypsin and elastase induced cell: cell adhesion in RPMI‐8866 cells. The same panel of proteases were screened against a range of CD23‐positive (Jijoye, AF‐10, T2, U937, ICH‐1) and CD23‐negative (RPMI‐8226, U266, MOLT‐4, Ramos) cell lines. It was found that chymotrypsin and elastase induce cell adhesion only in cells expressing CD23. Peptide mapping studies showed that chymotrypsin and elastase cleaved immunoprecipitated CD23 near the same site by which 37‐kDa S‐CD23 is released (Ala 80). Serum demonstrated no proteolytic activity towards CD23. However, it was found that cells grown in serum‐free medium released 25‐kDa S‐CD23 without the need for prior cleavage at the 37‐kDa cleavage site. To confirm the role of proteolysis in CD23‐mediated cell adhesion we screened a range of protease inhibitors for their ability to antagonize this process. It was found that tosyl‐lysine chloromethyl ketone inhibited CD23‐mediated cell adhesion. Lactoperoxidase treatment, which inhibits CD23 cleavage, also inhibited cell adhesion. Addition of chymotrypsin and elastase to lactoperoxidase‐treated cells induced cell adhesion. From these data we propose that intact CD23 has no demonstrable role in cell adhesion; instead, the portion of CD23 remaining on the cell surface following cleavage appears to mediate cell adhesion.
In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild‐type p53
Authors: Houbiers Jos G. A. , Nijman Hans W. , Van Der Burg Sjoerd H. , Drijfhout Jan Wouter , Kenemans Peter , Van De Velde Cornelis J. H. , Brand Anneke , Momburg Frank , Kast W. Martin , Melief Cornells J. M.
Published: March 2007

The central role of the p53 tumor suppressor gene product in oncogenesis is gradually being clarified. Point mutations in the p53 tumor suppressor gene are common in most human cancers and are often associated with p53 protein overexpression. Overexpressed wild‐type or mutant determinants of the p53 protein thus represent an attractive target for immunotherapy of cancer directed against a structure involved in malignant transformation. An important step towards this goal is identification of epitopes of p53 that can be recognized by human cytotoxic T lymphocytes. We identified peptides of (mutant) p53 capable of binding to HLA‐A2.1 in an in vitro assay. These HLA‐A2.1‐binding peptides were utilized for in vitro induction of primary cytotoxic T lymphocyte responses using a human processing‐defective cell line (174CEM.T2) as antigen‐presenting cell. These cells display “empty” HLA class I surface molecules, that can efficiently be loaded with a single peptide. We obtained CD8+ cytotoxic T lymphocyte clones capable of specifically lysing target cells loaded with wild‐type or tumor‐specific mutant p53 peptides. This strategy allows the in vitro initiation of human cytotoxic T lymphocyte responses against target molecules of choice.
Ontogeny of fetal CD8lo4lo thymocytes: Expression of CD44, CD25 and early expression of TcR α mRNA
Authors: Andjeli Sofija , Jain Nada , Nikolić‐Žugi Janko
Published: March 2007

CD8lo 4lo cells are the immediate precursors of immature CD8hi4lcTcRlo, CD8hi 4hiTcRlo and CD8hi4hiTcRlo double‐positive (DP) thymocytes in the adult murine thymus. These cells are the first subset in the adult thymus to express accessory CD8 and CD4 molecules, to rearrange the T cell receptor (TcR) a chain genes and to express the TcR αβ heterodimer at low levels at the surface. Here, we investigate the fetal ontogeny of CD8lo 4lo cells. We detect these cells on day 15 of fetal development. They dominate the thymus on day 15.5, to become progressively less prominent thereafter. An important characteristic of fetal CD8lo 4lo cells is the early expression of TcR α mRNA (on fetal day 15, 36–48 h earlier than reported previously). Our results also suggest, but do not prove, that the receptor may be expressed on the surface as early as day 15.5.

