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Chondrocyte apoptosis induced in vitro is caspase-3-mediated, but is not demonstrated in cartilage from patients with rheumatoid arthritis or osteoarthritis
J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2
1009 s,,,.o,. s.o,,,o,gE Against Carbonic Anhydrase in a Family with Tubular Renal Acidosis
J. C. Muifio z, L. I. Juncos 2, N. Garcfa 2, M. D. Romero-Piffeguer3, M. Ferrero 3, D. Beltramo4; JAllergy & Immunology Secci6n, Internal Medicine Service, Hospital Misericordia, C6rdoba, ARGENTINA, 2Clinical Departament of IPEM, IPEM, C6rdoba, ARGENTINA, 3Allergy & Immunology Secci6n, Internal Medicine Service, Hospital Misericordia, C6rdoba, ARGENTINA, 4Immunology Departament Ceprocor, Ceprocor, C6rdoba, ARGENTINA. RATIONALE: We previously reported a woman with renal tubular acidosis associated with high serum IgE level and specific IgE against a 31 kd kidney tubular protein (carbonic anhydrase II) demonstrated by both immunohistochemical and immunoblot procedures. This woman has one brother with similar kidney disease and high serum IgE level. The purpose of this study was to investigate if these patient relatives presented lgE with binding affinities to carbonic anhydrase II. METHODS: We obtain sera from this woman parents, brothers, sons, cousins and her husband (n: 10; 5 male and 5 female). We measured total IgE serum level by ELISA, and specific IgE to carbonic anhydrase by both ELISA and immunoblot assays in all the samples. RESULTS: Our patient's husband had an IgE serum level of 75 kU/L, while her relatives ranged from 580 to 3250 kU/L. The specific IgE to carbonic anhydrase by both ELISA and Immunoblot was positive in all her relatives CONCLUSIONS: The results presented in this study suggest the significance of specific IgE to carbonic anhydrase II in this family group.
Funding: Self-funded
1010
Inhibition of Tonsil-Derived (T-D) B-Cell Apoptosis Permits the Expression of Disease-Specific (D-S), IgM Rheumatoid Factor (RF) Expression
D. MilojeviO, V. R. Bonagura2; IDivision of Allergy and Immunology, Dept. of Pediatrics, Schneider Children's Hospital, LIJ Medical Center, New Hyde Park, NY, 2Division of Allergy and Immunology, Dept. of Pediatrics, Schneider Children's Hospital, LIJ Medical Center, New Hyde Park, NJ. RATIONALE: D-S IgM RFs that bind IgG3 are commonly expressed by RA-patients, but rarely by individuals without RA. To determine if defective peripheral regulation of D-S RF expression may be present in RA, we inhibited T-D B-cell apoptosis in an individual without RA and then screened for D-S RF expression by ELISA. METHODS: T-D B-cells were fused with NSO bcl-2 mouse myeloma cells, containing the anti-apoptotic bcl-2 gene, or NSO wild-type cells (NSO wt) without bcl-2, or were transformed with Epstein Barr Virus (EBV) containing "bcl-2 like" genes. RESULTS: 0.83% of NSObCl-2-B-cell hybrids produced RFs. D-S, lgG3 binding RFs were present in 85% of the hybrids. 35% exclusively bound IgG3, 20% bound IgGl, and 3 only, 20% were pan-binding (IgGl->4), 5% bound IgG3 and 4 only, and 5% bound IgG2, 3, and 4 only. 0.76% of the EBV transformed B-cells produced RFs. D-S, lgG3 binding was present in 64%. Of these. 7.3% bound IgG3 only, 14.7% bound IgGl->4, 19.1% bound IgGl, 3, and 4 only, 20.5% bound only IgGl and 3, 1.4% bound IgG 1, 2, and 3 only, and 1.4% bound IgG3 and 4 only. None of the NSO wt T-D B-cell hybrids expressed RFs. CONCLUSIONS: Inhibition of T-D B-cell apoptosis permits D-S RF expression. The RF repertoires produced by T-D B-celI-NSO bcl-2 hybrids and EBV transformed lymphoblasts differ dramatifcally from that expressed by T-D B-celI-NSO wt hybrids. Thus, defective peripheral regulation of D-S lgM RF production by RA patients may permit D-S RF expression, and implicate RF immune dysregulation in RA pathogenesis.
