bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactive oxygen species‐mediated regulation of eNOS and iNOS expression in multicellular prostate tumor spheroids
Authors: Wartenberg Maria , Schallenberg Maurice , Hescheler Jürgen , Sauer Heinrich
Published: February 2003

Nitric oxide (NO) generated by either endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) may be involved in prostate tumorigenesis through the inhibition of reactive oxygen species (ROS)‐induced apoptosis. Multicellular DU‐145 prostate tumor spheroids endogenously generated NO that paralleled the production of ROS. With increasing spheroid size, eNOS expression was downregulated, whereas an upregulation of iNOS expression was observed. In parallel, NO generation declined, as evaluated by the NO indicator diaminofluorescein‐2 diacetate (DAF‐2DA), suggesting that NO generation in DU‐145 tumor spheroids is mainly mediated by eNOS. Elevation of ROS by treatment of tumor spheroids with either buthionine sulfoximine (BSO) or hydrogen peroxide resulted in upregulation of eNOS, whereas iNOS was downregulated. Furthermore, eNOS expression was increased by epidermal growth factor (EGF) in a redox‐sensitive manner. Upregulation of eNOS after treatment with hydrogen peroxide was apparently transduced through receptor tyrosine kinase signaling pathways since it was abolished by the protein kinase C (PKC) inhibitor bisindolylmaleimide‐1 (BIM‐1), the p21ras inhibitor S‐trans‐trans‐farnesylthiosalicylic acid (FTS), the c‐Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1/2 activation. Endogenous NO may serve to escape from oxidative stress‐induced apoptosis since treatment of tumor spheroids with the NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethyl imidazoline‐1‐oxyl 3‐oxide (carboxy‐PTIO) as well as the NO synthase inhibitor N‐ω‐amino‐L‐arginine (L‐NAA) increased cleaved caspase‐3. Consequently, lowering intracellular NO levels with either L‐NAA or PTIO significantly raised ROS levels, indicating that endogenously generated NO may play a role as a ROS scavenger, thereby protecting exponentially growing tumor spheroids from ROS‐induced apoptosis. © 2003 Wiley‐Liss, Inc.
Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo
Authors: Kashima Takeshi , Nakamura Kazuya , Kawaguchi Jitsutaro , Takanashi Masakatsu , Ishida Tsuyoshi , Aburatani Hiroyuki , Kudo Akira , Fukayama Masashi , Grigoriadis Agamemnon E.
Published: February 2003

Osteosarcoma by nature shows aggressive pulmonary metastasis; however, the underlying molecular mechanisms remain unclear. We previously showed that N‐cadherin and cadherin‐11 (OB‐cadherin), which are highly expressed in normal osteoblasts, are anomalously expressed in human osteosarcoma (Kashima et al., Am J Pathol 1999;155:1549–55). In the present study, we examined the role of cadherins in osteosarcoma metastasis using the mouse osteosarcoma cell line Dunn and its highly metastatic subline LM8. Oligonucleotide array and RT‐PCR analyses demonstrated that Dunn and LM8 cells did not express appreciable levels of several members of the cadherin family, and Western blot analysis confirmed that Dunn and LM8 cells did not express P‐cadherin, E‐cadherin, N‐cadherin or cadherin‐11 protein. We therefore investigated the functional consequences of cadherin overexpression on cell migration and in vivo metastatic potential of LM8 cells. Several LM8 clones were isolated which expressed exogenous N‐cadherin and cadherin‐11 localized to the cell membrane and able to bind to β‐catenin. Overexpression of N‐cadherin or cadherin‐11 in LM8 cells did not affect cell proliferation but caused an inhibitory effect on cell migration in vitro. In vivo analysis showed that N‐cadherin‐ and cadherin‐11‐overexpressing cells exhibited a marked reduction in their ability to form pulmonary metastases, with significant decreases in lung weight and the number and weight of metastatic lesions, as well as the size and weight of primary lesions at the s.c.‐inoculated site. These observations demonstrate that disruption of N‐cadherin‐ and cadherin‐11‐mediated cell–cell adhesion is critical in the pulmonary metastasis of osteosarcoma. © 2002 Wiley‐Liss, Inc.
Differential expression of genes induced by resveratrol in LNCaP cells: P53‐mediated molecular targets
Authors: Narayanan Bhagavathi A. , Narayanan Narayanan K. , Re Gian G. , Nixon Daniel W.
Published: February 2003

Prostate cancer prevention by key elements present in human nutrients derived from plants and fruits has been confirmed in various cell cultures and tumor models. Resveratrol (RE), a phytoalexin, induces remarkable inhibitory effects in prostate carcinogenesis via diverse cellular mechanisms associated with tumor initiation, promotion and progression. Earlier studies have shown that RE alters the expression of genes involved in cell cycle regulation and apoptosis, including cyclins, cdks, p53 and cdk inhibitors. However, most of the p53‐controlled effects related to the role of RE in transcription either by activation or repression of a sizable number of primary and secondary target genes have not been investigated. Our study examined whether RE activates a cascade of p53‐directed genes that are involved in apoptosis mechanism(s) or whether it modifies the androgen receptor and its co‐activators directly or indirectly and induces cell growth inhibition. We demonstrate by DNA microarray, RT‐PCR, Western blot and immunofluorescence analyses that treatment of androgen‐sensitive prostate cancer cells (LNCaP) with 10−5 M RE for 48 hr downregulates prostate‐specific antigen (PSA), AR co‐activator ARA 24 and NF‐kB p65. Altered expression of these genes is associated with an activation of p53‐responsive genes such as p53, PIG 7, p21Waf1‐Cip1, p300/CBP and Apaf‐1. The effect of RE on p300/CBP plays a central role in its cancer preventive mechanisms in LNCaP cells. Our results implicate activation of more than one set of functionally related molecular targets. At this point we have identified some of the key molecular targets associated with AR and p53 target genes. These findings point to the need for further extensive studies on AR co‐activators, such as p300, its central role in post‐translational modifications such as acetylation of p53 and/or AR by RE in a time‐ and dose‐dependent manner at different stages of prostate cancer that will fully elucidate the role of RE as a chemopreventive agent for prostate cancer in humans. © 2003 Wiley‐Liss, Inc.
CYP enzyme polymorphisms and susceptibility to HCV‐related chronic liver disease and liver cancer
Authors: Silvestri Laura , Sonzogni Laura , De Silvestri Annalisa , Gritti Chiara , Foti Luciana , Zavaglia Claudio , Leveri Michela , Cividini Agostino , Mondelli Mario U. , Civardi Emilio , Silini Enrico M.
Published: February 2003