Fetal CD8lo 4lo cells, like their adult counterparts, become DP in vitro. However, early fetal CD8lo 4lo thymocytes express both CD44 and CD25 – unlike the adult subset ‐ and that links them to their putative precursors, fetal CD44+CD25+ double‐negative cells. This finding underscores the difference between adult and fetal thymocytes in turnover of membrane molecules and/or the kinetics of progression through phenotypic stages.
Release from a human monocyte‐like cell line of two different soluble forms of the lipopolysaccharide receptor, CD14
Authors: Labeta Mario O. , Durieux Jean‐Jacques , Fernandez Nelson , Herrmann Richard , Ferrara Pascual
Published: March 2007

Lipopolysaccharide (LPS) stimulates mononuclear phagocytes to synthesize and secrete immunoregulatory and inflammatory molecules such as interleukin‐1 (IL‐1), IL‐6, and tumor necrosis factor‐α (TNF‐α). LPS forms complexes with either the serum protein termed LPS‐binding protein or a serum factor, septin. These complexes are more stimulatory than LPS alone. The myeloid differentiation antigen CD14 is known to be the receptor for such complexes. In the present study, by using a monocytic cell line, we demonstrate the release of two different soluble forms of CD14 (sCD14) which are secreted by different mechanisms. We show that the two sCD14 forms differ in their electrophoretic mobility, two‐dimensional gel electrophoretic patterns, sensitivity to endoglycosidases and peptide maps. One of the sCD14 molecules, apparent molecular mass 48 kDa, was found in supernatants of both surface iodinated and [35S] methionine biosynthetically labeled cells. The other sCD14 molecule (56 kDa) was found labeled only in supernatants of [35S] methionine‐labeled cells. Furthermore, purified 48 kDa sCD14 enhanced the LPS‐induced TNF‐a and IL‐6 release by the monocytic cells suggesting that a cell‐surface signal transducer molecule may be involved in signaling. The data suggest a possible novel role for sCD14 in the monocyte response to LPS.
Somatic hypermutation in 5′ flanking regions of heavy chain antibody variable regions
Authors: Rothenfluh Harald S. , Taylor Linda , Bothwell Alfred L. M. , Both Gerald W. , Steele Edward J.
Published: March 2007

The aim of this study has been to determine the distribution of somatic mutations in the 5′ flanking regions of rearranged immunoglobulin heavy chain variable region genes (VDJ).We sequenced the 5′ flanking region in 12 secondary immune response antibodies produced in C57BL/6J mice against the hapten (4‐hydroxy‐3‐nitrophenyl)acetyl (NP) coupled to chicken‐Y‐globulin. In these and previously published sequences, almost 97 % of the mutations occurred in the transcribed region of the gene, and only a minority of genes (5/29) contained mutations upstream of the transcription start (cap) site. No potential germ‐line donor was found for a cluster of five base changes previously found in a single heavy chain gene, 3B62. However, the uniqueness of this mutational cluster and its distance from the normally mutated region suggests that the nucleotide changes may not be due to the normal mutator mechanism. Thus, as this was the only instance of somatic mutations that far upstream of the promoter/cap site region, the reverse transcriptase model for somatic hypermutation is still a possibility. The data are consistent with a mutational mechanism that requires transcription of the rearranged target V(D)J gene which appears to result in the generation of a positively skewed asymmetrical distribution of somatic mutations. A single mode is centered near the V(D)J and a long tail extends into the 3′ non‐translated region of the J‐C intron. Two classes of model could explain this mutation distribution pattern: those where transcription products (RNA, cDNA) are the direct mutational substrates, or those that postulate local unfolding of the chromatin around a V(D)J rearrangement directly exposing the DNA of the transcribed region to specific mutational enzymes.
The evolutionary origin of the major histocompatibility complex: Polymorphism of class II α chain genes in the cartilaginous fish
Authors: Kasahara Masanori , McKinney E. Churchill , Flajnik Martin F. , Ishibashi Teruo
Published: March 2007

T cells recognize antigen (Ag) in the form of peptides bound to the major histocompatibility complex (MHC) molecule. One of the important issues in evolutionary immunology is to identify the stage in phylogeny when this mode of Ag recognition emerged. At present, there is a considerable controversy as to whether the cartilaginous fish have the bona fide MHC. In our previous study, we showed that the nurse shark, a member of the cartilaginous fish, has (a) gene(s) capable of encoding MHC class II a chains. In the present study, we examined the polymorphism of nurse shark MHC class II a chain genes designated Gici‐DAA and Gici‐DBA using the polymerase chain reaction. The Gici‐DAA and Gici‐DBA genes had six and five alleles, respectively, and individual alleles usually differed by multiple nucleotides. In addition, most of the nucleotide substitutions were located at the putative Ag‐binding sites, where non‐synonymous substitutions occurred more frequently than synonymous substitutions. The fact that the Gici‐DAA and Gici‐DBA genes display a polymorphism pattern essentially similar to that of mammalian MHC genes playing a major role in Ag presentation suggests that the cartilaginous fish have the bona fide MHC. Thus, the MHC‐peptide‐based T cell recognition system appears to have arisen at or before the emergence of the cartilaginous fish.
C1q, the collagen‐like subcomponent of the first component of complement C1, is a membrane protein of guinea pig macrophages
Authors: Kaul Marcus , Loos Michael
Published: March 2007

