bluuebunny/crossref_metadata_2025 huggingface dataset:
Abnormal expression pattern of cyclin E in tumour cells
Authors: Erlandsson Fredrik , Wählby Carolina , Ekholm‐Reed Susanna , Hellström Ann‐Cathrin , Bengtsson Ewert , Zetterberg Anders
Published: February 2003

The expression pattern of cyclin E during the cell cycle was studied in normal and tumour cells in culture and in tumour biopsies. This pattern was found to be abnormal in tumour cells. A triple immunostaining protocol, digital microscopy and image analysis were used to find the position of the individual cells in the cell cycle and to measure the nuclear cyclin E levels. In normal cells, the number of cyclin E‐positive cells decreased rapidly when the cells entered the S‐phase. In the tumour cell lines, cyclin E was not downregulated in early S‐phase, as in normal cells. Instead the number of cyclin E‐positive cells remained high throughout S‐phase, and the cyclin E staining intensity per cell often increased during S‐phase. In about half of the analysed tumour cell lines, many cells stained positive for cyclin E even in the G2‐phase. This abnormal expression over the cell cycle of cyclin E was also found in tumour biopsies from cervical, breast and prostatic carcinomas, even though it varied greatly between individual tumours. In some tumours, the expression pattern of cyclin E was similar to that of normal cells in culture, whereas in others high cyclin E levels could be seen in S‐phase cells, as in the transformed cell lines. A high percentage of cells expressing cyclin E during S‐ or G2‐phase was found to be related to poor outcome (p < 0.025) in a small group of cervical carcinoma patients (n = 12). © 2003 Wiley‐Liss, Inc.
Structural analysis of the ErbB‐2 receptor using monoclonal antibodies: Implications for receptor signalling
Authors: Yip Yum L. , Novotny Jiri , Edwards Michael , Ward Robyn L.
Published: February 2003

The extracellular part of ErbB‐2 is formed by 4 domains, specifically, L1, L2 that adopt a β‐helical structure and S1, S2 that consist of several cysteine‐rich, EGF‐fold modules. These ectodomains mediate ErbB‐2 dimerisation with itself or with other members of the epidermal growth factor receptor (EGFR) family, events essential to both ErbB‐2 signaling and the development of certain malignancies. The anti‐ErbB‐2 monoclonal antibodies N12, N28 and L87 bind to the polypeptides C531‐A586, T216‐C235 and C220‐C235 respectively. In this study, glycine walking and random mutagenesis were used to further delineate the critical residues involved in antibody binding. A molecular model of ErbB‐2 ectodomains was then constructed based on the recently published coordinates of the EGFR (EGFR) model. This model rationalized successfully many features of our epitope mapping, including their location in modules within the S1 and S2 domains and the importance of Arg545, Gln548 and Leu561 for N12 binding. Further investigation of the functional effects of the anti‐ErbB‐2 monoclonal antibodies demonstrated that N28 strongly stimulated ErbB‐2 phosphorylation and MAPK activation whereas N12 had no effect. As bivalency is required for the action of these antibodies we propose that at least 2 different kinds of ErbB‐2 homodimers can be formed as relative rotational isomers and that the S1 and S2 domains are instrumental in determining the relative orientations of the ErbB‐2 homodimers, such that different signaling effects are induced. © 2003 Wiley‐Liss, Inc.
Anti‐tumor effect and increased survival after treatment with [177Lu‐DOTA0,Tyr3]octreotate in a rat liver micrometastases model
Authors: Breeman Wouter A.P. , Mearadji Amir , Capello Astrid , Bernard Bert F. , van Eijck Casper H.J. , Krenning Eric P. , de Jong Marion
Published: February 2003

Peptide receptor scintigraphy with [111In‐DTPA0]octreotide (a stabilized radiolabeled somatostatin (SS) analogue, OctreoScan®) is widely used for the visualization and staging of somatostatin receptor‐positive tumors. The application of likewise somatostatin analogues as vehicle for the deliverance of radionuclides to somatostatin receptor‐positive targets are now in use for peptide receptor‐targeted radionuclide therapy (PRRT). Currently preclinical and clinical investigation are ongoing trying to find the optimal combination of radionuclide and ligand. The anti‐tumoral effects of such combinations, like [90Y‐DOTA°, Tyr3]octreotide and [177Lu‐DOTA°, Tyr3]octreotate, on SSR‐positive solid tumors have been reported. In this study we present the anti‐tumor effects of 177Lu‐DOTA‐tate on: a) a single SSR‐positive cell model and b) on a SSR‐positive tumor in a rat liver micrometastatic model, mimicking disseminated disease. 177Lu‐DOTA‐tate showed anti‐tumoral effects in both cases and significant survival in the PRRT‐treated rats. 177Lu‐DOTA‐tate is a very promising new treatment modality for SSR‐positive tumors, including disseminated disease. © 2003 Wiley‐Liss, Inc.
A CD4+, HLA‐DR7‐restricted T‐helper lymphocyte clone recognizes an antigen shared by human malignant melanoma and glioma
Authors: Somasundaram Rajasekharan , Swoboda Rolf , Caputo Laura , Lee Allana , Jackson Naidine , Marincola Francesco M. , Guerry DuPont , Herlyn Dorothee
Published: February 2003