Cancer risk can be influenced by the exposure to endogenous or environmental toxins. Polymorphic enzymes involved in the metabolic activation/detoxification of carcinogens may account for individual variations of risk. We studied the polymorphisms of five enzymes of the P450 superfamily, CYP1A1, CYP1A2, CYP2D6, CYP2E1 and CY3A4, as risk factors for liver disease progression and cancer in hepatitis C virus‐infected patients. CYP genotyping was performed by polymerase chain reaction (PCR) restriction fragment length polymorphism or allele‐specific PCR. Different stages of disease were considered, as follows: 90 asymptomatic carriers and 87 chronic hepatitis, 92 cirrhosis and 91 hepatocellular carcinoma (HCC) cases. Reference allele frequencies were obtained from 99 blood donors. Allele distributions among categories were compared using the χ2 test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to express relative risks. Independent associations were modeled by correspondence analysis and logistic regression. Frequencies of the CYP1A1 highly inducible alleles, MspI m2 and Val, were increased in liver disease patients compared with carriers; no specific association with HCC was found. The high‐activity CYP2E1 c2 allele was underrepresented among HCC patients with respect to other HCV categories, including cirrhosis. CYP2D6 poor metabolizer (PM) genotypes were significantly more frequent in healthy subjects (7.1%) and carriers (11.1%) than in hepatitis/cirrhosis (4.6%) and HCC (1.2%) patients. This was confirmed by multivariable analysis. PM genotypes protected against progressive disease as ORs reduced proportionally to stage. The age at diagnosis for HCC was anticipated in non‐PM individuals. No differences were seen for CYP1A2 and CYP3A4 genes. Polymorphic variants of CYP genes may contribute to the progression of liver disease and HCC risk in HCV‐infected subjects. © 2003 Wiley‐Liss, Inc.
Polyamine biosynthesis inhibition enhances HSV‐1 thymidine kinase/ganciclovir‐mediated cytotoxicity in tumor cells
Authors: Pasanen Tiina , Karppinen Anne , Alhonen Leena , Jänne Juhani , Wahlfors Jarmo
Published: February 2003

Cancer gene therapy with the aid of herpes simplex virus type 1 thymidine kinase gene (HSV‐TK) and anti‐herpes drug ganciclovir (GCV) has been widely used and its efficacy has been demonstrated in a variety of different malignant cells and animal tumor models. It is also commonly accepted, however, that this gene therapy regimen needs to be enhanced for a true clinical success. We studied whether polyamine biosynthesis inhibition by 2‐difluoromethylornithine (DFMO), a clinically tested and well‐tolerated chemotherapeutic drug, can increase the cytotoxicity of HSV‐TK/GCV in 9L rat glioma cells. Our initial experiments showed that polyamine depletion actually protected the cells from cytotoxicity if GCV treatment was started too early after removal of DFMO. Analyses of cell growth, intracellular polyamine pools and cell cycle phase distribution suggested that later initiation of GCV treatment would be more beneficial due to increased proportion of cells in the middle of the cell cycle S phase. When the cells were exposed to GCV 3 or 4 days after removal of DFMO from growth medium, the cytotoxicity was increased up to 2.5‐fold. We also verified whether cell cycle blockage per se could yield similar effect as DFMO. Our results from serum deprivation experiments showed that, despite of apparent cell growth and cell cycle phase distribution effects, serum starvation was weaker enhancer of HSV‐TK/GCV cytotoxicity than DFMO. Finally, the general utility of HSV‐TK/GCV + DFMO combination was tested in another tumor cell type, human prostate carcinoma cell line DU‐145. DFMO sensitized these cells to HSV‐TK/GCV cytotoxicity, but the effect was less prominent than in 9L cells. In conclusion, we have demonstrated that a correctly timed induction of DFMO‐mediated polyamine biosynthesis inhibition can enhance the efficiency of HSV‐TK/GCV gene therapy in vitro. The observed synergistic effect is potentially useful in clinical trials because, as opposed to use of other cell cycle‐altering drugs, DFMO has already been tested in the treatment of human tumors and used as chemo preventive regimen with excellent tolerability. © 2003 Wiley‐Liss, Inc.
Effects of granulocyte‐macrophage colony‐stimulating factor and foreign helper protein as immunologic adjuvants on the T‐cell response to vaccination with tyrosinase peptides
Authors: Scheibenbogen Carmen , Schadendorf Dirk , Bechrakis Nikolaos E. , Nagorsen Dirk , Hofmann Udo , Servetopoulou Fotini , Letsch Anne , Philipp Armin , Foerster Michael H. , Schmittel Alexander , Thiel Eckhard , Keilholz Ulrich
Published: February 2003