C1q, a subcomponent of C1 ‐ the first component of complement, is synthesized by macrophages (Mϕ). Immunofluorescence and immunoperoxidase studies first indicated the presence of Clq on the surface of guinea pig (gp) and human peritoneal Mϕ (Loos, M., Storz, R., Müller, W. and Lemmel, E.M., Immunobiology 1981. 158: 213). In our study different methods for labeling of gp serum and gp Mϕ C1q were employed. The presence of C1q protein on the surface of gp peritoneal Mϕ is shown by cell surface labeling with the biotin derivative sulfosuccinimdyl–6–(biotinamido)‐hexanoate and subsequent immunoprecipitation. The mechanism by which Clq is attached to the cell membrane was also investigated. Intact cells were treated with acid stripping‐buffers or phosphati‐dylinositol‐specific phospholipase C and separated membranes were extracted with a buffer containing 1 M KC1 and 3 M urea. Regardless of which method was used, Clq remained attached to the membrane. When surface‐labeled cells were cultured, they were found to release the Clq from their surface membrane into the culture medium. Lysates of biosynthetically labeled cells were used to show that, like secreted or serum Clq, cellular Mϕ Clq binds to immobilized homologous IgG. This implies that the globular regions of the cellular Clq are functionally active. The results reveal that (i) cellular Mϕ C1q is firmly located in the membrane throughout the biosynthetic pathway, such that it is comparable with an integral membrane protein, (ii) cellular Mϕ C1q is not reversibly bound to the cell surface via a receptor. We suggest that C1q, as a membrane protein of Mϕ, serves as an Fc binding factor that also is secreted into the environment.
The B7 adhesion molecule is expressed on activated human T cells: Functional involvement in T‐T cell interactions
Authors: Wyss‐Coray Tony , Mauri‐Hellweg Daniela , Baumann Kaspar , Bettens Florence , Grunow Roland , Pichler Werner J.
Published: March 2007

The B cell antigen B7 delivers a strong co‐stimulatory signal for the activation of T cells by binding to its ligands CD28 and CTLA4. Here we demonstrate the surface expression of the B7 molecule on activated human T cells in vitro and under certain conditions in vivo and its functional importance in T‐T cell interactions. B7 was detected by flow cytometry on antigen‐specific CD4+ and allospecific CD8+ cloned T cells from different donors with anti‐B7 monoclonal antibody (mAb) or a soluble CTLA4‐Cγ1 chimera molecule and by reverse transcription‐polymerase chain reactions. The expression of B7 was up‐regulated following restimulation of the T cell clones and peaked after 7‐9 days. Moreover, we show that the B7 molecule on T cells is functionally involved in T‐T cell interactions: mAb to CD28 and the CTLA4‐Ig fusion protein could inhibit the proliferation of specific T cell clones in response to T cells as antigen‐presenting cells (APC) or the proliferation of peripheral blood mononuclear cells in a primary allostimulation with activated T cells as stimulator cells. Finally, we found that B7 can be expressed on freshly isolated circulating T cells since in a preliminary study with a limited number of patients, B7 was present on a subset of CD3+ cells. B7 was expressed on activated T cells (CD4+ and CD8+) of certain human immunodeficiency virus (HIV)‐infected individuals (0.5–20% B7+CD8+ cells) or some patients with autoimmune diseases whereas CD3+ cells of healthy individuals did not express B7. The coexpression of major histocompatibility complex class II molecules and B7 may be relevant for the capacity of activated T cells to function as APC. The expression of B7 on T cells in vivo in autoimmune diseases and in HIV infection may be important for a better understanding of these diseases.
Physical association of the cytoplasmic domain of CD2 with the tyrosine kinases p56lck and p59fyn
Authors: Carmo Alexandre M. , Mason Don W. , Beyers Albertus D.
Published: March 2007

