bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen‐specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection
Authors: Martin Stefan , Bevan Michael J.
Published: March 2007

Analysis of antigen‐induced negative selection of thymocytes in T cell receptor (TCR)‐transgenic mice is complicated by the presence of an antigen‐responsive peripheral T cell compartment. Our experiments address the question of whether and how peripheral T cell activation can affect immature thymocytes. Following three daily injections of peptide antigen into mice expressing a peptide‐specific transgenic TCR and deficient for TAP1, we and others have found profound deletion of the CD4+CD8+ (DP) thymocyte subset. However, our work shows that even though mature CD8+ T cells are inefficiently selected in TAP1‐deficient mice, there was a striking degree of peripheral expansion and activation of CD8+ peripheral T cells. Furthermore, when cells from TCR‐transgenic mice were adoptively transferred, we found that deletion of non‐transgenic DP thymocytes occurred in Thy‐1‐congenic and even more efficiently in TAP1‐deficient recipients after repeated peptide injection resulting in peripheral T cell activation. In the adoptive transfer experiments the degree of deletion of immature bystander thymocytes was decreased upon blocking of TNF. These data show that deletion of DP thymocytes can result from excessive peripheral T cell activation and identify TNF as an important effector molecule for this process. When steps are taken to avoid peripheral T cell activation, peptide antigen can induce TCR‐mediated thymocyte deletion, presumably in the thymus cortex, since injection of TAP1‐deficient TCR‐transgenic mice resulted in deletion of immature DP thymocytes prior to detectable peripheral T cell expansion and activation. This effect was not blocked by inhibiting tumor necrosis factor activity. In addition, DP depletion was seen in the absence of peripheral T cell activation when antibody‐mediated depletion of CD8+ T cells was performed. Our work clearly shows that two mechanisms for deletion of DP thymocytes exist: deletion induced by antigen presentation in the thymus and deletion as a consequence of repeated stimulation of mature peripheral T cells.
Susceptibility to hard metal lung disease is strongly associated with the presence of glutamate 69 in HLA‐DPβ chain
Authors: Potolicchio Ilaria , Mosconi Giovanni , Forni Alessandra , Nemery Benoit , Seghizzi Paolo , Sorrentino Rosa
Published: March 2007

Clinical, epidemiological and experimental data indicate that inhaled metal dust containing cobalt may produce an interstitial lung disease termed “hard metal disease” (HMD). Some aspects of this pathology such as the lack of correlation with dose exposure, the low frequency of the disease and the presence of T cells in the inflammation site, all suggest the existence of a genetic susceptibility, possibly to an immunological response to cobalt or to self proteins modified by cobalt. Here we report that HMD is strongly associated with residue Glu‐69 of the HLA‐DP beta chain. All patients, except for one with a rare genotype, possessed this marker as compared to 17 out of 35 exposed unaffected individuals (p = 0.0014). These data allow us to genetically distinguish a subgroup of cobalt‐exposed individuals at risk for HMD, independently from the more common allergic reaction.
Activation induces apoptosis in Herpesvirus saimiri‐transformed T cells independent of CD95 (Fas, APO‐1)
Authors: Bröker Barbara M. , Kraft Michael S. , Klauenberg Ulricke , Le Deist Françoise , de Villartay Jean‐Pierre , Fleckenstein Bernhard , Fleischer Bernhard , Meinl Edgar
Published: March 2007

Signaling via the T cell receptor (TCR)/CD3 complex of pre‐activated T cells induces apoptosis. Such an activation‐induced cell death (AICD) is thought to play an important role in the regulation of cellular immune responses. In this study we analyzed pathways of AICD by using human T cells transformed by Herpesvirus saimiri. These growth‐transformed T cells show the phenotype of activated mature T cells and continue to express a functionally intact TCR. We show that human H. saimiri‐transformed T cell clones readily undergo cell death upon signaling via the TCR/CD3 complex or via phorbol 12‐myristate 13‐acetate (PMA) + ionomycin. The AICD in H. saimiri‐transformed T cells was detectable a few hours after activation and it was not affected by the presence of interleukin (IL)‐2 or by anti‐CD4 cross‐linking. However, AICD required tyrosine phosphorylation, since it could be blocked by herbimycin A. Cyclosporin A (CsA) did not block the development of AICD, but other consequences of activation in H. saimiri‐transformed T cells like the production of interferon‐γ. Surprisingly, the development of AICD was not reduced by neutralizing antibodies to tumor necrosis factor (TNF)‐α or blocking antibodies directed to CD95 (Fas, APO‐1), although H. saimiri‐transformed T cells were sensitive to CD95 ligation. To confirm that this form of AICD is really independent of CD95, we have established an H. saimiri‐transformed T cell line from a patient with a homozygous deletion in the CD95 gene. This CD95‐deficient T cell line was as sensitive to AICD as other CD95‐expressing H. saimiri‐transformed T cells. In conclusion, we describe here a type of AICD in H. saimiri‐transformed T cells that is independent of CD95 and TNF‐α, not sensitive to CsA, but requires tyrosine phosphorylation. This system should be useful for the investigation of CD95‐independent forms of AICD.
Extracellular HIV‐1 Tat protein activates phosphatidylinositol 3‐ and Akt/PKB kinases in CD4+ T lymphoblastoid Jurkat cells
Authors: Borgatti Paola , Zauli Giorgio , Colamussi Maria Luisa , Gibellini Davide , Previati Maurizio , Cantley Lewis L. , Capitani Silvano
Published: March 2007

