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G C Sharp

Publications and source records attributed to G C Sharp.

At least 37 records · Page 2Linked to original sources
PubMed1998

Analysis of autoantibodies against RNA polymerases using immunoaffinity-purifed RNA polymerase I, II, and III antigen in an enzyme-linked immunosorbent assay.

Autoantibodies against RNA polymerases (RNAP) have been reported to occur in patients with a wide variety of connective tissue diseases (CTD), including systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). The frequency of anti-RNAP antibodies has been reported to vary widely between different CTD diseases in studies examining different patient populations. Furthermore, these studies have been limited by the fact that methods have not previously been available for detecting antibodies against RNAP which are both rapid and quantitative. We have developed an enzyme-linked immunosorbent assay (ELISA) for rapidly quantitating antibodies against RNAP I, II, and III. We have utilized both the ELISA and the immunoprecipitation of 35S-labeled HeLa cells to analyze sera from a large cohort of well-characterized Caucasian CTD patients for the presence of anti-RNAP antibodies. We found excellent concordance for the presence of anti-RNAP antibodies using immunoprecipitation and ELISA. Anti-RNAP antibodies occurred predominantly among female patients with the diffuse form of SSc and were detected in 8/36 (22%) of Caucasian patients with diffuse SSc and 1/53 (2%) with limited SSc. Anti-RNAP antibodies occurred in 1/42 (2%) of patients with SLE. Anti-RNAP antibodies did not occur in MCTD (0/49). Antibodies against RNAP were rare among antinucleolar-reactive sera, occurring in only 3/200 (1.5%). The RNAP ELISA provides a validated method which can be rapidly utilized in a clinical diagnostic laboratory setting to identify SSc patients who are at risk for developing diffuse SSc with multiorgan involvement and hypertensive renal crisis.

Adult↗
PubMed1998

The kinetics of cytokine gene expression in the thyroids of mice developing granulomatous experimental autoimmune thyroiditis.

To study the potential roles of cytokines in development and resolution of granulomatous experimental autoimmune thyroiditis (EAT), the kinetics of in vivo expression of cytokine genes in thyroid infiltrates was analysed using reverse transcriptase-PCR (RT-PCR). Both Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-10) cytokines as well as TGF-betaTNF-alphaIL-12 and IL-1beta were detected in thyroids during both the initial phase and peak of granulomatous EAT. Maximal expression of cytokine genes generally occurred 11-14 days after cell transfer, prior to maximal EAT severity, which occurred 19-21 days after cell transfer. The relative ratios of Th1:Th2 cytokines and mouse thyroglobulin-(MTg)-specific IgG1 and IgG2a autoantibody levels were similar during both the initial phase and peak of EAT. Depletion of CD8(+) T cells did not decrease the severity of EAT but delayed resolution of lesions. Cytokine gene expression in thyroids was not decreased by anti-CD8 treatment. Together, these data indicate that both Th1 and Th2 cytokines produced by CD4(+) T cells are involved in induction and development of granulomatous EAT, and CD8-dependent resolution of granulomatous EAT is apparently not mediated by these cytokines.

Animals↗
PubMed1998

Interleukin-12 promotes activation of effector cells that induce a severe destructive granulomatous form of murine experimental autoimmune thyroiditis.

