bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and sequence analysis of two isotypic IgM heavy chain genes from Atlantic salmon, Salmo salar L
Authors: Hordvik Ivar , Voie Ann Mari , Glette Johan , Male Rune , Endresen Curt
Published: March 2007

A leukocyte cDNA library from Atlantic salmon, based on oligo‐dT priming, was constructed in λ‐gt10. Immunoglobulin heavy chain (IgH) cDNA were isolated from the library using a specific probe generated by polymerase chain reaction (PCR) between two conserved areas within the variable region (second and fourth frame region). Two cDNA clones encoding the entire constant region of membrane‐bound IgH, and one cDNA encoding the entire constant region of secretory IgH were sequenced, revealing messages from two isotypic IgM genes. Both genes were shown to be present in haploid embryos and have been isolated from a genomic library, the exons and deduced amino acid sequences of which are presented here (salmon CHA and CHB). The splicing of transcripts encoding the membrane‐bound IgH excises the whole fourth exon as in other teleosts. The nucleotide and amino acid identity between salmon CHA and CHB are 98.2 %, and 96.2%, respectively. Two subfractions of IgM from Atlantic salmon separated by ion chromatography can be explained by a net exchange of basic residues in salmon CHB compared to CHA. The finding of two closely related salmon CH genes is in accordance with the quasi‐tetraploid state of the Atlantic salmon genome.
Altered major histocompatibility complex‐restricted antigen recognition by T cells from elderly humans
Authors: Schwab Risë , Russo Carlo , Weksler Marc E.
Published: March 2007

Positive selection of T cells within the thymus gland leads to major histocompatibility complex (MHC)‐restricted recognition of antigen by T lymphocytes. As the thymus gland involutes with age, altered MHC‐restricted antigen recognition by T cells from elderly humans would be expected. We have tested this hypothesis by comparing the proliferative response of T cells and T cell clones from aged and young subjects to influenza determinants presented by autologous or allogeneic antigen‐presenting cells (APC). Under conditions in which the allogeneic mixed lymphocyte reaction was minimal, T cells from six of seven aged donors but only one of seven young donors were stimulated by influenza vaccine presented by allogeneic APC. More importantly, one‐half of the influenza‐specific T cell clones derived from aged donors, but none of the clones derived from young donors, were activated by influenza vaccine presented by allogeneic APC. While 80% of the MHC‐nonrestricted influenza‐specific T cell clones expressed the γ/δ T cell receptor, 20% of these clones expressed the α/β T cell receptor. Thus, changes in MHC‐restricted antigen recognition by T cells and in altered distribution of α/β versus the γ/δ T cell receptor bearing antigen‐specific T cell clones occur with aging.
α Heavy chain disease αmRNA contain nucleotide sequences of unknown origins
Authors: Fakhfakh Faïza , Dellagi Koussay , Ayadi Hammadi , Bouguerra Annie , Fourati Rashid , Ayed Farhat Ben , Brouet Jean Claude , Tsapis Andréas
Published: March 2007

Human α heavy chain disease is characterized by the production of abnormally short α IgH chains. In previously published cases it has been found that the malignant cells produce abnormal α mRNA, lacking VH and CH1 sequences and composed of a leader sequence peptide, sequences of variable length (69 to 84 bp) and of unknown origin, followed by normal CH2 and CH3 sequences. In this study we established the nucleotide sequence of α mRNA for six cases of α heavy chain disease. We observed that all six α mRNA lack the VH and CH1 sequences as do those previously described. They also contain in‐frame inserts of unknown origin between the leader peptide and the normal CH2 and CH3 coding sequences. These inserts are of variable length (42 to 105 bp) and they are unrelated. These results suggest the existence of a common mechanism defect leading to deletions/insertions in α heavy chain disease rather than a specific interaction between α1 IgH gene with a unique defined molecular species.
Role of T cell subsets during the recall of immunologic memory to Leishmania major
Authors: Müller Ingrid
Published: March 2007

The contributions of different T cell subpopulations to the maintenance of immunity during secondary Leishmania major infections were analyzed in healed, resistant animals by depletion of T cell subsets in vivo. The strong delayed‐type hypersensitivity mounted in immune genetically resistant mice upon challenge with viable promastigotes was mediated by both CD4+ and CD8+ T cells. Each T cell subpopulation alone contributes, although to a different extent, to the resolution of secondary lesions; both subsets, however, are required for an efficient and rapid healing of the secondary lesions and the decrease in the parasite burden in infected tissues.

The results indicate that in immune, genetically resistant CBA mice, the activity of both T cell subsets is required for successful resistance to reinfection and an efficient maintenance of immunity.
Cryptic T cell epitopes in polymorphic immunoglobulin regions: Evidence for positive repertoire selection during fetal development
Authors: Chen Jian‐Jun , Kaveri Srinivas‐Venkatesh , Kohler Heinz
Published: March 2007

The adult T cell repertoire is the result of positive and negative intrathymic selective processes. Since non‐self antigens are not present in the thymus, self peptides are believed to play a role in the thymic T cell selection. T cell lines raised against a peptide from the second complementarity‐determining region (CDR2) of the S107 germ‐line immunoglobulin (Ig) do not respond to the intact Ig, indicating that in adult animals this CDR2 epitope is cryptic in the context of intact S107 Ig. To show that the response to self Ig peptide is the result of positive selection, the fetal expression of the S107 germ‐line Ig was suppressed by maternal anti‐idiotype (Id) injection. Id S107‐suppressed mice responded poorly to the CDR2 peptide, indicating that S107 Ig selects positively T cells responding to a polymorphic self epitope. Cross‐reactivity of cryptic self Ig with non‐self epitopes could be a mechanism to increase the T cell repertoire for foreign antigens.
Interferon‐α prevents endotoxin‐induced mortality in mice
Authors: Tzung Shie‐Pon , Mahl Thomas C. , Lance Peter , Andersen Valerie , Cohen Stefan A.
Published: March 2007

