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  • 1
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    Postranslationally Modified Proteins in the Central Nervous System (CNS) Are the Dominant Antigenic Target/Stimulus of the B-Cell Receptor (BCR) in Primary CNS Lymphomas (PCNSL) Providing Strong Evidence for the Role of Chronic Autoantigenic Stimulation As an Early Step in the Pathogenesis of Aggressive B-Cell Lymphomas
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 142-142
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 142-142
    Abstract: Background: Sequence analyses of variable immunoglobulin gene fragments of PCNSL from immunocompetent patients have shown a VH4-34 restriction raising speculations on a selection/stimulation of a functional BCR by an autoantigen in the central nervous system. The present study focused on the search for these hypothetic autoantigens presumably driving the malignant transformation of B-cells into PCNSL cells by chronic BCR stimulation in immunocompetent patients. Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen lymphoma specimens and checked for binding to proteins expressed on macroarrays of human cDNA expression libraries. Results: Recombinant BCR expression was attempted in 21 and successful in twelve PCNSL cases. The VH4-34 family was overrepresented, but was found in less than a quarter of the PCNSL patients (4/21). Screening of the recombinant BCRs on protein arrays revealed that 8 of 12 recombinant PCNSL-BCRs reacted with SAMD14 and the SAM domain of neurabin-1, two proteins with high homology and preferential expression in the CNS. Subsequent proteomic analysis of cryoconserved lymphoma specimens showed that SAMD14 and the SAM domain of neurabin-1 were alternatively N-glycosylated in patients with a PCNSL-BCR specific for SAMD14 and neurabin-1, but not in the remaining PCNSL patients with BCR specificities other than for SAMD14/neurabin-1. Compared to SAMD14 and neurabin-1 from healthy controls, Asn 339 of SAMD14 and Asn 1277 of neurabin-1 were shown to carry additionally glycosylated Asn residues. Of interest, both additional glycosylation sites belonged to atypical, non-canonical Asn-Leu-Glu-Gln (N-L-E-Q) sites instead of the Asn-X-Ser/Thre consensus sequence (N-X-S/T; where X can be any amino acid except proline), which is reported to constitute 97% of N-glycosylation sites under physiologic circumstances. These atypical N-hyperglycosylations were shown for every case with sufficient biopsy material for this proteomic analysis and a PCNSL-BCR specific for SAMD14 and neurabin-1 in their PCNSL and CNS, and to a lesser degree in their peripheral blood mononuclear cells and patient-derived EBV-transformed lymphoblastoid cell lines (LCLs). Of the recombinant BCRs of all cases with sufficient material to test for hyperglycosylation, only the BCRs of the 6 cases with hyperglycosylated SAMD14/neurabin-1 reacted against SAMD14/neurabin-1. Of note, glycosylation status of 2/8 cases with recombinant SAMD14/neurabin-1 reactive BCRs could not be analyzed due to insufficient cryomaterial left after variable region gene PCRs. No hyperglycosylation of SAMD14 and neurabin-1 was found in the peripheral blood of 400 healthy controls, 100 newborns and 50 nursery residents. Moreover, antibodies against SAMD14 and neurabin-1 were detected in the sera and cerebrospinal fluids of an independent second cohort of patients with PCNSL (8/22), but not in sera of patients with secondary CNS manifestations of systemic DLBCL (0/17) or of healthy controls (0/92). Conclusion: Our results strongly suggest that the atypical (NLEQ) glycosylation of the highly homologous SAMD14 and the SAM domain of neurabin-1 maintains a chronic autoimmunogenic stimulation in the CNS, ultimately leading to the malignant transformation of B-cells with a BCR specific for these atypically N-hyperglycosylated proteins into an aggressive B-cell lymphoma in the CNS in a majority of patients with PCNSL. The fact that the VH4-34 family represents only a minority of VH families recognizing SAMD14/neurabin-1 underlines the extraordinary autoimmunogenicity of these posttranslationally modified proteins in a broad range of individuals. Our results provide the first and strongest experimental evidence for the role of chronic autoantigenic stimulation as a first step in the pathogenesis of aggressive B-cell lymphomas. Supported by Deutsche Forschungsgemeinschaft DFG, Deutsche José Carreras Leukämie Stiftung, Wilhelm-Sander-Stiftung, Deutsche Krebshilfe e.V. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.142.142
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
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    Online Resource
    Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments
    Oxford University Press (OUP) ; 2006
    In:  Clinical Chemistry Vol. 52, No. 6 ( 2006-06-01), p. 1161-1167
    Details
    In: Clinical Chemistry, Oxford University Press (OUP), Vol. 52, No. 6 ( 2006-06-01), p. 1161-1167
    Abstract: Background: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification. Methods: We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART™ for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomer-spotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases. Results: Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases. Conclusions: Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when & lt;200 ng of total RNA is used as starting material, we suggest the use of SMART PCR for preamplification.
