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B Rapoport

Publications and source records attributed to B Rapoport.

At least 37 records · Page 2Linked to original sources
PubMed2001

Human monoclonal autoantibodies to B-cell epitopes outside the thyroid peroxidase autoantibody immunodominant region.

Human autoantibodies to thyroid peroxidase (TPO) interact with a restricted or immunodominant region (IDR) on intact TPO. However, a smaller proportion of polyclonal serum TPO autoantibodies bind outside this region. To isolate monoclonal nonimmunodominant region (non-IDR) TPO autoantibodies, we screened a thyroid-derived immunoglobulin gene phage display library while "epitope masking" the TPO IDR with four human TPO monoclonal autoantibodies that define the IDR. Among 31 non-IDR autoantibodies obtained (expressed as Fab), 8 representatives were analyzed further based on their restriction digestion profiles. All are encoded by almost identical H chains (VH3 family), with extremely long D regions, paired with three different types of light chains. In contrast, IDR TPO Fab from the same patient utilize seven different heavy chains (VH1 and VH5 families) paired nonpromiscuously with different light chains. Use of VH5 genes has not been reported previously for TPO autoantibodies. Both non-IDR and IDR Fab bind specifically to TPO and not to other proteins. The non-IDR Fab affinities for TPO are moderately high (Kd 1-2 x 10(-9) M), somewhat lower than those for most IDR Fab (Kd 1-4 x 10(-10) M). The epitopes of the three types of non-IDR Fab overlap with each other, indicating a major role for their heavy chain in TPO binding. Most importantly, the epitopes of non-IDR Fab are recognized by patients' serum autoantibodies. In summary, we provide the first insight into the immunoglobulin genes, affinities and epitopes of human monoclonal autoantibodies that bind outside the TPO-immunodominant region.

Amino Acid Sequence↗
PubMed2001

Does the autoantibody immunodominant region on thyroid peroxidase include amino acid residues 742-771?

Identification of the thyroid peroxidase (TPO) amino acid residues that comprise the autoantibody immunodominant region is an important goal that has proven difficult because of the conformational nature of the epitopes involved. Recent data suggest that the immunodominant region has been located. Thus, by autoantibody recognition of tryptic fragments of native TPO, as well as of conformational portions of TPO expressed as cell-free translates, the autoantibody immunodominant region appears to include amino acid residues 742-771, near the C terminus of the ectodomain. To evaluate this deduction, we expressed as cell-free translates the full TPO ectodomain, as well as TPO truncated after residues 741 and 771. The epitopic integrity of these molecules was first confirmed by immunoprecipitation by patient sera containing TPO autoantibodies. However, autoantibody recognition could involve a minority of TPO autoantibodies with the individual sera, not fulfilling the strict criteria for immunodominance. In order to obtain definitive data, we performed immunoprecipitations on these TPO variants with four recombinant human monoclonal autoantibodies that define the immunodominant region. All four monoclonal autoantibodies immunoprecipitated TPO 1-741 to the same extent as they did TPO 1-771 and the full TPO ectodomain, indicating that the immunodominant region comprises (at least in large part) amino acid residues upstream of residue 741.

Antibodies, Monoclonal↗
PubMed2001

Naked TSH receptor DNA vaccination: A TH1 T cell response in which interferon-gamma production, rather than antibody, dominates the immune response in mice.

Two approaches have been developed to induce TSH receptor antibodies in mice with properties resembling those in Graves' disease, the Shimojo model of injecting live fibroblasts coexpressing the TSH receptor and major histocompatibility complex antigen Class II, and TSH receptor-DNA vaccination. Thyroid-stimulating antibodies appear to occur less commonly after DNA vaccination, but there has been no direct comparison of these models. We performed a three-way comparison of 1) AKR/N and 2) BALB/c mice vaccinated with TSH receptor-DNA and 3) AKR/N mice injected with fibroblasts expressing the TSH receptor and the major histocompatibility complex antigen class II of AKR/N mice. TSH receptor-DNA vaccinated mice had low or undetectable levels of TSH receptor antibodies determined by ELISA or flow cytometry. Nonspecific binding precluded comparisons with sera from Shimojo mice by these assays. TSH binding inhibition and thyroid-stimulating antibody were undetectable in TSH receptor-DNA vaccinated mice. In Shimojo mice, TSH binding inhibition was positive in approximately 60%, and thyroid-stimulating antibodies were positive in hyperthyroid animals. Unlike the negative antibody data, splenocytes from TSH receptor-vaccinated (but not Shimojo) mice proliferated and produced the Th1 cytokine interferon-gamma in response to TSH receptor antigen. In conclusion, DNA vaccination is less effective at inducing TSH receptor antibodies than the Shimojo approach, but it permits the future characterization of TSH receptor-specific T cells generated without adjuvant.

