Biomedical subjects

Carl A K Borrebaeck

Publications and source records attributed to Carl A K Borrebaeck.

At least 19 recordsLinked to original sources

PubMed2007

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Affinity Labels↗

PubMed2006

The nuclear DNA repair protein Ku70/80 is a tumor-associated antigen displaying rapid receptor mediated endocytosis.

To be of therapeutic relevance, a tumor-associated antigen should be expressed on the surface of neoplastic cells but not, or to a significantly lower extent, on cells of non-transformed nature. The Ku heterodimer (Ku70/80) is involved in DNA double strand break recognition and repair and is ubiquitously expressed in the nucleus of all cells. However, its exclusive nuclear localization has been reassessed by studies that demonstrate Ku to be expressed on the surface of tumor cell lines, displaying functions in cell adhesion, migration and invasion. In this study, we add another feature to the pluripotent role of Ku70/80 by showing that, upon binding the novel human recombinant antibody INCA-X, the Ku70/80 heterodimer is internalized into pancreatic carcinoma cells. The receptor-mediated endocytosis of Ku70/80 is rapid (t(1/) (2) 12 min) and extensive (90% of the receptor pool inside the cell after 100 min) as measured by rotating radioimmunoassay. Ku70/80 was also successfully used as a port of entry for cytotoxic payloads to tumor cells of various origin, as determined by indirect immunotoxin administration of a saporin-conjugated, secondary anti-human antibody. Thus, the internalization properties of the Ku70/80 suggest a potential role of this tumor associated antigen in selective drug-delivery in several human malignancies.

Antigens, Neoplasm↗

PubMed2006

The human CD77- B cell population represents a heterogeneous subset of cells comprising centroblasts, centrocytes, and plasmablasts, prompting phenotypical revision.

The process of becoming an Ig-producing plasma cell takes the mature B cell through the germinal center, where Ig genes are diversified through somatic hypermutation and class switch recombination. To more clearly define functional characteristics of the germinal center dark zone centroblasts and the light zone centrocytes, we have performed expression analysis of the CD77(+) and CD77(-) populations, because CD77 has been accepted as a discriminator of centroblasts and centrocytes. Our results demonstrated that the CD77(+) and the CD77(-) populations lack functional associated expression programs discriminating the two populations. Both populations are shown to be actively cycling and to share common features associated with cell cycle regulation and DNA maintenance. They are also shown to have an equally active DNA repair program, as well as components involved in somatic hypermutation and class switch recombination. Moreover, the data also demonstrated that the CD77(-) population comprises cells with an already initiated plasma cell differentiation program. Together this demonstrates that CD77 does not discriminate centroblasts and centrocytes and that the CD77(-) population represents a heterogeneous subset of cells, comprising centroblasts, centrocytes, and plasmablast.

B-Lymphocyte Subsets↗

PubMed2006

The human IgE-encoding transcriptome to assess antibody repertoires and repertoire evolution.

Upon encounter with antigen, the B lymphocyte population responds by producing a diverse set of antigen-specific antibodies of various isotypes. The vast size of the responding populations makes it very difficult to study clonal evolution and repertoire composition occurring during these processes in humans. Here, we have explored an approach utilizing the H-EPSILON-encoding transcriptome to investigate aspects of repertoire diversity during the season of antigen exposure. We show through sequencing of randomly picked transcripts that the sizes of patients' repertoires are relatively small. This specific aspect of the transcriptome allows us to construct evolutionary trees pinpointing features of somatic hypermutation as it occurs in humans. Despite the small size of the repertoires, they are highly diverse with respect to VDJ gene usage, suggesting that the H-EPSILON-encoding transcriptome is a faithful mimic of other class-switched isotypes. Importantly, it is possible to use antibody library and selection technologies to define the specificity of clonotypes identified by random sequencing. The small size of the H-EPSILON-encoding transcriptome of peripheral blood B cells, the simple identification of clonally related sets of genes in this population, and the power of library and selection technologies ensure that this approach will allow us to investigate antibody evolution in human B lymphocytes of known specificity. As H-EPSILON repertoires show many of the hallmarks of repertoires encoding other isotypes, we suggest that studies of this type will have an impact on our understanding of human antibody evolution even beyond that occurring in the IgE-producing B cell population.

Amino Acid Sequence↗

PubMed2006

Rapid induction of apoptosis in B-cell lymphoma by functionally isolated human antibodies.