CD4+ Th cells that are restricted by MHC class II molecules play an important role in the induction of antitumor immune responses. We have established a stable CD4+ Th cell clone (Th35‐1A) from the PBMCs of a patient with primary cutaneous melanoma. The Th cell clone is noncytolytic and proliferates specifically in the presence of irradiated autologous melanoma cells or autologous EBV‐transformed B cells pulsed with melanoma tumor cell lysates. Th35‐1A produces IFN‐γ (a Th1‐type cytokine) after autologous tumor cell stimulation, and its proliferative reactivity is HLA class II–restricted. Th cells showed helper activity for PWM responses of PBMCs. Using a panel of HLA class II–matched and unmatched EBV‐B cells as APCs and allogeneic melanoma tumor cell lysate as stimulant, DR7 was delineated as the HLA class II restriction element used by the Th cell clone. In agreement with these results, transfection of an allogeneic melanoma cell line with HLA‐DR7 isolated from autologous EBV‐B cells rendered the cell line stimulatory for Th35‐1A cells. Specificity studies using autologous EBV‐B cells (EBV‐B35) pulsed with a panel of allogeneic tumor cell lysates of various tissue origins indicated that the Th cell clone recognizes an antigen shared by melanoma and glioma cells. The availability of the Th cell clone may lead to the development of new therapies against melanoma, using adoptive Th cell transfer and/or active immunization with a shared Th cell antigen. © 2003 Wiley‐Liss, Inc.
The interaction between NK cells and dendritic cells in bacterial infections results in rapid induction of NK cell activation and in the lysis of uninfected dendritic cells
Authors: Ferlazzo Guido , Morandi Barbara , D'Agostino Antonella , Meazza Raffaella , Melioli Giovanni , Moretta Alessandro , Moretta Lorenzo
Published: February 2003

NK and DC reciprocal interactions have only recently been investigated. In this study, we focused on the interplay between NK cells and DC in two models of bacterial infection. Immature monocyte‐derived DC were cultured in the presence of live Escherichia coli or bacillus Calmette–Guérin. Upon exposure to either extracellular or intracellular bacteria, DC underwent maturation as assessed by the increased levels of expression of CD80, CD86, and HLA molecules and the de novo expression of CD83 and CCR7. Significant amounts of TNF‐α and IL‐12 were released by DCupon infection, whereas IL‐2 and IL‐15 were barely detectable in culture supernatants. Both infected and uninfected DC were capable of inducing in fresh autologous NK cells the expression of CD69 and HLA‐DR and of inducing cell proliferation. Remarkably, however, infected DC were much stronger inducers of NK cell activation and proliferation than uninfected DC. Thus, after just 24 h of NK/DC coculture, only those NK cells that had been exposed to bacteria‐infected DC had acquired the ability to lyse autologous immature DC. In addition, infected DC were more resistant to NK‐mediated lysis as a consequence of the up‐regulation of HLA class I molecule expression on their surface. This study suggests a regulatory circuit involving NK cells and DC in which DC‐induced NK cell activationis effectively enhanced by the presence of pathogens. Activated NK cells, by limiting the supply of immature DC, may then exert a control on subsequent innate and adaptive immune responses.
Transgenic mice with hematopoietic and lymphoid specific expression of Cre
Authors: de Boer Jasper , Williams Adam , Skavdis George , Harker Nicola , Coles Mark , Tolaini Mauro , Norton Trisha , Williams Keith , Roderick Kathleen , Potocnik Alexandre J. , Kioussis Dimitris
Published: February 2003

Bacteriophage P1 Cre/loxP based systems can be used to manipulate the genomes of mice in vivo and in vitro, allowing the generation of tissue‐specific conditional mutants. Wehave generated mouse lines expressing Cre recombinase in hematopoietic tissues using the vav regulatory elements, or in lymphoid cells using the hCD2 promoter and locus control region (LCR). The R26R‐EYFP Cre reporter mouse line was used to determine the pattern of Cre expression in each line and enabled the assessment of Cre activity at a single‐cell level. Analysis showed that the vav promoter elements were able to direct Cre‐mediated recombination in all cells of the hematopoietic system. The hCD2 promoter and LCR on the other hand were able to drive Cre‐mediated recombination only in T cells and B cells, but not in other hematopoietic cell types. Furthermore, in the appropriate tissues, deletion of the floxed target was complete in all cells, thereby excluding the possibility of variegated expression of the Cre transgene. Both of these Cre‐transgenic lines will be useful in generating tissue‐specific gene deletions within all the cells of hematopoietic or lymphoid tissues.
Outer membrane protein A renders dendritic cells and macrophages responsive to CCL21 and triggers dendritic cell migration to secondary lymphoid organs
Authors: Jeannin Pascale , Magistrelli Giovanni , Herbault Nathalie , Goetsch Liliane , Godefroy Sylvie , Charbonnier Peggy , Gonzalez Alexandra , Delneste Yves
Published: February 2003

Outer membrane protein A (OmpA) is a class of bacterial cell wall protein that is immunogenic without adjuvant. As specific immune responses are initiated in the lymph nodes (LN), we analyzed the effect of the OmpA from Klebsiella pneumoniae (KpOmpA) on chemokine/chemokine receptor expression by APC and on cell migration to the LN. Upon contact with KpOmpA, human immature DC andmacrophages acquire CCR7 expression and responsiveness to CCL21. In parallel, CCR1 and CCR5 expression is down‐regulated and CXCL8, CCL2, CCL3 and CCL5 production is up‐regulated. Mice injected subcutaneously with KpOmpA present a transient inflammatory reaction at the site of injection accompanied by an enlargement of the draining LN with a higher proportion of DC and macrophages. Lastly, when exposed to KpOmpA prior injection, DC but not macrophages migrate to the draining LN. In conclusion, KpOmpA confers a migratory phenotype to DC and triggers their migration to the
regional LN. This property contributes to explain how innate cells initiate adaptive immune response upon recognition of conserved bacterial components and also why OmpA is immunogenic in the absence of adjuvant.
Somatic hypermutation does not require Rad54 and Rad54B‐mediated homologous recombination
Authors: Bross Linda , Wesoly Joanna , Buerstedde Jean‐Marie , Kanaar Roland , Jacobs Heinz
Published: February 2003