The biological basis for the pleiotropic activity of extracellular human immunodeficiency virus (HIV)‐1 Tat protein on lymphoid T cell survival is not well understood. We have here demonstrated that the addition in culture of 0.1–10 nM Tat protein to 36‐h serum‐starved lymphoblastoid Jurkat T cells rapidly stimulates the catalytic activity of phosphatidylinositol 3‐kinase (PI 3‐K). The peak of activation was observed 30 min after Tat addition. Extracellular Tat also stimulated the catalytic activity of the Akt/PKB kinase, a major target of PI 3‐K lipid products. Pretreatment of serum‐starved Jurkat cells with 100 nM wortmannin (WT) or 10 μM LY294002, two unrelated pharmacological inhibitors of PI 3‐K, markedly suppressed the catalytic activity of both PI 3‐K and Akt/PKB in Jurkat cells. Moreover, at low concentrations (0.1–1 nM), extracellular Tat showed a small but reproducible protection of Jurkat cells from apoptosis induced by serum deprivation (p < 0.05), while the combination of Tat plus 100 nM WT significantly (p < 0.05) increased the percentage of apoptosis with respect to cells left untreated or treated with Tat alone. Taken together, these data suggest that the anti‐apoptotic activity of low concentrations of Tat protein on Jurkat cells is mediated by a PI 3‐kinase/Akt pathway.
The central nervous system environment controls effector CD4+ T cell cytokine profile in experimental allergic encephalomyelitis
Authors: Krakowski Michelle L. , Owens Trevor
Published: March 2007

In experimental allergic encephalomyelitis (EAE), CD4+ T cells infiltrate the central nervous system (CNS). We derived CD4+ T cell lines from SJL/J mice that were specific for encephalitogenic myelin basic protein (MBP) peptides and produced both Th1 and Th2 cytokines. These lines transferred EAE to naive mice. Peptide‐specific cells re‐isolated from the CNS only produced Th1 cytokines, whereas T cells in the lymph nodes produced both Th1 and Th2 cytokines. Mononuclear cells isolated from the CNS, the majority of which were microglia, presented antigen to and stimulated MBP‐specific T cell lines in vitro. Although CNS antigen‐presenting cells (APC) supported increased production of interferon (IFN)‐γ mRNA by these T cells, there was no increase in the interleukin (IL)‐4 signal, whereas splenic APC induced increases in both IFN‐γ and IL‐4. mRNA for IL‐12 (p40 subunit) was up‐regulated in both infiltrating macrophages and resident microglia from mice with EAE. We have thus shown that a Th1 cytokine bias within the CNS can be induced by CNS APC, and that IL‐12 is up‐regulated in microglial cells within the CNS of mice with EAE. Microglia may therefore control Th1 cytokine responses within the CNS.
Nitric oxide and the immunomodulation of experimental allergic encephalomyelitis
Authors: Gold Daniel P. , Schroder Kim , Powell Henry C. , Kelly Carolyn J.
Published: March 2007

Previous studies examining the effect of nitric oxide synthase (NOS) inhibition on the course of experimental allergic encephalomyelitis (EAE) have yielded conflicting results. This may relate to the use of nonspecific inhibitors and to differences between active and adoptive EAE. We examined the effect of treatment with L‐N‐(1‐iminoethyl)lysine (L‐NIL), a selective inhibitor of the cytokine‐inducible isoform of NOS, on the clinical course of active and adoptive EAE in Lewis rats. We find that while L‐NIL treatment of recipients is protective in adoptive EAE, treatment of active EAE with L‐NIL leads to a marked accentuation of disease expression. In L‐NIL‐treated animals treated with myelin basic protein/complete Freund's adjuvant (MBP/CFA), disease onset is accelerated and clinical symptoms are more severe. Accentuation of integrated disease scores is seen even if L‐NIL treatment is started 5 days following immunization. The histological findings in involved spinal cords from L‐NIL‐treated animals with active EAE are similar to those from untreated animals with similar clinical scores. L‐NIL treatment of MBP/CFA‐immunized animals does not prevent recovery from clinical symptoms, nor does it allow for reinduction of disease in animals previously immunized with MBP/CFA. Treatment of F344 rats, a strain which is relatively nonsusceptible for EAE, with L‐NIL results in consistent evidence of EAE following immunization with MBP/CFA. These findings, together with our previous work on interstitial nephritis, support a role for endogenously generated NO in immunoregulation of T cell responses following immunization with antigen in CFA, and suggest that inducibility of NOS expression may be an important susceptibility factor for autoimmunity.
Expression of ε germ‐line gene transcripts and mRNA for the ε heavy chain of IgE in nasal B cells and the effects of topical corticosteroid
Authors: Durham Stephen R. , Gould Hannah J. , Thienes Cortlandt P. , Jacobson Mikila R. , Masuyama Keisuke , Rak Sabina , Lowhagen Olle , Schotman Elsa , Cameron Lisa , Hamid Qutayba A.
Published: March 2007