In T lymphocytes, CD2 forms part of a loosely associated membrane complex which includes the T cell receptor (TcR) for antigen, the CD3 subunits, CD4 or CD8, CD5 and the protein tyrosine kinases p56lck and p59fyn. The interaction of CD2 with tyrosine kinases in this complex provides a possible mechanism for transmembrane signal transduction by CD2. We have investigated whether the interaction of CD2 with the kinases is dependent on other known members of the complex, or whether an independent association can be observed. Using in vitro kinase assays with immune complexes precipitated from cell lysates, we demonstrate that CD2 can associate with p56lck and p59fyn in a rat thymoma line that does not express CD4 or CD8, and in a TcR‐negative Jurkat cell line. In TcR‐positive Jurkat cells that express rat CD2, interaction of CD2 with p56lck and p59fyn Was clearly seen, but it was absent in cells where the cytoplasmic tail of CD2 is truncated, indicating that the interactions are mediated by the cytoplasmic region of CD2. Furthermore, using cells expressing CD2 molecules with partial truncations in the cytoplasmic domain, we show that the association of CD2 with p56lck: is progressively lost as the cytoplasmic domain is shortened, and that the capacity of the mutants to associate with p56lck correlates with their capacity to transduce transmembrane signals.
KIM185, a monoclonal antibody to CD18 which induces a change in the conformation of CD18 and promotes both LFA‐1‐ and CR3‐dependent adhesion
Authors: Andrew David , Shock Anthony , Ball Elaine , Ortlepp Susan , Bell Joanne , Robinson Martyn
Published: March 2007

Unactivated peripheral blood leukocytes show little tendency to bind to other cells or matrix components, whilst, in the presence of inflammatory mediators, adhesive interactions can rapidly increase. The Leu‐CAM (β2 integrin) family of adhesion molecules have been shown to mediate a variety of these induced adhesion events. Here we describe a monoclonal antibody against CD18, KIM185, which stimulates JY cell homotypic aggregation by a CD11a pathway as well as inducing the adherence of neutrophils to protein‐coated plastic by a CD lib‐dependent mechanism. The antibody recognizes an epitope distinct from the previously described KIM127 antibody and evidence is presented that the binding of KIM185 can cause a change in the conformation of the CD18 molecule.
Vaccination with cytotoxic T lymphocyte epitope‐containing peptide protects against a tumor induced by human papillomavirus type 16‐transformed cells
Authors: Feltkamp Mariet C. W. , Smits Henk L. , Vierboom Michel P. M. , Minnaar René P. , De Jongh Barteld M. , Drijfhout Jan Wouter , Schegget Jan Ter , Melief Cornelis J. M. , Kast W. Martin
Published: March 2007

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus‐induced tumors we generated a B6 (H‐2b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H‐2Kb and H‐2Db MHC class I molecules. Binding peptides were compared with the presently known peptide‐binding motifs for H‐2Kb and H‐2Db and the predictive value of these motifs is shortly discussed. The high‐affinity H‐2Db‐binding peptide and putative CTL epitope E7 49‐57 (RAHYNIVTF) was used in vaccination studies against HPV 16‐transformed tumor cells. Immunization with peptide E7 49‐57 rendered mice insensitive to a subsequent challenge with HPV 16‐transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.
Human immunoglobulin class and IgG subclass regulation: Dual action of interleukin‐4
Authors: Kotowicz Karolena , Callard Robin E.
Published: March 2007

Epstein‐Barr virus (EBV) was used as a polyclonal human B cell mitogen to investigate the regulation of immunoglobulin class and IgG subclass responses by interleukin‐4 (IL‐4). Activation of tonsillar B cells with EBV resulted in an early peak of polyclonal immunoglobulin secretion between days 13 and 14 consisting of IgM, IgA, and IgG1, IgG2, IgG3 and IgG4, but not IgE. Addition of IL‐4 to EBV‐activated B cells at concentrations of 100 U/ml or greater induced the production of IgE and enhanced IgG4 secretion, but had no effect, or more often inhibited the other isotypes. In contrast, low concentrations of IL‐4 (1‐5 U/ml) significantly increased the production of IgM, IgA, IgG1, IgG2and IgG3, but had no effect on IgG4 or IgE. The increase in immunoglobulin secretion obtained with low concentrations of IL‐4 was found to occur only with high‐density (resting) B cells, suggesting that IL‐4 was not functioning simply as a late‐acting differentiation factor. Low concentrations of IL‐4 significantly increased IgG1, IgG2, IgG3, and IgA production by surface (s)IgM+ (sIgG−/sIgA−) B cells which is consistent with heavy chain switching. In some experiments, however, IL‐4 enhanced IgM secretion by sIgM+ B cells, and IgA, IgG1, IgG2, IgG3 by sIgM B cells, suggesting that it may have an additional B cell differentiation factor activity which was not isotype specific. The different effect of IL‐4 at high and low concentrations were similar to those observed in B cell activation experiments, and may be due to the existence of high‐ and low‐affinity IL‐4 receptors.
Homologous C3 deposition and homotypic cell adhesion in a human myeloid cell line, P39
Authors: Matsumoto Misako , Seya Tsukasa
Published: March 2007