Granulomatous inflammatory lesions are a major histopathological feature of a wide spectrum of human infectious and autoimmune diseases. Experimental autoimmune thyroiditis (EAT) with granulomatous histopathological features can be induced by mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg and anti-interleukin-2 receptor (anti-IL-2R), anti-IL-2, or anti-interferon-gamma (anti-IFN-gamma) monoclonal antibody (MAb). These studies suggested that IFN-gamma-producing T cells requiring IL-2 for growth may negatively regulate activation of granulomatous EAT effector cells. As IL-12 promotes activation of IFN-gamma-producing Th1 cells, the present study was undertaken to determine the role of IL-12 in activation of effector cells for granulomatous EAT. MTg-sensitized cells activated in vitro with MTg, anti-IL2R MAb, and IL-12 induced severe, destructive granulomatous thyroiditis with neutrophil inflammation, fibrin deposition, and necrosis. Many glands ultimately underwent atrophy and became fibrotic; some also showed fibrinoid necrosis and a mixed inflammatory cell infiltration of blood vessel walls indicative of a necrotizing vasculitis. Induction of severe granulomatous EAT by IL-12 required MTg in vitro and was unrelated to the IL-12-induced increase in IFN-gamma production. IL-12 markedly increased IFN-gamma production but did not induce a shift to a Th1-dominant phenotype, as other Th1 and Th2 cytokines were generally unaffected and both Th1 and Th2 cytokines were expressed in recipient thyroids. Addition of IL-12 or neutralization by anti-IL-12 at various times indicated that IL-12 exerted its primary effects in the final 24 hours of the 72-hour culture and was not required in recipient mice. Cells cultured with anti-IL-12, MTg, and anti-IL2R MAb transferred mild lymphocytic EAT but little or no granulomatous EAT. Thus, IL-12 profoundly regulates the in vitro activation of effector cells that induce histologically distinct autoimmune inflammatory lesions in the thyroid.

Animals↗
PubMed1997

Screening of SLE sera using purified recombinant Sm-D1 protein from a baculovirus expression system.

The Sm-D1 polypeptide is a major target of autoantibodies diagnostic for systemic lupus erythematosus (SLE). The cDNA encoding the human antigen was expressed as a full-length, nonfusion protein using a eukaryotic baculovirus expression system. This recombinant version of Sm-D1 (rSm-D1) was purified to apparent homogeneity by a combination of differential extraction steps and FPLC chromatography. A direct antibody-binding ELISA was developed using the purified antigen. There was 96% correlation between the rSm-D1 and bona fide Sm-D1 from either HeLa cells or rabbit thymus when tested against Sm-positive patient sera by ELISA. The baculovirus-expressed Sm-D1 is reactive not only with patient anti-Sm sera, but also with anti-Sm monoclonal antibodies. Our results suggest that this rSm-D1 mimics the bona fide sources, providing a valuable addition to the roster of antigens available for SLE screening, epitope mapping and overall structure study.

Antibodies, Monoclonal↗
PubMed1997

A thyroxine-containing thyroglobulin peptide induces both lymphocytic and granulomatous forms of experimental autoimmune thyroiditis.

Mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg can induce two histologically distinct forms of experimental autoimmune thyroiditis (EAT). MTg-sensitized cells activated with MTg alone induce a mild chronic form of EAT in which the thyroid infiltrate consists primarily of lymphocytes and other mononuclear cells (lymphocytic EAT). The same donor cells activated with MTg and anti-IL2R mAb induce a more severe and acute form of EAT with a thyroid inflammatory lesion having granulomatous histopathological features. A thyroxine-containing (T4) peptide, corresponding to positions 2549-2560 of human Tg, was shown by others to activate spleen cells of mouse thyroglobulin (MTg)-sensitized CBA/J mice to induce lymphocytic EAT. To determine if the CD4+ effector T cells that induce granulomatous EAT can respond to the same T-cell epitope, the present study was undertaken to determine if both forms of EAT could be induced by the 2549-2560 thyroxine (T4)-containing peptide. This peptide was very effective for activation of T cells from MTg-primed CBA/J donors to induce granulomatous EAT but, in contrast to MTg, did not activate T cells from AKR/J or DBA/1 mice to induce granulomatous EAT. The T4 peptide did not apparently activate peptide-specific B cells in vivo but did activate MTg-primed B cells in vitro to produce anti-MTg autoantibody in recipient mice. These results demonstrate that a single 12-amino-acid thyroxine-containing peptide can activate T cells from CBA/J mice to induce both lymphocytic and granulomatous EAT. However, this peptide does not activate T cells from some other EAT-susceptible strains of mice, suggesting that MTg contains multiple epitopes able to activate T cells to induce granulomatous EAT.

Animals↗
PubMed1997

Antiphospholipid antibodies among anti-U1-70 kDa autoantibody positive patients with mixed connective tissue disease.