Funding: University Monies
Abstracts
1011
$321
Chondrocyte Apoptosis Induced in Vitro is Caspase-3-Mediated, but is not Demonstrated in Cartilage from Patients with Rheumatoid Arthritis or Osteoarthritis
J. F. Van OffelL I. C. Koster ], G. J. Van Elsen I, A. J. Schuerwegh j, C. H. Bridts 1, M. M. Kockx 2, W. J. Stevens 1, L. S. De Clerckl; qmmunologyAllergology-Rheumatology, University of Antwerp, Antwerpen, BELGIUM, 2pathology, AZ Middelheim, Division of Pharmacology, University of Antwerp, Antwerpen, BELGIUM. RATIONALE: Chondrocyte apoptosis might be one of the mechanisms of cartilage destruction in rheumatoid arthritis (RA) and osteoarthritis (OA). First, an in vitro model of apoptosis in bovine chondrocytes was evaluated. Subsequently, we investigated whether chondrocyte apoptosis is present in cartilage of patients with RA or OA in a caspase-3-dependent way. METHODS: Bovine articular chondrocytes were cultured in vitro in the presence of IL-l-alfa, TNF-alfa and interferon-gamma. Apoptosis was induced by adding synovial fluid immune complexes of RA or OA patients. Caspase-3 activity was measured immunohistochemically. Cartilage of 11 RA and 44 OA patients were studied for the presence of chondrocyte apoptosis and compared with 6 normal human cartilages obtained after trauma. Apoptosis was evaluated microscopically by HE, Sirius Red and PAS Alcian blue staining and by DNA in situ end labeling (TUNEL). RESULTS: In vitro, bovine chondrocyte apoptosis was induced by 100% of the RA and 60% of the OA synovial fluids, the majority being caspase3-positive. There was a good correlation between caspase- and TUNELpositivity (r=0.82 for RA and 0.73 for OA). In contrast, in the human articular cartilage, no apoptotic chondrocytes could be demonstrated by any technique in the patients with RA, OA, nor in the control group. CONCLUSIONS: Apoptosis, induced in bovine chondrocytes in vitro, is caspase-3-mediated. However. we failed to demonstrate apoptosis in human RA or OA cartilage.
Funding: FWO
1012
Expression of Soluble High-Affinity ,gE Receptors and Its Interferences with Type 1 HypersensitivityReactions
S. Renshan; Department of Dermatology, Institute of Dermatovenerology in Gaotanyan, Chongqing, CHINA. RATIONALE: The ectodomain of the human alpha subunit (sFcERl~) has the potency to block the binding of IgE to Fc r~R1 on mast cells and thus to suppress the lgE-dependent degranulations of both mast cells and basophils. To obtain sufficient amounts of sFc~Rlct, we tried a baculovirus expression system for expression of sFcER 1c~. METHODS: Insect cells (Sf21) and baculovirus system were utilized to express the exodomain of the human sFcER I e~ chain (ctt). The recombinant baculobirus containing~t eDNA purified by visual plaque screening. The soluble proteins were purified by chromatography on Sepharose 4B, and its biological effects were judged by passive cutaneous anaphylaxis test in mice. RESULTS: The resultant transfer plasmid was sequenced and shown to have the correct open reading frames. It was found sFc~:Rl~2 was in the second UV-absorbed peak represented as one 60 kD band on SDS-PAGE analyse which can bind human IgE proved by western blot. Furthermore, injection of the protein in the second peak (20 I.tg per mouse) intraviously could restrict volumes of anaphylactic wheals by 19% compared to those on mice administrated with NaCI solution (n=10 animals in each group) (p<0.05), but can not inhibit the formation of wheals. CONCLUSIONS: Sf21 infected by the recombinant virus from our study can generated soluble Fc~Rla protiens which have the abilities to inhibit type one hypersensitivity reactions.