Secondary diversification of immunoglobulin (Ig) genes occurs through somatic hypermutation (SHM) in B cells of the germinal center (GC). The GC reaction is associated with a high frequency ofDNA double‐strand breaks (DSB) in the hypermutation domain of Ig genes. Homologous recombination (HR) is a prominent DSB repair pathway. Among the proteins involved in HR are the Rad‐54 paralogues,Rad54 and Rad54B. To investigate whether Rad54/Rad54B‐mediated HR is involved in SHM, we determined the ratio of mutated versus non‐mutated Vλ PCR products from memory (IgM–, IgD–, Vλ1+) and GC (PNAhigh, Vλ1+) B cells, the mutation load, the mutation frequency, the base exchange pattern and the distribution of somatic mutations along the rearranged Vλ light chain (VλLC) genes. All these parameters of SHM were unaltered in memory and GC B cells lacking one or both Rad54 paralogues. Thus, our data indicate that Rad54 and Rad54B‐mediated HR is not essential for SHM. In addition, the finding that the ablation of RAD51 paralogues causes an increase in SHM argues against a direct involvement of HR in promoting SHM.
Disparate peptide‐dependent thymic selection outcomes in β2M‐deficient mice versus TAP‐1‐deficient mice: implications for repertoire formation
Authors: Sasada Tetsuro , Yang Yuting , Lai Char‐Chang , Touma Maki , Clayton Linda K. , Liu Jin‐huan , Parisini Emilio , Wang Jia‐huai , Reinherz Ellis L.
Published: February 2003

Fetal thymic organ cultures of N15‐transgenic RAG‐2–/– H‐2b mice on normal, β‐2 microglobulin (β2M)–/– or transporter associated with antigen processing (TAP‐1)–/– MHCI‐deficient backgrounds were used to examine differentiation of thymocytes bearing a TCR specific for a viral peptide bound to H‐2Kb. Strong agonists mediate negative selection in all mice whereas weak agonists are positively selecting in β2M–/– mice but negatively selecting on TAP‐1–/– or normal backgrounds. Very weak agonists and very weak antagonists are generally without effect in β2M–/– mice yet foster differentiation in TAP‐1–/– animals. The 20–40‐fold reduction in β2M–/– thymic H‐2Kb surface expression suggests that the avidity of the TCR for peptide–MHCI accounts for these differences, consistent with effects of TCR density and individual thymic‐peptide abundance in peptide–MHC complexes. TCR–self‐MHC interaction dominates Kb‐based selection, subtly modulated by peptides as revealed by X‐ray crystallography.
Downmodulation of antigen presentation by H2‐O in B cell lines and primary B lymphocytes
Authors: Brocke Pascale , Armandola Elena , Garbi Natalio , Hämmerling Günter J.
Published: February 2003

Peptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory moleculeH2‐M, which helps to exchange the invariant chain‐derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2‐O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2‐M and selectively expressed in B cells. Using stable H2‐O ribozyme‐antisense transfectants, H2‐O overexpressing murine B cell lines, and H2‐O‐transgenic mice, we investigated the effects of H2‐O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levelsof H2‐O in the antigen‐presenting B cells. Thus, increased H2‐O expression downmodulated, whereas reduced H2‐O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2‐O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from Ab molecules were very similar in cells expressing different H2‐O levels. Theintracellular location of H2‐O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2‐O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2‐O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor.
IL‐4 regulates IL‐12 p40 expression post‐transcriptionally as well as via a promoter‐based mechanism
Authors: Seegmüller Irene , Häcker Hans , Wagner Hermann
Published: February 2003

IL‐12 and IL‐4, respectively, dominate Th1 versus Th2 polarization. Additionally IL‐4 can inhibit IL‐12 p40 production in DC. Here we show that macrophages respond to bacterial CpG‐DNA with IL‐12 p40 production in an IL‐4‐sensitive manner. Analysis of the molecular mechanism of this inhibition shows that IL‐4 acts by reducing the stability of IL‐12 p40 mRNA as well as by affecting promoter activity. IL‐4 did not affect early CpG‐DNA‐induced signal transduction. However, IL‐4 reduced the activity of the IL‐12 p40 promoter and when de novo transcription of IL‐12 p40 mRNA was blocked,IL‐4 led to acceleration of IL‐12 p40 mRNA degradation. These data show that IL‐4 regulates IL‐12 p40 expression by influencing promoter activity and by interfering with mRNA stability.
Mice transgenic for intracellular interleukin‐1 receptor antagonist type 1 are protected from collagen‐induced arthritis
Authors: Palmer Gaby , Talabot‐Ayer Dominique , Szalay‐Quinodoz Ildiko , Maret Michel , Arend William P. , Gabay Cem
Published: February 2003

