B. Hekking - Academia.edu (original) (raw)

Papers by B. Hekking

Research paper thumbnail of Characterization of in vivo expanded OspA-specific human T-cell clones☆

Clinical Immunology, 2005

A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from ... more A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from a patient with treatment-resistant Lyme arthritis using a DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with outer surface protein A (OspA) peptide164-175, an immunodominant epitope of Borrelia burgdorferi. Sequencing of the T-cell receptors of the OspA reactive clones showed significant skewing of the T-cell receptor repertoire. Of the 101 T-cell clones sequenced, 81 possessed TCR beta chains that were present in at least one other clone isolated. Complete sequencing of both alpha and beta chains of a subset of clones showed that at least two distinct T-cell clones were expanded in vivo. Binding studies using a panel of Ala-substituted peptide ligands were performed to determine potential MHC binding sites of the OspAp164-175 to DRB1*0401. In addition, T-cell clones were tested functionally for their reactivity to the wild-type peptide as well as to altered peptide ligands (APLs) and peptide libraries based on the OspA epitope in order to determine the TCR contact residues and the stringency in T cell recognition. We are among the first to define the characteristics of TCR usage of T cells isolated from an inflamed immune compartment in an individual with an autoimmune disease potentially triggered by a microbial antigen.

Research paper thumbnail of P216-T Beyond Differential In-Gel Electrophoresis: Top-down Multiplexed Analysis in Two-dimensional Gels Using an Isobaric Mass Tagging Approach

Large sets of biological samples are commonly encountered in biomedical research, and a rigorous,... more Large sets of biological samples are commonly encountered in biomedical research, and a rigorous, reliable top-down quantification method for proteins from multiple samples is required. With differential in-gel electrophoresis (DIGE), Cy3 and Cy5 dyes are used to fluorescently label two different protein samples prior to running them on the same 2D gel. Often, Cy2 dye is used as an internal standard as well. This approach permits analysis of two to three samples under identical conditions, eliminating the need to register and match the images as in traditional 2D gels. Though conceptually seductive, problems plague DIGE, especially when attempting to perform complex quantitative proteomics studies. Since the DIGE images are generated with two to three different dyes of differing molar extinction coefficients, quantum yields, and physicochemical properties, absolute quantification of small intensity differences is challenging. Also, DIGE is overly simplistic in its basic assumption t...

Research paper thumbnail of P216-T Beyond Differential In-Gel Electrophoresis: Top-down Multiplexed Analysis in Two-dimensional Gels Using an Isobaric Mass Tagging Approach

Research paper thumbnail of Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits

Chemistry & Biology

The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in... more The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible immunoproteasome by a different set of catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and su...

Research paper thumbnail of Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis

Research paper thumbnail of Calorie Restriction Promotes Mammalian Cell Survival by Inducing the SIRT1 Deacetylase

Research paper thumbnail of Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic β-subunits

Chemistry & Biology, 2001

Research paper thumbnail of Protein sulfation analysis—A primer

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006

Research paper thumbnail of Characterization of in vivo expanded OspA-specific human T-cell clones☆

Clinical Immunology, 2005

A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from ... more A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from a patient with treatment-resistant Lyme arthritis using a DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with outer surface protein A (OspA) peptide164-175, an immunodominant epitope of Borrelia burgdorferi. Sequencing of the T-cell receptors of the OspA reactive clones showed significant skewing of the T-cell receptor repertoire. Of the 101 T-cell clones sequenced, 81 possessed TCR beta chains that were present in at least one other clone isolated. Complete sequencing of both alpha and beta chains of a subset of clones showed that at least two distinct T-cell clones were expanded in vivo. Binding studies using a panel of Ala-substituted peptide ligands were performed to determine potential MHC binding sites of the OspAp164-175 to DRB1*0401. In addition, T-cell clones were tested functionally for their reactivity to the wild-type peptide as well as to altered peptide ligands (APLs) and peptide libraries based on the OspA epitope in order to determine the TCR contact residues and the stringency in T cell recognition. We are among the first to define the characteristics of TCR usage of T cells isolated from an inflamed immune compartment in an individual with an autoimmune disease potentially triggered by a microbial antigen.