Novel panning and screening methodology was devised to isolate high affinity human recombinant scFv antibody fragments with functionally associated properties in B lymphoma cells. The approach was used to generate a panel of apoptosis-inducing antibodies specific for antigens differentially expressed in B lymphoma vs. T leukaemia cells. The selections resulted in an antibody pool with near perfect selectivity (>99%) for the B lymphoma target cells. Randomly picked clones (72) revealed 7 unique antibody genotypes. Six of these rapidly induced apoptosis in target cells. Following the conversion to full IgGs, the antibodies were shown to be specific for HLA-DR/DP, the B-cell receptor mu chain and for CD54/ICAM-1. The latter receptor was not previously associated with apoptotic properties in B-cell lymphomas. Anti-ICAM-1 IgG induced apoptosis in a broad range of B lymphoma cell lines and were shown by immunohistochemistry to bind strongly to B lymphoma tissue obtained from 5 different B lymphoma patients. The recombinant IgG antibodies had affinities in the subnanomolar (0.3 nM) to nanomolar (3 nM) range. The described technology is generally applicable for the rapid isolation of high affinity human antibodies with specificity for differentially expressed cell surface receptors with intrinsic negative or positive signalling properties from naïve phage libraries.

Antibodies↗

PubMed2006

Identification of protein expression signatures associated with Helicobacter pylori infection and gastric adenocarcinoma using recombinant antibody microarrays.

Antibody microarray based technology is a powerful emerging tool in proteomics, target discovery, and differential analysis. Here, we report the first study where recombinant antibody fragments have been used to construct large scale antibody microarrays, composed of 127 different antibodies against mostly immunoregulatory antigens. The arrays were based on single framework recombinant antibody fragments (SinFabs) designed for high on-chip stability and functionality and were used for the analysis of malignant and normal stomach tissue samples from Helicobacter pylori-positive and -negative patients. Our results demonstrate that distinct tumor- as well as infection-associated protein expression signatures could be identified from these complex tissue proteomes, as well as biomarkers such as IL-9, IL-11, and MCP-4, previously not found in these diseases. In a longer perspective, this study may improve the understanding of H. pylori-induced stomach cancer and lead to development of improved diagnostics.

Adenocarcinoma↗

PubMed2006

Mantle cell lymphomas acquire increased expression of CCL4, CCL5 and 4-1BB-L implicated in cell survival.

We have analyzed mantle cell lymphomas (MCLs), using high-density DNA microarrays, and confirmed the expression of differentially regulated antigens, using flow cytometry and immunohistochemistry. The results show that MCLs acquire expression of molecules that normally are involved in interaction with other immune cells and, thus, might affect the ability of the tumor to survive. The MCL signature is represented by the overexpression of the chemokine CCL4 (MIP-1beta), implicated in the recruitment of regulatory T cells, as well as CCL5 and 4-1BB-L. The latter molecules are normally involved in chemotaxis of T cells and B cell activation, respectively. Signaling through 4-1BB-L allows B cells to proliferate and the expression of its ligand, by the intra-tumoral mesh of follicular dendritic cells (FDC), could thus serve as a paracrine loop facilitating growth and survival of MCL cells.

4-1BB Ligand↗

PubMed2006

From gene expression analysis to tissue microarrays: a rational approach to identify therapeutic and diagnostic targets in lymphoid malignancies.

Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy for which better treatment strategies are needed. To identify potential diagnostic and therapeutic targets, a signature consisting of MCL-associated genes was selected based on a comprehensive gene expression analysis of malignant and normal B cells. The corresponding protein epitope signature tags were identified and used to raise monospecific, polyclonal antibodies, which were subsequently analyzed on paraffin-embedded sections of malignant and normal tissue. In this study, we demonstrate that the initial selection strategy of MCL-associated genes successfully allows identification of protein antigens either uniquely expressed or overexpressed in MCL compared with normal lymphoid tissues. We propose that genome-based, affinity proteomics, using protein epitope signature tag-induced antibodies, is an efficient way to rapidly identify a number of disease-associated protein candidates of both previously known and unknown identities.

Antibodies, Neoplasm↗

PubMed2006

Improved affinity coupling for antibody microarrays: engineering of double-(His)6-tagged single framework recombinant antibody fragments.

Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density antibody microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni(2+)-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(His)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(His)(6)-tag recombinant antibodies for the facile fabrication of high-density antibody microarrays.

Antibodies↗

PubMed2006

One-step fractionation of complex proteomes enables detection of low abundant analytes using antibody-based microarrays.