    Type of Medium: Online Resource
    ISSN: 0009-9147 , 1530-8561
    URL: Article
    DOI: 10.1373/clinchem.2005.062406
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2006
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  • 3
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    Online Resource
    Identification of Posttranvslationally Modified Neoantigens As Targets of B Cell Receptors of Burkitt Lymphoma
    American Society of Hematology ; 2018
    In:  Blood Vol. 132, No. Supplement 1 ( 2018-11-29), p. 1588-1588
    Details
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 1588-1588
    Abstract: Introduction: Burkitt lymphoma represents the most aggressive neoplasm of mature sIg+ B cells. Besides the characterisitc translocation of the MYC gene with an immunoglobulin heavy or light chain gene locus, activating mutations in the TCF3 gene and inactivating mutations in the ID3 gene represent the key events in the pathogenesis of Burkitt lymphoma. These TCF3/ID3 mutations result in tonic and antigen-independent B cell receptor (BCR) pathway activation. Additionally, chronic BCR activation by antigens might play a role in Burkitt lymphoma pathogenesis and we set out to identify such potential target antigens of BCRs of Burkitt lymphoma. Methods: BCRs were expressed as recombinant Fabs in TG1 E.coli based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen sporadic Burkitt lymphoma specimens. Additionally, natural Fabs and recombinant Fabs were produced of 8 established Burkitt lymphoma cell lines by Papain digestion and BCR expression cloning. The purified pooled Fabs were screened for reactivities against non-modified and differently posttranslationally modified human protein macroarrays. Reactivities were verified by ELISA with coated N-terminally FLAG-tagged candidate antigens, each separately for the posttranslationally modified and non-modified isoforms. Recombinant Fabs derived of mantle cell lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma served as controls. Moreover the functional effects on proliferation and BCR pathway activation after addition of the identified target antigens to Burkitt lymphoma cell lines with and without reactive BCRs were analyzed by proliferation assays and western blots. Finally, mutation status, methylation status and expression level of identified target antigens were analyzed in ICGC MMML-Seq lymphoma databases for differences in Burkitt lymphoma versus distinct lymphoma entities. Results: The Burkitt lymphoma derived Fabs were tested on posttranslationally modified protein arrays. The BCR of CA46 line showed specific reactivity against sumoylated Bystin, the Fab of the BL41 line reacted specifically against acetylated HSP40. Recombinant Fabs of diffuse large B cell lymphoma, primary central nervous system lymphoma and mantle cell lymphoma did neither bind sumoylated Bystin nor acetylated HSP40. Addition of the posttranslationally modified cognate antigens to respective Burkitt lymphoma cell line with the reactive BCR induced proliferaton. The analysis of the ICGC MMML-Seq lymphoma databases (representing different cohorts) showed a higher expression of Bystin in Burkitt lymphoma compared to other aggressive B-cell lymphomas. However, the mutation and methylation status of Bystin and HSP40 in the ICGC MMML-seq cohort did not provide any direct indication of the origin of their immunogenic post-translational modifications. Conclusions: A subgroup of sporadic Burkitt lymphoma has autoreactive BCRs with specific affinity against posttranslationally modified self antigens, demonstrating a new aspect in the pathogenesis of Burkitt lymphoma. Specific secondary modifications, as sumolytation of Bystin or acetylation of HSP40 appear to evoke the immunogenicity. Future studies will focus on the functional consequences of the antigen/BCR interaction on Burkitt cells. Furthermore, the causes of these posttranslationally modified neoantigens will be investigated in more detail. Disclosures Stilgenbauer: Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2018-99-114209
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 4
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    Immunohistochemical assessment of PD-L1 expression using three different monoclonal antibodies in triple negative breast cancer patients
    Springer Science and Business Media LLC ; 2022