Endotoxins, the lipopolysaccharide (LPS) moieties on the bacterial cell wall, cause many of the pathological features of Gram‐negative septicemia. Tumor necrosis factor (TNF), primarily a product of monocyte/macrophages, has been shown to mediate many of the pathophysiological effects of endotoxin. Kupffer cells, the largest macrophage population in the body, release TNF when stimulated by LPS in vitro. A recombinant human hybrid interferon‐α A/D (rIFN‐α) markedly inhibited this LPS‐elicited TNF production by Kupffer cells. The effects of rIFN‐α were further tested in C57BL/6 mice receiving a lethal dose (400 μg/mouse) of LPS. All LPS‐treated mice died within 2 days. Pretreatment with rIFN‐α 1 h before LPS challenge improved the survival at 3 days to 22% (5/23, p < 0.04). In contrast, rIFN‐α was more effective when administered 20 min after LPS injection, increasing the survival rate to 81% (13/16, p < 0.0001). TNF mRNA expression in the liver and spleen 50 min after LPS challenge, and plasma TNF 1.5 h after LPS were also reduced by either pretreatment or post‐treatment with rIFN‐α. Subsequently, experiments were carried out to test the efficacy of delayed rIFN‐α treatment. A significant protective effect was still apparent when rIFN‐α was administered 6, 10 and even 14 h (81%, 62% and 28% survival, respectively) after LPS challenge when serum TNF levels had already returned to near baseline. These experimental results suggest that rIFN‐α might have a therapeutic potential for the prevention and treatment of the deleterious effects associated with endotoxemia besides mechanisms initially blocking TNF production.
Functional role of α2/β1 and α4/β1 integrins in leukocyte intercellular adhesion induced through the common β1 subunit
Authors: Campanero Miguel R. , Arroyo Alicia G. , Pulido Rafael , Ursa Angeles , de Matías Milagros S. , Sánchez‐Mateos Paloma , Kassner Paul D. , Chan Bosco M. C. , Hemler Martin E. , Corbí Angel L. , de Landázuri Manuel O. , Sánchez‐Madrid Francisco
Published: March 2007

Whereas all of the integrins in the VLA protein subfamily are involved in cell‐extracellular matrix interactions, only VLA‐4 (through the α4 subunit) has been implicated in the triggering of intercellular adhesion. Here we describe that the VLA protein β1 subunit (CD29) is also involved in the induction of homotypic cell aggregation. We have obtained three novel anti‐β1 monoclonal antibodies (mAb) with the ability to induce cell aggregation on different leukocyte cell types. These mAb recognize an antigenic site on the common β1 chain of VLA proteins which is topographically and/or functionally distinct from other epitopes previously defined by several prototype anti‐β1 mAb. Induction of cell aggregation by anti‐β1 mAb is epitope specific, isotype and Fc independent, and displays kinetics similar to α4‐mediated aggregation. This cell aggregation requires an intact cellular metabolism, the presence of divalent cations in the extracellular medium, and the integrity of the cytoskeleton. We also have found that the Na+/H+ antiporter may be essential for this process. For Ramos cells, which bear only the VLA α4/β1 heterodimer, intercellular adhesion induced through the VLA‐β1 chain could be selectively inhibited by other anti‐β1 mAb as well as by anti‐α4 mAb. Interestingly, anti‐β1 mAb which induced strong aggregation of VLA‐α2‐ or VLA‐α4‐transfected K562 cells, had minimal effect on the α2− α4− α5+ K562 cell line. Furthermore, the β1‐mediated induction of cell aggregation on α2‐K562‐ and α4‐K562‐transfected cells was blocked by preincubation with either anti‐α2 or anti‐α4 mAb, respectively, as well as by other anti‐β1 mAb. Interestingly, parental K562 cells were able to interact with both α2‐ and α4‐transfected K562 cells, thus suggesting that counter‐receptors for both integrins (VLA‐2 and VLA‐4) might exist on these cells. Together these results provide strong evidence supporting the involvement of α2/β1 and α4/β1 heterodimers in intercellular interactions and underline the pivotal role of the common β1 chain of VLA proteins in the integrin‐mediated induction of cell aggregation.
Short peptides assist the folding of free class I heavy chains in solution
Authors: Elliott Tim , Cerundolo Vincenzo , Townsend Alain
Published: March 2007

Previous experiments have shown that short peptides coresponding to naturally processed epitopes of viral antigens can induce a conformational change in the class I heavy chain (HC) to which they bind in the fully assembled molecule. Here, we present evidence that the mechanism for this conformational change may involve binding of peptide to a partially unfolded form of free HC, followed by its subsequent folding. These results may be important for understanding the way in which class I molecules are assembled in vivo, and how certain epitopes are selected for presentation to T cells.
Binding of T cell receptor to major histocompatibility complex class II‐peptide complexes at the single‐cell level results in the induction of antigen unresponsiveness (anergy)
Authors: Celis Esteban , Saibara Toshiji
Published: March 2007

Using dispersion cultures performed in semi‐solid medium we demonstrate here that the interaction of T cell antigen receptor molecules with Ia‐peptide complexes on the same cell surface results in T cell activation, without the production of lymphokines. This recognition of antigen at the single‐cell level, induced a state of anergy which was not due to a decrease of surface T cell antigen receptor/CD3 complexes. The induction of anergy by peptide could not be prevented by the addition of various co‐stimulatory signals, including antibodies to CD28, or CD2, high doses of interleukin‐2 or phorbol ester.
Relative roles of decay‐accelerating factor, membrane cofactor protein, and CD59 in the protection of human endothelial cells against complement‐mediated lysis
Authors: Brooimans Ricardo A. , van Wieringen Patrick A. M. , van Es Leendert A. , Daha Mohamed R.
Published: March 2007