We have studied the expression of the gene encoding the ε heavy chain of IgE in nasal B cells of hayfever patients. We developed probes to detect transcripts of the ε germ‐line gene and the rearranged gene by in situ hybridization of biopsy sections from the nasal mucosa. We compared tissue from hayfever patients out of season with that of normal controls, and also of hayfever patients treated with topical corticosteroid (fluticasone propionate) or placebo for 6 weeks and then challenged with antigen. ε chain mRNA was expressed in an unexpectedly high proportion of nasal B cells of both hayfever patients and normal subjects. However, although similar numbers of B cells were found in both groups, the proportion of cells that express ε chain mRNA was several times higher in the hayfever patients. No transcripts of the ε germ‐line gene were detected in either group before allergen challenge. When hayfever patients were administered antigen locally, early (10–30 min) and late (1–24 h) symptoms ensued. After 24 h, coincident with an increase in the number of cells expressing mRNA for IL‐4 in the tissue, ε germ‐line gene transcripts appeared in the nasal B cells. The induction by allergen of IL‐4 mRNA and ε germ‐line gene transcripts was suppressed by fluticasone propionate treatment. Our results suggest that local IgE synthesis and cytokine regulation of heavy chain switching to IgE occur in the nasal mucosa.
Nitric oxide pathway is induced by FcεRI and up‐regulated by stem cell factor in mouse mast cells
Authors: Bidri Mohamed , Ktorza Sandra , Vouldoukis Ioannis , Le Goff Liliane , Debré Patrice , Guillosson Jean‐Jacques , Arock Michel
Published: March 2007

Murine stem cell factor (SCF) induces the differentiation of mucosal mast cells (MMC) into connective tissue mast cells (CTMC) and potentiates mediator release induced by aggregation of high‐affinity IgE receptors (FcεRI). In the present work, we investigated the effect of FcεRI aggregation on nitric oxide (NO) pathway induction in the different subsets of mast cells, as well as the contribution of SCF in this induction. Inducible NO synthase (iNOs) expression was not evidenced in non‐stimulated MMC obtained by culture of hematopoietic progenitors in the presence of interleukin‐3, whereas IgE‐antigen‐stimulated MMC expressed iNOs mRNA and protein and synthesized nitrites. Long‐term treatment of MMC with SCF, allowing them to differentiate into CTMC, induced iNOs expression in non‐stimulated cells and up‐regulated iNOs expression and generation of NO derivatives induced by IgE‐antigen stimulation. Thus, NO derivatives generated by mast cells could participate in inflammatory reactions during allergic stimulation.
Expression and function of NKRP1A molecule on human monocytes and dendritic cells
Authors: Poggi Alessandro , Rubartelli Anna , Moretta Lorenzo , Zocchi Maria Raffaella
Published: March 2007

In this study, we analyzed the expression and function of the lymphocyte surface lectin NKRP1A on peripheral blood monocytes (Mo) or Mo and dendritic cells (DC) derived from thymic and bone marrow precursors. De novo expression of NKRP1A and CD14 molecules was detected upon culture of CD2− CD3− CD14− CD16− CD1a− NKRP1A− immature thymic precursors for 7 days in the presence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Under these culture conditions, by day 21, a fraction of cells had lost CD14 and acquired both CD80 (B7.1) and CD86 (B7.2) molecules. These cells displayed a DC‐like morphology and were surface NKRP1A positive. CD34+ NKRP1A− CD14− precursors, isolated from bone marrow and cultured in the presence of GM‐CSF, also expressed both NKRP1A and CD14: these antigens were newly expressed on about one third of cells which had lost the CD34 precursor marker. In addition, NKRP1A was constitutively present on resting CD14+ peripheral blood Mo. When these cells were cultured in the presence of GM‐CSF, the resulting DC population retained the expression of NKRP1A and acquired CD80, while they lost the CD14 antigen. Functional analysis revealed that the engagement of NKRP1A molecule leads to a strong intracellular calcium ([Ca2+]i) increase both in resting peripheral blood Mo and in vitro‐derived DC. [Ca2+]i increase was mainly due to extracellular calcium influx, as it was completely abrogated by the addition of EGTA. More importantly, the engagement of the NKRP1A molecule induced interleukin (IL)‐1β and IL‐12 production by resting Mo and DC, respectively. Altogether these data indicate that NKRP1A lectin is present at the surface of Mo and DC and may play a relevant role in the activation and function of both cell types.
Analysis of the tyrosine phosphorylation and calcium fluxing of human CD6 isoforms with different cytoplasmatic domains
Authors: Kobarg Jörg , Whitney Gena S. , Palmer Dawn , Aruffo Alejandro , Bowen Michael A.
Published: March 2007

CD6 is a cell surface glycoprotein that functions both as a co‐stimulatory and adhesion receptor on T cells. Recently we have described CD6 isoforms (CD6a, b, c, d, e) that arise via alternative splicing of exons encoding the cytoplasmic region of the molecule. CD6 becomes phosphorylated on tyrosine (Tyr) residues following stimulation through the T cell receptor (TCR) complex. Since the phosphorylation of Tyr residues renders some cell surface receptors competent for interactions with proteins of intracellular signaling pathways, we wanted to determine which region(s) and residues in the cytoplasmic domain of CD6 were important for phosphorylation on Tyr residues. We engineered and stably expressed chimeric receptors that consisted of the extracellular region of mouse CD6 and the cytoplasmic regions of either naturally occurring isoforms of human CD6, truncated proteins, or point mutants. We were able to demonstrate that of the nine Tyr residues in the cytoplasmic domain of the largest isoform CD6a, the two C‐terminal Tyr residues (Tyr 629/662) are critical for the phosphorylation of CD6 following TCR cross‐linking. Isoform CD6e, which is missing a region that contains two proline‐rich motifs, is not phosphorylated. We further analyzed the ability of the different CD6 isoforms and truncated receptors to mobilize intracellular calcium after CD6/TCR co‐ligation. All CD6 isoforms, including CD6e, as well as the truncation mutant Δ 555, which is missing approximately the C‐terminal half of the cytoplasmic domain, are able to increase Ca2+ influx. Taken together, these results suggest that the region of CD6 which is critical for Ca2+ mobilization is located N‐terminal from amino acid 555 and is therefore different from the region located at the C terminus of CD6, which is necessary for tyrosine phosphorylation.
Identification of a homolog of the Cα3′/hs3 enhancer and of an allelic variant of the 3′IgH/hs1,2 enhancer downstream the human immunoglobulin α1 gene
Authors: Pinaud Eric , Aupetit Corinne , Chauveau Christine , Cogné Michel
Published: March 2007