    In:  Archives of Gynecology and Obstetrics Vol. 306, No. 5 ( 2022-04-04), p. 1689-1695
    Details
    In: Archives of Gynecology and Obstetrics, Springer Science and Business Media LLC, Vol. 306, No. 5 ( 2022-04-04), p. 1689-1695
    Abstract: PD-L1 receptor expression in breast cancer tissue can be assessed with different anti-human PD-L1 monoclonal antibodies. The performance of three specific monoclonal antibodies in a head-to-head comparison is unknown. In addition, a potential correlation of PD-L1 expression and clinico-pathological parameters has not been investigated. Methods This was a retrospective study on tissue samples of patients with histologically confirmed triple negative breast cancer (TNBC). PD-L1 receptors were immune histochemically stained with three anti-human PD-L1 monoclonal antibodies: 22C3 and 28-8 for staining of tumor cell membranes (TC) and cytoplasm (Cyt), SP142 for immune cell staining (IC). Three different tissue samples of each patient were evaluated separately by two observers in a blinded fashion. The percentage of PD-L1 positive tumor cells in relation to the total number of tumor cells was determined. For antibodies 22C3 and 28-8 PD-L1 staining of 0 to 〈  1% of tumor cells was rated "negative", 1–50% was rated "positive" and  〉  50% was rated "strong positive". Cyt staining was defined as “negative” when no signal was observed and as “positive”, when any positive signal was observed. For IC staining with SP142 all samples with PD-L1 expression ≥ 1% were rated as “positive”. Finally, the relationship between PD-L1 expression and clinico-pathological parameters was analyzed. Results Tissue samples from 59 of 60 enrolled patients could be analyzed. Mean age was 55 years. Both the monoclonal antibodies 22C3 and 28-8 had similar properties, and were positive for both TC in 13 patients (22%) and for Cyt staining in 24 patients (40.7%). IC staining with antibody SP142 was positive in 24 patients (40.7%), who were also positive for Cyt staining. The differences between TC and Cyt staining and TC and IC staining were significant ( p  = 0.001). Cases with positive TC staining showed higher Ki67 expression compared to those with negative staining, 40 vs 30%, respectively ( p  = 0.05). None of the other clinico-pathological parameters showed any correlation with PDL1 expression. Conclusions Antibodies 22C3 and 28-8 can be used interchangeably for PD-L1 determination in tumor cells of TNBC patients. Results for Cyt staining with 22C3 or 28-8 and IC staining with SP142 were identical. In our study PD-L1 expression correlates with Ki67 expression but not with OS or DFS.
    Type of Medium: Online Resource
    ISSN: 1432-0711
    URL: Article
    DOI: 10.1007/s00404-022-06529-w
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
    detail.hit.zdb_id: 1458450-5
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  • 5
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    Identification of the atypically modified autoantigen Ars2 as the target of B-cell receptors from activated B-cell-type diffuse large B-cell lymphoma
    Ferrata Storti Foundation (Haematologica) ; 2020
    In:  Haematologica Vol. 106, No. 8 ( 2020-07-16), p. 2224-2232
    Details
    In: Haematologica, Ferrata Storti Foundation (Haematologica), Vol. 106, No. 8 ( 2020-07-16), p. 2224-2232
    Abstract: It has been suggested that B-cell receptor (BCRs) stimulation by specific antigens plays a pathogenic role in diffuse large B-cell lymphoma (DLBCL). Here, it was the aim to screen for specific reactivities of DLBCL-BCRs in the spectrum of autoantigens and antigens of infectious origin. Arsenite resistance protein 2 (Ars2) was identified as the BCR target of 3/5 ABC-type DLBCL cell lines and 2/11 primary DLBCL cases. Compared to controls, Ars2 was hypo-phosphorylated exclusively in cases and cell lines with Ars2-specific BCRs. In a validation cohort, hypo-phosphorylated Ars2 was found in 8/31 ABC-type, but only 1/20 germinal center B cell (GBC)-like type DLBCL. Incubation with Ars2 induced BCR-pathway activation and increased proliferation, while an Ars2/ETA-toxin conjugate induced killing of cell lines with Ars2-reactive BCRs. Ars2 appears to play a role in a subgroup of ABC-type DLBCLs. Moreover, transformed DLBCL lines with Ars2-reactive BCRs still show growth advantage after incubation with Ars2. These results provide knowledge about the pathogenic role of a specific antigen stimulating the BCR pathway in DLCBL.