Funding: Chongqing government
1009 s,,,.o,. s.o,,,o,gE Against Carbonic Anhydrase in a Family with Tubular Renal Acidosis
J. C. Muifio z, L. I. Juncos 2, N. Garcfa 2, M. D. Romero-Piffeguer3, M. Ferrero 3, D. Beltramo4; JAllergy & Immunology Secci6n, Internal Medicine Service, Hospital Misericordia, C6rdoba, ARGENTINA, 2Clinical Departament of IPEM, IPEM, C6rdoba, ARGENTINA, 3Allergy & Immunology Secci6n, Internal Medicine Service, Hospital Misericordia, C6rdoba, ARGENTINA, 4Immunology Departament Ceprocor, Ceprocor, C6rdoba, ARGENTINA. RATIONALE: We previously reported a woman with renal tubular acidosis associated with high serum IgE level and specific IgE against a 31 kd kidney tubular protein (carbonic anhydrase II) demonstrated by both immunohistochemical and immunoblot procedures. This woman has one brother with similar kidney disease and high serum IgE level. The purpose of this study was to investigate if these patient relatives presented lgE with binding affinities to carbonic anhydrase II. METHODS: We obtain sera from this woman parents, brothers, sons, cousins and her husband (n: 10; 5 male and 5 female). We measured total IgE serum level by ELISA, and specific IgE to carbonic anhydrase by both ELISA and immunoblot assays in all the samples. RESULTS: Our patient's husband had an IgE serum level of 75 kU/L, while her relatives ranged from 580 to 3250 kU/L. The specific IgE to carbonic anhydrase by both ELISA and Immunoblot was positive in all her relatives CONCLUSIONS: The results presented in this study suggest the significance of specific IgE to carbonic anhydrase II in this family group.
Funding: Self-funded
1010
Inhibition of Tonsil-Derived (T-D) B-Cell Apoptosis Permits the Expression of Disease-Specific (D-S), IgM Rheumatoid Factor (RF) Expression
D. MilojeviO, V. R. Bonagura2; IDivision of Allergy and Immunology, Dept. of Pediatrics, Schneider Children's Hospital, LIJ Medical Center, New Hyde Park, NY, 2Division of Allergy and Immunology, Dept. of Pediatrics, Schneider Children's Hospital, LIJ Medical Center, New Hyde Park, NJ. RATIONALE: D-S IgM RFs that bind IgG3 are commonly expressed by RA-patients, but rarely by individuals without RA. To determine if defective peripheral regulation of D-S RF expression may be present in RA, we inhibited T-D B-cell apoptosis in an individual without RA and then screened for D-S RF expression by ELISA. METHODS: T-D B-cells were fused with NSO bcl-2 mouse myeloma cells, containing the anti-apoptotic bcl-2 gene, or NSO wild-type cells (NSO wt) without bcl-2, or were transformed with Epstein Barr Virus (EBV) containing "bcl-2 like" genes. RESULTS: 0.83% of NSObCl-2-B-cell hybrids produced RFs. D-S, lgG3 binding RFs were present in 85% of the hybrids. 35% exclusively bound IgG3, 20% bound IgGl, and 3 only, 20% were pan-binding (IgGl->4), 5% bound IgG3 and 4 only, and 5% bound IgG2, 3, and 4 only. 0.76% of the EBV transformed B-cells produced RFs. D-S, lgG3 binding was present in 64%. Of these. 7.3% bound IgG3 only, 14.7% bound IgGl->4, 19.1% bound IgGl, 3, and 4 only, 20.5% bound only IgGl and 3, 1.4% bound IgG 1, 2, and 3 only, and 1.4% bound IgG3 and 4 only. None of the NSO wt T-D B-cell hybrids expressed RFs. CONCLUSIONS: Inhibition of T-D B-cell apoptosis permits D-S RF expression. The RF repertoires produced by T-D B-celI-NSO bcl-2 hybrids and EBV transformed lymphoblasts differ dramatifcally from that expressed by T-D B-celI-NSO wt hybrids. Thus, defective peripheral regulation of D-S lgM RF production by RA patients may permit D-S RF expression, and implicate RF immune dysregulation in RA pathogenesis.