Although four regulatory elements are known downstream the mouse IgH α gene, a single enhancer homologous to hs1,2 has been thus far described downstream each human α gene (Chen, C. and Birshtein, B. K., J. Immunol. 1997. 159: 1310). We characterized a 10‐kb region downstream the human α1 gene. Two B cell‐specific regulatory elements homologous to the murine Cα3′/hs3 and hs1,2 3′ enhancers were found, which are duplicated downstream α2. The hs1,2 element is in inverted orientation by comparison with a recently reported α1 hs1,2 element: it appears as a common allelic variant carrying an internal tandem repeat insertion and its prevalence in the human population is 60%. As in the mouse, the human hs1,2 enhancer is flanked with long inverted repeats which may have promoted inversion events through homologous recombination. Although the palindromic organization of the region is maintained in human, sequence identity with rodents focuses on core enhancer elements rather than on flanking repeats. Concerted divergence of both sides of the dyad symmetry suggests that inverted repeats are not just evolutionary remnants but rather play an architectural role in the LCR function.
Conservation of a master hematopoietic switch gene during vertebrate evolution: Isolation and characterization of Ikaros from teleost and amphibian species
Authors: Hansen John D. , Strassburger Pamela , Du Pasquier Louis
Published: March 2007

The generation of T, B and NK lymphocyte lineages from pluripotent hematopoietic stem cells is dependent upon the early expression of the Ikaros locus which by means of alternative splicing produces a variety of zinc finger DNA binding transcription factors. We assessed the general biological importance of Ikaros by studying its conservation and expression in teleost fish and amphibians. Portions of Ikaros cDNA from rainbow trout and Xenopus were amplified by reverse transcription‐polymerase chain reaction (RT‐PCR). They show roughly 75% conservation of the amino acid sequence with mammalian Ikaros. The trout fragment was then used to isolate full‐length Ikaros clones from a trout thymocyte cDNA library. In mice and humans, Ikaros produces six alternatively spliced isoforms, but in trout two additional novel splice variants designated Ik‐7 and Ik‐8 were also found. Ik‐7 is expressed in a similar fashion to Ik‐1 and Ik‐2, the predominant isoforms expressed in mammalian lymphocytes. In trout and zebrafish, as in mammals, Ikaros is a single‐copy gene, but in Xenopus segregation analysis demonstrates that Ikaros has been duplicated, most likely a result of polyploidization. We then examined the expression of Ikaros in trout and Xenopus tumor T cell lines via Northern blot, RT‐PCR, immunofluorescence and Western blot analysis. Overall, Ikaros is expressed in a lymphoid‐specific fashion similar to that found in mice and humans. In addition Ikaros is expressed early in trout ontogeny, beginning roughly at days 3–4 in the yolk sac and at day 5–6 in the embryo proper. The conservation of Ikaros structure and expression confirms it as a master switch of hematopoiesis.
Pro‐inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum‐free conditions
Authors: Jonuleit Helmut , Kühn Ulrich , Müller Gabriele , Steinbrink Kerstin , Paragnik Lydia , Schmitt Edgar , Knop Jürgen , Enk Alexander H.
Published: March 2007

Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte‐macrophage colony‐stimulating factor and interleukin (IL)‐4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte‐conditioned medium (MCM) and FCS‐free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)‐α, IL‐1β and IL‐6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF‐α/IL‐1/IL‐6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)‐γ and no IL‐4 or IL‐10. Production of IFN‐γ was significantly induced by addition of PGE2, while no effect on production of IL‐4 or IL‐10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN‐γlow, IL‐4neg cells, TNF‐α/IL‐1/IL‐6 promoted growth of IFN‐γintermediate, IL‐4neg CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN‐γ production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF‐α/IL‐1/IL‐6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF‐α/IL‐1, IL‐6 + PGE2‐cultured DC seem to be optimal for generation of IFN‐γ‐producing CD4/CD8+ T cells.
Antigen presentation by T cells: T cell receptor ligation promotes antigen acquisition from professional antigen‐presenting cells
Authors: Arnold Paula Y. , Davidian Daniel K. , Mannie Mark D.
Published: March 2007