Human umbilical vein endothelial cells (HUVEC) were found by Western blot analysis to express three membrane‐bound C regulatory proteins, decay‐accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. DAF was detected on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis as a 70‐kDa molecule under nonreducing conditions in 2% deoxycholate extracts of HUVEC, MCP as a 63‐kDa protein and CD59 as a 20‐kDa molecule. Northern blot analysis revealed the presence of two species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for DAF, with sizes of about 2.0 kb and 2.7 kb. MCP mRNA was detected at 4.2 kb and a CD59 cDNA probe hybridized with three mRNA species with sizes of about 800, 1400 and 2000 bp. DAF and CD59 were released from the surface of HUVEC by phosphatidylinositol‐phospholipase C, demonstrating that both are attached to the cell membrane by means of a glycolipid anchor. The relative contribution of DAF, MCP and CD59 in regulating the sensitivity to lysis of HUVEC by autologous complement was determined by incubation of sensitized endothelial cells with F(ab′)2 fragments of polyclonal antibodies raised against these proteins. The susceptibility of sensitized cells to lysis by homologous complement was markedly increased in the presence of F(ab′)2 anti‐CD59 and to a lesser, but significant, extent in the presence of F(ab′)2 anti‐DAF. F(ab′)2 anti‐MCP did not significantly alter the susceptibility of HUVEC to complement‐mediated lysis.
Regulation of tumor necrosis factor (TNF)‐α synthesis and TNF receptors expression in T lymphocytes through the CD2 activation pathway
Authors: Santis Ana G. , Campanero Miguel R. , Alonso Jose L. , Sánchez‐Madrid Francisco
Published: March 2007

The involvement of the CD2 (T11) molecule, an alternative activation pathway for T lymphocytes, in the regulation of tumor necrosis factor (TNF)‐α/TNF receptor system in human T lymphocytes has been investigated. It has been found that both TNF‐α synthesis and secretion were induced after incubation of purified T lymphocytes with the appropriate mitogenic combination of antibodies specific for two different epitopes on the CD2 molecule. Moreover, TNF‐α secretion was also observed by activation of T lymphocytes either through CD3 or CD69 molecular pathways, or with other stimulating agents such as Ca2+ ionophore in combination with phorbol esters. The expression of TNF receptors has been studied in both nonactivated and CD2‐activated T lymphocytes. Unstimulated T cells weakly expressed a functional 75‐kDa receptor form, whereas they lacked detectable levels of the 55‐kDa receptor form. Triggering of T cell activation through the CD2 molecule also markedly increased the expression of the p75‐kDa TNF receptor form, but did not exert any inductive effect on the expression of the p55‐kDa TNF receptor. In addition, we have found that TNF‐α enhanced the proliferative response triggered by the mixture of anti‐CD2 monoclonal antibodies. Taken together, these results support a role for the CD2 activation pathway in the functional regulation of TNF‐α/TNF receptor system in T lymphocytes, and reinforce the view of CD2 as an alternative pathway for regulation of the cytokine network that modulates the function of T lymphocytes.
Peritoneal sanctuary for human lymphopoiesis in SCID mice injected with human peripheral blood lymphocytes from Epstein‐Barr virus‐negative donors
Authors: Hoffmann‐Fezer Gertrud , Kranz Bernd , Gall Claudius , Thierfelder Stefan
Published: March 2007

The successful engraftment in SCID mice of intraperitoneally (i.p.) injected human lymphocytes (hu‐PBL‐SCID) and the failure of intravenously injected peripheral blood lymphocytes (PBL) directed the present study to investigate the early events of donor cell proliferation in the peritoneal cavity. We found focal lymphocyte engraftment together with histio‐monocytic interleukin (IL)‐6+ cell phenotypes which must have been transferred with the human cell inoculum, which could explain certain immune functions observed in hu‐PBL‐SCID chimeras. Following i.p. injection of 108 PBL, human cells suspended in peritoneal fluid as well as those adherent to the serosal peritoneum and abdominal organs were investigated by immunocytology and immunohistology. Human cells were found to form foci consisting predominantly of proliferating human lymphoblastoid CD3+ cells, which were mostly activated HLA‐DR+ CD8+ lymphocytes. Among the lymphoid cells larger epithelioid‐like cells were found to belong to the monocytic series and to stain strongly with anti‐HLA‐DR and anti‐CD11c antibodies. Some of these cells were also positive with anti‐ICAM and anti‐IL‐6. Congenic as well as allogeneic mouse PBL, injected i.p. into SCID mice, temporarily produced analogous foci, which shifted later on to foci similar in appearance to milky spots. However, the human cell foci appeared less compact, more closely resembling in vitro‐culture soft agar colonies. It is possible that cytokines in the human histio‐monocytic cells of the foci may have a feeder effect on the human lymphocytes and be a prerequisite for proliferation of human PBL in SCID mice. The observed early HLA‐DR activation of human lymphocytes in the peritoneal foci could reflect triggering of immune reactions like xenogeneic graft‐versus‐host reactions in the peritoneal site, where the human CD11c+ HLA‐DR+ histio‐monocytic cells may act as antigen‐presenting cells.
Induction of primary anti‐viral cytotoxic T cells by in vitro stimulation with short synthetic peptide and interleukin‐7
Authors: Kos Ferdynand J. , Müllbacher Arno
Published: March 2007