Animals↗
PubMed2001

A prion-like shift between two conformational forms of a recombinant thyrotropin receptor A-subunit module: purification and stabilization using chemical chaperones of the form reactive with Graves' autoantibodies.

A secreted recombinant TSH receptor (TSHR) ectodomain variant (TSHR-289) neutralizes TSHR autoantibodies in Graves' disease, but is heterogeneous in containing both immunologically active and inactive molecules and is also unstable. We have now purified each form of TSHR-289 using sequential affinity chromatography with a mouse mAb (3BD10) specific for the inactive form, and a mAb to C-terminal His residues that recognizes both forms. The immunological difference between active and inactive TSHR-289 was unrelated to primary amino acid sequence or carbohydrate content and was, therefore, attributable to its folded state. The epitopes for Graves' autoantibodies and 3BD10 overlap, and both are destroyed by denaturation. Therefore, reciprocal binding by autoantibodies and 3BD10 to conformational determinants involving the same TSHR segment suggests a prion-like shift between two folded states of the molecule. Despite purification, immunologically active TSHR-289 remained labile, as determined by loss of autoantibody, and gain of 3BD10, recognition. However, using chemical chaperones we have, for the first time, been able to stabilize purified TSHR antigen in immunologically intact form. In summary, purification of immunologically active and stable antigen in milligram quantities provides a powerful tool for future diagnostic and therapeutic studies in Graves' disease.

Amino Acid Sequence↗
PubMed2000

Role of MHC class I expression and CD8(+) T cells in the evolution of iodine-induced thyroiditis in NOD-H2(h4) and NOD mice.

Dietary iodine has long been known to influence the development of human autoimmune thyroid disease. In nonobese diabetic (NOD) and NOD-H2(h4) mice elevated dietary iodine has been shown to induce autoimmune thyroid disease. Immune responses to thyroid antigens can be detected in these mouse strains, including T cell responses in the NOD-H2(h4) mouse to thyroid peroxidase. Cell transfer studies and antibody depletion experiments reveal a requirement for both CD4(+) T cells and CD8(+) T cells in the development of thyroid autoimmunity. Histological analyses of the thyroids show that following 1 week of iodine administration MHC class I expression is elevated on thyroid follicular cells and CD4(+) and CD8(+) T cells have begun to infiltrate the gland. Although MHC class II expression on thyroid epithelial cells was also elevated, the tempo of expression was slower and the extent of expression was far less than that for MHC class I. Depletion of CD8(+) T cells at early stages of disease induction inhibited not only thyroid infiltration and autoantibody production but also reduced the levels of MHC expression in the thyroid, suggesting that cytokine production by infiltrating lymphocytes was responsible for the increased MHC expression.

Adoptive Transfer↗
PubMed2000

Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II.

Injection of AKR/N mice with fibroblasts co-expressing MHC class II and TPO in the absence of adjuvant induces IgG-class TPO antibodies that resemble spontaneously arising human thyroid autoantibodies. We have used this model to examine the effect of iodide on TPO antibody induction as well as to analyse the interaction between T and B cells. Despite its importance as a major environmental factor in thyroid autoimmunity, variable iodide intake had no detectable effects on TPO antibody levels, lymphocytic infiltration of the thyroid or thyroid hormone levels. In terms of T cell responsiveness, splenocytes from TPO fibroblast-injected mice, but not from control mice, proliferated in response to TPO. Intriguingly, B cell-depleted splenocytes (mainly T cells without reduction of macrophages) proliferated in response to TPO only when co-cultured with irradiated autologous splenocytes from TPO fibroblast-injected mice but not from control mice. These data suggest that TPO-specific B cells are involved in antigen presentation to sensitized T cells and are supported by the ability of spleen cells from TPO cell-injected (but not control) mice to secrete TPO antibodies spontaneously in culture. In conclusion, we provide the first evidence for the presence of thyroid autoantigen-specific B cells and their ability to present their autoantigen to sensitized T cells in mice induced to develop TPO antibodies resembling autoantibodies in humans.