OBJECTIVE: The association between antiphospholipid antibodies (aPL) and recurrent venous and/or arterial thrombotic events, fetal loss, and thrombocytopenia in systemic lupus erythematosus (SLE) has been well documented. Such an association has not been carefully assessed in mixed connective tissue disease (MCTD). Our aim was to assess the prevalence and clinical significance of aPL in anti-U1-70 kDa autoantibody positive patients with MCTD. METHODS: We compared 48 consecutive anti-U1-70 kDa autoantibody positive patients with MCTD versus 59 consecutive anti-U1-70 kDa autoantibody negative patients with SLE to determine the frequency of aPL and clinical features of the aPL syndrome. RESULTS: Among the patients with MCTD 7/48 (15%) had anticardiolipin antibodies (aCL) versus 24/59 (41%) patients with SLE (p < 0.005) and versus 2/150 (1%) apparently healthy blood donors (p < 0.001). Among patients with MCTD with aPL, 2 were IgG, 3 IgM, and 2 both IgG and IgM isotypes; among patients with SLE 5 were IgG, 11 IgM, and 8 both IgG and IgM isotypes. No clotting events or other features of the aPL syndrome were found among the patients with MCTD compared with 26 events documented among the group of aCL positive patients with SLE (p < 0.001). There were 10 patients with SLE with deep vein thrombosis, one with a pulmonary embolism, 2 with recurrent fetal loss, one with chorea, 2 with livedo reticularis, one with severe thrombocytopenia, and one with avascular necrosis. CONCLUSION: aCL were increased in patients with MCTD compared to controls. Furthermore, aCL were increased in SLE compared with both patients with MCTD and controls. Finally, while clotting events and other manifestations of the aPL syndrome occurred among the group of aCL positive patients with SLE these were distinctly absent from the aCL positive MCTD group.

Adolescent↗
PubMed1996

Detection of anticentromere antibodies using recombinant human CENP-A protein.

OBJECTIVE: To evaluate CENP-A reactivity with anticentromere antibodies (ACA) using recombinant protein (rCENP-A). METHODS: Human CENP-A antigen was overexpressed in insect cells using the baculovirus system. We tested for ACA activity against the full-length recombinant polypeptide by immunoblot and by enzyme-linked immunosorbent assay (ELISA). RESULTS: Of the ACA+ sera studied (n = 38), 95% were positive when tested against the rCENP-A in the ELISA system. Of the ACA- sera (n = 100), only 2% gave false-positive results in the assay. There was good correlation between the recombinant and bona fide antigens in assaying for ACA reactivity. CONCLUSION: CENP-A is a significant ACA target. The availability of the rCENP-A assay is a valuable adjunct to the previously described rCENP-B assay in analyses of the clinical significance of ACA.

Animals↗
PubMed1996

Prolactin levels and antinuclear antibody profiles in women tested for connective tissue disease.

Hyperprolactinemia has been reported in some patients with active systemic lupus erythematosus (SLE). To determine if there was an association between selected autoantibodies and hyperprolactinemia, we assayed prolactin concentrations in sera from women submitted to a reference antinuclear antibody laboratory. Autoantibody-positive samples were separated into groups that contained antibodies to double-stranded DNA (anti-DNA), antibodies to SSA/Ro (anti-SSA/Ro), or antibodies to both SSA/Ro and SSB/La (anti-SSA/Ro-SSB/La). Results were compared with autoantibody-negative sera from age-matched women, submitted to the same laboratory. We also compared the study groups with a separate cohort of 84 healthy women who were not referred for autoantibody testing. Elevated prolactin levels were clustered in 20% of sera from anti-DNA-positive women < or = 50 years of age. Twenty-one percent of anti-SSA/Ro-SSB/La-positive women < 50 years of age were hyperprolactinemic. Four of the 15 hyperprolactinemic women identified in this survey had no known cause of elevated prolactin. In the other 11 individuals secondary causes such as hypothyroidism, pregnancy, chronic renal failure, and medications may have accounted for high serum prolactin values. We also examined sera by Western blot, to determine if immunoblot patterns were associated with elevated serum prolactin concentrations. The hyperprolactinemic sera yielded novel bands migrating at 70 kd, 32 kd, and 16.5 kd. This study confirmed the reported associations of hyperprolactinemia with SLE and Sjögren's syndrome. Multiple factors appeared to contribute to elevated serum prolactin levels in women with connective tissue diseases, and the presence of hyperprolactinemia was related to unique findings on immunoblot analysis.