Interleukin‐1 receptor antagonist (IL‐1Ra) is a natural IL‐1 inhibitor, which competitively inhibits binding of IL‐1 to its receptors. IL‐1Ra is produced as four different isoforms, one secreted (sIL‐1Ra) and three intracellular (icIL‐1Ra1, 2, 3), derived from the same gene. We previously observed increased production of icIL‐1Ra1 in the joints of mice with collagen‐induced arthritis (CIA). However, due to its intracellular localization, the biological role of icIL‐1Ra1 remains unknown. The aim of the present study was to examine the effect of the icIL‐1Ra1 isoform, as compared to that of sIL‐1Ra, in the CIA model by comparing transgenic (tg) mice overexpressing icIL‐1Ra1 or sIL‐1Ra to their wild‐type littermates. Serum levels of tg human IL‐1Ra were elevated in sIL‐1Ra and, to a lesser extent, also in icIL‐1Ra1 mice. Clinical scoring indicated that none of the icIL‐1Ra1 or sIL‐1Ra tg mice developed CIA, whereas arthritis was present in, respectively, 60% and 100% of their wild‐type littermates. Histological and radiological analyses confirmed the absence of arthritis in icIL‐1Ra1 and sIL‐1Ra tg mice. Accordingly, circulating levels of the acute‐phase protein serum amyloid A tended to be lower in icIL‐1Ra1 tg mice than in their wild‐type littermates and were significantly lower in sIL‐1Ra tg mice than in controls. In contrast, no difference was observed between the groups regarding serum levels of anti‐type II collagen antibodies and ex vivo spleen cell proliferative response to collagen. In conclusion, icIL‐1Ra1, which is released into the extracellular space when produced in high amounts, has a similar anti‐arthritic effect as sIL‐1Ra.
Normal B‐1 cell development but defective BCR signaling in LCK–/– mice
Authors: Ulivieri Cristina , Valensin Silvia , Majolini M. Bernardetta , Matthews R. James , Baldari Cosima T.
Published: February 2003

Mature B cells are grouped into two major subsets, B‐1 and B‐2, believed to derive from separate lineages. We have recently shown that B‐1 cells, which are characterized by CD5 surface expression, specifically exhibit significant levels of the tyrosine kinase Lck in man. Here we show that also in mice Lck expression is restricted to B‐1 cells and address the potential role of Lck in B‐1 cell development and activation. Using as a model an Lck–/– mouse, we show that, while dispensable for B‐1 cell development, Lck is required for full and sustained activation of the tyrosine phosphorylation and MAP kinase cascades triggered by the BCR in CD5+, B‐1 cells. The data suggest a potential role for Lck in the achievement of the higher activation threshold required for productive BCR signaling in B‐1 as compared to B‐2 cells.
T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by Lck
Authors: Soldaini Elisabetta , Wack Andreas , D'Oro Ugo , Nuti Sandra , Ulivieri Cristina , Baldari Cosima T. , Abrignani Sergio
Published: February 2003

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross‐linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck‐deficient Jurkat T cells. In wild‐type Jurkat cells, Lck was activated by CD81 cross‐linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl‐β‐cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3‐deficient Jurkat cells. CD81 cross‐linking increased and prolonged the anti‐CD3‐induced tyrosine phosphorylation of TCRζ and of other proteins, indicating that the CD81‐mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross‐linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.
Involvement of macrophage mannose receptor in the binding and transmission of HIV by macrophages
Authors: Nguyen Deborah Greene , Hildreth James E. K.
Published: February 2003

The human immunodeficiency virus (HIV) is an enveloped virus whose surface glycoprotein gp120 binds CD4 on target cell membranes to initiate infection. About half of the carbohydrates on gp120are terminally mannosylated, a pattern common to many pathogens. We have examined the ability of macrophage mannose receptor (MMR) on primary monocyte‐derived macrophages to bind HIV and facilitateits transmission to T cells. We adapted the tyramide signal amplification system for fluorescence detection of HIV bound to macrophages. Our data show that approximately 60% of the initial association of HIV with macrophages that lack expression of DC‐SIGN (a dendritic cell‐specific ICAM‐3 receptor/HIV‐1‐binding protein) is MMR mediated, as evidenced by inhibition with mannan, D‐mannose, EDTA, and soluble mannose‐binding lectin, but not by D‐galactose. This inhibition is not seen in cells that lack MMR. Macrophages are able to mediate transmission of bound HIV to co‐cultured T cells, and this transmission is blocked up to 80% by inhibitors of MMR binding. Unlike virus bound to DC‐SIGN, macrophage‐bound HIV has a slightly lower half‐life compared to free virus, with no transmission in co‐culture observed beyond 24 h after virus binding to macrophages. Results obtained with endocytosis inhibitors indicate that this decrease in viral longevity is due to rapid internalization of macrophage‐bound HIV. Together, these results suggest a substantial role for MMR in the binding and transmission of HIV‐1 by macrophages.
CCR8‐dependent activation of the RAS/MAPK pathway mediates anti‐apoptotic activity of I‐309/CCL1 and vMIP‐I
Authors: Louahed Jamila , Struyf Sofie , Demoulin Jean‐Baptiste , Parmentier Marc , Snick Jacques Van , Damme Jo Van , Renauld Jean‐Christophe
Published: February 2003