Immunologic adjuvants are used to augment the immunogenicity of MHC class I–restricted peptide vaccines, but this effect has rarely been systematically evaluated in a clinical trial. We have investigated, in a phase I study, whether addition of the 2 adjuvants GM‐CSF and KLH can enhance the T‐cell response to MHC class I peptide vaccines. Forty‐three high‐risk melanoma patients who were clinically free of disease received 6 vaccinations with MHC class I–restricted tyrosinase peptides alone, with either GM‐CSF or KLH or with a combination of both adjuvants. The primary end point was induction of tyrosinase‐specific T cells, and serial T‐cell monitoring was performed in unstimulated peripheral blood samples before and after the second, fourth and sixth vaccinations by ELISPOT assay. Tyrosinase‐specific IFN‐γ‐producing T cells were detected as early as 2 weeks after the second vaccination in 5 of 9 patients vaccinated with tyrosinase peptides in combination with GM‐CSF and KLH but not in any patient vaccinated with tyrosinase peptides without adjuvants or in combination with either adjuvant alone. After 6 vaccinations, tyrosinase‐specific T cells were found in patients immunized with peptides either without adjuvants (3 of 9 patients) or in combination with the single adjuvant GM‐CSF (4 of 9 patients) but not with KLH (0 of 10 patients). Our results suggest that addition of either GM‐CSF or KLH as a single adjuvant has little impact on the immunogenicity of tyrosinase peptides. The combined application of GM‐CSF and KLH was associated with early induction of T‐cell responses. © 2003 Wiley‐Liss, Inc.
Frequent monoallelic deletion of PTEN and its reciprocal associatioin with PIK3CA amplification in gastric carcinoma
Authors: Byun Do‐Sun , Cho Kyucheol , Ryu Byung‐Kyu , Lee Min‐Goo , Park Jae‐Il , Chae Kwon‐Seok , Kim Hyo‐Jong , Chi Sung‐Gil
Published: February 2003

Mutational alterations of PTEN and PIK3CA, which negatively and positively regulate PI3‐kinase activity, respectively, have been observed in many types of human cancer. To explore the implication of PTEN and PIK3CA mutations in gastric tumorigenesis, we characterized the expression and mutation status of the genes in 126 gastric tissues and 15 cell lines. Expression of PTEN transcript was abnormally low in 5 of 15 (33%) cell lines and 20 of 55 (36%) primary carcinomas, whereas 0 of 71 noncancerous tissues including 16 benign tumors showed altered expression. Allelotyping analysis using an intragenic polymorphism (IVS4+109) revealed that 14 of 30 (47%) informative cases carried LOH of the gene, which is closely linked to low expression. The LOH rate was significantly higher in advanced tumors [12 of 19 (63%)] compared to early‐stage tumors [2 of 11 (18%)] and more frequent in poorly differentiated tumors [9 of 13 (69%)] than well‐ or moderately differentiated tumors [5 of 17 (29%)]. Interestingly, however, none of the LOH tumors carried mutational disruption of the remaining allele, suggesting haploinsufficiency of PTEN in gastric tumorigenesis. Methylation studies revealed that PTEN pseudogene, but not PTEN, is methylated in cell lines and primary tumors, indicating that PTEN is not a target of epigenetic silencing in gastric cancers and that the pseudogene should be considered more carefully in methylation analysis of the PTEN promoter. Genomic amplification of PIK3CA was found in 9 of 15 (60%) cell lines and 20 of 55 (36.4%) primary tumors but in no noncancerous tissues. Furthermore, PIK3CA amplification was predominantly detected in tumors with no PTEN alterations, suggesting that mutations of PTEN and PIK3CA are mutually exclusive events in gastric tumorigenesis. Amplification of PIK3CA was strongly associated with increased expression of PIK3CA transcript and elevated levels of phospho‐AKT. Collectively, our data reveal that 13 of 15 (87%) gastric cell lines and 31 of 55 (56%) primary carcinomas harbored either amplification of PIK3CA or abnormal reduction of PTEN. Mutually exclusive alterations of PTEN and PIK3CA also suggest that mutations of either gene could activate the PI3‐kinase/AKT signaling pathway, which is directly linked to the malignant progression of gastric tumor cells. © 2003 Wiley‐Liss, Inc.
Decreased DNA repair gene expression among individuals exposed to arsenic in United States drinking water
Authors: Andrew Angeline S. , Karagas Margaret R. , Hamilton Joshua W.
Published: February 2003

Arsenic is well established as a human carcinogen, but its precise mechanism of action remains unknown. Arsenic does not directly damage DNA, but may act as a carcinogen through inhibition of DNA repair mechanisms, leading indirectly to increased mutations from other DNA damaging agents. The molecular mechanism underlying arsenic inhibition of nucleotide excision repair after UV irradiation (Hartwig et al., Carcinogenesis 1997;18:399–405) is unknown, but could be due to decreased expression of critical genes involved in nucleotide excision repair of damaged DNA. This hypothesis was tested by analyzing expression of repair genes and arsenic exposure in a subset of 16 individuals enrolled in a population based case‐control study investigating arsenic exposure and cancer risk in New Hampshire. Toenail arsenic levels were inversely correlated with expression of critical members of the nucleotide excision repair complex, ERCC1 (r2 = 0.82, p < 0.0001), XPF (r2 = 0.56, p < 0.002), and XPB (r2 = 0.75, p < 0.0001). The internal dose marker, toenail arsenic level, was more strongly associated with changes in expression of these genes than drinking water arsenic concentration. Our findings, based on human exposure to arsenic in a US population, show an association between biomarkers of arsenic exposure and expression of DNA repair genes. Although our findings need verification in a larger study group, they are consistent with the hypothesis that inhibition of DNA repair capacity is a potential mechanism for the co‐carcinogenic activity of arsenic. © 2003 Wiley‐Liss, Inc.
Recirculating and germinal center B cells differentiate into cells responsive to polysaccharide antigens
Authors: Vinuesa Carola G. , Sze Daniel M‐Y. , Cook Matthew C. , Toellner Kai‐Michael , Klaus Gerry G. B. , Ball Jennifer , MacLennan Ian C. M.
Published: February 2003

