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Homozygous C4A gene deletion and absence of spinal fluid oligoclonal bands in a multiple sclerosis patient
C4 AND BF POLYMORPHISMS IN AN ALBANIAN POPULATION OF CALABRIA (ITALY) M. Cuccia I , C. Cereda 1, M. Fazzari 2, G. Ruberto 3, V. Misefari 4, A. Veratti 2. 1 Dip. Genetica e Microbiologia e 3 Clinica Medica Univ. di Pavia, 2 Div. Nefrologia, Ospedale Reggio Calabria, 4 Centro Trasfusionale Ospedale Pugliese - Cosenza (Italy) Populations genetics in an essential approach for investigation of ethnic relationships and extremely important for disease association studies. In order to provide data on populations up to now not deeply analyzed we present BF, C4A and C4B allele frequencies of an Albanian population (N=92) of Calabria region (the village is named Caraffa); these Albanians escaped from Turkish invasion of South Europa on XV century. C4A and C4B alleles were analyzed at proteinic level by agarose gel immunoelectrophoresis and with western blot for part of the samples. At DNA level the short or long form of C4B gene was defined by Southern blot technique with specific probes. BFF was split by isoelectrofocusing methods into FB and FA subtypes. The data obtained are compared and appear to differ from those obtained in individuals of South Calabria (Stretto di Messina) and of other Mediterranean population. BF, C4A, C4B frequencies are presented: Albanians of C.% Calabria % Lombardy % BFS 79,59 72,00 77,09 BFF 19,39 23,70 20,16 BF1 2,30 1,18 BF SO7 1,02 2,30 1,57 C4A 1 C4A2 C4A3 C4A4 C4A5 C4A6 C4A0 C4B 1 C4B2 C4B3 C4B4 C4B 5 C4B0
10,00 80,00 4,29 1,43 4,29 82,86 7,14 1,43 8,57
0,46 7,80 82,60 1,37 0,00 1,83 5,96
8,11 76,35 5 0,68 1,89 7,84
85,78 4,13 0,46 0,46 0,46 8,72
82,57 6,35 0,41 0,14 0,14 9,73
HOMOZYGOUS C4A GENE DELETION AND ABSENCE OF SPINAL FLUID OLIGOCLONAL BANDS I N A M U L T I P L E S C L E R O S I S P A T I E N T . M. Cuccia 1, E Dondi I , D. Franciotta 2, G. Piccolo, 2 K. Bergamaschi 2, G.V. Melzi D'Eri|2, V. Cost 2. l Dip. Genetica e Microbiologia, 2 Clinica Neurologica Istituto "C. Mondino" Universit~ di Pavia,(Italy) The detection of Ig oligoclonal bands (OCB) in the cerebrospinal fluid is a sensitive tool for multiple sclerosis(MS) diagnosis. Using highly sensitive tecnique OCB can be detected in up to 100% of MS patients. BF, C4A and C4B complement genes polymorphisms were investigated in 80 Italian patients with MS in order to describe immunogenetic disease heterogeneity. We observed an increase of C4AQ0 allele frequency, due to structural gene deletion or absence of protein only, particularly in the group of patients with relapsing-remitting form of disease. We here report the co-occurrence of the serum complement C4A protein absence and the persistant absence of OCB in one MS patient out of 80 studied. The C4A polymorphism was analyzed with immunoelectrophoretic methods and then the DNA sample was analyzed in Southern blotting with C4 specific probes. The C4A protein deficiency can alter the development of both primary and secondary humoral immune response, thus limiting OCB production. Our observation support the biological role of the C4A protein in antibody production(l ). ( 1) Finco et al.: Journal of Experimental Medicine, 175: 537-543, 1992
C Y T O K I N E S AND DIBUTRYL CYCLIC AMP (dbcAMP) INDUCE C3 PRODUCTION IN RAT SCHWANN CELLS (SchC). S. Dashiell, P. Vanguri, and C.L. Koski. University of Maryland,Baltimore, MD 21201, USA Astrocytes constitutively express C3 which can be modulated by cytokines. We investigated whether SchC could also express C3 using Northern analysis and a sandwich ELISA. Differentiated SchC purified from 5-6 day-old rat sciatic nerve were expanded in vitro in 10% fetal bovine serum (FBS) and 4~M forskolin, then treated with or without 10% FBS for 5 days (SchC-C) prior to stimulation with dbcAMP, "yinterferon (IFN), and/or TNFo~ for 0-120 hrs. Primary SchC of 5-6 day-old rats and unstimulated SchC-C do not constitutively express C3 nor secrete C3 protein. Low levels of C3 mRNA and protein were induced after 72 hrs by 0.1 mM and 1.0mM dbcAMP + FBS, in a dose-dependent manner. TNFa alone did not induce C3 but IFN with 50-450ng TNFo~ showed a dose-dependent enhancement of C3 mRNA and protein induction that was detectable by 48 hrs. C3 protein secretion was increased 10-fold at 48 hrs and 40-fold by 72 hrs by 450 ng TNF~+ IFN. These data suggest that enhanced C3 production in SchC by inflammatory cytokines requires elevated levels of cAMP, a second messenger system involved in SchC differentiation. Supported by NIH# P50 NS20022.