We have previously shown that the CC‐chemokine I‐309 (CCL1) protects mouse thymic lymphomas against corticoid‐induced apoptosis. Here, we analyzed the signal transduction pathways involved in this activity on BW5147 lymphoma. Inhibition of the CCL1 activity by pertussis toxin suggested the involvement of a G protein‐coupled chemokine receptor. The role of CCR8 was supported by the observation that vMIP‐I, another CCR8‐ligand identified from the genome of a T cell transforming herpes virus, shared CCL1 anti‐apoptotic activity. In addition to CCR8, BW5147 cells also expressed the CXCR4 receptor but its ligand, SDF‐1 (CXCL12) showed only a modest anti‐apoptotic activity. Other chemokines acting on CCR2, CCR4 and CCR5 failed to protect against apoptosis and to induce BW5147 chemotaxis, suggesting that these receptors were not functionally expressed. By contrast, both CCL1 and vMIP‐I up‐regulated ERK1/2 MAPK phosphorylation in BW5147 cells. Further analysis demonstrated thatCCL1 activates the MAPK pathway in CCR8‐transfected CHO cells. The implication of this pathway was confirmed by the fact that PD98059, an inhibitor of MEK kinases, as well as a dominant negative isoform of the M‐RAS protein specifically blocked the anti‐apoptotic activity of CCL1.
High antigen dose and activated dendritic cells enable Th cells to escape regulatory T cell‐mediated suppression in vitro
Authors: George Thaddeus C. , Bilsborough Janine , Viney Joanne L. , Norment Anne M.
Published: February 2003

CD4+CD25+ regulatory T cells (Tregs) are critical for peripheral tolerance and prevention of autoimmunity. In vitro coculture studies have revealed that increased costimulation breaks Treg‐mediated suppression in response to anti‐CD3 or antigen. However, it was unclear whether loss of suppression arose from inactivation of Tregs or whether increased stimulationcaused Th cells to escape suppression. We have investigated conditions that allow or override Treg‐mediated suppression using DO11.10 TCR‐transgenic T cells and chicken ovalbumin peptide 323–339‐pulsed antigen‐presenting cells. Treg suppression of Th proliferation is broken with potent stimulation, using activated spleen cells and high antigen dose, but is intact at low antigen dose. Costimulation with CD80 and CD86 expressed on activated dendritic cells was essential for Th cell escape from suppression at a high antigen dose. Potently stimulated Tregs were functional since they reducedlevels of IL‐2, IFN‐γ, IL‐4 and Th CD25 expression in cocultures. Furthermore, Tregs responding to high antigen dose and activated splenocytes retained the ability to suppress proliferation, but only of Th cells responding to a sub‐optimal dose of independent antigen. Together, our results demonstrate that under conditions of strong antigen‐specific stimulation, Tregs remain functional, but Th cells escape Treg‐mediated suppression.
Characterization of H+,K+‐ATPase T cell epitopes in human autoimmune gastritis
Authors: Bergman Mathijs P. , Amedei Amedeo , D'Elios Mario M. , Azzurri Annalisa , Benagiano Marisa , Tamburini Carlo , van der Zee Ruurd , Vandenbroucke‐Grauls Christina M. , Appelmelk Ben J. , Prete Gianfranco Del
Published: February 2003

Human autoimmune gastritis (AIG) is an organ‐specific inflammatory disorder leading to gastric atrophy and pernicious anemia. Gastric H+,K+‐ATPase was identified as the autoantigen in both human disease and experimental murine AIG (EAIG). Studies of EAIG significantly contributed to current knowledge of human AIG, but to what extent EAIG mimics AIG is still debated, andthe autoantigenic epitopes in AIG are yet unknown. This study aimed to identify the H+,K+‐ATPase epitopes recognized by gastric T cell clones from AIG patients, to define their TCR Vβ usage and epitope‐induced cytokine response. Sixteen H+,K+‐ATPase‐reactive CD4+ gastric T cell clones of four AIG patients were tested for proliferation to overlapping 15‐mer peptides spanning the α and β chains of H+,K+‐ATPase. We identified 6 epitopes in the α chain and 5 in the β chain; TCR Vβ usage was not restricted. Four (36%) of the 11 H+,K+‐ATPase epitopes recognized in AIG were found to overlap with epitopes that are relevant in EAIG, including a previously described gastritogenic epitope. Gastric T cell recognition of the peptide epitopes resulted in secretion of Th1 cytokines. Our data suggest a striking similarity between human AIG and EAIG, at the epitope level, with regard to cytokinesecretion and likely also with regard to pathogenic mechanisms.
Adoptive transfer of an anti‐MART‐127–35‐specific CD8+ T cell clone leads to immunoselection of human melanoma antigen‐loss variants in SCID mice
Authors: Lozupone Francesco , Rivoltini Licia , Luciani Francesca , Venditti Massimo , Lugini Luana , Cova Agata , Squarcina Paola , Parmiani Giorgio , Belardelli Filippo , Fais Stefano
Published: February 2003

The identification of appropriate mouse models could be useful in carefully evaluating the actual role of the in vivo development of antigen‐loss variants during antigen‐specific vaccine therapy of human tumors. In this study we investigated the level of efficacy of a MART‐1/Melan‐A‐specific CD8+ T cell clone against its autologous melanoma in a severe combined immunodeficiency (SCID) mouse model, in which the tumor cells expressed in vivo heterogeneous and suboptimal levels of MART‐1. The subcutaneous co‐injection of the MART‐1/Melan‐A‐reactive T cell clone A42 with MART‐1/Melan‐A+ autologous human melanoma cells into SCID mice caused a total inhibition of tumor growth. However, the systemic treatment with A42 clone lymphocytes resulted inonly 50–60% inhibition of tumor growth, although the T cell clone targeted the tumors and the MART‐1+ cells virtually disappeared from the tumors. This study suggests that an immunotherapybased on the expansion of an antigen‐specific T cell clone generated in vitro is highly efficient in abolishing tumor growth when the target antigen is fully expressed, but leads to in vivoimmunoselection of antigen‐loss variants in the presence of suboptimal levels of antigen expression. Furthermore, this work shows that human tumors/SCID mouse models may be useful in evaluating thein vivo efficacy of adoptive immunotherapies.
The human TREM gene cluster at 6p21.1 encodes both activating and inhibitory single IgV domain receptors and includes NKp44
Authors: Allcock Richard J. N. , Barrow Alexander D. , Forbes Simon , Beck Stephan , Trowsdale John
Published: February 2003