Antibody-based microarray is a novel technology with great promise within high-throughput proteomics. The tremendous complexity of all proteomes will, however, pose major technological challenges, especially when targeting low-abundant analytes that remains to be resolved. In this paper, we have shown that antibody microarrays readily could be used for screening of low-abundant low molecular weight analytes in complex proteomes by optimizing the sample format. Focused antibody microarrays, based on human recombinant single-chain Fv anti-cytokine antibodies on Ni2+-NTA functionalized glass slides or black polymer Maxisorp substrates, and crude cell supernatants from activated dendritic cells, containing low levels of secreted cytokines, was used for evaluation. The proteome was pre-fractionated based on size in a simple one-step procedure using centrifugal filter devices of various molecular weight cutoffs. The results showed that the generation of a nondiluted low molecular weight (LMW) fraction, corresponding to less than 2% of the original protein content, was critical for the successful screening of cytokines in the sub pg/mL range. The reduced complexity of the LMW fraction significantly improved the assay sensitivity, by improving the fluorescent tagging step and/or reducing the nonspecific binding to the substrates.

Chemical Fractionation↗

PubMed2006

Antibody microarrays: current status and key technological advances.

Antibody-based microarrays are among the novel classes of rapidly evolving proteomic technologies that holds great promise in biomedicine. Miniaturized microarrays (< 1 cm2) can be printed with thousands of individual antibodies carrying the desired specificities, and with biological sample (e.g., an entire proteome) added, virtually any specifically bound analytes can be detected. While consuming only minute amounts (< microL scale) of reagents, ultra- sensitive assays (zeptomol range) can readily be performed in a highly multiplexed manner. The microarray patterns generated can then be transformed into proteomic maps, or detailed molecular fingerprints, revealing the composition of the proteome. Thus, protein expression profiling and global proteome analysis using this tool will offer new opportunities for drug target and biomarker discovery, disease diagnostics, and insights into disease biology. Adopting the antibody microarray technology platform, several biomedical applications, ranging from focused assays to proteome-scale analysis will be rapidly emerging in the coming years. This review will discuss the current status of the antibody microarray technology focusing on recent technological advances and key issues in the process of evolving the methodology into a high-performing proteomic research tool.

Animals↗

PubMed2006

Functional and transcriptional profiling of MUTZ-3, a myeloid cell line acting as a model for dendritic cells.

The incidence of allergy is steadily increasing, but the molecular mechanisms involved in the allergic immune response are still not fully understood. In particular, further investigations focusing on dendritic cells, which are central in orchestrating the immune response, are needed. The objective of this study was to investigate the ability of myeloid leukaemia-derived cell lines, such as KG-1, THP-1 and MUTZ-3, to serve as in vitro models for dendritic cells. The ability of these cell lines to mature into functional dendritic cells, expressing costimulatory molecules, was assessed by functional and transcriptional profiling and compared with that of monocyte-derived dendritic cells, which are now used as a standard source of dendritic cells. High-density microarray analysis was utilized to study the transcriptional activity and kinetics of activation of the differentiated MUTZ-3 cell line, in response to a cocktail of inflammatory cytokines. The data obtained clearly demonstrate that MUTZ-3 cells have the ability to induce antigen-independent proliferation in CD4(+) CD45RA(+) T cells, whereas KG-1 and THP-1 only induced a marginal response. Furthermore, MUTZ-3 displayed the phenotypic and transcriptional profiles of immature dendritic cells, after differentiation with granulocyte-macrophage colony-stimulating factor and interleukin-4. Upon activation with inflammatory cytokines, MUTZ-3 matured phenotypically and exhibited a gene induction similar to that of monocyte-derived dendritic cells. This delineation of the cellular and transcriptional activity of MUTZ-3, in response to maturational stimuli, demonstrates the significance of this cell line as a model for functional studies of inflammatory responses.

CD4-Positive T-Lymphocytes↗

PubMed2006

Escherichia coli TolA tolerates multiple amino-acid substitutions as revealed by screening randomized variants for membrane integrity and phage receptor function.

Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at positions 415-420. These libraries were introduced into the tolA strain K17DE3tolA/F(+) and several variants, containing complementing, multiple amino-acid substitutions, were identified. However, most randomized variants did not complement the tolA strain K17DE3tolA/F(+). The TolA variants that restored sensitivity to phage infection displayed a considerable sequence variation, while the few variants that restored tolerance to detergent were from the same library. A comparison of the generated residue variation and natural variation, suggests that structural dependence overrides contact residue dependence. Thus, library screening can be efficient in identifying TolA variants with different functionally associated characteristics.

Amino Acid Sequence↗

PubMed2006

Antibody microarray-based oncoproteomics.

The driving force behind oncoproteomics is the belief that certain protein signatures or patterns exist that are associated with a particular malignancy. If so, the correlation of clinical parameters with defined protein expression patterns would allow us to predict disease progression and perhaps even postulate improved therapeutic modalities. The technological challenges to achieve these goals are significant, as the human proteome is not defined. No general methodological approach exists today, and human cancer can, furthermore, be divided into several disease subgroups. One potential solution to finding cancer-associated protein signatures is the emerging technology of affinity proteomics. This approach addresses some of the shortcomings of traditional proteomics and combines it with the power of microarrays. The present review focuses on the role of antibody microarrays in oncoproteomics and its potential to provide a truly proteome-wide analytical approach.