The present study investigated whether a short synthetic peptide NPP, with a modified sequence (147–158 R156−) derived from influenza A virus nucleoprotein with high affinity for Kd major histocompatibillity complex class I molecules, could induce primary influenza virus‐specific cytotoxic T (Tc) cells in vitro. Naive BALB/c (H‐2d) splenocytes did not respond to the stimulation with only NPP with the generation of effector Tc cells specific for influenza A virus‐infected target cells in vitro. However, they were able to do so if cultured with NPP in the presence of IL‐7. IL‐7 activity in this system differed significantly from IL‐2 activity in the specificity of the effect. The use of exogenous IL‐2, instead of IL‐7, with NPP resulted in the induction of lytic cells that lysed both influenza virus‐infected and uninfected syngeneic target cells. These results suggest that IL‐7 is a potent regulatory cytokine in the antigen‐specific activation of resting naive Tc cell precursors and may provide the necessary conditions for the induction of human primary Tc cells in vitro.
Cloning of TRAP, a ligand for CD40 on human T cells
Authors: Graf Daniel , Korthäuer Ulf , Mages Hans W. , Senger Gabriele , Kroczek Richard A.
Published: March 2007

A cDNA clone, designated TRAP (TNF‐related activation protein) was isolated from a collection of T cell activation genes. The polypeptide encoded by a mRNA of approx. 2.3 kb is 261 amino acids long with a calculated Mr of 29.3 kDa. The structural features predict a type II transmembrane protein, but are also compatible with a secreted form. TRAP is highly similar to an identified murine CD40 ligand both at the cDNA (82.8% identity) and the protein (77.4% identity) levels, and related to tumor necrosis factor/lymphotoxin. Expressed in a murine myeloma, TRAP was identified as a ligand for CD40 by binding to a soluble CD40 construct. TRAP mRNA is expressed in a T cell‐specific fashion with a maximum at 8 h after stimulation. The TRAP gene is located in the q26.3–q27.1 region of the X chromosome.
T helper type 2‐like cells and therapeutic effects of interferon‐γ in combined immunodeficiency with hypereosinophilia (Omenn's syndrome)
Authors: Schandené Liliane , Ferster Alina , Mascart‐Lemone Françoise , Crusiaux Alain , Gérard Catherine , Marchant Arnaud , Lybin Myriam , Velu Thierry , Sariban Eric , Goldman Michel
Published: March 2007

We characterized the defects of CD4+ cells in a 17‐month‐old girl suffering from combined immunodeficiency with hypereosinophilia (Omenn's syndrome). Because the vast majority of peripheral blood CD4+ cells expressed the CD45R0 isoform, we purified circulating CD4+ CD45R0+ cells from the patient and healthy individuals in order to compare their production of cytokines. The patient's CD4+ CD45R0+ cells spontaneously produced high levels of interleukin‐5 (IL‐5) in vitro (1600 pg/ml after 24 h of culture) and this was associated with the presence of IL‐5 in serum (323 pg/ml). After stimulation with phorbol 12‐myristate 13‐acetate (PMA) and calcium ionophore A23187, they produced higher levels of IL‐4 (306 vs. 55 ± 4 pg/ml) and IL‐5 (2900 vs. 213 ± 72 pg/ml) and lower levels of IL‐2 (17 vs. 63 ± 17 IU/ml) and interferon‐γ (IFN‐γ) (16 vs. 299 ± 70 IU/ml) than controls CD4+ CD45R0+ cells. This T helper type 2 (Th2) pattern was confirmed by the detection using reverse polymerase chain reaction of IL‐4, IL‐5 and IL‐10 mRNA within peripheral blood mononuclear cells. During a therapeutic trial with human IFN‐γ (40 μg/day) which ameliorated the clinical status of the patient, we observed a down‐regulation of the in vivo expression of IL‐5 and IL‐10, a normalization of the eosinophil count and an improvement of the Tcell response to phytohemagglutinin. This observation indicates for the first time that Th2‐like cells might be involved in certain forms of congenital immunodeficiency and that IFN‐γ might down‐regulate their activities in vivo.
Comparison of three different forms of HLA‐DR4Dw4 proteins
Authors: Buelow Roland , Paborsky Lisa R. , van Schooten Wim C. A. , Kummerehl Tammy J. , Schreifels Robert , Marshall Keith , Mayer John , Rothbard Jonathan B.
Published: March 2007

The biochemical behavior and peptide binding properties of a soluble form of the human class II DR4Dw4 molecule (PI‐DR4Dw4) were compared to DR4Dw4 molecules containing the transmembrane and cytoplasmic domains that were purified both from B and transfected Chinese hamster ovary cells. Recombinant and B cell‐derived DR4Dw4 molecules bound monoclonal anti‐DR4Dw4 antibodies with different affinities and varied in their stability in the presence of sodium dodecyl sulfate. The three forms of DR4Dw4 bound peptides with a similar apparent affinity constant, but soluble class II molecules bound up to ten times more peptide than DR4Dw4 containing a transmembrane region. Peptide binding kinetics for soluble DR4Dw4 molecules were 10‐20 times faster than for the other two forms of DR4Dw4 molecules. Finally, soluble PI‐DR4Dw4/peptide complexes were shown to stimulate T cell proliferation.
Inhibition of activation‐induced changes in the structure of the T cell interleukin‐7 receptor by cyclosporin A and FK506
Authors: Foxwell Brian M. J. , L. Willcocks Joanie , Taylor‐Fishwick David A. , Kulig Kimary , Ryffel Bernhard , Londei Marco
Published: March 2007