Antibodies against bacterial capsular polysaccharides play a critical protective role. Responses to these antigens can occur without the help or control of T cells and are associated with marginal zone (MZ) B cells. Capsular antigens are diverse and some cross‐react with self‐carbohydrate epitopes. This diversity may explain the recruitment of non‐autoreactive recirculating B cells and memory B cells to the MZ in addition to other B cells, some of which are weakly autoreactive cells, that are recruited to the MZ without entering the recirculating pool. To test whether memory B cells respond to polysaccharide‐based antigens, mice with hapten‐specific memory B cells were challenged with hapten‐polysaccharide. Hapten‐specific plasma cells producing high affinity antibody with IgV‐region mutations were induced. To test whether naive recirculating B cells can form MZ cells that respond to polysaccharide, recirculating B cells from lymph nodes were transferred into Rag‐1‐deficient mice. MZ cells differentiated from the donor cells without proliferation or T cell help and responded to polysaccharide‐based antigen. The differentiation of B cells both from germinal centers and the recirculating pool to the MZ phenotype is likely to make an important contribution to the repertoire of B cells that respond to polysaccharide antigens.
TRANCE together with IL‐7 induces pre‐B cells to proliferate
Authors: Kato Ibuki , Sato Hiromu , Kudo Akira
Published: February 2003

TRANCE (TNF‐related activation‐induced cytokine)‐deficient mice completely lack osteoclasts, and develop severe osteopetrosis. These mice also show a defect in their pre‐B cell differentiation. In the present study, the expression of TRANCE was examined in pre‐B cell lines using flow cytometry and reverse transcription‐PCR. Three pre‐B cell lines, 18‐81, B3P816‐1, and 38B9, expressed TRANCE on their surface, and two pre‐B cell lines, 70Z/3 and NFS5, at the late pre‐B cell stage, expressed it at low levels, although their mRNA expression was normal. Another pre‐B cell line, 38‐C‐13, at the intermediate stage between pre‐B and immature B cells, did not express TRANCE. The IL‐7‐dependent pre‐B cell line PreBR, which expresses the pre‐B cell receptor on the cellsurface, also expressed TRANCE. When differentiation of PreBR cells was induced in vitro by removing IL‐7 from cultures, TRANCE expression dropped; it was restored by the addition of IL‐7, suggesting that TRANCE functions in cooperation with IL‐7. To examine the function of TRANCE, we introduced the TRANCE gene into PreBR cells and established two transfectants that constitutively expressed TRANCE, even in the absence of IL‐7. In these transfectants, after removal of IL‐7, the number of cells that succeeded in κ chain rearrangement was decreased to one third; and CD40 expression decreased to less than one tenth. Moreover, the percentage of cells in the S/G2/M phase was increased by 50% over the mock transfectant. These findings indicate that, before κ chain rearrangement occurs, TRANCE together with IL‐7 induces pre‐B cells to proliferate and makes this rearrangement more efficient.
Molecular characterization of the novel rat NK receptor 1C7
Authors: Bäckman‐Petersson Eva , Miller J. Ross , Hollyoake Martine , Aguado Begoña , Butcher Geoffrey W.
Published: February 2003

A novel receptor, named 1C7 or NKp30 and involved in natural cytotoxicity, was recently identified. This receptor is encoded by the 1C7 gene, which is located within the class III region of the human MHC, HLA. It is a member of the immunoglobulin gene superfamily (IgSF) and, remarkably, is expressed at the mRNA level as six different splice variants in human. Recent investigations have indicated that the 1c7 gene of the mouse is silenced by in‐frame stop codons. In this study, the molecular characterization of the rat 1c7 gene is described. cDNA derived from this gene encode a protein of 192 amino acid residues predicted to contain a single IgV‐set domain in the extracellular region and a positively charged residue in the transmembrane region. Expression of the gene was detected in freshly isolated rat Natural Killer (NK) and T splenocytes. Transfection of rat 1C7 into the NK cell line RNK‐16 induced cytolytic activity against glioma as well as lymphoma tumor cells. In addition, binding of a r1C7‐Fc fusion protein by a panel of target cells correlated with susceptibility to killing by RNK‐16–1C7 effector cells. These results indicate that the r1C7 molecule could function as an NK activating receptor as previously reported for the human NKp30 receptor molecule.
CD2+/CD14+ monocytes rapidly differentiate into CD83+ dendritic cells
Authors: Pucchio Tiziana Di , Lapenta Caterina , Santini Stefano Maria , Logozzi Mariantonia , Parlato Stefania , Belardelli Filippo
Published: February 2003

Since denditric cells (DC) represent the main players linking innate and adaptive immunity, their prompt generation from blood cells would be instrumental for an efficient immune response to infections. Consistent with this, CD2+ monocytes were found to express the DC maturation marker CD83, along with acquisition of high antigen‐presenting activity, after a surprisingly short time in culture. This rapid process is associated with expression of IFN‐α/β genes and secretion of low levels of pro‐inflammatory cytokines. Exposure of monocytes to IFN‐α, but not to IL‐4, induced persistence of CD2+/CD83+ cells, which were fully competent in stimulating primary responses by naive T cells. These results unravel the natural pathway by which infection‐induced signals rapidly transform pre‐armed monocytes into active DC.
A novel ligand for the NKG2D receptor activates NK cells and macrophages and induces tumor immunity
Authors: Diefenbach Andreas , Hsia Jennifer K. , Hsiung Ming‐Yu B. , Raulet David H.
Published: February 2003