We have characterized a cluster of single immunoglobulin variable (IgV) domain receptors centromeric of the major histocompatibility complex (MHC) on human chromosome 6. In addition to triggering receptor expressed on myeloid cells (TREM)‐1 and TREM2, the cluster contains NKp44, a triggering receptor whose expression is limited to NK cells. We identified three new related genes and two gene fragments within a cluster of ∼200 kb. Two of the three new genes lack charged residues in their transmembrane domain tails. Further, one of the genes contains two potential immunotyrosine inhibitory motifs in its cytoplasmic tail, suggesting that it delivers inhibitory signals. The human and mouse TREM clusters appear to have diverged such that there are unique sequences in each species. Finally, each gene in the TREM cluster was expressed in a different range of cell types.
Gender identity, ethnicity, acculturation, and drug use: Exploring differences among adolescents in the Southwest
Authors: Kulis Stephen , Marsiglia Flavio Francisco , Hurdle Donna
Published: February 2003

This article presents the findings of a survey completed by 1351 predominantly Mexican American middle school students residing in a large urban center in the U.S. Southwest. The study explores possible associations between drug use attitudes and behaviors and gender (biological sex), gender identity, ethnicity, and acculturation status. Based on the concepts of “machismo” and “marianismo” that have been used to describe Mexican populations, four dimensions of gender identity were measured: aggressive masculinity, assertive masculinity, affective femininity, and submissive femininity. In explaining a variety of indicators of drug use behaviors and anti‐drug norms, gender alone had limited explanatory power, while gender identity—often regardless of gender—was a better predictor. Aggressive masculinity was generally associated with higher risk of drug use, while the other three gender identity measures had selected protective effects. However, the impact of gender identity was strongly mediated by acculturation. Less acculturated Mexican American students reported lower aggressive masculinity scores than non‐Latinos. Less acculturated Mexican American girls reported both the lowest aggressive masculinity scores and the highest submissive femininity scores. More acculturated Mexican American students, along with the less acculturated Mexican American boys, did not appear to be following a polarized approach to gender identity (machismo and marianismo) as was expected. The findings suggest that some aspects of culturally prescribed gender roles can have a protective effect against drug use behaviors and attitudes, possibly for both girls and boys. © 2003 Wiley Periodicals, Inc.
Raman spectroscopy of II–VI semiconductor nanostructures: CdS quantum dots
Authors: Schreder B. , Dem C. , Schmitt M. , Materny A. , Kiefer W. , Winkler U. , Umbach E.
Published: February 2003

Information about confinement effects and dot–matrix interactions of CdS nanoparticles was obtained from resonance Raman spectroscopy. The quantum dots had diameters of 3 and 5 nm and were prepared with and without organic spacer groups. It was found that the spacer improves the quality of the nanocrystallites. No phonon confinement shift could be observed even for the small quantum dots. The linewidths of the overtone series point to a mechanism of vibrational relaxation which is dominated by the decay of the LO phonons into acoustic phonons. Copyright © 2003 John Wiley & Sons, Ltd.
Spectral investigations on 2‐methyl‐1,4‐naphthoquinone: solvent effects, host–guest interactions and SERS
Authors: Umadevi M. , Ramasubbu A. , Vanelle P. , Ramakrishnan V.
Published: February 2003

Optical absorption and fluorescence emission spectroscopic techniques were employed to investigate the solvent effects on 2‐methyl‐1,4‐naphthoquinone (2‐mNQ) and the molecular recognition of 2‐mNQ in calix(8)arene. The orientation of 2‐mNQ on a silver surface was analysed by SERS. The optical absorption and fluorescence emission spectra of 2‐mNQ in different solvents reveal the n → π* transition and the observed features show that the singlet excited state is more stabilized in polar protic solvents by intermolecular hydrogen bonding and in polar aprotic solvents by dipole–dipole interaction. The evidence for the formation of inclusion complexes of calix(8)arene with 2‐mNQ in dichloromethane was obtained by optical absorption and fluorescence emission studies. From the observations, the existence of association of molecules is proposed and the association constant is evaluated. The orientation of 2‐mNQ on a silver surface was studied by SERS measurements, and was found to be ‘flat‐on.’ Copyright © 2003 John Wiley & Sons, Ltd.
Hyper‐spectral Raman imaging using a diffractive optical element
Authors: Thomson G. A. , Thorley F. C. , Batchelder D. N.
Published: February 2003

A modification to a conventional imaging Raman microscope to allow the acquisition of quasi‐confocal spectra from multiple discrete Raman sampling volumes is reported. Preliminary performance was demonstrated by generating an extended area hyper‐spectral chemical map of a number of ground sulfur crystals deposited on an aluminium mirror. Images generated using this multiplexed sampling arrangement were found to contain a number of weak ghost images (cross‐talk), corresponding to inadequate separation of the focused laser spots at the sample. Copyright © 2003 John Wiley & Sons, Ltd.
Lattice dynamics and temperature dependence of the first‐order Raman spectra for α‐SiO2 crystals
Authors: Ichikawa Shinya , Suda Jun , Sato Tsutomu , Suzuki Yushi
Published: February 2003

