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Article ; Online: Analysis by Flow Cytometry of B-Cell Activation and Antibody Responses Induced by Toll-Like Receptors.

Pone, Egest J

Methods in molecular biology (Clifton, N.J.)

2016 Volume 1390, Page(s) 229–248

Abstract: Toll-like receptors (TLRs) are expressed in B lymphocytes and contribute to B-cell activation, antibody responses, and their maturation. TLR stimulation of mouse B cells induces class switch DNA recombination (CSR) to isotypes specified by cytokines, and ...

Abstract	Toll-like receptors (TLRs) are expressed in B lymphocytes and contribute to B-cell activation, antibody responses, and their maturation. TLR stimulation of mouse B cells induces class switch DNA recombination (CSR) to isotypes specified by cytokines, and also induces formation of IgM(+) as well as class-switched plasma cells. B-cell receptor (BCR) signaling, while on its own inducing limited B-cell proliferation and no CSR, can enhance CSR driven by TLRs. Particular synergistic or antagonistic interactions among TLR pathways, BCR, and cytokine signaling can have important consequences for B-cell activation, CSR, and plasma cell formation. This chapter outlines protocols for the induction and analysis of B-cell activation and antibody production by TLRs with or without other stimuli.
MeSH term(s)	Animals ; Antibody Formation/immunology ; B-Lymphocytes/immunology ; B-Lymphocytes/metabolism ; Biomarkers ; Flow Cytometry/methods ; Immunomagnetic Separation ; Immunophenotyping ; Lymphocyte Activation/immunology ; Mice ; Toll-Like Receptors/metabolism
Chemical Substances	Biomarkers ; Toll-Like Receptors
Language	English
Publishing date	2016
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-3335-8_15
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Article ; Online: Multimericity Amplifies the Synergy of BCR and TLR4 for B Cell Activation and Antibody Class Switching.

Pone, Egest J / Hernandez-Davies, Jenny E / Jan, Sharon / Silzel, Emily / Felgner, Philip L / Davies, D Huw

Frontiers in immunology

2022 Volume 13, Page(s) 882502

Abstract: Sustained signaling through the B cell antigen receptor (BCR) is thought to occur only when antigen(s) crosslink or disperse multiple BCR units, such as by multimeric antigens found on the surfaces of viruses or bacteria. B cell-intrinsic Toll-like ... ...

Abstract	Sustained signaling through the B cell antigen receptor (BCR) is thought to occur only when antigen(s) crosslink or disperse multiple BCR units, such as by multimeric antigens found on the surfaces of viruses or bacteria. B cell-intrinsic Toll-like receptor (TLR) signaling synergizes with the BCR to induce and shape antibody production, hallmarked by immunoglobulin (Ig) class switch recombination (CSR) of constant heavy chains from IgM/IgD to IgG, IgA or IgE isotypes, and somatic hypermutation (SHM) of variable heavy and light chains. Full B cell differentiation is essential for protective immunity, where class switched high affinity antibodies neutralize present pathogens, memory B cells are held in reserve for future encounters, and activated B cells also serve as semi-professional APCs for T cells. But the rules that fine-tune B cell differentiation remain partially understood, despite their being essential for naturally acquired immunity and for guiding vaccine development. To address this in part, we have developed a cell culture system using splenic B cells from naive mice stimulated with several biotinylated ligands and antibodies crosslinked by streptavidin reagents. In particular, biotinylated lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, and biotinylated anti-IgM were pre-assembled (multimerized) using streptavidin, or immobilized on nanoparticles coated with streptavidin, and used to active B cells in this precisely controlled, high throughput assay. Using B cell proliferation and Ig class switching as metrics for successful B cell activation, we show that the stimuli are both synergistic and dose-dependent. Crucially, the multimerized immunoconjugates are most active over a narrow concentration range. These data suggest that multimericity is an essential requirement for B cell BCR/TLRs ligands, and clarify basic rules for B cell activation. Such studies highlight the importance in determining the choice of single vs multimeric formats of antigen and PAMP agonists during vaccine design and development.
MeSH term(s)	Animals ; Immunoglobulin Class Switching ; Immunoglobulin Isotypes ; Ligands ; Mice ; Streptavidin ; Toll-Like Receptor 4
Chemical Substances	Immunoglobulin Isotypes ; Ligands ; Tlr4 protein, mouse ; Toll-Like Receptor 4 ; Streptavidin (9013-20-1)
Language	English
Publishing date	2022-05-19
Publishing country	Switzerland
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2022.882502
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Article ; Online: Stem Cell Extracellular Vesicles: Extended Messages of Regeneration.