Animals↗
PubMed2000

Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens.

AKR/N mice injected with fibroblasts expressing MHC class II (RT4.15HP cells) and the TSH receptor (TSHR) develop antibodies similar to those in Graves' disease. We were unable to analyse the subclass of these antibodies because of unexpectedly high non-specific binding by ELISA or flow cytometry. The non-specific binding reflected generalized immune activation which occurred even when the fibroblasts did not express the TSHR. However, the IgG subclasses were determined for thyroid peroxidase (TPO) antibodies induced using TPO-expressing RT4.14HP cells and found to be IgG2a > IgG1. This Thl pattern is consistent with spontaneous secretion of interferon-gamma (but not IL-4 or IL-10) by splenocytes from injected mice. The Th1 bias was related to fibroblast injection because conventional immunization of the same mouse strain with purified TPO and adjuvant induced a Th2 response (IgG1 >> IgG2a). Further, untransfected fibroblasts themselves induced powerful, non-specific proliferative responses when used as antigen-presenting cells (APC) in vitro. Flow cytometry revealed that the RT4.15HP fibroblasts (and TSHR- and TPO-transfected derivatives) expressed B7-1. Unexpected constitutive expression of this key molecule may bypass the requirement for up-regulation of other costimulatory molecules involved in T cell stimulation. Our data support the concept that RT4.15HP fibroblasts present the TSHR (or TPO), at least for initiating the immune response. However, the accompanying generalized immune stimulation creates difficulties for analysis of TSHR-specific T and B lymphocytes. On the other hand, extension of the model to TPO, an easier antigen to study, will facilitate analysis of murine T cell responses likely to resemble those in human thyroid autoimmunity.

Animals↗
PubMed2000

Elephantiasic pretibial myxedema: insight into and a hypothesis regarding the pathogenesis of the extrathyroidal manifestations of Graves' disease.

The basis for the extrathyroidal manifestations of Graves' ophthalmopathy (GO) and dermopathy are not well understood. We describe immunohistochemical studies on the skin of a patient with an extreme, elephantiasic form of Graves' dermopathy that developed after periods of prolonged standing with dependent edema. Excision of part of the lesion with subsequent skin grafting from a normal donor site resulted in recurrence of the disease at the original site as well as in development of disease at the donor site. A murine monoclonal antibody reacted with the thyrotropin receptor (TSHR) or a cross-reacting protein in fibroblast-like cells in the patient's upper dermis and, surprisingly, with dermal cells from unaffected individuals. The patient's dermis containing lymphoid follicles comprising B cells and CD3+, CD4+ T cells, with few CD8+ T cells. CD21+ cells (most likely follicular dendritic cells) were also present in the dermis. Based on past and present observations, we raise an unifying hypothesis to explain the diverse extrathyroidal manifestations of Graves' disease and their apparent lack of association with TSHR autoantibodies. As opposed to the present concept that these phenomena relate to site-specific properties on preadipocytes or fibroblasts, we suggest that clinically evidence GO and dermopathy are primarily caused by local factors (particularly in the orbit) superimposed on a systemic, low-grade connective tissue inflammation.

Antibodies, Monoclonal↗
PubMed2000

The Multinational Association for Supportive Care in Cancer risk index: A multinational scoring system for identifying low-risk febrile neutropenic cancer patients.