The purpose of this study was to determine whether the clonotypic specificity of the T cell receptor influences the specificity of T cell‐mediated antigen presentation. We have previously shown that myelin basic protein (MBP)‐specific Lewis rat GP2.E5/R1 (R1) T cells cultured with antigen, irradiated syngeneic splenocytes (IrrSPL) and tolerogenic monoclonal antibody become highly effective antigen‐presenting cells (APC). In the current studies, we investigated the transfer of specific (MBP) and unrelated (conalbumin) antigens from antigen‐pulsed SPL to R1 T cells. R1 T cells cultured with IrrSPL that were pulsed simultaneously with both MBP and conalbumin acquired and presented both antigens to the appropriate T cell responders in a secondary assay. These results suggested a physical transfer of major histocompatibility complex (MHC)/peptide complexes from professional APC to R1 T cells. Transfer of conalbumin from professional APC to R1 T cells required specific recognition of MBP and was optimal when both conalbumin and MBP were presented on the same group of professional APC. Antigens transfer did not occur when allogeneic SPL were used as APC. The anti‐I‐A mAb OX6 inhibited antigen transfer but only when added during the initiation of culture. OX6 also inhibited antigen acquisition by R1‐trans, a variant of the R1 T cell line which constitutively synthesizes high levels of I‐A, from MBP‐pulsed IrrSPL but blockade of I‐A did not inhibit antigen acquisition when soluble MBP was added directly to the culture. Despite constitutive synthesis of I‐A, R1‐trans T cells did not acquire guinea pig MBP from pulsed allogeneic APC. These studies demonstrate that although T cells of a particular specificity can present unrelated antigens, the cognate interaction of the T cell antigen receptor with the appropriate antigen/self‐MHC complex strongly promotes acquisition of these complexes from professional APC.
The minimal number of antigen‐major histocompatibility complex class II complexes required for activation of naive and primed T cells
Authors: Kimachi Kazuhiko , Croft Michael , Grey Howard M.
Published: March 2007

Although previous studies have shown that 50–200 antigen‐major histocompatibility complex complexes (Ag‐MHC) are sufficient to stimulate significant secretion of interleukin (IL)‐2 from MHC class II‐restricted T cell hybridomas, there have been no studies of this nature on more physiologically relevant T cell populations. In this study we have analyzed the ligand requirements for stimulation of responses from naive and previously primed T cells derived from T cell receptor (TCR)‐transgenic animals whose TCR is specific for the pigeon cytochrome c (PCC) 88–104 peptide presented by I‐Ek. Primed T cells were as sensitive as the previously reported T cell hybridomas, requiring about 100 Ag‐MHC complexes to synthesize readily detectable quantities of IL‐2, whereas naive T cells required 15 times more ligand to produce equivalent quantities of IL‐2. Similarly, primed T cells required about 40 Ag‐MHC complexes to produce a significant proliferative response, whereas naive T cells required about 400 complexes. In contrast to these results, naive and primed T cells showed similar ligand requirements when early events in the T cell activation pathway were analyzed; i.e. TCR down‐modulation, CD69 and CD25 expression, and blast transformation. A further analysis of IL‐2 and IL‐2R expression indicated: 1) The first synthesis of IL‐2 was detected at the same ligand concentration in both primed and naive T cells, but primed T cells made much more IL‐2 as the ligand concentrations increased; 2) primed T cells expressed about fivefold more IL‐2 receptor (R) than naive T cells, despite the fact that the antigen doseresponse curves with respect to the percentage of cells expressing IL‐2R were identical. These results suggest that naive and primed T cells have the same threshold with respect to the number of Ag‐MHC complexes required to initiate T cell activation, but that due to the inefficient expression of IL‐2 and IL‐2R, engagement of more complexes is needed to enable naive T cells to synthesize the necessary amounts of these two molecules to allow T cells to go through a complete cycle of replication.
A dual participation of ZAP‐70 and scr protein tyrosine kinases is required for TCR‐induced tyrosine phosphorylation of Sam68 in Jurkat T cells
Authors: Lang Valérie , Mège Dominique , Semichon Monique , Gary‐Gouy Hélène , Bismuth Georges
Published: March 2007

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59fyn and p56lck, as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cγ‐1 and Grb2, has been shown. In this study we analyze the contribution of p56lck, as well as the role of ZAP‐70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4‐amino‐5‐(4‐methylphenyl)7‐(t‐butyl) pyrazolo [3,4‐d] pyrymidine] and cell variants with defective expression of p56lck or expressing a dominant negative form of ZAP‐70, we demonstrate that, while both p56lck and ZAP‐70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56lck and ZAP‐70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56lck activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59lck and p56lck differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP‐70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.
Critical test of hot spot motifs for immunoglobulin hypermutation
Authors: Bachl Juergen , Steinberg Charles , Wabl Matthias
Published: March 2007

In hypermutation at the immunoglobulin loci, some bases are much more mutable than others. The increased mutability of the hot spots has been attributed to their being embedded in short sequence motifs. Among the suggested motifs are palindromes, TAA and RGYW (i.e. A/G G C/T A/T). We have tested these proposed motifs in a transfection system in vitro, which ordinarily uses the hypermutable stop codon TAG. The stop codon TAA is not hypermutable in our system, even when embedded in the pentamer and hexamer palindromes TAATA and ATTAAT; in fact, the revertants isolated were due to deletions. Single or double base changes in an RGYW motif containing a hypermutable stop codon result in a reduction of one order of magnitude or more in point mutation frequency. When the nonamer GACTAGTAT, which includes the same RGYW motif, was moved over hundred base pairs upstream, hypermutability was reduced by an order of magnitude. Thus, while RGYW apparently is a hypermutability motif, it cannot be the sole determinant of mutability.
Immunostimulatory DNA: Sequence‐dependent production of potentially harmful or useful cytokines
Authors: Lipford Grayson B. , Sparwasser Tim , Bauer Marc , Zimmermann Stefan , Koch Eva‐Sophie , Heeg Klaus , Wagner Hermann
Published: March 2007