NK cells are involved in the immune response against viral and microbial infections and tumors. In contrast to B and T cells, NK cells employ various modes of immune recognition. An important mode of immune recognition employed by NK cells is "induced self recognition" exemplified by the NKG2D receptor–ligand system. The NKG2D immunoreceptor, expressed by NK cells, and by activated CD8+ T cells and macrophages, recognizes one of several cell surface ligands that are distantly related to MHC class I molecules (i.e. H60 and Rae1 proteins in mice, and MHC class I chain‐related proteins and UL‐16‐binding proteins in humans). These ligands are not expressed abundantly by most normal cells but are up‐regulated on cells exposed to various forms of cellular insults.Here we report the cloning of another ligand for NKG2D; transcripts of this ligand are found in a wide variety of tissues and in various tumor cells. Cross‐linking of NKG2D with the novel ligand potently activated NK cells and macrophages. Tumor cells ectopically expressing the molecule were efficiently rejected by naive mice, and induced strong protective immunity to the parental, ligand‐negative tumor cells.
Costimulation of human CD28– T cells by 4‐1BB ligand
Authors: Bukczynski Jacob , Wen Tao , Watts Tania H.
Published: February 2003

The T cell surface protein CD28 provides a critical costimulatory signal for T cell activation. With age, humans accumulate increasing numbers of CD28– T cells, and this loss of CD28 expression is exacerbated certain disease states, such as HIV infection, autoimmune conditions or cancer. It is unclear whether CD28– T cells represent terminally differentiated effector cells or whether they remain sensitive to costimulation by CD28‐independent pathways. Here, we demonstrate that 4–1BB ligand can costimulate human CD28– T cells, resulting in cell division, inflammatory cytokine production, increased perforin levels, enhancement of cytolytic effector function, as well as the up‐regulation of the anti‐apoptotic protein Bcl‐XL. Thus, human CD28– T cells can respond to costimulatory signals and as such become attractive targets for therapeutic intervention, particularly in chronic infectious and inflammatory diseases where large numbers of these cells accumulate.
Biochemical and functional characterization of the interaction between pentraxin 3 and C1q
Authors: Nauta Alma J. , Bottazzi Barbara , Mantovani Alberto , Salvatori Giovanni , Kishore Uday , Schwaeble Wilhelm J. , Gingras Alexandre R. , Tzima Sotiria , Vivanco Fernando , Egido Jesús , Tijsma Odette , Hack Erik C. , Daha Mohamed R. , Roos Anja
Published: February 2003

Pentraxin 3 (PTX3) is a recently characterized member of the pentraxin family of acute‐phase proteins produced during inflammation. Classical short pentraxins, C‐reactive protein, and serum amyloid P component can bind to C1q and thereby activate the classical complement pathway. Since PTX3 can also bind C1q, the present study was designed to define the interaction between PTX3 and C1q and to examine the functional consequences of this interaction. A dose‐dependent binding of both C1q and the C1 complex to PTX3 was observed. Experiments with recombinant globular head domains of human C1q A, B, and C chains indicated that C1q interacts with PTX3 via its globular head region. Binding of C1q to immobilized PTX3 induced activation of the classical complement pathway as assessed by C4 deposition. Furthermore, PTX3 enhanced C1q binding and complement activation on apoptotic cells. However, in the fluid‐phase, pre‐incubation of PTX3 with C1q resulted in inhibition of complement activation by blocking the interaction of C1q with immunoglobulins. These results indicate that PTX3 can both inhibit and activate the classical complement pathway by binding C1q, depending on the way it is presented. PTX3 may therefore be involved in the regulation of the innate immune response.
Kinetics and expression patterns of chemokine receptors in human CD4+ T lymphocytes primed by myeloid or plasmacytoid dendritic cells
Authors: Langenkamp Anja , Nagata Kinya , Murphy Kristine , Wu Lijun , Lanzavecchia Antonio , Sallusto Federica
Published: February 2003

We investigated the kinetics of expression of 12 chemoattractant receptors as a function of cell division following priming of human naive CD4+ T cells by different populations of dendritic cells (DC) and under conditions favoring Th1 or Th2 differentiation. Two chemokine receptors, CXCR3 and CXCR5, were rapidly up‐regulated following T cell activation by either monocyte‐derivedDC, myeloid DC (mDC) or plasmacytoid DC (pDC). While CXCR5 expression was transient, expression of CXCR3 at advanced cell divisions was dependent on differentiation, being expressed at high levels on Th1 cells. Several other receptors (CCR2, CCR3, CCR4, CCR5, CXCR6 and CRTh2) were acquired progressively as a function of cell division and in a fashion that was influenced by polarizing cytokines. The Th2‐associated chemoattractant receptors CRTh2 and CCR3 were up‐regulated with slower kinetics compared to the Th1‐associated receptors CXCR3 and CXCR6, consistent with a different kinetics and efficiency of polarization. Moreover, CCR4 and CXCR6 were preferentially induced in T cells activated by mDC and pDC, respectively. Finally, CXCR5 and CCR7 were also rapidly and transiently up‐regulated in memory T cells following TCR stimulation. These results indicate a complex chemokine receptor regulation dependent on both T cell activation and differentiation state. In addition, they reveal the existence of DC‐specific cues for the regulation of T cell migratory capacity.
Antigen‐presenting cell exosomes are protected from complement‐mediated lysis by expression of CD55 and CD59
Authors: Clayton Aled , Harris Claire L. , Court Jacquelyn , Mason Malcolm D. , Morgan B. Paul
Published: February 2003