Riazifar, Milad / Pone, Egest J / Lötvall, Jan / Zhao, Weian

Annual review of pharmacology and toxicology

2017 Volume 57, Page(s) 125–154

Abstract: Stem cells are critical to maintaining steady-state organ homeostasis and regenerating injured tissues. Recent intriguing reports implicate extracellular vesicles (EVs) as carriers for the distribution of morphogens and growth and differentiation factors ...

Abstract	Stem cells are critical to maintaining steady-state organ homeostasis and regenerating injured tissues. Recent intriguing reports implicate extracellular vesicles (EVs) as carriers for the distribution of morphogens and growth and differentiation factors from tissue parenchymal cells to stem cells, and conversely, stem cell-derived EVs carrying certain proteins and nucleic acids can support healing of injured tissues. We describe approaches to make use of engineered EVs as technology platforms in therapeutics and diagnostics in the context of stem cells. For some regenerative therapies, natural and engineered EVs from stem cells may be superior to single-molecule drugs, biologics, whole cells, and synthetic liposome or nanoparticle formulations because of the ease of bioengineering with multiple factors while retaining superior biocompatibility and biostability and posing fewer risks for abnormal differentiation or neoplastic transformation. Finally, we provide an overview of current challenges and future directions of EVs as potential therapeutic alternatives to cells for clinical applications.
Language	English
Publishing date	2017-01-06
Publishing country	United States
Document type	Journal Article
ZDB-ID	196587-6
ISSN	1545-4304 ; 0362-1642
ISSN (online)	1545-4304
ISSN	0362-1642
DOI	10.1146/annurev-pharmtox-061616-030146
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Article: Approaches in Immunotherapy, Regenerative Medicine, and Bioengineering for Type 1 Diabetes.

Kopan, Christopher / Tucker, Tori / Alexander, Michael / Mohammadi, M Rezaa / Pone, Egest J / Lakey, Jonathan Robert Todd

Frontiers in immunology

2018 Volume 9, Page(s) 1354

Abstract: Recent advances on using immune and stem cells as two-pronged approaches for type 1 diabetes mellitus (T1DM) treatment show promise for advancement into clinical practice. As T1DM is thought to arise from autoimmune attack destroying pancreatic β-cells, ... ...

Abstract	Recent advances on using immune and stem cells as two-pronged approaches for type 1 diabetes mellitus (T1DM) treatment show promise for advancement into clinical practice. As T1DM is thought to arise from autoimmune attack destroying pancreatic β-cells, increasing treatments that use biologics and cells to manipulate the immune system are achieving better results in pre-clinical and clinical studies. Increasingly, focus has shifted from small molecule drugs that suppress the immune system nonspecifically to more complex biologics that show enhanced efficacy due to their selectivity for specific types of immune cells. Approaches that seek to inhibit only autoreactive effector T cells or enhance the suppressive regulatory T cell subset are showing remarkable promise. These modern immune interventions are also enabling the transplantation of pancreatic islets or β-like cells derived from stem cells. While complete immune tolerance and body acceptance of grafted islets and cells is still challenging, bioengineering approaches that shield the implanted cells are also advancing. Integrating immunotherapy, stem cell-mediated β-cell or islet production and bioengineering to interface with the patient is expected to lead to a durable cure or pave the way for a clinical solution for T1DM.
Language	English
Publishing date	2018-06-12
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2606827-8
ISSN	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2018.01354
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Article ; Online: Magnitude and breadth of antibody cross-reactivity induced by recombinant influenza hemagglutinin trimer vaccine is enhanced by combination adjuvants.

Hernandez-Davies, Jenny E / Dollinger, Emmanuel P / Pone, Egest J / Felgner, Jiin / Liang, Li / Strohmeier, Shirin / Jan, Sharon / Albin, Tyler J / Jain, Aarti / Nakajima, Rie / Jasinskas, Algimantas / Krammer, Florian / Esser-Kahn, Aaron / Felgner, Philip L / Nie, Qing / Davies, D Huw

Scientific reports

2022 Volume 12, Issue 1, Page(s) 9198

Abstract: The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen ... ...

