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J L Fan

Publications and source records attributed to J L Fan.

At least 19 recordsLinked to original sources
PubMed2000

Adjuvant effects of cholera toxin b subunit on immune response to recombinant thyrotropin receptor in mice.

We had previously shown that BALB/c mice immunized with the extracellular domain of human thyrotropin receptor (ETSHR) developed moderate hyperthyroxinemia. The antibody responses in these mice were predominantly of the IgG1 subclass. Since cholera toxin B subunit (CT-B) has direct effects on the thyroid, and is known to activate B lymphocytes and cause enhanced IgG1 production, we tested the ability of CT-B to modulate the antibody response to ETSHR. CT-B is unique in that it not only elicits a strong immune response to itself, but more importantly, when given with other antigens acts as a potent adjuvant. In the present study, BALB/c mice given ETSHR with CFA or CT-B via ip route showed higher titers of antibodies to ETSHR when compared to mice similarly immunized with ETSHR alone, or with IFA. Antibodies in ETSHR+CT-B immunized mice were mostly of the IgG1 subclass and reacted predominantly with ETSHR peptides 1 (aa 22-41), 21 (aa 322-341), and 23 (352-371). In contrast, animals immunized with ETSHR+CFA showed IgG1, IgG2a and IgG2b responses and reacted with peptides 1 and 21. Furthermore, mice immunized with ETSHR along with CT-B showed significantly higher levels of thyrotropin (TSH) binding inhibitory immunoglobulins (TBII) compared to those that did not receive CT-B. None of the mice immunized with a control antigen showed antibody response to ETSHR. These results suggested that CT-B could enhance and modulate immune response to ETSHR.

Adjuvants, Immunologic↗
PubMed1999

BALB/c mice produce blister-causing antibodies upon immunization with a recombinant human desmoglein 3.

Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.

Animals↗
PubMed1999

Comparison of immune responses to extracellular domains of mouse and human thyrotropin receptor.

The mouse and human thyrotropin receptors show greater than 87% homology in their amino acid sequences. However, glycosylated extracellular domains of mouse (mET-gp) and human (hET-gp) thyrotropin receptors showed differences in their ability to react with patient autoantibodies to thyrotropin receptor (TSHR). To test for potential differences in their immunogenicity, we immunized BALB/c mice with either gel pure non-glycosylated ectodomain of human TSHR (ETSHR II), or hET-gp (hET-gp III), or mET-gp (mET-gp III). Alternatively, mice were primed with gel pure hET-gp or mET-gp and subsequently immunized with insect cells expressing hET-gp (hET-gp II) or mET-gp (mET-gp II) respectively. All groups of mice immunized with TSHR developed high titers of antibodies against the respective immunogens. As shown earlier, sera obtained from mice immunized with ETSHR showed strong reactivity to peptide 1 (aa 22-41) and weak reactivity to peptides 23 (aa 352-371), 24 (aa 367-386), 25 (aa 382-401), and 26 (aa 397-415). Mice immunized with hET-gp or mET-gp showed comparable titers to peptides 1 and 23 and lower reactivity to other peptides. Mice immunized with hET-gp showed higher TBII reactivity (52.2%) compared to mice immunized with either ETSHR (20.9%) or mET-gp (34.5%). Peptides from the C-terminal region of ETSHR could neutralize the TBII activities of sera from mice immunized with ETSHR or hET-gp but not mET-gp. Compared to corresponding control mice, T4 levels in mET-gp II mice were only marginally higher. These data suggested that outcome of immunization with mouse ETSHR is comparable to that seen after immunization with human ETSHR.

Amino Acid Sequence↗
PubMed1997

Thyrotropin-receptor-mediated diseases: a paradigm for receptor autoimmunity.

Autoantibodies to the thyrotropin receptor (TSHR) can act as thyrotropin agonists or antagonists, or can cause thyroid hypertrophy. Neither the autoantibody-binding sites on the TSHR nor the intracellular mechanisms by which the autoantibodies mediate their diverse functional effects are completely understood. This article reviews how cloning of the TSHR has contributed to our understanding of its structure and function, and has allowed induction of experimental autoimmunity to the TSHR.