Exosomes are secreted nanometer‐sized vesicles derived from antigen‐presenting cells, which have attracted recent interest as they likely play important roles in immune regulation, and their use as cell‐free tools for immunotherapy has been proposed. Liposomes used clinically as transport vehicles can activate the complement system, leading to their rapid degradation and significant inflammatory toxicity. The use of isolated exosomes in therapy, therefore, may also elicit complement activation, reducing their potential efficacy. We have examined the expression and functional roles of the membrane regulators of complement (CD46, CD55 and CD59) on antigen‐presenting cell‐derived exosomes. Exosomes express the glycosylphosphatidylinositol (GPI)‐anchored regulators CD55 and CD59,but not the transmembrane protein CD46. Antibody blocking of CD55 in the presence of sensitizing antibody (w6/32) and human serum resulted in increased C3b deposition and significantly increased exosome lysis. Blockade of CD59 also resulted in significant lysis, while blocking both CD55 and CD59 increased lysis still further. We conclude that exosomes express GPI‐anchored complement regulators in order to permit their survival in the extracellular environment.
Deviation of islet autoreactivity to cryptic epitopes protects NOD mice from diabetes
Authors: Song Aihua , Winer Shawn , Tsui Hubert , Sampson Anastazia , Pasceri Peter , Ellis James , Elliott John F. , Dosch H.‐Michael
Published: February 2003

To better understand loss of self‐tolerance in diabetes‐prone NOD mice, we are generating ICA69 transgenes under control of the tetracycline‐regulated tet07 minimal promoter. In vitro pilot studies showed leaky transgene expression, but addition of β‐globin genomic insulator flanks prevented leakage and dramatically enhanced transgene expression even in transient transfection, with excellent suppression by Doxycycline. In vivo, the accidental loss of insulator flanks during transgene insertion in one transgenic NOD founder, tet1, re‐established leakiness with high level, exclusive ICA69‐transgene expression in stromal elements of thymus and spleen. This led to persistent deletion of T cells targeting the immunodominant ICA69 epitope, Tep69, but emergenceof T cell pools targeting cryptic ICA69 epitopes not normally generated in sufficient density to select and maintain ICA69‐autoreactive T cells. This subtle modification of T cell repertoires reduced insulitis, and protected from diabetes in transgenics and in wild‐type mice carrying irradiated tet1 thymus grafts. The low pathogenicity of T cells targeting cryptic epitopes likely reflects the fact that the major ICA69 determinant presented in the islet milieu remains Tep69, while cryptic epitopes are under‐represented. Deviation of T cell autoreactivity from major to cryptic target epitopes in tet1 mice provides a fortuitous model to explain previously observed diabetes protection by immunotherapy or autoantigen transgenes despite apparent failure to achieve tolerance to the full length islet antigens.
Attitudes and reactions to media coverage of terrorist acts
Authors: Keinan Giora , Sadeh Avi , Rosen Sefi
Published: February 2003

The present study examined attitudes and reactions of individuals towards media coverage of terrorist acts. Shortly after a series of severe terrorist attacks had taken place in Israel, 534 people were asked to fill out a questionnaire that assessed their attitudes and reactions to the media's coverage of these acts as well as a questionnaire that examined their information‐seeking style. The results suggested that although a considerable proportion of media consumers preferred detailed coverage of terrorist acts, when the coverage included horrifying details, the readiness for receiving detailed information declined. In addition, the results indicated that exposure to such coverage was associated with the development of symptoms similar to those of Post‐Traumatic Stress Disorder. Finally, individual differences in both attitudes and reactions towards media coverage were found as a function of participants' gender, political orientation, and information‐seeking style. The theoretical and empirical implications of these findings are discussed. © 2003 Wiley Periodicals, Inc.
Action research on leadership for community development in West Africa and North America: A joining of liberation theology and community psychology
Authors: Trout John , Dokecki Paul R. , Newbrough J. R. , O'Gorman Robert T.
Published: February 2003

Our purpose is to enhance the field's understanding of the nature of community leadership, the psychological sense of community, and the spiritual dimensions of community life. We describe an action research community development project conducted in a low‐income Catholic parish in Lagos, Nigeria, in Western Africa. After describing the community development process that resulted in the establishment of 40 small/street basic communities (SBCs), we report an evaluation study in which 70 SBC leaders were surveyed regarding (1) SBC background characteristics (socioeconomic and environmental conditions and statistical information), (2) the state of community life in the SBCs (spirit/sense of community, participation in activities, benefits of community life), and (3) challenges for the future (what SBC members and leaders can do to improve the community, problems influencing community development). We then reflect on the Lagos findings in light of leadership‐for‐community and spirituality frameworks developed in an action research project conducted in a Catholic parish in the United States. © 2003 Wiley Periodicals, Inc.
Mass spectrometry of polyaromatic sulfur compounds in the presence of palladium(II)
Authors: Rudzinski Walter E. , Zhang Yi , Luo Xuemei
Published: February 2003

Electrospray ionization mass spectrometry (ESI‐MS) and tandem MS in an ion trap were used to explore the reduction of a PdCl2 solution and the role of the metal in the formation of organosulfur radical cations. For tandem MS experiments, a 32 Da neutral loss (M − S), was observed for dibenzothiophene, thianthrene and benzonaphthothiophene, whereas both 32 and 33 Da neutral losses were observed for 2‐methyldibenzothiophene and 4,6‐dimethyldibenzothiophene. The effects of several variables on the formation of molecular ions or molecular fragments were studied to optimize radical cation formation, including the concentration ratio between PdCl2 and sulfur and the collision energy. Electrochemical and chromatographic data were used to provide an explanation for the relative sensitivity observed for a mixture of polyaromatic sulfur heterocycles. Copyright © 2003 John Wiley & Sons, Ltd.
Non‐linearity in the quadrupole detector system: implications for the determination of the 13C mass distribution of an ion fragment
Authors: Vogt Josef A. , Wachter Ulrich , Georgieff Michael
Published: February 2003

