A study of human-human hybridomas from patients with autoimmune thyroid disease

Bernardo, Erica De; Davies, Terry F.

doi:10.1007/bf00915428

Cited by 10 publications

(5 citation statements)

References 18 publications

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“…Fibroblast feeder cells were essential for the establishment of the clones, just as feeder cells are required in many other cloning procedures (5,6), and is further evidence for the FRTL-5 dependence on a variety of growth factors secreted by fibroblasts and other support cells. Hence, FRTL-5 cells have not only growth responsiveness to autocrine factors, as demonstrated by the ease with which FRTL-5 cells can be passaged, but also responsiveness to many other thyroid cell growth stimulators, which may be provided by irradiated fibroblast feeder cells, and as determined by examination of specific growth factors in the recent studies of Tramontano et al (10) and Mak et al (11).…”

Section: Discussionmentioning

confidence: 79%

“…However, after approximately 2 yr of continuous culture we became concerned about the heterogeneity of the cell cultures, since differences in cell types became visible on light microscopy. For this reason, we have investigated the cloning potential of FRTL-5 cells using the type of approach previously used in our laboratory for the production of monoclonal antibodies and T cell clones (5,6). We find that, under appropriate conditions, it is possible to clone FRTL-5 cells by limiting dilution with the production of individual cell clones which respond to TSH, in terms of growth and cAMP generation, but with remarkable variations in sensitivity and degree of responsiveness.…”

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confidence: 99%

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Cloning the Fisher Rat Thyroid Cell Line (FRTL-5): Variability in Clonal Growth and 3′5′-Cyclic Adenosine Monophosphate Response to Thyrotropin*

1987

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To investigate the stability of FRTL-5 cells we cloned stock cells by limiting dilution in a 6-hormone medium with bovine TSH (bTSH) (10 mU/ml) (6H medium). One third of the wells with irradiated fibroblast feeder cells developed FRTL-5 colonies. One hundred and twenty clones were obtained, of which 29 (8%) developed sufficiently for functional analysis. Clones were tested for growth responses to bTSH stimulation as evidenced by 72-h [3H]thymidine uptake and TSH receptor activation by cAMP generation. Clonal growth responses to bTSH were of three types: dose-related growth increase, absence of growth stimulation, and stimulation by bTSH in concentrations up to 100 microU/ml and inhibition of growth above 100 microU/ml. All clones tested showed evidence of extracellular cAMP accumulation in response to bTSH. However, sensitivity to bTSH varied from 1 to 100 microU/ml and maximum cAMP secretion with 1 mU/ml bTSH varied from 2 to 13 pmol/ml. Whereas certain clones showed high sensitivity to bTSH with respect to both growth and cAMP responses (e.g. 1B-6), there were clones which showed disparity in this relationship, as evidenced by poor growth dependency but high cAMP responses or by growth stimulation yet insensitivity with respect to cAMP secretion. These data demonstrate that the FRTL-5 line contains cells with variable responsiveness to bTSH. Whereas the FRTL-5 line is heterogeneous and subject to developmental variation, cloning by limiting dilution allows the derivation of highly bTSH-sensitive cells, such as 1B-6, from the stock cultures.

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Section: Discussionmentioning

confidence: 79%

mentioning

confidence: 99%

Cloning the Fisher Rat Thyroid Cell Line (FRTL-5): Variability in Clonal Growth and 3′5′-Cyclic Adenosine Monophosphate Response to Thyrotropin*

1987

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“…The establishment of B cell lines and clones would provide a useful tool to study further the role and biological functions of autoimmune myelin‐specific B cells and would also facilitate studies on B–T‐cell interactions in the pathogenesis of PN‐MGUS. Epstein–Barr virus (EBV) transformation of B cells, as a method [19,20], has been used to obtain autoantibody‐producing B cell lines in a number of autoimmune diseases, such as systemic lupus erythematosus [21], myasthenia gravis [22], multiple sclerosis [23], and autoimmune thyroiditis [24,25]. In MGUS, where a clone of B cells already exists in vivo , the experience of establishing B cell clones is limited.…”

Section: Introductionmentioning

confidence: 99%

Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS)

