PubMed Health⌕ Search

Biomedical subjects

B S Prabhakar

Publications and source records attributed to B S Prabhakar.

At least 55 records · Page 3Linked to original sources
PubMed1994

High frequency of B cells capable of producing anti-thyrotropin receptor antibodies in patients with Graves' disease.

Hyperthyroidism in Graves' disease (GD) is mediated by antibodies to the thyrotropin receptor (TSHr). Patients that go into remission show a decline in antibody titer. However, upon cessation of treatment with anti-thyroid drugs a significant proportion of patients relapse and TSHr antibodies (TSHrAb) are present in their circulation. This suggests that B cells capable of producing TSHrAb persist despite treatment. To determine the frequency of these cells, B cells from six patients with GD and four healthy controls were infected with Epstein-Barr virus and cultured in 96-well plates at varying cell concentrations. A higher frequency of B cells capable of producing TSHrAbs was detected in patients with GD, relative to normal controls. For example, at 2 x 10(5) cells per well, 100% of wells containing cells from either patients with GD or controls were positive for immunoglobulin (Ig) production. In contrast, 27% of the wells containing cells from Graves' patients, and only 3% from controls, were positive for TSHrAb. Higher titers of TSHrAbs were produced in cultures containing lymphocytes from patients with GD and were predominantly of IgG isotype. All patients with GD who had high thyrotropin binding inhibitory immunoglobulins also had higher frequencies of TSHr-specific B cells. These findings show that TSHrAb-producing B cells are present at a higher frequency in the peripheral circulation of patients with GD.

Adolescent↗
PubMed1994

A region on the human thyrotropin receptor which can induce antibodies that inhibit thyrotropin-mediated activation of in vitro thyroid cell function also contains a highly immunogenic epitope.

Autoantibodies to the thyrotropin receptor (TSHr) bind to the extracellular domain of the TSHr (ETSHr) and either stimulate or inhibit thyroid cell function and/or growth. In order to investigate the regulation and the specificity of the immune response to the TSHr, our laboratory recently produced recombinant human ETSHr protein by using the baculovirus expression system. In the present study, we used the recombinant ETSHr protein, a panel of overlapping synthetic peptides derived from the TSHr, and polyclonal rabbit antibodies produced against recombinant ETSHr and synthetic peptides to define a highly immunogenic region (aa 352-388) of the TSHr. Moreover, we used competitive inhibition studies to identify a dominant epitope (aa 367-372) within this region to which ETSHr antibodies react. This immunodominant epitope lies within a region unique to the TSHr when compared to the other glycoprotein hormone receptors. These data, together with the earlier observation that antibodies against aa region 357-372 can inhibit thyrotropin (TSH)-mediated activation of thyroid cells in culture, show that aa 367-372 represents, an immunodominant epitope within a functionally important region which is unique to the TSHr. Therefore, this region may play an important role in the induction or modulation of the specific immune response against the TSHr.

Amino Acid Sequence↗
PubMed1994

A recombinant extracellular domain of the thyrotropin (TSH) receptor binds TSH in the absence of membranes.

We produced large quantities of the extracellular domain of the human TSH receptor (ETSHR) using the baculovirus expression system. Insect cells containing the ETSHR protein were sequentially extracted using lysis, nuclease, and high salt buffers to enrich for recombinant protein. The ETSHR protein was purified to homogeneity on a C4 reverse phase semipreparative column using HPLC. The recombinant protein was identified as ETSHR by immunoreactivity with antibodies prepared against TSHR-derived synthetic peptides. The identity of the ETSHR was further confirmed by amino acid compositional analyses, which agreed with the amino acid composition predicted from reported cDNA sequence analyses. Protein sequence analyses confirmed that the first 26 amino acids of the N-terminal region and the C-terminal amino acid were identical to the predicted amino acid sequence. The purified ETSHR was refolded in the presence of 1.5 M guanidine-HCl and 1 mM each of cystine and cysteine. [125I] TSH bound to the refolded ETSHR in vitro in a dose-dependent manner and was specifically blocked by unlabeled TSH, but not by LH or FSH. It was notable that a membrane requirement was not essential for TSH to bind to ETSHR.

Amino Acid Sequence↗
PubMed1994

Thyrotropin (TSH) interacts with multiple discrete regions of the TSH receptor: polyclonal rabbit antibodies to one or more of these regions can inhibit TSH binding and function.