The phonon dispersion curves for α‐SiO2 crystals were calculated on the basis of a rigid‐ion model. The first‐order Raman spectra of the A1 mode of the α‐SiO2 crystal were measured in the temperature range from 303.1 to 773.1 K. The temperature dependence of the linewidth was analyzed by using the phonon dispersion curves, and the results showed that it was caused approximately by the cubic anharmonic term in the crystal potential energy. The temperature dependence of the frequency shift of the A1 mode was analyzed by using the lattice dynamic perturbative treatment. We found that the quartic anharmonic term of the first‐order perturbation and also the cubic term of the second‐order perturbation contribute to the temperature dependence of the frequency shift of the A1 mode in the α‐SiO2 crystal. Copyright © 2003 John Wiley & Sons, Ltd.
High‐pressure Raman scattering study of the A1 mode of CuInS2
Authors: Pinnick D. A. , Lee S. A.
Published: February 2003

Raman spectroscopy was used to measure the wavenumber of the A1 mode of CuInS2 as a function of hydrostatic pressure up to about 5 GPa. This mode was found to have a linear pressure dependence of 5.17 ± 0.12 cm−1 GPa−1. The Grüneisen parameter for this mode was determined as 1.33. Copyright © 2003 John Wiley & Sons, Ltd.
Raman spectroscopic study of the basic copper sulphates—implications for copper corrosion and ‘bronze disease’
Authors: Martens Wayde , Frost Ray L. , Kloprogge J. Theo , Williams Peter A.
Published: February 2003

The basic copper sulphates are of interest because of their appearance in many environmental situations such as copper pipe corrosion, restoration of brass and bronze objects, leaching from waste mineral dumps and the restoration of frescoes. The Raman spectra of the basic copper sulphate minerals antlerite, brochantite, posnjakite and langite are reported using a diode laser operating at an excitation wavelength of 785 nm. In line with their crystal structures, each basic copper sulphate mineral has its own characteristic Raman spectrum, which allows their identification. Except for brochantite, multiple bands are observed for the SO stretching vibration. Similarly, multiple bands are observed for the antisymmetric SO and OSO bending regions. Hydroxyl deformation modes in the 730–790 cm−1 region are observed. The use of an He–Ne laser operating at an excitation wavelength of 633 nm allowed the hydroxyl stretching bands of the minerals to be obtained. Antlerite and brochantite are characterized by hydroxyl stretching bands at 3580 and 3488 cm−1. The minerals posnjakite and langite display Raman hydroxyl stretching vibrations at 3588 and 3564 cm−1. The Raman spectra of these two minerals show water OH stretching bands at 3405, 3372 and 3260 cm−1. Raman spectroscopy allows the ready identification of these minerals. Copyright © 2003 John Wiley & Sons, Ltd.
Study of ordered nano‐regions in Pb(Zn1/3Nb2/3)0.915Ti0.085O3 single crystal using Raman spectroscopy
Authors: Dobal P. S. , Katiyar R. S. , Tu C.‐S.
Published: February 2003

A Raman spectroscopic study was performed on a Pb(Zn1/3Nb2/3)0.915Ti0.085O3 crystal to study the ordered nano‐regions. The variation of the Raman spectra with temperature in the range from −200 to 1000°C clearly exhibited a rhombohedral–tetragonal transition at about 75°C followed by a tetragonal–cubic transition at about 175°C. The polarization behavior of the 777 cm−1 octahedral mode suggested the existence of Fm3m nano‐ordered regions in Pm3m cubic symmetry. Thermal switching of nano‐region polarization was observed above 388°C as the thermal energy became greater than the activation energy of nano‐clusters and resulted in a decrease in the polarized intensity of the 777 cm−1 mode. However, both the 51 and 777 cm−1 modes were observed up to 1000°C. Copyright © 2003 John Wiley & Sons, Ltd.
Fourier transform Raman spectroscopic data on the fossil resin siegburgite
Authors: Winkler Waltraud , Musso Maurizio , Kirchner Elisabeth Ch.
Published: February 2003

The fossil resin siegburgite is known from two localities in Europe: Siegburg, near Bonn, and the ‘Goitsche,’ near Bitterfeld, both in Germany. Raman spectroscopic data on the material proved the polystyrene character of siegburgite and therefore supported the chemical investigations conducted previously. Cinnamic acid, which was identified as a major constituent in recent resins of Liquidambar ssp. (Hamamelidaceae), was included as a standard pointing towards the origin of siegburgite in the Hamamelidaceae. The rapid and non‐destructive method used permits a quick distinction of fossil resins, especially when different resin types occur together in the same deposit, as in Bitterfeld. Copyright © 2003 John Wiley & Sons, Ltd.
Combination of GC‐MS with local resolution for determining volatile components in si‐wu decoction
Authors: Gong Fan , Liang Yi‐Zeng , Chau Foo‐Tim
Published: February 2003

In this paper, the combination of gas chromatography‐mass spectrometry with chemometric local resolution techniques such as subwindow factor analysis (SFA) and orthogonal projection resolution (OPR) is investigated as a method of determining volatile components present in a traditional Chinese medicinal formulation known as si‐wu decoction and its two individual herbs Rhizoma chuanxiong and Radix angelicae sinensis. In order to validate the reliability of the results obtained, the volatile components of interest were further separated on open glass columns and then analyzed in the same way as above. With the help of SFA and OPR approaches, the purity of chromatographic peaks can first be identified. Then, the pure chromatogram and mass spectrum of each component involved in a target peak cluster can be easily resolved and subsequently subjected to similarity searches in the NIST MS database to qualitatively and quantitatively determine the volatile components. Our results showed that about 127, 80, and 97 chemical components could be separated and 81, 49, and 55 of them identified, representing 83.95%, 91.86%, and 85.11% of the total relative content of volatile components from Rhizoma chuanxiong, Radix angelicae sinensis, and si‐wu decoction, respectively. The results obtained in this work strongly indicate that the combination of GC‐MS with chemometric local resolution methods could greatly improve the chromatographic separation ability by means of mathematical approaches. Moreover, they indicated the reliability and practicability of this combined technique.
Comparison of micellar electrokinetic chromatography and microemulsion electrokinetic chromatography for separation of pharmacologically active xanthones from Securidaca inappendiculata
Authors: Bo Tao , Yang Xuedong , Li Ke An , Xu Lizhen , Liu Huwei
Published: February 2003

Micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) have been investigated for the separation of nine xanthones from Securidaca inappendiculata, based on the systematic optimization of such parameters as pH, concentration of borate buffer, and sodium dodecyl sulfate (SDS) concentration. The study indicated that, in MEKC, although the addition of modifiers including organic solvents and cyclodextrins (CDs) could greatly affect the separation selectivity of xanthones, the baseline separation for nine xanthones could not be completed. As a special variant of electrokinetic chromatography (EKC), MEEKC successfully achieved the baseline separation of nine xanthones with satisfactory repeatability. Compared with MEKC, MEEKC showed better separation selectivity and higher separation efficiency.
Forensic analysis of hair surface components using off‐line supercritical fluid extraction and large volume injection
Authors: Goodpaster John V. , Bishop Jeremiah J. , Benner Bruce A.
Published: February 2003

The analysis of organic material on the surface of human hair may indicate various subject characteristics such as age, race, gender, or the use of hair products. In addition, comparisons of hair chemical composition may be used to assess the potential for a common origin between a known and unknown sample. However, evidentiary hair samples are often extremely small, necessitating highly sensitive extraction and analysis techniques. In this communication, off‐line supercritical fluid extraction (SFE) followed by gas chromatography/mass spectrometry (GC/MS) using large volume injection (LVI) is demonstrated as a potential method for extracting and analyzing hair surface components. The retention gap technique was used to achieve large volume injections, allowing up to 30 μL of the off‐line extracts to be analyzed. Subsequent chemical analysis by GC/MS identified various natural components on the hair surface such as saturated and unsaturated free fatty acids, squalene, and cholesterol. Overall, off‐line SFE was quite efficient, allowing the measurement of surface components of sample amounts ranging from 0.22 mg to 0.24 mg. In addition, derivatization of free fatty acids using a silylating agent increased both chromatographic resolution and sensitivity. Results from two hair samples are discussed to demonstrate the technique.
Synthesis and characteristics of composite chiral stationary phases based on cellulose derivatives
Authors: Chen Xiaoming , Zou Hanfa , Ni Jianyi , Feng Shun
Published: February 2003

Composite chiral stationary phases (CSPs) were prepared on the basis of cellulose derivatives coated or bonded onto silica. “Molecular exterior” type CSPs were prepared by mixing together two different cellulose tris‐derivatives before or after being coated or bonded onto silica, and the “molecular interior” type was obtained by synthesizing non‐regioselectively heterosubstituted cellulose derivatives coated or bonded onto silica. For the sake of comparison, the individual phases were also prepared with corresponding cellulose derivatives by coating or bonding approaches, respectively. All of the prepared CSPs were characterized and their chiral recognition properties were evaluated by HPLC with several test racemates. The experimental results demonstrated that the “molecular exterior” CSPs generally exhibit chiral recognition capacities intermediate between those of the two individual phases. However, in the separation of some racemates higher enantioselectivity may be achieved on the “molecular interior” phases than on individual phases, thus broadening the application range of a single cellulose‐based CSP.
Separation of basic central nervous system drugs by capillary electrochromatography
Authors: Jiskra Jan , Claessens Henk A. , Cramers Carel A.
Published: February 2003

This article describes the sample preparation and separation of three strongly basic central nervous system (CNS) drugs by capillary electrochromatography. The separation was developed on Hypersil C8 MOS and Hypersil Phenyl stationary phases using acetonitrile (ACN) as organic modifier together with mobile phase additives, ammonia, ethylenediamine, or 1,3‐diaminopropane. A successful, fast separation after a simple derivatization procedure of the compound was achieved on the Hypersil Phenyl stationary phase using ethylenediamine as mobile phase additive. The general approach to method development for complex mixtures is discussed.
Analysis of chemical processes at single bovine adrenergic chromaffin cells with micellar electrokinetic capillary chromatography and electrochemical detection
Authors: Suljak Steven W. , Swanek Franklin D. , Gavin Peter F. , Ewing Andrew G.
Published: February 2003

Micellar electrokinetic capillary chromatography with electrochemical detection (MEKC‐EC) has been applied to the investigation of single cell systems. Separations of bovine adrenergic chromaffin cells reveal average contents of 33 ± 14 fmol of norepinephrine and 24 ± 11 fmol of epinephrine. The resolution benefits of MEKC‐EC have been more fully realized in the analysis of a complex mixture resulting when adrenal cells are incubated with the dopamine precursor, L‐dihydroxyphenylalanine (L‐DOPA). Dopamine has been unambiguously identified in these cells, supporting the hypothesis that transport of dopamine into vesicles is a slow, rate‐limiting process. In addition, MEKC‐EC has been used to monitor the release of catecholamines from single adrenal cells when stimulated by nicotine. Low‐fmol quantities of norepinephrine and epinephrine have been detected in the extracellular environment following stimulation.