It has been accepted that decay‐accelerating factor (DAF) and membrane cofactor protein (MCP) on human cells block C3 deposition, thereby preventing homologous complement attack. In this study, we discovered that a human myeloid cell line, P39, was a target for human C3 even though it expressed normal DAF and MCP. This homologous C3 deposition was induced by serum containing Mg2+ and EGTA (Mg2+‐EGTA serum) selectively on a P39 subline [P39(+) cells] having the capacity to form cell aggregates. Another P39 subline [P39(‐)cells] growing as a separated form did not induce homologous C3 deposition. Multiple C3 fragments, C3b and C3bi, were fixed on P39(+) cells and a significant amount of C5a was released. Several distinct C3 fragment‐membrane acceptor molecule complexes were immunoprecipitated with anti‐C3c antibody from surface‐labeled P39 (+) cells treated with Mg2+‐EGTA serum and from unlabeled cells incubated with 125I‐labeled C3 and Mg2+‐EGTA serum. Two‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed 90,60 and ≦ 40 kDa C3 acceptors on P39(+) cells. On these cells, some of the C3 bound to these acceptors remained in the form of C3b, which can form C3/C5 convertases. P39(+) cells differed phenotypically from P39(‐) cells in that P39(+) cells expressed intercellular adhesion molecule‐1 (ICAM‐1), whereas P39(‐) cells did not. Both cells were lymphocyte function‐associated antigen 1 (LFA‐1)+/CR3− counterreceptors for ICAM‐1. However, homotypic cell adhesion was not completely inhibited by antibodies against ICAM‐1 and LFA‐1, suggesting that the homotypic cell aggregation of P39(+) cells is due only in part to ICAM‐1 and LFA‐1. In addition, C3 deposition, the expression of ICAM‐1, and cell aggregation were enhanced by both tumor necrosis factor‐α and interferon‐γ. Although the principal causative mechanisms remain obscure, C3 deposition and cell adhesion appear in parallel in this cell line and may be involved in the modulation of cell‐mediated immune reactions.

We are grateful to Dr. Iida for the generous gift of the mAb against human C3c and C3bi, to Dr. Yamada for anti‐ICAM‐1 mAb, and to Drs. Nakai, Kondo, Hamuro, Nihira, and Matsuda for cytokines. We also thank the JCRB for providing P39 cells, Drs. Akedo, Miyagawa, and Hatanaka (Center for Adult Diseases Osaka) for valuable discussions, Dr. S. Hori (Osaka Prefectural Institute of Public Health) for providing FACS can, Ms H. Fukuda for the photographs in phase‐contrast microscopy, Ms. F. Yamashita, A. Kojima and T. Hara for technical help, and Ms. A. Itoh for secretarial assistance.
Induction by anti‐CD40 antibody or soluble CD40 ligand and cytokines of IgG, IgA and IgE production by B cells from patients with X‐linked hyper IgM syndrome
Authors: Durandy Anne , Schiff Claudine , Bonnefoy Jean Yves , Forveille Monique , Rousset Françoise , Mazzei Gonzalo , Milili Michèle , Fischer Alain
Published: March 2007

We studied the ability of B lymphocytes from patients with X‐linked hyper IgM syndrome (HIGM1) to be activated via the CD40 membrane receptor. HIGM1 is caused by a CD40 ligand gene mutation, leading to defective expression on the membrane of activated T lymphocytes. We found that triggering of B cells by an anti‐CD40 monoclonal antibody or the soluble CD40 ligand plus interleukin (IL)‐4 or IL‐10 led to B cell proliferation and/or differentiation towards IgG, IgA and IgE secretion. This was reflected by transcription of Cγ, α and ε membrane isotype expression and IgG, IgA and IgE production. These results confirm the integrity of B cells in patients with the HIGM1 immunodeficiency and open up new therapeutic possibilities.
Epitope analysis of the T cell response to a complex antigen: Proliferative responses to human rhinovirus capsids
Authors: Hastings Gillian Z. , Francis Michael J. , Rowlands D. J. , Chain Benjamin M.
Published: March 2007