As a means of identifying functional regions of the TSH receptor (TSHr), we immunized four rabbits with recombinant extracellular TSHr (ETSHr) protein and systematically evaluated their antibody response. The antibody response was characterized by testing serial serum samples for immunoglobulin G (IgG) against ETSHr protein and 26 synthetic peptides which span the entire ETSHr. Sera were also tested for their ability to block TSH binding to native TSHr. All four rabbits developed high serum IgG titers (>1:100,000) to ETSHr. None of the rabbits developed significant IgG titers against 11 of the peptides, but each showed persistent high titers against several of the others. After multiple inoculations of antigen, sera from 3 rabbits showed significant ability to block TSH binding. Based on the ability of peptides to reverse this blocking activity, we identified 3 regions of the TSHr (i.e. amino acids 292-311, 367-386, and 397-415) through which antibodies can block TSH binding. Moreover, antibodies purified on either peptide 292-311 or peptide 367-386 affinity columns could block both TSH binding and TSH-mediated activation of thyroid cells in culture. These studies show ETSHr protein is sufficient to induce production of functionally relevant antibodies. Furthermore, we have identified several sites on the TSHr through which antibodies can inhibit TSH binding, thus leading to identification of several potential TSH-binding regions.

Amino Acid Sequence↗
PubMed1994

Multiple herpesviruses in saliva of HIV-infected individuals.

Oral infections with human herpesviruses cause increased morbidity in patients infected with HIV. In this study, multiple HHVs were often isolated from the saliva of HIV-seropositive dental patients, but their isolation rate did not differ substantially from rates reported for the general population, except for human cytomegalovirus.

Adult↗
PubMed1994

Induction of hyperthyroxinemia in BALB/C but not in several other strains of mice.

We recently expressed the extracellular domain of the human TSHR (ETSHR) protein using a baculovirus expression system and purified it to homogeneity. The ETSHR specifically binds both TSH and antibodies to TSHR. In the present study, C57BL/6J, SJL/J, BALB/cJ and B10BR.SgSnJ mice were immunized with the recombinant ETSHR or an equivalent amount of control antigen. All strains of mice produced high titers of antibody against the TSHR protein which were capable of blocking the binding of TSH to native TSHR. However, only BALB/cJ mice showed significantly elevated levels of thyroxine in their sera compared to the control mice. Similarly, BALB/cJ mice primed with ETSHR and then challenged with thyroid membranes showed significantly elevated levels of thyroxine. In addition, histopathological examination of thyroid glands from affected mice showed morphological changes characterized by hydropic and subnuclear vacuolar changes and focal scalloping, with no apparent inflammation or glandular destruction. Moreover, mice with elevated thyroxine levels showed increased in vivo thyroidal uptake of 131Iodine. Together, these data suggest that BALB/cJ mice are susceptible to the induction of hyperthyroxinemia.

Animals↗
PubMed1994

Identification of epitopes and affinity purification of thyroid stimulating auto-antibodies using synthetic human TSH receptor peptides.

We prepared a series of overlapping peptides (29 in total, 20 amino acids each) containing the sequence of the entire extracellular domain of the human TSH receptor. Three peptides (181-200, 376-394, and EC3 (629-639)) bound IgG from patients with Graves' disease in an enzyme linked immunoassay. Peptide 181-200 bound IgG from 9 of 10, EC3 from 8 of 10, and 376-394 from 6 of 10 patients respectively, compared to 0 of 9 controls. We affinity purified TSHr auto-antibodies from four Graves' patients using the three above noted peptides bound to epoxy-activated sepharose. Thyroid stimulating activity was enriched in the bound fraction from at least two of the three peptide affinity columns in each of the four patients, although the pattern of affinity enrichment differed between patients. One patient was found to possess a combination of stimulatory and inhibitory TSHr antibodies and, after affinity purification, the anti-376-394 and anti-EC3 fractions were enriched in stimulatory activity, suggesting that those regions of the receptor were epitopes for stimulatory antibodies. However, affinity purification against peptide 181-200 produced an IgG preparation that was not stimulatory, but was a potent thyroid inhibitor. Thus, we have not only partially purified TSHr auto-antibodies, but also successfully separated stimulatory and inhibitory antibodies from a single patient using combination TSHr peptide affinity.

Blotting, Western↗
PubMed1993

Immunization of mice with Yersinia enterocolitica leads to the induction of antithyrotropin receptor antibodies.