PURPOSE: Febrile neutropenia remains a potentially life-threatening complication of anticancer chemotherapy, but some patients are at low risk for serious medical complications. The purpose of this study was to develop an internationally validated scoring system to identify these patients. MATERIALS AND METHODS: Febrile neutropenic cancer patients were observed in a prospective multinational study. Independent factors assessable at fever onset, predicting low risk of complications, on a randomly selected derivation set, were assigned integer weights to develop a risk-index score, which was subsequently tested on a validation set. RESULTS: On the derivation set (756 patients), predictive factors were a burden of illness indicating absence of symptoms or mild symptoms (weight, 5; odds ratio [OR], 8.21; 95% confidence interval [CI], 4.15 to 16.38) or moderate symptoms (weight, 3; OR, 3.70; 95% CI, 2.18 to 6.29); absence of hypotension (weight, 5; OR, 7.62; 95% CI, 2.91 to 19.89); absence of chronic obstructive pulmonary disease (weight, 4; OR, 5. 35; 95% CI, 1.86 to 15.46); presence of solid tumor or absence of previous fungal infection in patients with hematologic malignancies (weight, 4; OR, 5.07; 95% CI, 1.97 to 12.95); outpatient status (weight, 3; OR, 3.51; 95% CI, 2.02 to 6.04); absence of dehydration (weight, 3; OR, 3.81; 95% CI, 1.89 to 7.73); and age less than 60 years (weight, 2; OR, 2.45; 95% CI, 1.51 to 4.01). On the validation set, a Multinational Association for Supportive Care in Cancer risk-index score >/= 21 identified low-risk patients with a positive predictive value of 91%, specificity of 68%, and sensitivity of 71%. CONCLUSION: The risk index accurately identifies patients at low risk for complications and may be used to select patients for testing therapeutic strategies that may be more convenient or cost-effective.

Anti-Bacterial Agents↗
PubMed2000

Evidence that cleavage of the thyrotropin receptor involves a "molecular ruler" mechanism: deletion of amino acid residues 305-320 causes a spatial shift in cleavage site 1 independent of amino acid motif.

Some TSH receptors (TSHR) on the cell surface cleave into A and B subunits. Cleavage at upstream Site 1 is followed by the proteolytic excision of an intervening C peptide region terminating at a downstream Site 2. Although present evidence suggests that Site 1 lies between amino acid residues 303 and 317, the mechanism and exact amino acid(s) involved in cleavage are unknown. Previous amino acid substitutions at Site 1 failed to abrogate cleavage. We, therefore, performed deletion mutations within this region. Cleavage of cell surface TSHR, detected by 125I-TSH cross-linking to intact cells, was not prevented by deletion of four individual segments within the Site 1 cleavage region (delta305-308, delta309-312, delta313-316, delta317-320). However, deletion of the entire region (delta305-320) reduced the extent of cleavage and shifted the cleavage site upstream of the glycan at amino acid residue N302. Elimination of this glycan (N302Q substitution) reversed the effect of deleting amino acid residues 305-320 on TSHR cleavage, suggesting that reduced cleavage at the new, upstream cleavage site was caused by steric hindrance by the glycan at N302. In summary, deletion, as opposed to mutagenesis, of the TSHR cleavage Site 1 region produces a spatial shift in TSHR cleavage Site 1 from downstream to upstream of the glycan at N302. These observations provide strong evidence that TSHR cleavage at this site does not occur at a particular amino acid motif and suggests that cleavage involves a "molecular ruler" mechanism involving cleavage at a fixed distance from a protease attachment site.

Amino Acid Motifs↗
PubMed2000

Autoimmune response to the thyroid in humans: thyroid peroxidase--the common autoantigenic denominator.

Autoimmunity to thyroid peroxidase (TPO), manifest as high affinity IgG class autoantibodies, is the common denominator of human thyroid autoimmunity, encompassing patients with overt hyper- or hypothyroidism as well as euthyroid individuals with subclinical disease. The identification and cloning of TPO (the "thyroid microsomal antigen") provided the critical tool for analyzing B and T cell reactivity to this major thyroid autoantigen. In particular, the availability of immunoreactive TPO permitted the isolation of essentially the entire repertoire of human monoclonal antibodies, a feat unparalled in an organ-specific autoimmune disease. These recombinant autoantibodies (expressed as Fab) provide insight into the genes encoding their H and L chains as well as the conformational epitopes on TPO with which serum autoantibodies interact. Analyses of TPO autoantibody epitopic "fingerprints" indicate a lack of epitope spreading as well as a genetic basis for their inheritance. Limited data are available for the responses and cytokine profiles of T cells to endogenously processed TPO. Moreover, the role of thyroid cells in initiating the autoimmune response to TPO, and of B cells in expanding and/or modulating the response of sensitized T cells, has yet to be established. Finally, because autoantibody (and likely T cell) responses to TPO parallel those to TSH receptor and thyroglobulin, manipulation of T and B cell responses to TPO may provide the basis for the development of immunospecific therapy for autoimmune thyroid disease in general.