Understanding the factors which regulate the repertoire of a T cell response is important when selecting T helper cell epitopes for inclusion in synthetic viral vaccines. In this study we have examined the T cell response to human rhinovirus (HRV) type 1A in a mouse model system, using a comprehensive set of synthetic peptides which span all four of the proteins which make up the HRV capsid. This constitutes the first study to use a set of peptides covering the entire sequence of all structural proteins of any virus. This study identifies the major proliferative (CD4) T cell epitopes within the minor receptor group HRV 1 A, and analyzes these epitopes with relation to their location within the three‐dimensional structure of the virus. The proliferative response to HRV is highly selective, with strong responses to only a very small number of epitopes, many of which are grouped together within restricted areas of the primary structure of the HRV proteins. The repertoire of the response is almost entirely specific to the major histocompatibility complex haplotype of the host. The major T cell epitopes are spatially distinct from the sites of the major antibody recognition sites, and are buried within the viral capsid. In striking contrast to the antibody responses, the T cell responses are highly cross‐reactive against a wide variety of viral serotypes.
Antigens targeted to the Leishmania phagolysosome are processed for CD4+ T cell recognition
Authors: Kaye Paul M. , Coburn Cara , McCrossan Maria , Beverley Stephen M.
Published: March 2007

Processing of antigen for recognition by class II‐restricted CD4+ T cells occurs within acidic compartments of the antigen‐presenting cell. The exact nature of this compartment has yet to be precisely defined, however, but may vary depending upon the cell type studied and the antigen used. The acidic compartments of macrophages are also responsible for the degradation of ingested micro‐organisms and play host to others which are adapted to an intracellular existance. To determine whether the phagolysosome (PL) formed in activated macrophages after ingestion of Leishmania parasites is also a site for entry of antigen into the class II presentation pathway, we have used the approach of genetic transformation. Hence, Leishmania were transfected with the genes for the protein antigens ovalbumin (OVA) and β‐galactosidase (β‐gal) and after infection were able to deliver these antigens specifically into the PL. Delivery of antigen to this site resulted in the ability of infected macrophages to present these antigens to antigen‐specific CD4+ T cells. After taking into account the absolute levels of antigen uptake by macrophages, a 4‐h processing period for OVA delivered by this or a soluble route led to equivalent levels of T cell activation. Unlike macrophages pulsed with soluble OVA, those with PL‐targeted OVA still retained the ability to stimulate T cells after a 24‐h processing period. This enhanced lifespan of antigen in macrophages corresponded to the kinetics of degradation of the parasite, suggesting slow release of antigen into the processing pathway. β‐gal presentation from the PL was tenfold less efficient under the same conditions. In addition to providing the first information on antigen processing in a protozoan PL, these studies highlight the usefulness of genetically transformed parasites for these types of studies.
A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily
Authors: Law S. K. Alex , Micklem Kingsley J. , Shaw Jacqueline M. , Zhang Xi‐Ping , Dong Yin , Willis Antony C. , Mason David Y.
Published: March 2007

The antibodies Ki‐M8, Ber‐Mac3, GHI/61 and SM4 define a human macrophage‐associated antigen with a relative molecular mass of 130000 which we designate M130. The protein was purified by immunoaffinity chromatography and an N‐terminal and three internal amino acid sequences were obtained. A cDNA fragment was initially obtained by polymerase chain reaction (PCR) using reverse‐translated primers. Several variant cDNA clones, derived from alternative spliced messages, were obtained from a lipopolysaccharide‐stimulated human monocyte library and were sequenced. The relative abundance of these variants was evaluated by a series of overlapping PCR reactions. The size of the most representative cDNA is 3.7 kb and closely agrees with the mRNA size of 3.8 kb determined by Northern blot analysis. The membrane protein encoded contains a leader peptide of 40 residues, a putative extracellular domain of 1003 residues, followed by a hydrophobic segment of 24 residues and a cytoplasmic domain of 49 residues. The extracellular domain was found to contain nine repeating elements, of about 110 residues, which are similar to those of the scavenger receptor superfamily.
Construction, expression and functional analysis of a glycolipid‐linked form of CR1
Authors: Clissold Patricia M. , Ebling Hazel J. , Lachmann Peter J.
Published: March 2007