Allergy and Immunology↗
PubMed1996

Induction of blister-causing antibodies by a recombinant full-length, but not the extracellular, domain of the pemphigus vulgaris antigen (desmoglein 3).

Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells.

Thyroid peroxidase (TPO) is an essential enzyme for thyroid hormone biosynthesis and is an autoantigen against which antibodies are found in a number of autoimmune thyroid disorders. Large quantities of pure TPO are essential for understanding its structure and role in normal thyroid function and thyroid diseases. In this study, we describe the production of human TPO (hTPO) using a baculovirus expression vector in insect cells. TPO was sequentially extracted from insect cells using various buffers and the protein was purified to homogeneity on a C4 reversed-phase semipreparative column using high-performance liquid chromatography. The purified protein was identified as hTPO by enzyme-linked immunosorbent assay, Western blot, and amino acid sequence analyses. Carbohydrate analysis of the recombinant hTPO showed that the protein is glycosylated and mannose is the major oligosaccharide. We have extended the carbohydrate analysis by establishing the occurrence of N-acetyl galactosamine which suggested that the recombinant hTPO might contain O-glycosyl moieties. Purified hTPO reacted specifically with sera from patients with Hashimoto's thyroiditis. Crude as well as purified hTPO did not show any enzymatic activity when produced in Sf9 insect cells grown in serum free medium. In contrast, hTPO produced in the presence of 10% fetal bovine serum containing 1 microgram/ml of haematin was enzymatically active. However, the enzymatic activity of the recombinant hTPO was lower than that often found with hTPO purified from thyroid tissue. Availability of purified hTPO in relatively large quantities should allow further structural and immunological studies.

Amino Acid Sequence↗
PubMed1996

Experimental autoimmunity to thyrotropin receptor.

Since the cloning of a full length cDNA encoding the thyrotropin receptor (TSHr), several laboratories have been actively trying to develop an optimal animal model to understand the pathogenesis of TSHr mediated autoimmune diseases and have made considerable progress. To date, results from our laboratory have indicated that the nature of the antigen, and the adjuvant used for immunization, immunogenetic background of the animal and fine specificities of antibodies elicited might play an important role in determining the qualitative nature of the antibody response. Although an ideal animal model for either Graves' disease or primary myxedema is not yet available, ongoing studies in our laboratory and elsewhere hold promise for establishing animal models for various TSHr mediated autoimmune diseases in the near future.

Amino Acid Sequence↗
PubMed1996

Recombinant desmoglein 3 has the necessary epitopes to adsorb and induce blister-causing antibodies.

The development of an animal model for studying the pathogenesis of pemphigus vulgaris (PV) has been hampered by the unavailability of the purified full-length autoantigen desmoglein 3 (Dsg 3).Therefore, we expressed Dsg 3 using a baculovirus expressed system. The expressed protein was identified as Dgs 3 by its reactivity with a pan-cadherin anti-serum, an anti-serum to a Dsg 3 synthetic peptide, or patient serum, and by amino-terminal sequencing. Carbohydrate analysis showed that recombinant Dsg 3 was glycosylated. While a majority of the recombinant protein was cell associated, by immunoprecipitation, some Dsg 3 was demonstrated in the medium. The Dgs 3 could adsorb out blister-causing antibodies from patient sera. Rabbit anti- Dsg 3 antibodies induced by the recombinant Dsg 3 showed specific binding to intercellular spaces of monkeys esophagus by indirect immunofluorescence. Moreover, these antibodies induced PV-like blisters in neonatal mice and weakly bound perilesional epidermis. Availability of large quantities of relatively pure Dsg 3 should now facilitate studies aimed at understanding Dsg 3 structure and pathogenesis of PV, with implications for developing specific immunotherapies.

Animals↗
PubMed1996

Influence of adjuvants on the induction of autoantibodies to the thyrotropin receptor.