Certain bacterial immunostimulatory (i.s.) DNA sequences containing unmethylated CpG motifs stimulate antigen‐presenting cells (APC) to express a full complement of costimulatory molecules and to produce cytokines including interleukin (IL)‐12 and tumor necrosis factor (TNF)‐α. While IL‐12 is key to their T helper cell (Th)1‐promoting adjuvant activity, secretion of toxic levels of TNF‐α is harmful in that it promotes toxic shock. Given the beneficial as well as harmful consequences of i.s. DNA, we investigated the possibility of identifying DNA sequences, i.e. CpG oligodeoxynucleotides (ODN) which differentially activate IL‐12 versus TNF‐α cytokine production in APC. Here, we describe an i.s. DNA sequence with these characteristics. While its potential to induce IL‐12 is preserved, its ability to trigger TNF‐α release is strongly curtailed both in vitro and in vivo. I.s. DNA could be segregated into lethal and non‐lethal in a mouse toxic shock model. The non‐toxic i.s. DNA was useful as an adjuvant, thus allowing cytotoxic T cell responses to the soluble protein ovalbumin and conferring a resistant Th 1 phenotype to BALB/c mice lethally infected with Leishmania major. This i.s. CpG motif may thus be prototypic for a useful immunostimulating DNA sequence that lacks harmful side effects.
Immunoregulation by interleukin‐12 in MB49.1 tumor‐bearing mice: Cellular and cytokine‐mediated effector mechanisms
Authors: Hunter Sharon E. , Waldburger Kristine E. , Thibodeaux Deborah K. , Schaub Robert G. , Goldman Samuel J. , Leonard John P.
Published: March 2007

Administration of recombinant murine interleukin (rmlL)‐12 to MB49.1 tumor‐bearing mice results in dose‐dependent regression of the primary tumor and the generation of protective antitumor immunity in the majority of animals. rmlL‐12 administration is associated with a marked increase in lymph node cellularity that is predominantly due to the expansion of B220+ B cells as well as CD8+ T cells. Stimulation of lymph node cells from rmlL‐12‐treated, but not control tumor‐bearing mice, with MB49.1 tumor cells in vitro was shown to enhance the secretion of interferon (IFN)‐γ. The magnitude of this in vitro response was dependent on the dose of rmlL‐12 administered in vivo and mirrored the change in circulating serum IFN‐γ. Furthermore, at the height of the in vitro response to tumor stimulation, the addition of a neutralizing antibody to murine IL‐12 suppressed IFN‐γ production, indicating a role for endogenous IL‐12 in this antigen‐specific cytokine response. Although studies in SCID mice confirmed that an appropriate T cell response was required for rmlL‐12‐mediated antitumor activity, in immunocompetent animals early tumor regression was not accompanied by cellular infiltration of the tumor. In contrast, a profound increase in tumor‐associated inducible nitric oxide synthase (iNOS) was observed in mice receiving rmlL‐12 which preceded T cell infiltration of the tumor which could be detected during the second week of IL‐12 treatment. Direct tumor killing through the cytotoxic actions of NO via the iNOS pathway may serve as a way of generating tumor antigen which enables the host to mount a subsequent T cell response against the tumor.
Ectopic expression of BcI‐2, but not BcI‐xL rescues Ramos B cells from Fas‐mediated apoptosis
Authors: Alam Mohammad K. , Davison Stephen , Siddiqui Nisha , Norton John D. , Murphy John J.
Published: March 2007

The human Burkitt lymphoma Ramos B cell line can be induced to undergo apoptosis in response to a variety of different agents, including calcium ionophores, anti‐immunoglobulin (Ig) and macromolecular synthesis inhibitors. In addition, following up‐regulation of the Fas (CD95) surface receptor by CD40 ligation, these cells also become susceptible to apoptosis induction by Fas ligation. We have previously shown that protection from calcium ionophore‐ and macromolecular synthesis inhibitor‐induced apoptosis by CD40 ligation is associated with a rapid up‐regulation of BcI‐xL followed by a more moderate and delayed up‐regulation of BcI‐2. We show here that overexpression of BcI‐xL, like BcI‐2, protects Ramos cells from apoptosis induction in response to calcium ionophore, anti‐Ig and macromolecular synthesis inhibition. However, in contrast to BcI‐2, ectopic overexpression of BcI‐xL does not rescue from Fas‐mediated apoptosis. Thus, in Ramos B cells, the Fas apoptotic pathway exhibits differential sensitivity to inhibition by BcI‐2 family members. These findings also suggest that CD40 signaling provides a switch which renders the cells susceptible to Fas‐ligand mediated apoptosis through up‐regulation of Fas whilst affording protection from anti‐Ig‐induced apoptosis through up‐regulation of BcI‐xL.
Synthesis, Characterization and Redox Behaviour of Mono‐ and Dicarbonyl Phosphane Rhenium(I) Complexes Bearing N‐, N,N‐ and N,O‐Type Ligands
Authors: Kirillov Alexander M. , Haukka Matti , Guedes da Silva M. Fátima C. , Pombeiro Armando J. L.
Published: March 2007