We have recently shown that activation of T cells causes structural changes in the interleukin‐7 receptor (IL‐7R) (Foxwell et al. Int. Immunol. 1992. 4: 277). Unactivated cells expressed a receptor characterized as a cross‐linked protein of 107‐kDa whereas activated cells had reduced levels of this 107‐kDa complex and now express a major cross‐linked product of 93 kDa. These changes in receptor expression were concomitant with the acquisition of IL‐7 growth responsiveness by activated T cells. In this study, the effect of the potent immunosuppressive agents cyclosporin A and FK506 on the activation‐induced responsiveness to IL‐7‐driven proliferation and the concomitant changes in receptor structure have been investigated. Cyclosporin A and FK506 suppressed the expression of the 93‐kDa complex and the loss of the 107‐kDa complex on activated cells. The presence of exogenous IL‐7 inhibited the effects of the drugs on IL‐7R structure, allowing expression of the 93‐kDa complex. Expression of the 93‐kDa complex could also be induced either by ionomycin or phorbol esters. As observed for other T cell activation parameters, only those which induced a calcium signal (ionomycin) but not protein kinase C (phorbol esters) were sensitive to the drugs. In all studies, the expression of the 93‐kDa complex correlated with the ability of cells to proliferate to IL‐7, and thus these results further support the hypothesis that the 93‐kDa form of the IL‐7R is required to transmit the cytokine's growth signal. Moreover, these data suggest that activation‐induced transcriptional events are required for the expression of the 93‐kDa complex and the down‐regulation of the 107‐kDa complex. As reported for IL‐2R and IL‐4R, our data also show that the expression of another T cell growth factor receptor is sensitive to the effects of cyclosporin A and FK506. These observations also have important implications for reported cyclosporin A effects on the thymus where IL‐7 can act as a growth factor for thymocytes.
Rapid re‐expression of CD45RC on rat CD4 T cells in vitro correlates with a change in function
Authors: Sarawar Sally R. , Sparshott Sheila M. , Sutton Philip , Yang Chun‐Ping , Hutchinson Ian V. , Bell Eric B.
Published: March 2007

Rat CD4+ T cells are divided phenotypically by the anti‐CD45RC monoclonal antibody OX22 into subsets with contrasting functions. Stimulation of T cells in vitro is known to induce a change in isoform from CD45RC+ to CD45RC−. We have investigated the in vitro conditions which promote a switch in isoform in the opposite direction. We observed that a majority of CD45RC− CD4 T cells (> 90%) spontaneously re‐expressed CD45RC during the first 1‐3 days of culture in both the presence and absence of alloantigen. The T cells remained CD45RC+ when cultured for 7 days in serum‐free growth medium. However, alloantigen‐activated lymphocytes, expressing the interleukin‐2 receptor (IL‐2R), down‐regulated CD45RC by day 4 and remained CD45RC− during the course of the experiment. Using mixtures of allotype‐marked CD45RC+ and CD45RC−T cells, it was demonstrated that each subset showed comparable survival, IL‐2R expression and time courses of activation in response to alloantigen. The repertoire of neither subset was, therefore, deficient in terms of allorecognition. The rapid re‐expression of CD45RC in culture was accompanied by a change in function: CD45RC+ “converts”, obtained by overnight culture of CD45RC− T cells, induced significantly higher graft‐versus‐host responses. Thus, the transition in culture from CD45RC− to CD45RC+ reflects a major functional reprogramming of the cell and not a trivial modulation of a surface antigen.
Protein tyrosine kinases couple the interleukin‐2 receptor to p21ras
Authors: Izquierdo Manuel , Cantrell Doreen A.
Published: March 2007

The T cell growth factor interleukin‐2 (IL‐2) induces p21ras activation in T lymphocytes. We have previously shown that a protein kinase C (PKC)‐mediated pathway for p21ras regulation exists in T cells and that the IL‐2 receptor (IL‐2R) can couple to p21ras independently of the presence of the PKC pathway for p21ras regulation. Our data show that in conditions where cellular protein tyrosine kinases (PTK) were efficiently down‐regulated by pretreatment with the specific PTK inhibitor herbimycin, the IL‐2‐induced activation of p21ras was blocked. Herbimycin did not inhibit the PKC‐mediated pathway for p21ras regulation. Thus, the data indicate that PTK are involved in the coupling of the IL‐2R to p21ras.
Phosphorylation of surface E‐selectin and the effect of soluble ligand (Sialyl Lewisx) on the half‐life of E‐selectin
Authors: Smeets Edgar F. , De Vries Theodora , Leeuwenberg Jet F. M. , van den Eijnden Dirk H. , Buurman Wim A. , Neefjes Jacques J.
Published: March 2007

E‐selectin (ELAM‐1) is an adhesion molecule for leukocytes that is transiently expressed on endothelial cells. Following cell surface expression of E‐selectin on human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor, the induced E‐selectin molecules are rapidly degraded. The kinetics of turnover of surface disposed E‐selectin were investigated. The rapid disappearance of surface E‐selectin is temperature dependent and sensitive to the lysosomotropic agent chloroquine. The half‐life of E‐selectin is not affected by inclusion of soluble sialyl Lewis x (sLex) ligands in the medium. Surface E‐selectin is phosphorylated on one or more serine residues, but this modification is not obviously related to internalization.
Regulation of T helper cell cytokine expression: functional dichotomy of antigen‐presenting cells
Authors: Schmitz Jürgen , Assenmacher Mario , Radbruch Andreas
Published: March 2007

A bias to either cell‐mediated or antibody‐mediated effector mechanisms is induced in an immune response against a pathogen, if activated T helper cells (Th) predominantly express Th1 [interleukin (IL)‐2, interferon (IFN)‐γ, and tumor necrosis factor (TNF)‐β] or Th2 (IL‐4, IL‐5, IL‐6 and IL‐10) cytokines. Here we provide evidence that, due to the capability to secrete IL‐1, macrophages, but not B cells, as antigen‐presenting cells (APC) induce production of IFN‐γ in resting Th cells. Normal murine splenic Th cells were activated in vitro with the superantigen Staphylococcus aureus enterotoxin B (SEB) presented by macrophages as compared to other APC from murine spleen. As determined by immunofluorescence, Th cells producing IL‐2 but almost none producing IL‐4 and IL‐5 are generated, irrespective of the type of APC. Generation of IFN‐γ‐producing Th cells is largely dependent on presentation of SEB by macrophages. The requirement for macrophages, however, is overcome if IL‐1 is provided. Expression of IFN‐y by Th cells is not induced, if production of IL‐1 by macrophages is inhibited by IL‐10. Our results suggest a functional dichotomy of APC: normal resting Th cells differentiate into IL‐2 and IFN‐γ secreting cells (Th1 cells) if antigen is presented by macrophages, whereas presentation by B cells generates Th cells secreting IL‐2, which might differentiate into Th2 cells upon re‐stimulation.
Human IgE mediates stimulus secretion coupling in rat basophilic leukemia cells transfected with the α chain of the human high‐affinity receptor
Authors: Wilson A. Penelope M. , Pullar Christine E. , Camp Andrew M. , Helm Birgit A.
Published: March 2007