By genetic engineering of human CR1 cDNA and its stable transfection into cells we have produced a cell line which expresses CR1 anchored to the cell surface by a glycolipid anchor. The glycosyl‐phosphatidylinositol (GPI)‐CRl protects cells intrinsically from damage mediated by complement activated through the classical pathway. Cell surface GPI‐CR1 is more efficient on a molar basis than soluble CR1 in the assay, but extrinsic protection of other cells was not obtained. Soluble CRl‐protected cells extrinsically in the assay but was required at nearly ten fold higher amounts than the intrinsic protection conferred by GPI‐anchored CR1. Additionally, GPI‐CR1 was shown to act as a co‐factor to Factor I in the generation of C3c from iC3b. Since GPI‐anchored proteins can incorporate spontaneously into the membranes of living cells by virtue of their lipid tails, the isolated GPI‐CR1 will be used to introduce CR1 on to the surfaces of many different types of cell so that its role in immunity can be further investigated.
Cloning of a human immunoglobulin gene fragment containing both VH‐D and D‐JH rearrangements: Implication for VH‐D as an intermediate to VH‐D‐JH formation
Authors: Shin Euy Kyun , Matsuda Fumihiko , Fujikura Junji , Akamizu Takashi , Sugawa Hideo , Mori Toru , Honjo Tasuku
Published: March 2007

In an Epstein‐Barr virus‐transformed human B cell line we found an unusual immunoglobulin heavy chain gene rearrangement. Restriction mapping and sequencing analysis led us to conclude that VH‐D and D‐JH recombination took place in a single allele. Both VH‐D and D‐JH complexes still had their recombination signal sequences adjacent and the DNA sandwiched by these two complexes retained a germ line configuration, suggesting the potential for a secondary rearrangement resulting in a VH‐D(‐D)‐JH formation. With this finding, we propose a novel pathway, in which the VH‐D complex is an intermediate in the formation of a functional VH exon.
Suppression of apoptosis in normal and neoplastic human B lymphocytes by CD40 ligand is independent of Bcl‐2 induction
Authors: Holder Michelle J. , Wang Hong , Milner Anne E. , Casamayor Monserat , Armitage Richard , Spriggs Melanie K. , Fanslow William C. , Maclennan Ian C. M. , Gregory Christopher D. , Gordon John
Published: March 2007

The tendency of isolated germinal center (GC) B cells to undergo apoptosis was suppressed by recombinant cell‐bound CD40 ligand (CD40L): after 2 days at 37°C, > 80 % of cells remained viable in the presence of CD40L as compared to < 1 % in control cultures. CD40L sustained a high rate of DNA synthesis in GC cells and was more effective than monoclonal antibody to CD40 in this regard. Group I Burkitt lymphoma (BL) cell lines induced to undergo apoptosis with anti‐immunoglobulin or calcium ionophore were also protected by CD40L. In BL cells, this route of rescue was not accompanied by induction of Bcl‐2 protein, the expression of which has been linked to hemopoietic cell survival. Bcl‐2 was induced in GC cells responding to CD40L, but its appearance was a relatively late event not reaching significant levels over controls until day 2 of culture. Thus induction of Bcl‐2 appears to be secondary to the survival signal imparted by CD40L. These findings are discussed in relation to a potential role for CD40L in supporting B cell tumors in vivo and the discovery that the molecular defect in the X‐linked Hyper‐IgM syndrome is targeted to the CD40L gene.
Linomide, a novel immunomodulator that prevents death in four models of septic shock
Authors: Gonzalo José Angel , González‐García Ana , Kalland Terje , Hedlund Gunnar , Martínez‐A Carlos , Kroemer Guido
Published: March 2007