Abstract	The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed 'IVAX-1') appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.
MeSH term(s)	Adjuvants, Immunologic/pharmacology ; Adjuvants, Pharmaceutic ; Animals ; Antibodies, Viral ; Hemagglutinins ; Humans ; Immunoglobulin G/chemistry ; Influenza Vaccines/immunology ; Influenza, Human ; Mice ; Mice, Inbred BALB C ; Vaccines, Synthetic/immunology
Chemical Substances	Adjuvants, Immunologic ; Adjuvants, Pharmaceutic ; Antibodies, Viral ; Hemagglutinins ; Immunoglobulin G ; Influenza Vaccines ; Vaccines, Synthetic
Language	English
Publishing date	2022-06-02
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-12727-y
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Article ; Online: Magnitude and breadth of antibody cross-reactivity induced by recombinant influenza hemagglutinin trimer vaccine is enhanced by combination adjuvants

Jenny E. Hernandez-Davies / Emmanuel P. Dollinger / Egest J. Pone / Jiin Felgner / Li Liang / Shirin Strohmeier / Sharon Jan / Tyler J. Albin / Aarti Jain / Rie Nakajima / Algimantas Jasinskas / Florian Krammer / Aaron Esser-Kahn / Philip L. Felgner / Qing Nie / D. Huw Davies

Scientific Reports, Vol 12, Iss 1, Pp 1-

2022 Volume 20

Abstract: Abstract The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic ...

Abstract	Abstract The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed ‘IVAX-1’) appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.
Keywords	Medicine ; R ; Science ; Q
Subject code	570
Language	English
Publishing date	2022-06-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Transplantation of stem cells from umbilical cord blood as therapy for type I diabetes.

Stiner, Rachel / Alexander, Michael / Liu, Guangyang / Liao, Wenbin / Liu, Yongjun / Yu, Jingxia / Pone, Egest J / Zhao, Weian / Lakey, Jonathan R T

Cell and tissue research

2019 Volume 378, Issue 2, Page(s) 155–162

Abstract: In recent years, human umbilical cord blood has emerged as a rich source of stem, stromal and immune cells for cell-based therapy. Among the stem cells from umbilical cord blood, CD45+ multipotent stem cells and CD90+ mesenchymal stem cells have the ... ...

Abstract	In recent years, human umbilical cord blood has emerged as a rich source of stem, stromal and immune cells for cell-based therapy. Among the stem cells from umbilical cord blood, CD45+ multipotent stem cells and CD90+ mesenchymal stem cells have the potential to treat type I diabetes mellitus (T1DM), to correct autoimmune dysfunction and replenish β-cell numbers and function. In this review, we compare the general characteristics of umbilical cord blood-derived multipotent stem cells (UCB-SCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and introduce their applications in T1DM. Although there are some differences in surface marker expression between UCB-SCs and UCB-MSCs, the two cell types display similar functions such as suppressing function of stimulated lymphocytes and imparting differentiation potential to insulin-producing cells (IPCs) in the setting of low immunogenicity, thereby providing a promising and safe approach for T1DM therapy.
MeSH term(s)	Animals ; Biomarkers/metabolism ; Cell Differentiation ; Diabetes Mellitus, Type 1/therapy ; Humans ; Insulin-Secreting Cells/metabolism ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stem Cells/cytology ; Mesenchymal Stem Cells/metabolism ; Mice ; Rats ; Umbilical Cord/cytology
Chemical Substances	Biomarkers
Language	English
Publishing date	2019-06-17
Publishing country	Germany
Document type	Journal Article ; Review
ZDB-ID	125067-x
ISSN	1432-0878 ; 0302-766X
ISSN (online)	1432-0878
ISSN	0302-766X
DOI	10.1007/s00441-019-03046-2
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Article ; Online: Isolation and characterization of microvesicles from mesenchymal stem cells.

Mohammadi, M Rezaa / Riazifar, Milad / Pone, Egest J / Yeri, Ashish / Van Keuren-Jensen, Kendall / Lässer, Cecilia / Lotvall, Jan / Zhao, Weian

Methods (San Diego, Calif.)

2019 Volume 177, Page(s) 50–57

Abstract: Mesenchymal stem or stromal cells are currently under clinical investigation for multiple diseases. While their mechanism of action is still not fully elucidated, vesicles secreted by MSCs are believed to recapitulate their therapeutic potentials to some ...