New mono‐ and dicarbonyl phosphane ReI complexes [ReCl(L)2(CO)(PPh3)2] {L = 3,5‐Me2‐Hpz (3), quin (4)} and [Re(L)n(CO)2(PPh3)2][BPh4]m {L = pz/Hpz, n = 2, m = 0 (2); L = 4,7‐Ph2‐phen, n = 1, m = 1 (5); L = 2‐Me‐dipic, n = 1, m = 0 (6)} were prepared by reaction of the dinitrogen ReI precursor [ReCl(N2)(CO)2(PPh3)2] (1), in refluxing methanol or methanol/benzene mixture, with 3,5‐dimethylpyrazole (3,5‐Me2‐Hpz), quinoline (quin), pyrazole (Hpz), 4,7‐diphenyl‐1,10‐phenanthroline (4,7‐Ph2‐phen) and dipicolinic acid (H2dipic), respectively, with esterification of the latter in 6 to afford the 2‐methoxycarbonyl‐6‐pyridinecarboxylate (2‐Me‐dipic) moiety. Complexes 2–6 were characterized by IR spectroscopy, 1H‐, 31P{1H}‐ and 13C{1H} NMR spectroscopy, FAB‐MS, cyclic voltammetry, elemental analyses and single‐crystal X‐ray diffraction (for 2, 5 and 6) analyses which indicate mutually trans triphenylphosphane ligands. The redox behaviour of the obtained complexes in solution was studied, and the Lever electrochemical ligand parameter EL was determined, for the first time, for 4,7‐Ph2‐phen. Compounds 2–6 represent the first examples of mono‐ and dicarbonyl Re complexes bearing these types of ligands.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)
The Synthesis of 6‐Amidino‐2‐oxopurine Revisited: New Evidence for the Reaction Mechanism
Authors: Dias Alice M. , Cabral Isabel , Vila‐Chã A. Sofia , Costa Daniela S. , Proença M. Fernanda
Published: March 2007

Treatment of N‐aryl‐ or N‐alkyl‐5‐amino‐4‐(cyanoformimidoyl)‐1H‐imidazoles 1 with benzoyl or ethoxycarbonyl isocyanates resulted in the formation of 5‐amino‐4‐[N‐benzoyl‐ or N‐(ethoxycarbonyl)carbamoylcyanoformimidoyl]‐1H‐imidazoles 2. In the presence of catalytic amounts of DBU (1,8‐diazabicyclo[5.4.0]undec‐7‐ene), these compounds cyclized to give the 5′‐amino‐5‐imino‐4,4′‐bi‐1H‐imidazol‐2(5H)‐ones 3. Compounds 3 efficiently rearrange to yield 6‐amidino‐2‐oxopurines 5 in ethanol or DMF solution. The formation of purines 5 both from imidazoles 2 and from bi‐imidazoles 3 was followed by 1H NMR, allowing a deeper understanding of the reaction mechanism. The rearrangements are acid‐catalysed (trifluoroacetic acid), but the use of one equivalent of acid produced different products, identified as the bi‐imidazoles 8.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)
Novel Spiroheterocycles by Aziridination of α‐Methylene‐γ‐ and ‐δ‐lactams
Authors: Loreto M. Antonietta , Migliorini Antonella , Tardella P. Antonio , Gambacorta Augusto
Published: March 2007

New potentially bioactive α‐spiroaziridino‐γ‐ and ‐δ‐lactams have been prepared by treatment of α‐methylene‐γ‐ and ‐δ‐lactams with ethyl N‐{[(4‐nitrophenyl)sulfonyl]oxy}carbamate (NsONHCO2Et) in the presence of CaO. These compounds, through reductive aziridine ring opening, can be intermediates for the synthesis of α‐ and β‐aminolactams, which are useful as conformational constraints in peptides. The above procedure has been successfully extended to one α‐methyleneoxindole to obtain a new spirooxindole derivative, a potential precursor of natural alkaloids. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)
Synthesis of a Spore Surface Pentasaccharide of Bacillus anthracis
Authors: Werz Daniel B. , Adibekian Alexander , Seeberger Peter H.
Published: March 2007

An analogue 2 of the pentasaccharide 1 found on the spore surface protein BclA of Bacillus anthracis was synthesized by a (3+2) glycosylation approach. A robust building block for 3‐linked α‐rhamnose is presented. Benzoate groups were used to ensure α‐selectivity for the rhamnose units. A radical‐initiated reduction with tributyltin hydride was shown to be able to convert the NHTCA group into an acetylated amine as well as an azido group into a free amine on one strike. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)
Cover Picture: The Structural Identification and Conformational Analysis of the Products from the Reaction of Acrolein with 2′‐Deoxycytidine, 1‐Methylcytosine and Calf Thymus DNA (Eur. J. Org. Chem. 9/2007)
Authors: Pawłowicz Agnieszka, J. , Klika Karel D. , Kronberg Leif
Published: March 2007

The cover picture shows that acrolein, a mutagenic environmental pollutant formed during incomplete combustion, reacts with the cytosine base to yield exocyclic ring adducts. Identification and characterization of adducts are discussed in detail in the article by A. J. Pawłowicz, K. D. Klika, and L. Kronberg on p. 1429 ff.
Wavelet transformation of mode shape difference function for structural damage location identification
Authors: Poudel Upendra P. , Fu Gongkang , Ye Jian
Published: March 2007

In this paper, a new approach to structural damage detection is presented using mode shape difference between the reference and damage states. In order to capture and display the signal of damage, wavelet transformation is performed upon the mode shape difference function. Results show that this presentation of signal is effective for damage detection. Furthermore, practical aspects of damage identification are investigated, including understanding and using the frequency contents of the mode shape difference function, the mother wavelet function and the resulting wavelet coefficients due to the band‐limited filtering of wavelet transformation. This proposition is experimented. Results are assertive of the proposed concept. Copyright © 2007 John Wiley & Sons, Ltd.
What can we learn from two‐center three‐electron bonding with the topological analysis of ELF?
Authors: Fourré I. , Silvi B.
Published: March 2007