Animals↗
PubMed2000

Presence of interleukin 4 or interleukin 10, but not both cytokines, in pancreatic tissue of two patients with recently diagnosed diabetes mellitus type I.

Studies in the NOD mouse model suggest that development of diabetes mellitus type I can be prevented and established disease cured by deviation towards a Th2-type response. To obtain insight into whether this approach may be applicable to human disease, we investigated the Th1/Th2 cytokine balance in pancreatic tissue from two patients with diabetes of recent onset (Case 1, accidental death; Case 2, ketoacidosis). Using the polymerase chain reaction to amplify reverse-transcribed cDNA, signals for actin and CD36 confirmed mRNA integrity and the presence of T cells in pancreatic tissue from both patients and from a control. IFN-gamma cDNA was also amplified from all three tissues. However, IL-4 (but not IL-10) cDNA, was amplified from the pancreas of Case 1. Conversely, IL-10 (but not IL-4) cDNA was amplified from the the pancreas of Case 2. The control pancreas yielded specific signals for both IL-4 and IL-10. Our data extend the limited database on Th1 and Th2 cytokine expression in human pancreatic tissue from recently diagnosed diabetics. Moreover, together with previous observations, our findings raise the possibility that the lack of both IL-4 and IL-10 may be associated with the development of IDDM in humans.

Adolescent↗
PubMed1999

Subunit structure of thyrotropin receptors expressed on the cell surface.

We studied cell surface thyrotropin receptor (TSHR) by biotinylating proteins on the surface of metabolically labeled, intact cells. In addition to TSHR cleaved into A and B subunits, mature single-chain receptors with complex carbohydrate were also present on the cell surface. A low A/B subunit ratio indicated partial shedding of extracellular A subunits from transmembrane B subunits. TSHR cleavage at upstream site 1 (within amino acid residues 305-316) would generate a B subunit of 51-52 kDa. However, only smaller B subunits (40-46 kDa) were detected, corresponding to N termini from residues approximately 370 (site 2) extending downstream to the region of B subunit insertion into the plasma membrane. The intervening C peptide region between sites 1 and 2 could not be purified from TSHR epitope-tagged (c-myc) within this region. However, the small proportion of B subunits recovered with a c-myc antibody were larger (45-52 kDa) than the majority of B subunits recovered with a C-terminal antibody. In conclusion, our study provides the first characterization of cell surface TSHR including their A and B subunits. Single-chain, mature TSHR do exist on the cell surface. The C peptide lost during intramolecular cleavage disintegrates rapidly following cleavage at upstream site 1 of the single-chain TSHR into A and B subunits. N-terminal disintegration of the B subunit pauses at site 2, but then progresses downstream to the vicinity of the plasma membrane, revealing a novel mechanism for A subunit shedding.

Animals↗
PubMed1999

The shed thyrotropin receptor is primarily a carboxyl terminal truncated form of the A subunit, not the entire A subunit.

The TSH receptor (TSHR) sheds its A subunit, particularly when cells are cultured in serum-poor medium. This shed A subunit is reported to be smaller than the cell-associated receptor because of the loss of glycan without change in its polypeptide core. Contrary to previous deductions, we now find that the 'small' shed A subunit has lost a glycan moiety because of the proteolytic clipping of a small C-terminal fragment containing an Asn-linked glycan. Moreover, this lost peptide fragment contains cysteine residues likely involved in A subunit linkage to the membrane-associated B subunit. Progressive lowering of the serum concentration in culture medium accentuates the process. Therefore, 'small' A subunit shedding does not appear related to a physiological mechanism involving disulfide bond reduction. On the other hand, we detected, for the first time, shedding of a lesser amount of normal-sized, in addition to small, A subunits, especially by cells cultured in standard serum concentrations.

Amino Acid Sequence↗