To determine the influence of adjuvant on the induction of antibodies to thyrotropin receptor (TSHR), we immunized BALB/c mice with a extracellular domain of the TSHR (ETSHR) protein in complete Freund's adjuvant (CFA), Titer Max (TM) and Gerbu. Similarly, control groups of mice were immunized with bovine serum albumin (BSA) in each of the different adjuvants. As determined by ELISA, ETSHR given along with CFA elicited high titers of antibodies to ETSHR which were mainly restricted to the IgG1 subclass. Mice immunized with ETSHR in TM also developed high titers of anti-ETSHR antibodies but had higher levels of both IgG1 and IgG2a. However, immunization with ETSHR in Gerbu resulted in low titers of antibodies, restricted to IgG1 subclass. Immunization of mice with BSA in each of the three adjuvants induced higher antibody titers to BSA. The subclass of antibodies in mice immunized with BSA in CFA and TM were predominantly IgG1 and IgG2a with lower levels of IgG2b, whereas in Gerbu treated group, antibody to BSA was restricted to IgG1 subclass. Analysis of specificity of antibodies against ETSHR, in mice immunized with ETSHR, revealed that irrespective of the adjuvant used, the dominant reactivity was against peptide 1 (AA 22-41) with weaker reactivity against several other. peptides. The only exception was in mice immunized with ETSHR in TM which also showed significant reactivity against peptide 23 (AA 352-371). Mice immunized with the ETSHR in CFA or in TM showed elevated levels of serum TSH binding inhibitory immunoglobulins (TBII). However, mice immunized with ETSHR in Gerbu, which had lower titers of antibodies to ETSHR, showed normal TBII levels. These studies showed that adjuvant composition could influence the titer, subclass and fine specificity of antibodies to ETSHR which in turn could affect the development of TBII activity.

Adjuvants, Immunologic↗
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

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↗
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

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

Abnormalities in platelets and vascular endothelial cells induced by glycated lipoproteins.

We studied the effects of glycated lipoproteins of low- and high-density (LDL and HDL) on platelets and vascular endothelial cells. After pretreatment for 5 minutes at 37 degrees C, the thrombin-induced synthesis of thromboxane B2 in washed platelets was significantly increased by glycated LDL as compared with native LDL (198.9 +/- 16.2 vs 90.3 +/- 29.4 ng/10(9) platelets, n = 8, p less than 0.01). Platelet aggregation was also increased by glycated LDL as compared with native LDL. After treatment with platelet-rich plasma for 5 hours at 37 degrees C, these values were suppressed by native HDL vs the control (buffer), but not by glycated HDL. Abnormalities in the release of 6-keto prostaglandin F1 alpha and lactate dehydrogenase from vascular endothelial cells were also induced by glycated LDL and/or HDL. These observations suggest that abnormalities induced in platelets and vascular endothelial cells by glycated lipoproteins may play an important role in the development of atherosclerosis in patients with diabetes mellitus.

6-Ketoprostaglandin F1 alpha↗
PubMed1992

[Establishment of the vascular intimal model in vitro and its application in studying atherosclerosis].

An endothelial culture model in vitro mimicking the vascular intima in vivo was designed which was composed of an upper and a lower well separated by a layer of amnion membrane, and upon which endothelial cells (ECs) isolated from the human umbilical vein were cultured. The upper well, the subendothelial amnion, and the lower well were analogical to the vascular lumen, the subendothelial tissue and the extravascular space, respectively. In comparison with the ECs cultured on the plastic, dishes, ECs cultured on the amnion membrane maintained more morphologic features as in vivo and could be cultured for up to 15 days without apparent detachment. Monocytes, loaded in the upper wells, were able to adhere to the cytoplasmic membrane of ECs. Furthermore, with the presence of the chemotactic factor (fMLP) in the lower well, monocytes showed active migrating ability passing through the EC junctions. If LDL (100 micrograms/ml) was added in the media simultaneously, monocytes might be aggregated beneath the subendothelial space and some of them took an foamy appearance. In conclusion, the culture model is of value in the study of experimental inflammation and atherosclerosis.

Amnion↗