In this paper, a review is presented of the abundant literature on the two‐center three‐electron (2c‐3e) bonding, which plays a crucial role in electron transfer and radical chemistry. Important questions regarding this peculiar type of interaction are (1) Is a three‐electron bonded complex a molecule or an assembly of molecules?, (2) Since an unpaired electron is involved, where is the spin density located?, and (3) Is there a descriptor of electron fluctuation, which is a central phenomenon in this type of bonding? We demonstrate that the topological analysis of the electron localization function (ELF), which provides a convenient mathematical framework to study chemical bond in molecules and solids, is able to answer these questions. First, examples of potentially 3e‐bonded complexes proposed by Pauling are reinvestigated. Second, the electron attachment process on molecules of the HnXYHmtype with X,Y = Cl, S, P, Si andn, m= 0–2 is considered. Finally, the ELF‐based topology of some prototypical radicals containing the SO bond is examined. From these studies, several topological signatures of the 3e bonding have been elaborated. No disynaptic basin, well known as the topological signature of a covalent shared‐electron pair interaction, is found between two 3e‐bonded atoms. Therefore, to distinguish 3e bonds from ionic or hydrogen bonds, a core valence bifurcation (CVB) index has been introduced, similar to the one previously defined by Fuster and Silvi (Theor Chem Acc 2000,104,13) to differentiate weak and medium H‐bonds. Moreover, in 3e‐bonded systems, the spin density is mainly localized within the lone‐pair basins of the heavy atoms. To quantify the electron fluctuation, a topological delocalization index has been defined. An unambiguous characterization of the 3e bonding is thus obtained, which is in agreement with other theoretical approaches, such as the valence bond method. © 2007 Wiley Periodicals, Inc. Heteroatom Chem 18:135–160, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.20325
The role of functional nitro and cyano groups in the self‐assembly of 1,3‐dithiole‐2‐thione–halogen adducts
Authors: Skabara Peter J. , Kanibolotsky Alexander , Render Simon , Berridge Rory , Crouch David J. , Bricklebank Neil , Coles Simon J. , Gelbrich Thomas , Hursthouse M. B.
Published: March 2007

The synthesis of five new dihalogen/interhalogen charge transfer adducts featuring cyano and nitro functionalized 1,3‐dithiole‐2‐thiones have been prepared and characterized by x‐ray crystallography. The structures feature a complex series of intermolecular close contacts between heteroatoms, as well as hydrogen bonding, to give highly ordered, polymeric assemblies. The adduct 4.IBr crystallizes in a noncentrosymmetric chiral space group and features a stack of molecules assembled through hydrogen bonds. The functional groups of all adducts participate in networks of close contacts, but do not affect the coordination environment of the dihalogen/interhalogen species. © 2007 Wiley Periodicals, Inc. Heteroatom Chem 18:176–184, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.20327
Synthesis and cellular biocompatibility of two kinds of HAP with different nanocrystal morphology
Authors: Zhao Yantao , Zhang Yumei , Ning Fang , Guo Dagang , Xu Zhixuan
Published: March 2007

Two kinds of hydroxyapatite (HA) with different nanocrystal morphology were obtained via a simple aqueous precipitation method under different reactants molar ratios. Under Ca/P molar ratio of 1.67/1, rod‐like crystal was produced, while under Ca/P molar ratio of 1.80/1, spherical crystal was produced. The spherical crystal was 40–60 nm in diameter, while the rod‐like crystal was 40–55 nm in diameter and 79–100 nm in length. The influence of HA nanocrystal morphology on osteoblasts growth was assayed by MTT method and SEM. The results indicated that there was a significantly higher absorbency value on the surface of HA with spherical crystal in MTT assay than the latter. In the process of SEM observation, it is found that osteoblasts spread out a large quantity of nano‐filopodias on spherical crystal surface, thus exhibiting much more active cell morphology. In conclusion, HA with spherical nanocrystal showed more favorable properties than that with rod‐like one for osteoblasts. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007
Characterization, quantification, and isolation of aluminum oxide particles on grit blasted titanium aluminum alloy hip implants
Authors: Grübl Alexander , Kolb Alexander , Reinisch Georg , Fafilek Günter , Skrbensky Gobert , Kotz Rainer
Published: March 2007

Background: This study was undertaken to verify whether or not the microstructure of aluminum alloy implants interferes with the characterization and quantification of aluminum inclusions on their surfaces, resulting from grit blasting. Methods: Four factory‐fresh prostheses were investigated by scanning electron microscopy and X‐ray microanalysis. Specimens were cut out of the stems and the cross‐sections analyzed. The specimens were etched in hot 25% hydrochloric acid. The hydrochloric acid was subsequently filtered with a 0.2‐μm‐pore filter. The filters were scanned using electron microscopy and X‐ray microanalysis. Results: Aluminum oxide particles were found on all investigated stems; the diameter of the particles ranged from 4 to 100 μm. One hundred fifty‐four particles were counted per mm2. No particles were seen on the cross‐sections of the implants. Scanning electron microscopy of the Millipore filters revealed aluminum oxide particles. Conclusion: Remnants of grit blasting were found only on the surfaces; none were observed on cross‐sections. We conclude that the microstructure of titanium aluminum alloy does not interfere with the identification and quantification of particles. Particles were identified on the filters by electron microscopy and X‐ray microanalysis. Aluminum oxide on the surface of grit‐blasted titanium aluminum alloy implants is, in fact, a residue of grit blasting. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007