DIFFERENTIAL BINDING OF TERMINAL COMPLEMENT COMPONENTS TO A NUCLEATED CELL AFTER CLASSICAL ALTERNATIVE PATHWAY ACTIVATION.
AND
Kerstin David, Markus W. Ollert, Reinhard Bredehorst, and Cart-Wilhelm Vogel. Department of Biochemistry and Molecular Biology, University of Hamburg, Germany. We compared the complement-mediated killing of human neuroblastoma cells using the complement-activating monoclonal antibody BW 704 and its F(ab')2 fragment conjugated to the complement-activating protein cobra venom factor (CVF). The BW 704 antibody belongs to the murine IgG3 subclass and is directed against the GD2 ganglioside (Bosslet et aL, Cancer Immunol. Immunother. (1989) 29, 171-178). The BW 704 F(ab')2 fragment was prepared by pepsin digestion and subsequently linked to purified CVF using the heterobifunctional crosslinking agent SPDP. Complement cytctoxicity using the intact BW 704 antibody reached 98%. The kinetics of binding of complement components C4, C3, C5, and C9 were determined using flow cytometry. Maximum binding of all four components was reached within 5 to 10 min of incubation and decreased during further incubation. Using the BW 704 F(ab')2-CVF conjugate, maximum killing reached 70%. Striking differences were observed in the binding of complement components. Maximum binding of C3, C5, and C9 occurred after approximately 90 rain of incubation. The total amounts of C3 and C5 were significantly below those observed with classical pathway activation by intact antibody, with the amounts of C5 at the limit of detectability. In contrast, the total amount of C9 bound to the cells after alternative pathway activation using the BW 704 F(ab')2-CVF conjugate was similar to that observed with intact antibody. These results indicate significant differences in the mechanism ot complement killing and/or cellular repair mechanisms between cytotoxic monoclonal antibodies and antibody-CVF conjugates.