    Type of Medium: Online Resource
    ISSN: 1592-8721 , 0390-6078
    URL: Article
    DOI: 10.3324/haematol.2019.241653
    Language: Unknown
    Publisher: Ferrata Storti Foundation (Haematologica)
    Publication Date: 2020
    detail.hit.zdb_id: 2186022-1
    detail.hit.zdb_id: 2030158-3
    detail.hit.zdb_id: 2805244-4
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  • 6
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    Online Resource
    Immunostaining and Laser-Assisted Cell Picking for mRNA Analysis
    Elsevier BV ; 2000
    In:  Laboratory Investigation Vol. 80, No. 3 ( 2000-03), p. 327-333
    Details
    In: Laboratory Investigation, Elsevier BV, Vol. 80, No. 3 ( 2000-03), p. 327-333
    Type of Medium: Online Resource
    ISSN: 0023-6837
    URL: Article
    DOI: 10.1038/labinvest.3780037
    RVK:
    WA 15000
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2000
    detail.hit.zdb_id: 2041329-4
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  • 7
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    Lymphocyte predominant cells detect Moraxella catarrhalis-derived antigens in nodular lymphocyte-predominant Hodgkin lymphoma
    Springer Science and Business Media LLC ; 2020
    In:  Nature Communications Vol. 11, No. 1 ( 2020-05-18)
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    In: Nature Communications, Springer Science and Business Media LLC, Vol. 11, No. 1 ( 2020-05-18)
    Abstract: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma of B-cell origin with frequent expression of functional B-cell receptors (BCRs). Here we report that expression cloning followed by antigen screening identifies DNA-directed RNA polymerase beta’ (RpoC) from Moraxella catarrhalis as frequent antigen of BCRs of IgD + LP cells. Patients show predominance of HLA-DRB1*04/07 and the IgVH genes encode extraordinarily long CDR3s. High-titer, light-chain-restricted anti-RpoC IgG1/κ-type serum-antibodies are additionally found in these patients. RpoC and MID/hag, a superantigen co-expressed by Moraxella catarrhalis that is known to activate IgD + B cells by binding to the Fc domain of IgD, have additive activation effects on the BCR, the NF-κB pathway and the proliferation of IgD + DEV cells expressing RpoC-specific BCRs. This suggests an additive antigenic and superantigenic stimulation of B cells with RpoC-specific IgD + BCRs under conditions of a permissive MHC-II haplotype as a model of NLPHL lymphomagenesis, implying future treatment strategies.
    Type of Medium: Online Resource
    ISSN: 2041-1723
    URL: Article
    DOI: 10.1038/s41467-020-16375-6
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2020
    detail.hit.zdb_id: 2553671-0
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  • 8
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    Online Resource
    LRPAP1 Is a Frequent B-Cell Receptor Antigen in Mantle Cell Lymphoma (MCL)
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 2685-2685
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 2685-2685
    Abstract: Introduction: Chronic antigenic stimulation may play an important role in the pathogenesis of malignant lymphomas. Although most MCL cases are believed to have an antigen-naive B cell as cell of origin, overrepresentation of certain VH genes has been reported. Therefore we screened BCRs from MCLs for possible antigens. Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen MCL specimens and established MCL cell lines. The purified BCR-Fabs were checked for binding to proteins expressed on macroarrays of human cDNA expression libraries and on bacterial lysates. In addition, sera from patients with MCL were screened for antibodies against respective BCR antigens. Results: The recombinant MCL-BCR derived Fabs from 9 patients and of four established MCL cell lines were tested on protein arrays. Recombinant lymphoma-BCR-derived Fabs from 4/9 patients and from 1/4 MCL cell lines reacted with human low density lipoprotein receptor-related protein associated protein 1 (LRPAP1). Specific secondary modifications of LRPAP1 explaining its autoimmunogenicity were not found. 8/30 patients with MCL had anti-LRPAP1-antibodies in their serum, which was the case in only 1/200 healthy controls. Finally, LRPAP1 specifically induced proliferation of Maver1 cells that express a BCR with specificity for LRPAP1. Conclusions: LRPAP1 is the first molecularly defined antigenic target of MCL-BCRs. The high frequency of LRPAP1-reactive BCRs in MCL suggests a role of LRPAP1 in the pathogenesis of MCL, even in cases with unmutated VH genes. The prevalence of LRPAP1-antibodies in MCL patients and healthy controls identifies LRPAP1-antibody as the first serologic risk factor for MCL (odds ratio: 72.36) Supported by Wilhelm-Sander-Stiftung Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.2685.2685
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 9
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    Online Resource
    LRPAP1 is a frequent proliferation-inducing antigen of BCRs of mantle cell lymphomas and can be used for specific therapeutic targeting
    Springer Science and Business Media LLC ; 2019
    In:  Leukemia Vol. 33, No. 1 ( 2019-1), p. 148-158
    Details
    In: Leukemia, Springer Science and Business Media LLC, Vol. 33, No. 1 ( 2019-1), p. 148-158
    Type of Medium: Online Resource
    ISSN: 0887-6924 , 1476-5551
    URL: Article
    DOI: 10.1038/s41375-018-0182-1
    RVK:
    WA 15000
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2019
    detail.hit.zdb_id: 2008023-2
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  • 10
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    Online Resource
    cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue
    Elsevier BV ; 2002
    In:  The American Journal of Pathology Vol. 160, No. 1 ( 2002-01), p. 81-90
    Details
    In: The American Journal of Pathology, Elsevier BV, Vol. 160, No. 1 ( 2002-01), p. 81-90
    Type of Medium: Online Resource
    ISSN: 0002-9440
    URL: Article
    DOI: 10.1016/S0002-9440(10)64352-0
    RVK:
    XA 10000
    RVK:
    XA 19800
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2002
    detail.hit.zdb_id: 1480207-7
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