Cell surface expression of the a chain of the human (h) Fee receptor type 1 (FceR1) can be observed following stable transfection of RBL‐2H3 cells with the ha chain of the FceR1 complex. Association and dissociation constants for hIgE are similar to those previously reported for the ligand and its receptor. The demonstration of mediator release following the cross‐linking of hFcϵR1α by hIgE and antigen or anti‐hFcϵR1α antibody suggests that this system will assist the identification of structural motifs and specific amino acids that are involved in the generation of the signal(s) that will initiate and/or propagate the secretion of mediators from mast cells and basophils.
The involvement of CD14 in stimulation of cytokine production by uronic acid polymers
Authors: Espevik Terje , Otterlei Mark , Skjåk‐Braek Gudmund , Ryan Liv , Wright Samuel D. , Sundan Anders
Published: March 2007

In this study the molecular mechanisms behind the stimulatory activities of the uronic acid polymers poly mannuronic acid (poly M), high M alginate and oxidized cellulose (C60XY) were investigated and compared with lipopolysaccharide (LPS). The cytokine‐inducing abilities of the uronic acid polymers and LPS were examined on CD14‐positive human monocytes and CD14‐negative U373 astrocytoma cells. It was found that LPS induced monocytes and U373 cells to produce tumor necrosis factor (TNF) and interleukin(IL)‐6, respectively, by different mechanisms. The poly uronic acids induced monocytes to produce TNF, but with 100‐1000 times less potency compared to LPS. On U373 cells, LPS at concentrations ? 32 ng/ml resulted in a dose‐related IL‐6 production, whereas the poly uronic acids had negligible effects even at 1 mg/ml. The binding data demonstrate that only the CD14‐positive monocytes in the peripheral blood mononuclear cells population bound poly M. Furthermore, poly M was found to bind to CD14 in the presence of serum. Antibodies against CD14 also inhibited the TNF‐inducing activity of the three uronic acid polymers tested. In conclusion, these results demonstrate that uronic acid polymers induce TNF production through mechanisms which involve CD 14.
Neutrophil attractant/activation protein‐1 (interleukin‐8) induces in vitro neutrophil migration by haptotactic mechanism
Authors: Rot Antal
Published: March 2007

The role of leukocyte migration induced by the substrate‐bound attractants is obscured by the inability of standard methods for the study of leukocyte migration to dissociate chemotaxis and haptotaxis, migration in response to soluble and surface‐bound gradients of attractants. Here we show that the gradient of neutrophil attractant/activation protein‐1 (interleukin‐8, NAP‐1/ IL‐8) induces directed in vitro neutrophil migration when bound to the polycarbonate filter. In addition, we suggest that haptotaxis is responsible for neutrophil migration induced by NAP‐l/IL‐8 in standard Boyden‐type chemotaxis assays and, in light of the ability of NAP‐l/IL‐8 to bind to the endothelial cell surface and the extracellular matrix, could also be of great significance in vivo.
Differential cytokine expression in acute and chronic murine graft‐versus‐host‐disease
Authors: Allen Ruth D. , Staley Tabitha A. , Sidman Charles L.
Published: March 2007

The relationship between acute and chronic graft ‐versus‐host disease (GVHD) is not well understood. While both syndromes appear to result from recognition of host antigens by donor T cells, their pathological changes differ markedly. In light of the recent concept that helper T cells (Th) may be divided into two types based on their cytokine secretion profile and their ability to mediate cellular (Th1) or humoral (Th2) immunity, and considering the inflammatory nature of acute GVHD and the occurrence of significant B cell activation in chronic GVHD, we hypothesized that acute and chronic GVHD may be associated with differential cytokine production by activated T cells. To evaluate this hypothesis, we assessed expression of a range of cytokines in (C57BL/6 × DBA/2 )F1 (B6D2F1) recipients of C57BL/6 (acute GVHD), DBA/2 (chronic GVHD) or B6D2F1 (control) spleen cells. The results reported here indicate that a wide range of cytokines, including interleukin (IL)‐4, IL‐10, interferon‐γ, tumor necrosis factor β and macrophage inflammatory protein‐1α, are indeed differentially expressed in acute and chronic GVHD and support the concept that the pathology peculiar to acute or chronic GVHD may arise due to differential cytokine expression by activated Tcells.
Cross‐reactivity among evolutionarily distant major histocompatibility complex class I molecules (HLA‐B27 and H‐2Kk) revealed by xenoreactive T lymphocytes
Authors: Frangoulis Bernard , Reboul Murielle , Rocca Anna , Pla Marika
Published: March 2007