10,00 80,00 4,29 1,43 4,29 82,86 7,14 1,43 8,57
0,46 7,80 82,60 1,37 0,00 1,83 5,96
8,11 76,35 5 0,68 1,89 7,84
85,78 4,13 0,46 0,46 0,46 8,72
82,57 6,35 0,41 0,14 0,14 9,73
HOMOZYGOUS C4A GENE DELETION AND ABSENCE OF SPINAL FLUID OLIGOCLONAL BANDS I N A M U L T I P L E S C L E R O S I S P A T I E N T . M. Cuccia 1, E Dondi I , D. Franciotta 2, G. Piccolo, 2 K. Bergamaschi 2, G.V. Melzi D'Eri|2, V. Cost 2. l Dip. Genetica e Microbiologia, 2 Clinica Neurologica Istituto "C. Mondino" Universit~ di Pavia,(Italy) The detection of Ig oligoclonal bands (OCB) in the cerebrospinal fluid is a sensitive tool for multiple sclerosis(MS) diagnosis. Using highly sensitive tecnique OCB can be detected in up to 100% of MS patients. BF, C4A and C4B complement genes polymorphisms were investigated in 80 Italian patients with MS in order to describe immunogenetic disease heterogeneity. We observed an increase of C4AQ0 allele frequency, due to structural gene deletion or absence of protein only, particularly in the group of patients with relapsing-remitting form of disease. We here report the co-occurrence of the serum complement C4A protein absence and the persistant absence of OCB in one MS patient out of 80 studied. The C4A polymorphism was analyzed with immunoelectrophoretic methods and then the DNA sample was analyzed in Southern blotting with C4 specific probes. The C4A protein deficiency can alter the development of both primary and secondary humoral immune response, thus limiting OCB production. Our observation support the biological role of the C4A protein in antibody production(l ). ( 1) Finco et al.: Journal of Experimental Medicine, 175: 537-543, 1992
C Y T O K I N E S AND DIBUTRYL CYCLIC AMP (dbcAMP) INDUCE C3 PRODUCTION IN RAT SCHWANN CELLS (SchC). S. Dashiell, P. Vanguri, and C.L. Koski. University of Maryland,Baltimore, MD 21201, USA Astrocytes constitutively express C3 which can be modulated by cytokines. We investigated whether SchC could also express C3 using Northern analysis and a sandwich ELISA. Differentiated SchC purified from 5-6 day-old rat sciatic nerve were expanded in vitro in 10% fetal bovine serum (FBS) and 4~M forskolin, then treated with or without 10% FBS for 5 days (SchC-C) prior to stimulation with dbcAMP, "yinterferon (IFN), and/or TNFo~ for 0-120 hrs. Primary SchC of 5-6 day-old rats and unstimulated SchC-C do not constitutively express C3 nor secrete C3 protein. Low levels of C3 mRNA and protein were induced after 72 hrs by 0.1 mM and 1.0mM dbcAMP + FBS, in a dose-dependent manner. TNFa alone did not induce C3 but IFN with 50-450ng TNFo~ showed a dose-dependent enhancement of C3 mRNA and protein induction that was detectable by 48 hrs. C3 protein secretion was increased 10-fold at 48 hrs and 40-fold by 72 hrs by 450 ng TNF~+ IFN. These data suggest that enhanced C3 production in SchC by inflammatory cytokines requires elevated levels of cAMP, a second messenger system involved in SchC differentiation. Supported by NIH# P50 NS20022.
DIFFERENTIAL BINDING OF TERMINAL COMPLEMENT COMPONENTS TO A NUCLEATED CELL AFTER CLASSICAL ALTERNATIVE PATHWAY ACTIVATION.
AND
Kerstin David, Markus W. Ollert, Reinhard Bredehorst, and Cart-Wilhelm Vogel. Department of Biochemistry and Molecular Biology, University of Hamburg, Germany. We compared the complement-mediated killing of human neuroblastoma cells using the complement-activating monoclonal antibody BW 704 and its F(ab')2 fragment conjugated to the complement-activating protein cobra venom factor (CVF). The BW 704 antibody belongs to the murine IgG3 subclass and is directed against the GD2 ganglioside (Bosslet et aL, Cancer Immunol. Immunother. (1989) 29, 171-178). The BW 704 F(ab')2 fragment was prepared by pepsin digestion and subsequently linked to purified CVF using the heterobifunctional crosslinking agent SPDP. Complement cytctoxicity using the intact BW 704 antibody reached 98%. The kinetics of binding of complement components C4, C3, C5, and C9 were determined using flow cytometry. Maximum binding of all four components was reached within 5 to 10 min of incubation and decreased during further incubation. Using the BW 704 F(ab')2-CVF conjugate, maximum killing reached 70%. Striking differences were observed in the binding of complement components. Maximum binding of C3, C5, and C9 occurred after approximately 90 rain of incubation. The total amounts of C3 and C5 were significantly below those observed with classical pathway activation by intact antibody, with the amounts of C5 at the limit of detectability. In contrast, the total amount of C9 bound to the cells after alternative pathway activation using the BW 704 F(ab')2-CVF conjugate was similar to that observed with intact antibody. These results indicate significant differences in the mechanism ot complement killing and/or cellular repair mechanisms between cytotoxic monoclonal antibodies and antibody-CVF conjugates.