It is well established that autoimmune Graves' disease, which is characterized by hyperthyroidism, is mediated by autoantibodies to the thyrotropin receptor (TSHr). Although what initially triggers this autoantibody response is not known, a number of studies have suggested that Yersinia enterocolitica, an enterobacteria, could initiate the immune response against the TSHr. In this study, we produced antibodies against purified extracellular domain of human TSHr (ETSHr) and showed that anti-ETSHr antibodies reacted with envelope preparations from Y. enterocolitica. This reactivity was specifically blocked by preincubating sera with purified ETSHr. Moreover, antibodies reactive with ETSHr were induced by immunizing mice with Y. enterocolitica but not with Shigella flexneri SA100, Salmonella typhimurium TML, and Listeria monocytogenes. Anti-Y. enterocolitica antisera specifically reacted with the ETSHr protein and the reactivity could be blocked both by ETSHr and Y. enterocolitica envelope proteins. Our studies provide the first direct evidence that immunization with Y. enterocolitica can lead to the production of antibodies capable of reacting with TSHr and might provide the initial stimulus necessary for breakdown of self-tolerance to TSHr, eventually leading to the development of autoimmunity to TSHr.

Animals↗
PubMed1993

Heterogeneity in cellular and antibody responses against thyrotropin receptor in patients with Graves' disease detected using synthetic peptides.

Graves' disease (GD) is characterized by the presence of autoantibodies to the thyrotropin receptor (TSHr). These antibodies bind to the TSHr and stimulate thyroid cells, thus causing hyperthyroidism. To understand the regulation of TSHr-specific immune responses in Graves' disease, it is important to evaluate the T-cell response in patients with GD against TSHr. In this study we used 11 different peptides that were derived from two regions (i.e. amino acid, AA 12-46 and 316-397) unique to the TSHr when compared to other glycoprotein hormone receptors, and which also have the highest predicted immunogenicity. We evaluated both lymphocyte proliferation as a measure of T-cell response and antibody binding to each of these peptides in nine patients with GD and eight healthy subjects. Patients with GD showed considerable lymphocyte proliferative and antibody responses against several of these peptides. There was considerable heterogeneity in immune responses amongst the patients. Moreover, our data suggested that several peptides contained both T cell and antibody reactive epitopes and might represent some of the highly immunogenic regions of the TSHr.

Adolescent↗
PubMed1993

Dual mechanism of perturbation of thyrotropin-mediated activation of thyroid cells by antibodies to the thyrotropin receptor (TSHR) and TSHR-derived peptides.

To further define the epitopes with which anti-TSH receptor (anti-TSHR) antibodies react and mediate their biological effects, we used antibodies against the extracellular domain of TSHR (ETSHR) protein and nine peptides derived from the ETSHR. Peptides were chosen based on their predicted immunogenicity as well as their uniqueness to the TSHR. Antipeptide antibodies showed varying degrees of reactivity against ETSHR, with antipeptide-2-(352-366) and -3A-(357-372) showing relatively stronger reactivity with the receptor. Antibodies were tested for their ability to stimulate thyroid cells and were found to be ineffective in causing both cAMP release and iodide uptake. However, anti-3A and anti-ETSHR showed blocking TSHR antibody (TSHRAb) activities of 76.9% and 79.7%, respectively, which were significantly different (P < 0.005) compared to that of preimmune serum. Anti-2 and -91 (AA 32-46) also showed blocking TSHRAb activities of 37.5% and 35.6%, respectively (P < 0.05). Antisera were also tested for their ability to block TSH binding to thyroid membranes in a RRA. Anti-ETSHR, but not any of the antipeptide antibodies, displayed TSH binding inhibitory immunoglobulin activity. These findings suggest that there might be different mechanisms that mediate blocking TSHR antibody activity. One mechanism involves the inhibition of TSH binding to the receptor, and the other probably involves a step subsequent to TSH binding.

Adenylyl Cyclases↗
PubMed1993

Induction of TSH binding inhibitory immunoglobulins with the extracellular domain of human thyrotropin receptor produced using baculovirus expression system.

A cDNA encoding the extracellular domain of human TSHr (ETSHr) was expressed in large quantities using the baculovirus expression system. Maximum level of protein was produced at 60 hr post-infection and represented approximately 20% of the total cellular protein. The identity of the protein as ETSHr was confirmed by Western blot using antibodies to synthetic peptides derived from the TSHr. The protein has an apparent molecular weight of 50 kDa and is larger than the predicted size of 44 kDa, suggesting that the protein is glycosylated. Polyclonal antibodies raised in rabbits against gel purified ETSHr blocked the binding of 125I-TSH to native TSHr in solubilized porcine thyroid membranes in a radioreceptor assay. The availability of this antigenically active protein will facilitate further characterization of the protein and analysis of immune response against TSHr in experimental animals as well as in patients with autoimmune Graves' disease.