Intravenous injections of 50 μ.g Staphylococcus aureus enterotoxin B (SEB) or bacterial lipopolysaccharide (LPS) are lethal, provided that mice are simultaneously sensitized with either N‐galactosamine (GalN) or the anti‐glucocorticoid RU‐38486. Similar to the synthetic glucocorticoid (GC) receptor agonist dexamethasone, pharmacological doses of the immunomodulator linomide (quinoline‐3‐carboxamide) prevent death in all four models of lethal septic shock (LPS + GalN, LPS + RU‐38486, SEB + GalN, and SEB + RU‐38486) and inhibit the secretion of tumor necrosis factor, one of the major intermediate effector molecules of SEB and LPS toxicity. In this system, cyclosporine A (CsA), although effective in suppressing SEB toxicity, fails to counteract the lethal effect of LPS. This observation, together with the fact that linomide acts in the presence of excess amounts of GC receptor antagonist, indicates that linomide functions in a different way to that of known immunosuppressive agents like CsA and GC.
Increased cytokine production in mononuclear cells of healthy elderly people
Authors: Fagiolo Umberto , Cossarizza Andrea , Scala Enrico , Fanales‐Belasio Emanuele , Ortolani Claudio , Cozzi Emanuele , Monti Daniela , Franceschi Claudio , Paganelli Roberto
Published: March 2007

The production of cytokines during aging, except interleukin (IL)‐2, has been neglected in humans. We measured the in vitro production of IL‐6, tumor necrosis factor (TNF)‐α, interferon (IFN)‐γ and IL‐1β by peripheral mononuclear cells from selected healthy young (mean age 26.8 years) and aged (mean age 80.2 years) subjects. Significant increases of IL‐6, TNF‐α and IL‐1bT levels were found in mitogen‐stimulated cultures from aged donors, occurring at 24 to 72 h after stimulation. No significant differences were observed for IFN‐γ production. Proliferative capability of cells stimulated with PHA was not impaired in aged subjects. Since the amounts of all cytokines studied were similar in unstimulated cultures from young and aged subjects, and also serum levels of TNF‐a did not differ, these data indicate that the cellular machinery for the production of these cytokines is well preserved in aging, and also that cells from old people are able to up‐regulate their production in response to appropriate stimuli. The increases in cytokine synthesis were not dependent on changes in the number of monocytes, nor were they related to the significant rise of CD45RO+, and the concomitant decrease of CD45RA+ occurring in both CD4+ and CD8+ lymphocytes from aged subjects. The increased production of pro‐inflammatory cytokines by stimulated mononuclear cells of healthy aged subjects may be relevant to several aspects of age‐associated pathological events, including atherosclerosis, osteoporosis, fibrosis and dementia.
Hyper‐reactivity of mouse CD45RA − T cells
Authors: Lightstone Elizabeth , Marvel Jacqueline , Mitchison Avrion
Published: March 2007

Mouse CD4 T cells have been partitioned into CD45RA and CD45RA− subpopulations by means ot the monoclonal antibody 14.8. The CD45RA− subpopulation proliferated more actively and generated more interleukin‐4 (IL‐4) in response to stimulation with anti‐CD3 antibody and phytohemaglutinin, and more IL‐2 in response to anti‐CD3. This subpopulation is therefore hyper‐reactive to these polyclonal stimulators, but does not show the bias towards T helper type 2 activity that has been found in studies with other related CD45 isoforms. No evidence of suppression was obtained by comparing proliferation of CD45RA− cells in the presence and absence of CD45RA cells. Thus mouse CD4 T cells behave in these respects similarly to those of man, as is evident in a brief review of the quiescence‐activation‐quiescence cycle in the two species.
Expressions of cytokine genes during development of autoimmune sialadenitis in MRL/Ipr mice
Authors: Hamano Hironori , Saito Ichiro , Haneji Norio , Mitsuhashi Yuriko , Miyasaka Nobuyuki , Hayashi Yoshio
Published: March 2007

Local cytokine gene expression in vivo was analyzed by direct analysis of RNA obtained from salivary gland tissues of MRL/lpr mice with autoimmune sialadenitis. The expression of cytokine genes were assessed by the reverse‐transcriptase polymerase chain reaction, and by immunohistochemical analysis. The expression of interleukin‐1(IL‐1)β and tumor necrosis factor was detected before the onset of inflammatory lesions in the salivary glands of mice of 1 or 2 months of age, and IL‐6 mRNA expression was clearly detected at the time of onset of typical autoimmune sialadenitis at 3 months of age in MRL/lpr mice, and was up‐regulated with advancing age. These results suggest that the overexpression of these inflammatory cytokine genes is involved in the development and progression of organ‐localized autoimmunity in the salivary glands of MRL/lpr mice.