Funding: University Monies
Abstracts
1011
$321
Chondrocyte Apoptosis Induced in Vitro is Caspase-3-Mediated, but is not Demonstrated in Cartilage from Patients with Rheumatoid Arthritis or Osteoarthritis
J. F. Van OffelL I. C. Koster ], G. J. Van Elsen I, A. J. Schuerwegh j, C. H. Bridts 1, M. M. Kockx 2, W. J. Stevens 1, L. S. De Clerckl; qmmunologyAllergology-Rheumatology, University of Antwerp, Antwerpen, BELGIUM, 2pathology, AZ Middelheim, Division of Pharmacology, University of Antwerp, Antwerpen, BELGIUM. RATIONALE: Chondrocyte apoptosis might be one of the mechanisms of cartilage destruction in rheumatoid arthritis (RA) and osteoarthritis (OA). First, an in vitro model of apoptosis in bovine chondrocytes was evaluated. Subsequently, we investigated whether chondrocyte apoptosis is present in cartilage of patients with RA or OA in a caspase-3-dependent way. METHODS: Bovine articular chondrocytes were cultured in vitro in the presence of IL-l-alfa, TNF-alfa and interferon-gamma. Apoptosis was induced by adding synovial fluid immune complexes of RA or OA patients. Caspase-3 activity was measured immunohistochemically. Cartilage of 11 RA and 44 OA patients were studied for the presence of chondrocyte apoptosis and compared with 6 normal human cartilages obtained after trauma. Apoptosis was evaluated microscopically by HE, Sirius Red and PAS Alcian blue staining and by DNA in situ end labeling (TUNEL). RESULTS: In vitro, bovine chondrocyte apoptosis was induced by 100% of the RA and 60% of the OA synovial fluids, the majority being caspase3-positive. There was a good correlation between caspase- and TUNELpositivity (r=0.82 for RA and 0.73 for OA). In contrast, in the human articular cartilage, no apoptotic chondrocytes could be demonstrated by any technique in the patients with RA, OA, nor in the control group. CONCLUSIONS: Apoptosis, induced in bovine chondrocytes in vitro, is caspase-3-mediated. However. we failed to demonstrate apoptosis in human RA or OA cartilage.
Funding: FWO
1012
Expression of Soluble High-Affinity ,gE Receptors and Its Interferences with Type 1 HypersensitivityReactions
S. Renshan; Department of Dermatology, Institute of Dermatovenerology in Gaotanyan, Chongqing, CHINA. RATIONALE: The ectodomain of the human alpha subunit (sFcERl~) has the potency to block the binding of IgE to Fc r~R1 on mast cells and thus to suppress the lgE-dependent degranulations of both mast cells and basophils. To obtain sufficient amounts of sFc~Rlct, we tried a baculovirus expression system for expression of sFcER 1c~. METHODS: Insect cells (Sf21) and baculovirus system were utilized to express the exodomain of the human sFcER I e~ chain (ctt). The recombinant baculobirus containing~t eDNA purified by visual plaque screening. The soluble proteins were purified by chromatography on Sepharose 4B, and its biological effects were judged by passive cutaneous anaphylaxis test in mice. RESULTS: The resultant transfer plasmid was sequenced and shown to have the correct open reading frames. It was found sFc~:Rl~2 was in the second UV-absorbed peak represented as one 60 kD band on SDS-PAGE analyse which can bind human IgE proved by western blot. Furthermore, injection of the protein in the second peak (20 I.tg per mouse) intraviously could restrict volumes of anaphylactic wheals by 19% compared to those on mice administrated with NaCI solution (n=10 animals in each group) (p<0.05), but can not inhibit the formation of wheals. CONCLUSIONS: Sf21 infected by the recombinant virus from our study can generated soluble Fc~Rla protiens which have the abilities to inhibit type one hypersensitivity reactions.
Funding: Chongqing government