Animals↗
PubMed1993

Analysis of autoantibody reactivity in patients with Graves' disease using recombinant extracellular domain of the human thyrotropin receptor and synthetic peptides.

Graves' disease is characterized by hyperthyroidism leading to enhanced production of thyroid hormones. Hyperthyroidism is primarily mediated by the binding of autoantibodies to the thyrotropin receptor (TSHr). In the past, either thyroid cells or thyroid membranes were used as a source of TSHr to detect anti-TSHr antibodies. Recently, we expressed the extracellular domain of the human TSHr (ETSHr) using the baculovirus expression system. In this study, we used ETSHr protein in an ELISA to detect anti-TSHr antibodies. Our data show that this assay can be used to analyze and quantitate isotype specific antibodies against the TSHr. To map immunogenic epitopes on the TSHr, we tested patients sera against synthetic peptides derived from two highly immunogenic regions (amino acid, AA 12-46 and 316-397) of the receptor. Although sera from patients with Graves' disease reacted with several peptides, they showed particularly strong reactivity against peptides from a relatively narrow region (i.e. AA 352-394) of the TSHr. The present study demonstrates the usefulness of the recombinant ETSHr to detect and characterize anti-TSHr antibodies in a simple and sensitive ELISA, and has lead to the identification of some of the immunoreactive epitopes on the TSHr.

Amino Acid Sequence↗
PubMed1992

A novel human insulinoma-associated cDNA, IA-1, encodes a protein with "zinc-finger" DNA-binding motifs.

A subtraction library was constructed from human insulinoma (beta cell tumor) and glucagonoma (alpha cell tumor) cDNA phagemid libraries. Differential screening of 153 clones with end-labeled mRNAs from insulinoma, glucagonoma, and HeLa cells resulted in the isolation of a novel cDNA clone designated IA-1. This cDNA clone has a 2838-base pair sequence consisting of an open reading frame of 1530 nucleotides, which translates into a protein of 510 amino acids with a pI value of 9.1 and a molecular mass of 52,923 daltons. At the 3'-untranslated region there are seven ATTTA sequences between two polyadenylation signals (AATAAA). The IA-1 protein can be divided into two domains based upon the features of its amino acid sequence. The NH2-terminal domain of the deduced protein sequence (amino acids 1-250) has four classical pro-hormone dibasic conversion sites and an amidation signal sequence, Pro-Gly-Lys-Arg. The COOH-terminal domain (amino acids 251-510) contains five putative "zinc-finger" DNA-binding motifs of the form X3-Cys-X2-4-Cys-X12-His-X3-4-His-X4 which has been described as a consensus sequence for members of the Cys2-His2 DNA-binding protein class. Northern blot analysis revealed IA-1 mRNA in five of five human insulinoma and three of three murine insulinoma cell lines. Expression of this gene was undetectable in normal tissues. Additional tissue studies revealed that the message is expressed in several tumor cell lines of neuroendocrine origin including pheochromocytoma, medullary thyroid carcinoma, insulinoma, pituitary tumor, and small cell lung carcinoma. The restricted tissue distribution and unique sequence motifs suggest that this novel cDNA clone may encode a protein associated with the transformation of neuroendocrine cells.

Amino Acid Sequence↗
PubMed1991

Prokaryotic expression of the thyrotropin receptor and identification of an immunogenic region of the protein using synthetic peptides.

Graves' disease is characterized by hypersecretion of thyroid hormones due to binding of autoantibodies to the thyrotropin receptor (TSHR). In order to study immunological aspects of the TSHR we expressed the extracellular domain of the rat TSHR (ETSHR) as a fusion protein with beta-galactosidase in a prokaryotic system. The identity of this ETSHR-fusion protein was confirmed by Western blot, using antibodies to synthetic peptides derived from TSHR. Patients' sera reacted to a significantly greater extent with the affinity purified ETSHR relative to control sera. Similarly, sera from patients with Graves' disease displayed significant reactivity with only one of five peptides, RH2 (residues 352-366), when compared with normal sera. These data, together with the predicted hydrophilicity of the peptide RH2, suggest that amino acids 352-366 which lie within one of the unique regions of the extracellular domain of the TSHR may be important for antibody binding.