Some biomedical 13C tracer applications to study glucose/lactate metabolism require the determination of up to 7 mass isotopomers of glucose, generated by the 13C‐labeling. Isotopomer ratios determined by GC‐Quadrupole‐MS can depend on the analyte amount being measured and may differ from expected theoretical values. A calibration is necessary to correct these effects. However, the large number of different isotopomers prevents an empirical calibration, that is based on known isotopomer mixtures. Neither insufficient mass separation in the quadrupole nor proton adducts or losses of the ionized fragment can account for the observed deviation from expected values and the dependency on the sample size. We explain the deviations with a nonlinear mapping of the isotopomer amount being analyzed to the area under the elution curve for the corresponding mass channel. The nonlinear relation allows to estimate potential errors in the determination of isotopomer ratios. It also allows to define a dynamic range as the range of signal areas, for which the systematic error in the isotopomer ratio determination is below 5% of the nominal value. It is 1 : 200 and 1 : 500 for two different fragments of glucose, measured under EI and CI and is smaller than the range imposed by the measurement precision. After correcting the nonlinearities the dynamic range improved by the factor 10 in both cases. Copyright © 2003 John Wiley & Sons, Ltd.
Analysis of the pigments on painted pottery figurines from the Han Dynasty's Yangling Tombs by Raman microscopy
Authors: Zuo Jian , Zhao Xichen , Wu Ruo , Du Guangfen , Xu Cunyi , Wang Changsui
Published: February 2003

The pigments on painted pottery figurines from the Han Dynasty's Yangling Tombs were analysed by Raman microscopy. It was found that BaCuSi2O6 is included in the purple pigment, which was first synthesized by ancient people 1845 years ago. The red pigments were identified as haematite and cinnabar, the yellow pigment as goethite, the white pigment as calcite, the blue pigment as azurite and the black pigment as soot. Copyright © 2003 John Wiley & Sons, Ltd.
Structural phase transition in (n‐C4H9NH3)2SiF6: DSC and Raman studies
Authors: Jeghnou H. , Ouasri A. , Rhandour A. , Dhamelincourt M.‐C. , Dhamelincourt P. , Mazzah A.
Published: February 2003

Butylammonium hexafluorosilicate, (n‐C4H9NH3)2SiF6, crystallizes at room temperature in the trigonal system with a probable space group P321. A new structural phase transition was detected at low temperatures for this compound by means of differential scanning calorimetry (DSC) and the basic thermodynamic data for this transition were determined. The internal vibrational modes of (n‐C4H9NH3)2SiF6 were studied at ambient temperature and as a function of the temperature, through the phase transition, by means of Raman spectroscopy. The observed phase transition is associated with the dynamics of the system and is of the order–disorder type. Copyright © 2003 John Wiley & Sons, Ltd.
Three‐layer substrates for surface‐enhanced Raman scattering: preparation and preliminary evaluation
Authors: Mulvaney Shawn P. , He Lin , Natan Michael J. , Keating Christine D.
Published: February 2003

In surface‐enhanced Raman scattering (SERS), the magnitude of enhancement is highly dependent on the nanometer‐scale morphological and optical properties of the substrate employed. This paper reports the development of an improved SERS‐active substrate that is based on a combination of nanoparticle assembly and evaporated thin films. Highly enhancing, three‐layer SERS‐active substrates were constructed by evaporation of an Ag film over an Ag‐clad colloidal Au submonolayer; the distinguishing feature of the three‐layer substrate is the formation of evaporated Ag islands between the surface‐bound colloidal particles. UV–visible absorption spectroscopy, atomic force microscopy and field emission scanning electron microscopy were used to characterize the substrate morphology. The thickness of the evaporated layer was varied in order to determine the optimal substrate architecture. The estimated enhancement factor for the optimized substrate architecture was 7 × 104. Furthermore, better than 15% reproducibility in the Raman spectra for adsorbed analytes was observed from spot to spot on a given substrate, and also from substrate to substrate. Finally, a sub‐picogram detection limit was demonstrated with the environmental pollutant 1,3,5‐triazine. Copyright © 2003 John Wiley & Sons, Ltd.
Capillary zone electrophoretic assay of biologically active thioacridine derivatives
Authors: Matalová Radka , Jelínek Ivan , Pumera Martin , Barbe Jacques
Published: February 2003

A capillary zone electrophoretic method was developed for the analytical evaluation of newly synthesized biologically active thioacridine derivatives. Based on background electrolyte pH optimization, the experimental conditions ensuring satisfactory resolution of the studied set of thioacridine derivatives and their accompanying synthetic impurities were found. Optimum background electrolyte consists of 30 mM citric acid and 30 mM β‐alanine (pH 3.74). Calibration data confirmed linear response for all compounds of interest in the concentration range 1–500 μg/mL, sufficiently high sensitivity, and limits of detection not exceeding 0.16 μg/mL. The described assay proved to be suitable for rapid identification and purity evaluation of studied thioacridine derivatives. The qualitative and quantitative results have been compared with those obtained using conventional liquid chromatography. The differences were not generally significant.
Facile monitoring and evaluation of bacteria in a fermentation process using perfusion chromatography and polarimetry detection
Authors: Berthod Alain , Zhang Bo , Armstrong Daniel W.
Published: February 2003