A set of mouse HLA‐B27‐reactive cytotoxic T lymphocyte clones were found to recognize the HLA‐B27 molecule in an H‐2‐unrestricted manner, i.e. independently of any mouse major histocompatibility complex (MHC) molecule. The reactivity patterns of these clones on HLA‐B27 variants (positive only on HLA‐B*2702 and HLA‐B*2701) allowed the identification of residues N77 and A81 of the HLA‐B27 molecule as important for their reactivity. The location of these residues in the peptide‐binding groove (specificity pocket F) suggested that the reactivity of the clones is dependent on HLA‐B27‐bound peptide(s). However, several other class I molecules sharing these residues (N77 and A81) were not recognized, indicating that other residues might also be involved. One of the clones was found to display an interesting cross‐reactivity with allogeneic H‐2Kk molecules, sharing N77 and A81 with HLA‐B*2702. Sequence comparison suggested the involvement of residue H9, located in specificity pocket B of the peptide‐binding groove, and revealed some similarity of pockets B in HLA‐B27 and H‐2Kk. The structural basis of such T cell‐mediated MHC cross‐reactions across species barriers is discussed.
Selective engraftment of memory CD4+ T cells with an unusual recirculation pattern and a diverse T cell receptor‐Vβ repertoire into scid mice
Authors: Reimann Jörg , Rudoiphi Angelika , Tscherning Thomas , Claesson Mogens H.
Published: March 2007

Young (H‐2d, Ld+) severe combined immunodeficiency (scid) mice were injected intravenously with 105 CD4+CD8− T cells purified from spleen, thymus or lymph nodes (LN) of dm2 (H‐2d, Ld ) donor mice. In the immunodeficient recipients, the lymphoid compartment in the splenic white pulp was repopulated with donor‐type T cells and cellularity in the red pulp was increased. In addition, donor‐type CD4+ T cells repopulated the peritoneal cavity, mesenteric LN and the lamina propria of the small intestine of scid mice, but were undetectable in thymus and peripheral (inguinal, axillary) LN. Histological examination of repopulated mesenteric LN showed expanded subcapsular sinuses, repopulated cortical areas, but poorly developed high endothelial venules (HEV) indicating deficient blood‐LN lymphocyte recirculation. The engrafted CD4+ T cell population had the surface phenotype of memory T cells (CD44/Pgp‐lhigh CD45RBlow) and expressed the Peyer's patch HEV‐specific homing receptor CD49d (LPAM‐1), but not the LN HEV‐specific homing receptor LECAM‐l. The CD4+ T cell population in spleen and mesenteric LN of transplanted scid mice displayed a diverse T cell receptor‐V(3 repertoire. Transfer of titrated numbers (103, 104, 105 cells per mouse) of CD4+ T cells into scid mice established donor‐type T cell populations with this unusual homing pattern in all recipients. Repeated serial transfers of dm2 CD4+ T cells through young scid mice revealed an extensive in vivo expansion potential of transferred cells for > 18 months. The experimental system described represents an in vivo model to study the functional competence and the differentiation potential of a murine memory CD4+ T cell subset.
A human myeloma IgA with a hybrid heavy chain resulting from putative somatic gene conversion
Authors: Silvain Christine , Aucouturier Pierre , Leduc Isabelle , Mihaesco Edith , Homme Jean‐Louis Preud' , Cogné Michel
Published: March 2007

A human monoclonal IgA1‐IgA2λ. hybrid molecule was detected in a myeloma patient homozygous for the A2m(1) allotype during a systematic study of monoclonal IgA with subclass‐specific monoclonal antibodies (mAb) and the lectin jacalin. This monoclonal immunoglobulin (GAU) reacted with both, although not with all, anti‐α1 and anti‐α2 mAb. Its heavy (H) chain contained an al hinge region as shown by jacalin reactivity, the presence of disulfide‐linked H and light chains in spite of its belonging to the IgA2m(1) allotype and amino acid composition of the isolated hinge region. The complete sequence of the H chain was deduced from that of complementary DNA clones from bone marrow cells. The CHI domain, hinge region and beginning of the CH2 domain and the membrane peptide were encoded by the al gene, with an insertion of an α2m(1) gene sequence accounting for the end of the CH2 and part or all of the CH3 domain (sequence identity between the two normal genes precludes a precise definition of breakpoints). The region of the 5′ recombination site included a repeat of a six base pair sequence which might play a role in the genetic exchange. The GAU hybrid a gene was undetectable in the patient's genomic DNA from polymorphonuclear cells. The genetic event which occurred at the level of the proliferating plasma cell clone is most likely to be a gene conversion.
Cross‐reactions of anti‐DNA autoantibodies with cell surface proteins
Authors: Raz Eyal , Ben‐Bassat Hanna , Davidi Talia , Shlomai Zipora , Eilat Dan
Published: March 2007

Anti‐DNA autoantibodies are thought to play a major role in the pathogenesis of systemic lupus erythematosus. However, the mechanism(s) by which they participate in tissue and organ damage is not well understood. It has been suggested that these antibodies combine with DNA or DNA‐histone complexes to produce circulating immune complexes which may deposit in various tissues. Alternatively, anti‐DNA autoantibodies could interact directly with tissue components by way of immunological cross‐reaction.

In this study we have used a panel of mouse monoclonal autoantibodies with anti‐nuclear specificity and measured their binding to membrane proteins of several tissues and cell lines. We show that the anti‐DNA antibodies, but not anti‐RNA or anti‐histone antibodies bind to membrane proteins of molecular weights 102,80,42,35 and 31 kDa, which are expressed in different combinations on several cell types. The binding of anti‐DNA antibodies to these cell surface proteins was not affected by DNase treatment of the target cells, was increased by DNase treatment of the antibody preparations and was completely inhibited by DNA, indicating a true cross‐reaction and not an indirect interaction of antibody and membrane proteins through a DNA bridge.