Adolescent↗
PubMed1995

Analysis of anti-U1 RNA antibodies in patients with connective tissue disease. Association with HLA and clinical manifestations of disease.

OBJECTIVE: To determine the prevalence of anti-U1 RNA antibodies in connective tissue disease (CTD) patients and evaluate immunogenetic and clinical features of patients possessing these antibodies. METHODS: RNA immunoprecipitation was used to analyze patient and healthy control sera for the presence of anti-R1 RNA antibodies. Enzyme-linked immunosorbent assay and immunoblotting were used to determine small nuclear RNP (snRNP) polypeptide antibodies. HLA polymorphisms were determined by microcytotoxicity and DNA typing. RESULTS: Anti-U1 RNA IgM and IgG antibodies were found in 60% of anti-RNP positive patients. All of the anti-U1 RNA positive patients had anti-70K, and most had anti-A, (U1)snRNP polypeptide antibodies. HLA-DR2/DR4, as well as Raynaud's phenomenon and synovitis, were significantly increased in the anti-U1 RNA positive group. CONCLUSION: The presence of anti-U1 RNA antibodies correlates with anti-70K and anti-A polypeptide antibodies. In addition, the anti-U1 RNA positive CTD patient group is immunogenetically and clinically distinctive from the anti-U1 RNA negative patient group.

Adolescent↗
PubMed1995

Association of systemic lupus erythematosus and gluten enteropathy.

Systemic lupus erythematosus presents various clinical manifestations. The coexistence of systemic lupus erythematosus and celiac sprue has been rarely reported. We present a patient who had systemic lupus erythematosus with malabsorption demonstrating characteristic clinical and pathologic findings of celiac sprue (gluten enteropathy).

Celiac Disease↗
PubMed1994

Differential requirement for autoantibody-producing B cells for induction of lymphocytic versus granulomatous experimental autoimmune thyroiditis.

Mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTG transfer experimental autoimmune thyroiditis (EAT) in which the thyroid cellular infiltrate consists primarily of mononuclear cells (lymphocytic EAT). Addition of anti-IL2R antibody to cultures with MTg leads to activation of cells that induce granulomatous EAT, accompanied by high serum anti-MTg autoantibody responses, in recipient mice. CD4+ T cells are required to induce both forms of EAT; whether B cells and/or autoantibodies produced by MTg-sensitized B cells also contribute to disease severity or the type of thyroid histopathology is unknown. In our study, B cells and autoantibody responses produced in recipient mice were reduced either by column removal of B cells from donor spleen cells or by treatment of recipient mice with anti-I-AK mAb at the time of cell transfer. These maneuvers only slightly reduced the severity of lymphocytic EAT but markedly reduced the severity and incidence of granulomatous EAT developing in recipient mice. Delaying the initiation of anti-I-AK treatment until 6 days after cell transfer was less effective in reducing anti-MTg autoantibody responses or granulomatous EAT. These studies all suggested that anti-MTg autoantibodies were required for development of granulomatous but not lymphocytic EAT. However anti-I-AK-treated recipients receiving injections of anti-MTg antibody or having serum antibody induced by prior immunization with MTg and LPS also developed less severe granulomatous EAT than controls. These results suggest that sensitized CD4+ T cells and circulating anti-MTg autoantibody are not sufficient for development of granulomatous thyroid lesions. It is possible that antibodies having a unique function or specificity are produced in mice developing granulomatous EAT or thyroid-infiltrating B cells may directly contribute to the granulomatous inflammatory response.

Animals↗
PubMed1994

Regulation of the induction and resolution of granulomatous experimental autoimmune thyroiditis in mice by CD8+ T cells.