Abstract	Mesenchymal stem or stromal cells are currently under clinical investigation for multiple diseases. While their mechanism of action is still not fully elucidated, vesicles secreted by MSCs are believed to recapitulate their therapeutic potentials to some extent. Microvesicles (MVs), also called as microparticles or ectosome, are among secreted vesicles that could transfer cytoplasmic cargo, including RNA and proteins, from emitting (source) cells to recipient cells. Given the importance of MVs, we here attempted to establish a method to isolate and characterize MVs secreted from unmodified human bone marrow derived MSCs (referred to as native MSCs, and their microvesicles as Native-MVs) and IFNγ stimulated MSCs (referred to as IFNγ-MSCs, and their microvesicles as IFNγ-MVs). We first describe an ultracentrifugation technique to isolate MVs from the conditioned cell culture media of MSCs. Next, we describe characterization and quality control steps to analyze the protein and RNA content of MVs. Finally, we examined the potential of MVs to exert immunomodulatory effects through induction of regulatory T cells (Tregs). Secretory vesicles from MSCs are promising alternatives for cell therapy with applications in drug delivery, regenerative medicine, and immunotherapy.
MeSH term(s)	Animals ; Bone Marrow Cells/chemistry ; Bone Marrow Cells/cytology ; Bone Marrow Cells/drug effects ; Bone Marrow Cells/immunology ; Cell Separation/methods ; Cell-Derived Microparticles/chemistry ; Cell-Derived Microparticles/immunology ; Culture Media, Conditioned/chemistry ; Drug Delivery Systems/methods ; Humans ; Immunotherapy/methods ; Interferon-gamma/pharmacology ; Mesenchymal Stem Cells/chemistry ; Mesenchymal Stem Cells/cytology ; Mesenchymal Stem Cells/drug effects ; Mesenchymal Stem Cells/immunology ; Proteins/classification ; Proteins/isolation & purification ; Proteomics/methods ; RNA/classification ; RNA/isolation & purification ; Regenerative Medicine/methods ; T-Lymphocytes, Regulatory/drug effects ; T-Lymphocytes, Regulatory/immunology
Chemical Substances	Culture Media, Conditioned ; Proteins ; RNA (63231-63-0) ; Interferon-gamma (82115-62-6)
Language	English
Publishing date	2019-10-25
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2019.10.010
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Article ; Online: Correction: Scaffold Functions of 14-3-3 Adaptors in B Cell Immunoglobulin Class Switch DNA Recombination.

Lam, Tonika / Thomas, Lisa M / White, Clayton A / Li, Guideng / Pone, Egest J / Xu, Zhenming / Casali, Paolo

PloS one

2017 Volume 12, Issue 3, Page(s) e0174195

Abstract: This corrects the article DOI: 10.1371/journal.pone.0080414.]. ...

Abstract	[This corrects the article DOI: 10.1371/journal.pone.0080414.].
Language	English
Publishing date	2017-03-15
Publishing country	United States
Document type	Published Erratum
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0174195
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Isolation and characterization of microvesicles from mesenchymal stem cells

Mohammadi, M. Rezaa / Riazifar, Milad / Pone, Egest J / Yeri, Ashish / Van Keuren-Jensen, Kendall / Lässer, Cecilia / Lotvall, Jan / Zhao, Weian

Methods. 2020 May 01, v. 177

2020

Abstract	Mesenchymal stem or stromal cells are currently under clinical investigation for multiple diseases. While their mechanism of action is still not fully elucidated, vesicles secreted by MSCs are believed to recapitulate their therapeutic potentials to some extent. Microvesicles (MVs), also called as microparticles or ectosome, are among secreted vesicles that could transfer cytoplasmic cargo, including RNA and proteins, from emitting (source) cells to recipient cells. Given the importance of MVs, we here attempted to establish a method to isolate and characterize MVs secreted from unmodified human bone marrow derived MSCs (referred to as native MSCs, and their microvesicles as Native-MVs) and IFNγ stimulated MSCs (referred to as IFNγ-MSCs, and their microvesicles as IFNγ-MVs). We first describe an ultracentrifugation technique to isolate MVs from the conditioned cell culture media of MSCs. Next, we describe characterization and quality control steps to analyze the protein and RNA content of MVs. Finally, we examined the potential of MVs to exert immunomodulatory effects through induction of regulatory T cells (Tregs). Secretory vesicles from MSCs are promising alternatives for cell therapy with applications in drug delivery, regenerative medicine, and immunotherapy.
Keywords	RNA ; T-lymphocytes ; bone marrow ; cell culture ; culture media ; humans ; immunomodulators ; immunotherapy ; mechanism of action ; medicine ; mesenchymal stromal cells ; microparticles ; proteins ; quality control ; secretory granules ; ultracentrifugation
Language	English
Dates of publication	2020-0501
Size	p. 50-57.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2019.10.010
Database	NAL-Catalogue (AGRICOLA)

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