Anita Söderberg

Microanalysis of catecholamines in human plasma by high-performance liquid chromatography with amperometric detection as compared with a radioenzymatic method

Abstract A high-performance liquid chromatographic procedure is described for the quantitative determination of epinephrine, norepinephrine, and dopamine in human plasma. The method, which is based on adsorption of the catecholamines to alumina and, after liberation, separation on a microparticulate bonded strong cation-exchange resin and amperometric detection, has been optimized to give complete baseline separation of the substances of interest. Dihydroxybenzylamine, a nonendogenous catecholamine, is used as the internal standard. The detection limit is about 0.1 pmol for dopamine. Analysis of data obtained for norepinephrine and epinephrine from a total of 59 plasma samples showed a good correlation to the corresponding values obtained with a radioenzymatic method. Some results from normal and pathological conditions are compared.

A Protein Disulfide Isomerase/Thioredoxin-1 Complex Is Physically Attached to Exofacial Membrane Tumor Necrosis Factor Receptors: Overexpression in Chronic Lymphocytic Leukemia Cells

AIMS The 3D structures and functions of cysteine-rich receptors such as tumor necrosis factor receptors (TNFRs) are redox-modulated by dithiol-disulfide exchange. TNFR superfamily members participate in growth regulation in B-cell chronic lymphocytic leukemia (CLL), and tissue stromal cells interact with leukemia cells, profoundly affecting their viability via release of redox-active components, including cysteine, thioredoxin-1 (Trx1), and Trx reductase. Trx1 was previously shown to enhance release of TNF, which acts as an autocrine/paracrine growth factor in CLL. The nature of the mechanism is not known, however. Here, we investigated whether Trx1 and protein disulfide isomerase (PDI), a chaperone and Trx-family member, may interact with TNFRs. RESULTS We found direct physical association between PDI and TNFR1 or TNFR2 by coclustering and affinity isolation. PDI (57 kDa) formed covalent/reduction-sensitive 69-kDa complexes with Trx1 (12 kDa) in a majority of CLL cell samples, detected at low levels only in control B-cells. Functionally, the TNF/TNFR signaling via the nuclear factor kappa B-driven autocrine loop was disrupted in a dose-dependent fashion by PDI-inhibitors bacitracin, anti-PDI, or anti-Trx1 antibodies, resulting in reduced viability. PDI was significantly overexpressed in immunoglobulin heavy-chain variable (IGHV) unmutated versus mutated CLL (p=0.0102), and amplified TNF release was observed in the former group. INNOVATION This study points out a previously unrecognized physical and functional association of TNFRs with the redox-active proteins PDI and Trx1. CONCLUSION We describe here a new level of TNF regulation, in which membrane TNFRs are redox controlled at the exofacial surface by PDI/Trx1. These findings shed new light on the observed survival benefit in CLL B-cells exerted by TNFR-superfamily ligands and point at potential therapeutic strategies.

Enzyme-linked immunospot assay for detection of thioredoxin and thioredoxin reductase secretion from cells.

Oxidative stress response was determined in this study by enzyme-linked immunospot (ELISpot) assays for thioredoxin (Trx) and Trx reductase (TrxR). On exposure to oxidative stress, cells can launch a variety of defense mechanisms, including release of antioxidant proteins. The Trx system, consisting of Trx, TrxR, and NADPH, constitutes one of these cellular defense systems for maintenance of a healthy reduction-oxidation (redox) balance. Trx and TrxR are rapidly upregulated and released from monocytes, lymphocytes, and other normal and neoplastic cells on exposure. Secreted Trx and TrxR have proved to be eminent indicators of oxidative stress. Trx is a small, 12-kDa protein released through a leaderless pathway, whereas TrxR, which is a 116-kDa selenoprotein and required for regeneration of Trx, is secreted through the Golgi pathway. In this chapter we present a detailed laboratory bench protocol for enumeration of single cells secreting redox-active Trx and TrxR after oxidative stress exposure. Physiological stimuli (such as interferon gamma, lipopolysaccharide, interleukin 1, and CD23 ligation; and phorbol 12-myristate 13-acetate and ionophore) as well as UV light and hydrogen peroxide were used to generate oxidative stress, and some are presented in detail. The protocol includes a description of cell isolation, preparation, handling, and development of ELISpot plates, troubleshooting notes, presentation of results, statistical evaluation, and comments on alternative sources of materials and manufacturer Web addresses. We concluded that the ELISpot assay is a useful method for detection of single cells secreting the redox-active proteins Trx and TrxR after oxidative stress exposure.

Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG oligodeoxynucleotides of class C

Significance Release of pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) contributes to inflammatory responses and antiviral signaling. Mitochondrial DNA (mtDNA) is a potent DAMP molecule observed in blood circulation of trauma, autoimmune, HIV, and certain cancer patients. Here, we report a previously unrecognized lymphocyte feature that CpG and non-CpG oligodeoxynucleotides of class C promptly induce release of mtDNA as extracellular web-like structures. Lymphocyte mtDNA webs provoked antiviral type I IFN production in peripheral blood mononuclear cells but were devoid of bactericidal proteins. Notably, cells remained viable after the release. Our findings imply an alternative role for lymphocytes in antiviral signaling by utilizing their mtDNA as a rapid signaling molecule to communicate danger. Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.

Myelin protein zero is naturally processed in the B cells of monoclonal gammopathy of undetermined significance of immunoglobulin M isotype: aberrant triggering of a patient’s T cells

Background Monoclonal gammopathy of undetermined significance of immunoglobulin M isotype is a condition with clonally expanded B cells, recently suggested to have an infectious origin. This monoclonal gammopathy is frequently associated with polyneuropathy and antibodies against myelin protein zero, whereas the role of the T cells remains largely unknown. We analyzed protein zero-specific B cells, as antigen-presenting cells, and their capacity to activate T helper cells. Design and Methods We used a well-characterized monoclonal gammopathy of undetermined significance-derived B-cell line, TJ2, expressing anti-protein zero immunoglobulin M. The ability of TJ2 cells to bind, endocytose, process, and present protein zero was investigated by receptor-clustering and immunofluorescence. The activation of protein zero-specific autologous T cells was studied by measuring interleukin-2 and interferon-γ with flow cytometry, immunobeads, and enzyme-linked immunospot assays. Results Surface-receptor clustering and endocytosis of receptor-ligand (immunoglobulin M/protein zero) complexes were pronounced after exposure to protein zero. Naturally processed or synthetic protein zero peptide (194–208)-pulsed TJ2 cells significantly induced interleukin-2 secretion from autologous T cells compared to control antigen-pulsed cells (P<0.001). The numbers of interferon-γ-producing T helper cells, including CD4+/CD8+ cells, were also significantly increased (P=0.0152). Affinity-isolated naturally processed myelin peptides were potent interferon-γ stimulators for autologous peripheral blood mononuclear cells, but not for control peripheral blood mononuclear cells. Conclusions We show for the first time that myelin protein zero is naturally processed in B cells from monoclonal gammopathy of undetermined significance of immunoglobulin M isotype, acting as aberrant antigen-presenting cells in activation of a patient’s T helper cells. Our findings cast new light on the important role of autoreactive protein zero-specific B cells in the induction of the pathogenic T-cell responses found in nerve lesions of patients with monoclonal gammopathy of undetermined significance with peripheral neuropathy.