Mouse thyroglobulin (MTg)--sensitized spleen cells activated in vitro with MTg induce experimental autoimmune thyroiditis (EAT) in which the thyroid cellular infiltrate consists primarily of mononuclear cells (lymphocytic EAT). Cells cultured with MTg plus anti-IL2R antibody induce EAT having a granulomatous histopathology. CD4+ T cells are required to induce both forms of EAT. The potential contribution of CD8+ T cells in the pathology of lymphocytic vs granulomatous EAT was addressed using anti-CD8 mAb to deplete CD8+ T cells from donor or recipient mice. Depletion of donor CD8+ T cells had little effect on EAT severity induced by cells cultured with MTg or MTg plus anti-IL2R mAb. However, recipients of CD8-depleted cells activated with MTg induced granulomatous EAT whereas cells from untreated donors activated with MTg alone induced lymphocytic EAT. Similar results were obtained when anti-CD8 mAb was added to cultures to block activation of CD8+ T cells. Injection of anti-CD8 mAb into recipient mice slightly increased the severity of EAT induced by cells cultured with MTg or MTg and anti-IL2R mAb when EAT was assessed 19-20 days after cell transfer. Cells cultured with MTg alone induced mild granulomatous EAT in most anti-CD8-treated recipients. When thyroids of mice which had granulomatous EAT on Day 19 were examined 47 or 60 days after cell transfer, the granulomatous inflammatory response in untreated recipients had almost completely resolved. In contrast, granulomatous EAT persisted and often became more severe in anti-CD8-treated recipients. Anti-CD8 treatment did not influence lymphocytic EAT assessed late after cell transfer. These results indicate that CD8+ T cells are required for the resolution of granulomatous EAT.

Animals↗
PubMed1994

Increased frequency of mutations in the hprt gene of T cells isolated from patients with anti-U1-70kD-autoantibody-positive connective tissue disease.

Mixed connective tissue disease (MCTD) is characterized by the presence of high titers of anti-U1-70kD autoantibodies which are the result of substantial B cell activation. The hprt gene encodes the constitutively expressed enzyme hypoxanthine-guanine phosphoribosyl transferase which is active in the purine salvage pathway. Rapidly dividing cells randomly accumulate gene mutations, including mutations in the hprt gene. These mutations may be used to identify activated cells. If activated T cells play a role in the pathogenesis of MCTD, an increased frequency of mutations in the hprt gene might be expected among T cells isolated from such patients. To examine this hypothesis, we isolated and cloned T cells from 10 anti-U1-70kD-autoantibody-positive MCTD patients and determined the precursor frequencies of cells possessing mutations in hprt by comparing the frequency of cells grown in the presence and absence of the purine analogue 6-thioguanine. We found that the frequency of 6-thioguanine-resistant hprt-negative T cells was significantly increased among MCTD patients (mean 566/10(6); range 122-2,845/10(6)) versus age- and sex-matched controls (mean 42/10(6); range 21-78/10(6); p < 0.003). These results demonstrate that there is an increase in the measured mutant frequency of T cells from MCTD patients. Such T cells may play a role in the pathogenesis of this disease.

Adult↗
PubMed1994

Intrathyroidal cell phenotype in murine lymphocytic and granulomatous experimental autoimmune thyroiditis.

In-vitro mouse thyroglobulin (MTg) activated spleen cells from immunized donor mice can induce experimental autoimmune thyroiditis (EAT) after transfer to recipient mice. The intrathyroidal cellular infiltrate consists primarily of mononuclear cells (lymphocytic EAT). Cells cultured with MTg together with anti-IL2R antibody induce EAT with a granulomatous histopathology in which the thyroid infiltrate contains mononuclear cells (MNC) in addition to PMN's histiocytes, and multinucleated giant cells. Flow cytometric analysis of intrathyroidal MNC infiltrates demonstrated that both CD4+ and CD8+ T cells infiltrate the thyroid in both lymphocytic and granulomatous EAT and that CD8+ T cells outnumber CD4+ T cells. There were usually increased numbers of PMN's in the granulomatous thyroids, but low number of Ig+ and F4/80+ cells (macrophages) in the intrathyroidal infiltrate of both disease types. IL2R and Pgp-1 were expressed on both CD4+ and CD8+ intrathyroidal T cells. The majority of CD8+ cells were ICAM+, LFA-1+, and CD45RB+ whereas only a small percentage of CD4+ intrathyroidal T cells expressed these markers. There were no major differences in intrathyroidal MNC phenotype between lymphocytic and granulomatous EAT. Depletion of CD8+ T cells in recipient mice did not reduce EAT severity and resulted in an increased percentage of intrathyroidal CD4+ T cells expressing IL2R. These results suggest that CD8+ T cells are not functioning as effector cells in lymphocytic or granulomatous EAT.