Amino Acid Sequence↗
PubMed1991

Detection of proviral sequences in saliva of patients infected with human immunodeficiency virus type 1.

Single samples of saliva collected from 20 human immunodeficiency virus type I (HIV-1) seropositive patients were tested by the polymerase chain reaction for HIV-1 proviral sequences using primers from the long terminal repeat (LTR), gag, and env regions of the virus. Proviral sequences were detected in the saliva of 50% of the patients. Sequential samples of saliva, collected at four different times, from each of six additional patients led to the detection of proviral sequences in 100% of the patients. Since, however, the detection of HIV-1 required not only the highly sensitive polymerase chain reaction, but also multiple samples, it appears that under ordinary circumstances infected cells are present in saliva in low numbers. Although this may explain the lack of transmission of HIV-1 by casual contact through the salivary route to household members and health-care workers, the presence of infected cells in the saliva of a high percentage of patients argues for avoidance of sexually intimate situations involving prolonged and repeated contact with saliva.

Adult↗
PubMed1991

The extracellular domain of the TSH receptor has an immunogenic epitope reactive with Graves' IgG but unrelated to receptor function as well as determinants having different roles for high affinity TSH binding and the activity of thyroid-stimulating autoantibodies.

The possibility that thyroid-stimulating antibodies (TSAbs) might interact with receptor determinants different from those important for high affinity TSH binding has been evaluated. Deletion mutants of the extracellular domain of the rat TSH receptor as well as point mutations of potential N-linked glycosylation sites were created. TSH binding and the ability of TSH or a TSAb to increase cAMP levels after transfection in Cos-7 cells were then measured. Mutation of two glycosylation sites (residues 77 and 198) was shown to significantly decrease high affinity TSH binding but not the activity of a TSAb. A third glycosylation site mutant (residue 302) was identified that enhanced TSAb activity but had no effect on high affinity TSH binding, and a deletion mutant (residues 308-410) lost TSAb activity but preserved TSH binding. The last two mutations are within a region having low homology with gonadotropin receptors. This same region has, in addition, a determinant that is not important for receptor activity, yet is reactive with Graves' IgG. Thus, a deletion of residues 339-367 has no effect on TSH binding or TSH/TSAb activity, yet contains a peptide (residues 352-367) reactive in ELISA assays with IgG from greater than 80% of Graves' patients but not with IgG from normal individuals, patients with nonautoimmune thyroid disease, or patients with autoimmune disease not related to the thyroid. We, therefore, identify different receptor determinants for TSAb and high affinity TSH binding, consistent with predictions from TSH receptor monoclonal antibody studies. In addition, we identify a receptor peptide that is reactive with TSH receptor antibodies in Graves' patients, despite its having no determinants important for TSH or autoantibody activity in functional assays.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of a heart antigen that cross-reacts with a neutralizing antibody to Coxsackievirus B4.

A panel of coxsackievirus B4 (CVB4) neutralizing monoclonal antibodies was tested against a panel of normal mouse tissues. One mAb, 356-1, reacted specifically with murine heart tissue. Examination of the reactivity of 356-1 with CVB4 polypeptides using Western immunoblotting revealed that 356-1 binds to the VP-1 capsid protein. Immunohistochemical studies revealed an A band pattern of staining of the heart by this antibody. Western immunoblotting of sequential differential extracts of heart showed that 356-1 predominantly reacted with the murine cardiac myosin heavy chain. A rather weak cross-reaction was found with actin. These observations were confirmed by the binding of 356-1 to purified cardiac myosin and actin. This antibody showed a higher affinity for murine cardiac muscle myosin than for skeletal muscle myosin. The monoclonal antibody 356-1 was then used to screen a lambda gt11 CD1 mouse heart cDNA expression library. Forty-eight positive plaques were obtained, 14 of which reacted strongly with the antibody and were selected for additional studies. In 13/14 clones, inserts were amplified using the polymerase chain reaction (PCR). The PCR products ranged in size from approximately 150 to 1400 bp. Northern hybridization using these inserts demonstrated that 10/13 recognized a approximately 6.5 kb message in mouse heart total RNA and not in liver total RNA. These amplified inserts were sequenced and were found to contain sequences which encode approximately 1/3 of the amino-terminal end of light meromyosin. By comparison with known sequences of rat alpha and beta cardiac myosin heavy chains, we were able to identify sequences specific for alpha chain as potential cross-reactive autoantigenic epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