Kvarnström¹,

Sidorova²,

Nilsson³

et al. 2002

Clinical and Experimental Immunology

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Polyneuropathy associated with monoclonal gammopathy of undetermined significance (PN-MGUS) is regarded as an immune-mediated disorder, where an expanded B cell clone secretes monoclonal IgM or IgG/A M-components (for review see [1,2]). About 50% of the patients with M-component of IgM isotype display a binding specificity for peripheral nerve myelin [3]. The target auto-antigen for these antibodies is mainly represented by carbohydrate epitopes shared by glycoproteins, like the myelin protein P 0 [4] and myelin-associated glycoprotein (MAG) [5], and glycolipids [6,7], like the sulphated glucuronic acid epitope present on, for example, sulphated glucuronyl paragloboside (SGPG). A direct pathogenic role of the antibodies against peripheral nerve myelin has been suggested based on findings that demyelination is associated with deposits of monoclonal antibodies and complement [8] SUMMARYMonoclonal expansion of B cells and plasma cells, producing antibodies against 'self' molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström's macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P 0 , using beads coated with P 0 . P 0 -coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein-Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P 0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5 + positive, although the expression declined in vitro over time. The anti-P 0 antibodies were of IgM-l type. The antibodies belonged to the V H 3 gene family with presence of somatic mutations. The IgM reacted with P 0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5 + clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P 0 -specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation.

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“…The establishment of B cell lines and clones would provide a useful tool to study further the role and biological functions of autoimmune myelin-specific B cells and would also facilitate studies on B-T-cell interactions in the pathogenesis of PN-MGUS. Epstein-Barr virus (EBV) transformation of B cells, as a method [19,20], has been used to obtain autoantibody-producing B cell lines in a number of autoimmune diseases, such as systemic lupus erythematosus [21], myasthenia gravis [22], multiple sclerosis [23], and autoimmune thyroiditis [24,25]. In MGUS, where a clone of B cells already exists in vivo, the experience of establishing B cell clones is limited.…”

Section: Introductionmentioning

confidence: 99%

Myelin Protein P‐0‐specific Igm Producing Monoclonal B Cell Lines Were Established From Polyneuropathy Patients With Monoclonal Gammopathy of Undetermined Significance (Mgus)

Kvarnström¹,

Sidorova²,

Nilsson³

et al. 2002

J Peripheral Nervous Sys

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Polyneuropathy associated with monoclonal gammopathy of undetermined significance (PN-MGUS) is regarded as an immune-mediated disorder, where an expanded B cell clone secretes monoclonal IgM or IgG/A M-components (for review see [1,2]). About 50% of the patients with M-component of IgM isotype display a binding specificity for peripheral nerve myelin [3]. The target auto-antigen for these antibodies is mainly represented by carbohydrate epitopes shared by glycoproteins, like the myelin protein P 0 [4] and myelin-associated glycoprotein (MAG) [5], and glycolipids [6,7], like the sulphated glucuronic acid epitope present on, for example, sulphated glucuronyl paragloboside (SGPG). A direct pathogenic role of the antibodies against peripheral nerve myelin has been suggested based on findings that demyelination is associated with deposits of monoclonal antibodies and complement [8]. The antibodies are mainly produced by B-1, CD5 + B lymphocytes and they are polyreactive and have low SUMMARY Monoclonal expansion of B cells and plasma cells, producing antibodies against 'self' molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström's macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P 0 , using beads coated with P 0 . P 0 -coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein-Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P 0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5 + positive, although the expression declined in vitro over time. The anti-P 0 antibodies were of IgM-l type. The antibodies belonged to the V H 3 gene family with presence of somatic mutations. The IgM reacted with P 0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5 + clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P 0 -specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation.

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A study of human-human hybridomas from patients with autoimmune thyroid disease

Cited by 10 publications

References 18 publications

Cloning the Fisher Rat Thyroid Cell Line (FRTL-5): Variability in Clonal Growth and 3′5′-Cyclic Adenosine Monophosphate Response to Thyrotropin*

Cloning the Fisher Rat Thyroid Cell Line (FRTL-5): Variability in Clonal Growth and 3′5′-Cyclic Adenosine Monophosphate Response to Thyrotropin*

Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS)

Myelin Protein P‐0‐specific Igm Producing Monoclonal B Cell Lines Were Established From Polyneuropathy Patients With Monoclonal Gammopathy of Undetermined Significance (Mgus)

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