Research paper thumbnail of Characterization of in vivo expanded OspA-specific human T-cell clones☆

Clinical Immunology, 2005

A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from ... more A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from a patient with treatment-resistant Lyme arthritis using a DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with outer surface protein A (OspA) peptide164-175, an immunodominant epitope of Borrelia burgdorferi. Sequencing of the T-cell receptors of the OspA reactive clones showed significant skewing of the T-cell receptor repertoire. Of the 101 T-cell clones sequenced, 81 possessed TCR beta chains that were present in at least one other clone isolated. Complete sequencing of both alpha and beta chains of a subset of clones showed that at least two distinct T-cell clones were expanded in vivo. Binding studies using a panel of Ala-substituted peptide ligands were performed to determine potential MHC binding sites of the OspAp164-175 to DRB1*0401. In addition, T-cell clones were tested functionally for their reactivity to the wild-type peptide as well as to altered peptide ligands (APLs) and peptide libraries based on the OspA epitope in order to determine the TCR contact residues and the stringency in T cell recognition. We are among the first to define the characteristics of TCR usage of T cells isolated from an inflamed immune compartment in an individual with an autoimmune disease potentially triggered by a microbial antigen.

Research paper thumbnail of P216-T Beyond Differential In-Gel Electrophoresis: Top-down Multiplexed Analysis in Two-dimensional Gels Using an Isobaric Mass Tagging Approach

Large sets of biological samples are commonly encountered in biomedical research, and a rigorous,... more Large sets of biological samples are commonly encountered in biomedical research, and a rigorous, reliable top-down quantification method for proteins from multiple samples is required. With differential in-gel electrophoresis (DIGE), Cy3 and Cy5 dyes are used to fluorescently label two different protein samples prior to running them on the same 2D gel. Often, Cy2 dye is used as an internal standard as well. This approach permits analysis of two to three samples under identical conditions, eliminating the need to register and match the images as in traditional 2D gels. Though conceptually seductive, problems plague DIGE, especially when attempting to perform complex quantitative proteomics studies. Since the DIGE images are generated with two to three different dyes of differing molar extinction coefficients, quantum yields, and physicochemical properties, absolute quantification of small intensity differences is challenging. Also, DIGE is overly simplistic in its basic assumption t...

Research paper thumbnail of P216-T Beyond Differential In-Gel Electrophoresis: Top-down Multiplexed Analysis in Two-dimensional Gels Using an Isobaric Mass Tagging Approach

Research paper thumbnail of Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits

Chemistry & Biology

The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in... more The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible immunoproteasome by a different set of catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and su...

Research paper thumbnail of Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis

Research paper thumbnail of Calorie Restriction Promotes Mammalian Cell Survival by Inducing the SIRT1 Deacetylase

Research paper thumbnail of Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic β-subunits

Chemistry & Biology, 2001

Research paper thumbnail of Protein sulfation analysis—A primer

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006

Research paper thumbnail of Characterization of in vivo expanded OspA-specific human T-cell clones☆

Clinical Immunology, 2005

A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from ... more A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from a patient with treatment-resistant Lyme arthritis using a DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with outer surface protein A (OspA) peptide164-175, an immunodominant epitope of Borrelia burgdorferi. Sequencing of the T-cell receptors of the OspA reactive clones showed significant skewing of the T-cell receptor repertoire. Of the 101 T-cell clones sequenced, 81 possessed TCR beta chains that were present in at least one other clone isolated. Complete sequencing of both alpha and beta chains of a subset of clones showed that at least two distinct T-cell clones were expanded in vivo. Binding studies using a panel of Ala-substituted peptide ligands were performed to determine potential MHC binding sites of the OspAp164-175 to DRB1*0401. In addition, T-cell clones were tested functionally for their reactivity to the wild-type peptide as well as to altered peptide ligands (APLs) and peptide libraries based on the OspA epitope in order to determine the TCR contact residues and the stringency in T cell recognition. We are among the first to define the characteristics of TCR usage of T cells isolated from an inflamed immune compartment in an individual with an autoimmune disease potentially triggered by a microbial antigen.