Our results suggest that direct binding of anti‐DNA autoantibodies to cell surface membrane proteins may play an important role in the induction of the pleomorphic tissue damage in systemic lupus erythematosus.
Vχ gene segments rearranged in chronic lymphocytic leukemia are distributed over a large portion of the Vχ locus and do not show somatic mutation
Authors: Wagner Simon D. , Luzzatto Lucio
Published: March 2007

The structure of the human Vχ locus has been thoroughly investigated, but how the germ‐line Vχ gene segment repertoire is actually sampled in χ chain gene rearrangements is not known. In order to begin to answer this question we have polymerase chain reaction (PCR) amplified the rearranged Vχ genes from 26χ‐expressing cases of chronic lymphocytic leukemia (CLL), followed by cloning and sequencing of the PCR product. All four Vχ gene families were represented amongst rearranged genes. In 25 out of 32 cases, the sequence of the rearranged gene matches perfectly that of 1 of 11 different known germ‐line Vχ genes, indicating that no somatic mutation has occurred. Of the remaining 7 rearranged Vχ genes, 4 differ from known germ‐line genes by only one or two amino acid residues; and 3 differ from each other and from all known sequences by 5 or more residues, suggesting that somatic mutation has occurred in these 3 cases. We conclude that: (a) in at least three‐squarters of cases the rearranged genes are unmutated; (b) there is preferential usage of individual Vχ genes but not of Vχ gene families; and (c) the Vχ genes used are widely dispersed in the Vχ locus.
Complement enhances the elimination of soluble aggregates of IgG by rat liver endothelial cells in vivo
Authors: Bogers Willy M. J. M. , Van Rooijen Nico , Janssen Daniëlle J. , Van Es Leendert A. , Daha Mohamed R.
Published: March 2007

In the present study, we have investigated the role of complement (C) and possible C receptors present on rat liver endothelial cells (EC) in the clearance and tissue distribution of soluble aggregates of IgG (AIgG). To study the effect of elimination of AIgG by EC in vivo, Kupffer cell (KC)‐depleted rats were used, with or without an intact C system (These rats will be referred to throughout this report as EC‐rats.) In EC‐rats with an intact C system, clearance of AIgG (2000‐3000 kDa, 20‐27 IgG molecules/aggregate) occurred in a biphasic manner with a first T½(T1)of 9.4 ± 2.3 min and a second T½(T2) of 44.7 ± 16.0 min. In EC‐rats without an intact C system [cobra venom factor (COVF)‐treated group], clearance of AIgG was significantly delayed with a T1 of 25.3 ± 9.9 min (p 0.005) and aT2 of 124.5 ± 18.4 min (p 0.001). There were less degradation products of AIgG in the circulation in EC‐rats treated with COVF as compared to EC‐rats with an intact C system. Eight minutes after injection, 27.5 ± 11.6% of the injected AIgG was found in the livers of EC‐rats while 15.1 ± 3.2% was found in the livers of the COVF‐treated group. Double immunofluorescence studies indicated that AIgG in the liver was associated with EC in the rats with an intact C system. Clear deposits of C3 and lesser amounts of Clq accompanied the deposition of AIgG. In COVF‐treated EC‐rats, AIgG together with Clq was also associated with EC but no detectable C3 was seen. These data suggest clearance of AIgG via Fc and C receptors present on EC in vivo.
Characterization of T cell receptor assembly and expression in a Ti γδ‐positive cell line
Authors: Kuhlmann Jan , Caspar‐Bauguil Sylvie , Geisler Carsten , Rubin Bent
Published: March 2007

Tcell antigen receptor (TcR) heterodimers of both the Ti‐αβ and Ti‐γδ types are expressed at the surface of T cells noncovalently associated with the CD3 complex composed of the monomorphic chains γ, δ, ε and ξ. The structural relationship and assembly of the various components of this multimeric protein complex is still not fully understood.

In this report, the human leukemic Tcell line Lyon which expresses a Ti‐γδ/CD3 complex, was characterized and compared to another human leukemic Tcell line Jurkat (Ti‐αβ/CD3). Membrane TCR−/CD3− variants of the Tcell Lyon were induced and found to produce all of the Ti/CD3 components, with the exception of Ti‐δ.

Biochemical analysis indicated that: (1) Ti‐γ/CD3γ, δ, ε complexes were formed in the endoplasmic reticulum in the absence of Ti‐δ; (2) the CD3‐ξ chain did not associate with the Ti‐γ/CD3γδε complex and (3) the Ti‐δ chain was required for cell surface expression of the Ti‐γδ/CD3 complex. Introduction of Jurkat wild‐type Ti‐α cDNA into Lyon T cells resulted in Ti‐αβ/CD3 expression and abrogated Ti‐γδ/CD3 expression. In contrast, the expression of the Ti‐γδ/CD3 complex was not affected by transfection of a mutated Ti‐α cDN A into Lyon cells. The mutated Ti‐α chain formed complexes with Ti‐β and CD3γδε, but the CD3‐ξ; chain did not associate with these complexes. Taken together analysis of Lyon cells transfected with either wild‐type or mutated Ti‐α suggested that the CD3‐ξ, chain may have higher affinity for Ti‐αβ/CD3 complexes than for Ti‐γδ/CD3 complexes.
T cell receptor heterogeneity in γδ T cell clones from intestinal biopsies of patients with celiac disease
Authors: De Libero Gennaro , Rocci Maria Paola , Casorati Giulia , Giachino Claudia , Oderda Giuseppina , Tavassoli Kaveth , Migone Nicola
Published: March 2007

In celiac disease large numbers of γδ T lymphocytes infiltrate the intestinal epithelia. We have isolated intestinal γδ T cell clones from patients with celiac disease and have analyzed their T cell receptor repertoire. T cell lines and clones were obtained from jejunal biopsies of 14 celiac patients and 12 individuals without celiac disease. These were analyzed by staining with monoclonal antibodies against CD3, αβ and γδ T cell receptor, by Southern blot with γ and δ specific probes and by polymerase chain reaction using Vδ‐specific oligonucleotides. Intestinal γδ cells from patients with celiac disease differed from those of controls with normal jejunal histology in that Vδ1+ cells and Vδ Vδ2 cells were significantly increased. There was no evidence of the expansion of one or more clones expressing particular types of γδ T cell receptor.