Animals↗
PubMed1993

Human T cell clones reactive against U-small nuclear ribonucleoprotein autoantigens from connective tissue disease patients and healthy individuals.

SLE and mixed connective tissue disease (MCTD) are characterized by the presence of high titers of autoantibodies against uridine-rich RNA-small nuclear ribonucleoprotein (snRNP) Ag. Because the presence of such snRNP-reactive autoantibodies has recently been shown to be associated with polymorphisms of HLA, this study was undertaken to determine whether snRNP-reactive T cells could be identified and characterized from patients. PBMC were stimulated with affinity-purified snRNP Ag and cloned by limiting dilution in the presence of rIL-2 and rIL-4, snRNP-reactive human T cell clones were generated from three patients and two healthy blood donors who possessed disease-associated HLA genotypes. The cell surface phenotype of clones determined by flow cytometry was CD3+, CD4+, CD45RO+, TCR V alpha beta+. TCR V beta analysis, performed using V beta-specific primers and polymerase chain reaction, revealed that the T cell lines generated were clonal; a limited number of TCR V beta genes were expressed among the clones tested. All clones tested by mAb blocking of Ag-induced proliferation were restricted by HLA-DR. Several T cell clones were identified that were specific for B'/B or D polypeptides. These results demonstrate that snRNP-reactive T cells can be isolated from SLE and MCTD patients in vitro, and that Ag-driven expansion of such T cells could play a role in the immunopathogenesis of these diseases in vivo.

Adult↗
PubMed1993

AI/LEARN/Rheumatology. A comparative study of computer-assisted instruction for rheumatology.

OBJECTIVE: To assess the effectiveness of AI/LEARN/Rheumatology, a computer-controlled interactive videodisc system for teaching. METHODS: We assessed improvement in knowledge about rheumatic diseases, using a pretest and posttest in a control year and a treatment year. The subjects were medical students and postgraduate trainees taking the rheumatology elective. The control year used traditional lectures and the standard rheumatology curriculum. The treatment year used AI/LEARN/Rheumatology in place of lectures on rheumatoid arthritis, ankylosing spondylitis, and osteoarthritis. RESULTS: The trainees showed significant improvement in knowledge in both the control year and the treatment year (P < 0.0001 for both). The average time spent using AI/LEARN/Rheumatology was similar to the time spent in lectures (3 hours). The number of patient consultations in which trainees participated was lower in the treatment year than in the control year; however, the adjusted posttest scores using the pretest as a covariate tended to be higher in the treatment year (P = 0.10). Analysis of covariance of the adjusted posttest scores for the treatment year only showed that the trainees who spent more time using AI/LEARN/Rheumatology learned more (r = 0.57). Trainees felt that AI/LEARN/Rheumatology was the most helpful educational experience of the elective. CONCLUSION: AI/LEARN/Rheumatology is an effective means of teaching about the rheumatic diseases. It has many advantages: availability for independent study, effective use of trainee's time, and liberation of faculty time from lectures. Trainees enjoyed using AI/LEARN/Rheumatology.

Computer-Assisted Instruction↗
PubMed1993

U1-70-kd autoantibody-positive mixed connective tissue disease in children. A longitudinal clinical and serologic analysis.

OBJECTIVE: To obtain longitudinal data on the clinical, serologic, and immunogenetic features of children with mixed connective tissue disease (MCTD). METHODS: Eleven children with MCTD were followed up for a mean of 9.8 years. Enzyme-linked immunosorbent assay and immunoblotting were used to analyze sera for autoantibodies to small nuclear ribonucleoprotein polypeptides. HLA types were determined in 9 patients, by microcytotoxicity and DNA typing. RESULTS: All 11 children had anti-U1-70-kd auto-antibodies. Six of 9 were positive for HLA-DR2, 4 of 9 for HLA-DR4, and 9 of 9 for either HLA-DR2 or DR4. Outcomes were favorable with no functional impairment in 8 of the 11 children and were poor in 3. CONCLUSION: The frequency of HLA-DR2/DR4 is increased among children with anti-U1-70-kd autoantibody positive MCTD.

Autoantibodies↗