A 670 nm diode laser polarimeter was used as a detector for living cells in perfusion chromatography. It was found that all microorganisms examined, including vegetal organisms such as algae and fungi as well as several strains of different bacteria, gave a levorotatory signal, producing negative peaks in the chromatograms. Most naturally occurring chiral molecules, such as sugars and amino acids are dextrorotatory and give positive polarimetric peaks. A hydrophilic OH‐functionalized polystyrene divinylbenzene spherical porous particle packing was used as a sieving and size exclusion material and packed in 27×0.95 cm columns. The theory of the close‐packing of spheres is summarized to show that common bacteria should be able to perfuse in the interparticle voids of the 50 μm packing. It is shown that bacteria are eluted with the exclusion volume; a selective permeation of polymer with molecular weight ranging from 103 to 2×106 is obtained, and total permeation occurs for small molecules. The restricted access columns are used to follow the changes in cell concentration in bacteria cultures. The growth of three different bacteria was studied using calibrated polarimetric signals.
Quantitative sample injection for capillary electrophoresis
Authors: Yao Chuanyi , Gao Ruyu , Yan Chao
Published: February 2003

An apparatus including a rotary‐type injector was designed for quantitative sample injection in capillary electrophoresis (CE), in which both pressurized flow and electroosmotic flow were used to drive the background electrolyte solution. A relative standard deviation of peak area of lower than 1% was achieved by using this apparatus. The effects of back‐pressure regulator, restrictor, and applied voltage on separation efficiency and resolution were investigated. The utility of this apparatus in both micro‐HPLC and pressurized capillary electrochromatography (pCEC) was also demonstrated.
Measurement of the binding of retinoic acid to β‐lactoglobulin B by affinity capillary electrophoresis
Authors: Lynen Frédéric , Van Thuyne Wim , Borremans Frans , Vanhoenacker Gerd , Sandra Pat
Published: February 2003

The quantification of the binding strength between retinoic acid and the primary binding site of β‐lactoglobulin B by dynamic equilibrium affinity capillary electrophoresis (DE‐ACE) is described. Although the peaks for retinoic acid were broad, a distinctive shift in migration time could be observed upon complexation allowing the construction of a binding curve and three linearised plots. By performing the corresponding linear and non‐linear regression analyses, an apparent dissociation constant varying between 1.4 and 2.2 μM was measured in a 100 mM Tris‐acetate buffer at pH 8.2 with 30 mM Na2SO4 and 1% EtOH as additives. The sample was prepared by adding a concentrated solution of retinoic acid in ethanol to the background electrolyte, such as to obtain a final 1% solution of ethanol and 5 μM retinoic acid in the buffer. It was shown that an MEKC‐based approach, attempted to improve the solubility of retinoic acid in the aqueous buffer, could not be used to perform a binding study.
Determination of metabolites of benzene, toluene, ethylbenzene, and xylene by β‐cyclodextrin modified capillary electrophoresis
Authors: Wang Chin‐Yu , Huang Ching‐Te , Hsieh You‐Zung
Published: February 2003

A β‐cyclodextrin modified capillary electrophoresis method for determining the metabolites of benzene, toluene, ethyl benzene, and xylene (BTEX) is described. Creatinine and uric acid were also examined as reference compounds. By employing a 20 mM, pH 10.0 borax‐NaOH buffer containing 7‐mM of β‐CD, ten analytes could be successfully separated within 10 min on a 50 μm capillary at an electric potential of 25 kV. The effects of pH and the concentration of β‐CD on the separation of the ten analytes are discussed. Under optimized separation conditions, the relative standard deviations for the migration times of the analytes were less than 0.93% and the correlation coefficients of the analyte linear calibration graphs exceeded 0.998 in the range from 1 to 300 μg/mL. Analyses of BTEX metabolites in urine samples from both occupationally exposed and non‐exposed subjects are shown herein. BTEX metabolites were determined in the urine sample of a gas station attendant.
Determination of low concentrations of organochlorine pesticides and PCBs in fish feed and fish tissues from aquaculture activities by gas chromatography with tandem mass spectrometry
Authors: Serrano Roque , Barreda Mercedes , Pitarch Elena , Hernández Félix
Published: February 2003

A sensitive and rapid multiresidual method has been developed for the determination of organochlorine compounds in fish feed and fish tissues. The method is based on automated off‐line normal‐phase liquid chromatography (NPLC) clean‐up prior to GC‐MS/MS. Analytes are extracted by refluxing in n‐hexane and fats present in the hexanic extracts are removed by automated NPLC, using n‐hexane and ethyl acetate as mobile phase. Both NPLC and acid digestion with sulphuric acid have been evaluated as clean‐up techniques for the analysis of these kinds of fatty samples. Thus, acid digestion is recommended before NPLC when large amounts of fat are present in the extracts. Purified extracts are then analysed by GC‐MS/MS (electron impact, EI) using the extracted ion mode. The high efficiency of elimination of fats from the extracts together with optimised MS/MS detection leads to high sensitivity in the multiresidue determination of analytes, reaching limits of detection at the sub‐ppb level in this type of fatty samples (up to 19% lipids fresh weight). The developed procedure represents an improvement over current methodologies for the analysis of organochlorine compounds (OCs) in fatty samples as regards to solvent consumption and automation, allowing the application of automated off‐line NPLC (26 min per sample) prior to GC determination. Besides, changes of solvent are not necessary during analysis. The application of the developed procedure to real world samples of fish feed and cultured fish revealed the presence of HCB, DDTs, and PCBs at ng g–1 levels.