Antibodies↗

PubMed2005

Gene family clustering identifies functionally associated subsets of human in vivo blood and tonsillar dendritic cells.

Human dendritic cells (DCs) are a distinct but heterogeneous lineage of APCs operating as the link between innate and adaptive immune responses, with the function to either maintain tolerance or trigger immunity. The DC lineage consists of several subpopulations with unique phenotypes; however, their functional characteristics and transcriptional similarities remain largely unknown. To further characterize the phenotypes and transcriptomes of the subsets, we purified myeloid CD16(+), blood DC Ag 1(+) (BDCA1(+)), and BDCA3(+) DC populations, as well as plasmacytoid CD123(+) DCs, from tonsillar tissue and peripheral blood. Transcriptional profiling and hierarchical clustering visualized that BDCA1(+) DCs clustered with BDCA3(+) DCs, whereas CD16(+) DCs and CD123(+) DCs clustered as distinct populations in blood. Differential expression levels of chemokines, ILs, and pattern recognition receptors were demonstrated, which emphasize innate DC subset specialization. Even though highly BDCA1(+) and BDCA3(+) DC-specific gene expression was identified in blood, the BDCA1(+) DCs and BDCA3(+) DCs from tonsils displayed similar transcriptional activity, most likely due to the pathogenic or inflammatory maturational signals present in tonsillar tissues. Of note, plasmacytoid DCs displayed less plasticity in their transcriptional activity compared with myeloid DCs. The data demonstrated a functionally distinct association of each of the seven subsets based on their signatures, involving regulatory genes in adaptive and innate immunity.

Antigens, CD↗

PubMed2005

Biocompatibility of surfaces for antibody microarrays: design of macroporous silicon substrates.

Major efforts to develop antibody microarray technology to enable global proteome analysis to be performed in a facile manner are under way. In this process, the design and the properties of the substrate will play crucial roles. In the present study, we have developed novel, highly biocompatible solid supports for microarrays, using adsorbed recombinant human single-framework antibody fragments as probes. Several silicon-based supports, including planar silicon, micro- and macroporous silicon, and nitrocellulose-coated variants thereof, were designed and evaluated in a stepwise procedure. The surfaces were scored based on biocompatibility and probe binding capacity as judged by spot morphology, signal intensities, signal to noise ratios, dynamic range, sensitivity, and reproducibility. A set of five commercially available substrates, selected to represent a set of supports providing different surface and coupling chemistries, was used as reference surfaces. The results showed that several well-performing silicon-based supports could be designed; in particular, a nitrocellulose-coated macroporous variant, MAP3-NC7, received the highest scores. In comparison, MAP3-NC7 displayed properties equal to or better than those of the reference substrates. Taken together, designed surfaces based on silicon can undoubtedly meet the requirements of the next generation of solid supports for antibody microarrays.

Antibodies↗

PubMed2005

Genomic and functional delineation of dendritic cells and memory T cells derived from grass pollen-allergic patients and healthy individuals.

Dendritic cells (DCIs) possess a potent ability to modulate and activate specific T-cell responses to allergens, which play a pivotal role in allergic inflammation by secreting cytokines and other mediators. However, the molecular mechanisms by which allergen-challenged DCs regulate specific T-cell responses are still not well characterized. This study aims at elucidating the molecular mechanisms underlying the DC-T-cell interaction during an allergic immune response to grass pollen, using a genomic and functional approach. Transcriptional analysis was performed on grass allergen Phleum pratense-stimulated DCs and on autologous memory CD4(+) T cells co-cultured with allergen-challenged DCs from healthy and allergic donors. DCs from the allergic donors were potent inducers of T-cell proliferation and T(h)2 polarization, as demonstrated by high IL-4, IL-5 and IL-13, and low IFN-gamma production. A gradual up-regulation of activation markers on both DCs and T cells was evident during the co-culture period, demonstrating an educational element of the DC-T-cell interaction. The global transcriptional analysis revealed a differential gene regulation in DCs and T cells derived from allergic donors after stimulation with allergen, as compared with the healthy donors. Peripheral memory CD4(+) T cells from healthy and allergic donors also responded differently after stimulation with allergen-loaded DCs with respect to cytokine production, proliferation, surface marker expression and gene transcription. We found up-regulated genes involved in T(h)2 cell biology, such as genes important for homing, adhesion, signaling and transcription, in addition to genes previously not described in the context of allergy. The panel of differentially expressed genes in the allergic group will form the basis for an increased understanding of the molecular mechanisms in allergy.

Biomarkers↗

PubMed2005

Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes.

Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics.

Humans↗