- Email: [email protected]
lpr mice
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083- ACCELERATED NEPHROTOXIC NEPHRITIS: INCREASED SEVERITY OF DISEASE IN Clq DEFICIENT MICE M.G.Robson , H, T.Cook’, P Taylor, M.Botto, M.J.Walport and K.A. Davies, Division of Medicine and Dept Histopathology’, ICSM, Hammersmith Hospital, Du Cane Road, London, UK Complement deficiency in humans predisposes to severe SLE. We have studied the development of accelerated nephrotoxic nephritis in complement-deficient mice. Glomerulonephritis was induced by immunisation with 200pg rabbit IgG in Freund’s complete adjuvant i/p and 7 days later, i/v administration of nephrotoxic globulin (Img). Clq-/-, Bf/CZ-I- and Clq/Bf/CZ-/mice were studied. Animals (7- I2 per group) of mixed (129/SVxC57BL/6) genetic background were compared. Renal histology (day 4) showed mild proliferative changes in wildtype (WT), and Bf/C2/- animals, but severe glomerular thrombosis in Clq-/- and Clq/Bf/CZ-/- mice. Experiments were also performed in mice of C57BLi6 background (n=9 per group). At day 1I, mice had proliferative nephritis with significantly more neutrophils (median I per glomerular cross section compared to 0.14 (p< 0.05)), and macrophages (5.4 vs 2.7) in Clq-i- than WT mice (p< 0.05). In addition, by quantitative immunofluorescence, Clq-i- mice had more glomerular IgG2a (median 42 arbitrary fluoresence units v 19) and IgG3 (57 vs 31), but not IgG2b (70 v 86) than WT mice (~~0.05 for IgG2a and IgG3). Electron microscopy showed findings similar to human lupus nephritis with large subendothelial deposits in Clq-/- but not WT mice. These data suggest that Clq may protect from immunemediated renal damage, which is independent of alternative pathway activation. The results in C57BL/6 mice suggest that the increased disease in Clq-i- mice may reflect a failure to clear immune complexes, and the electron microsopy suggests important parallels with human lupus nephritis.
084-
EXPRESSION
OF PLASMA
SOLUBLE
CR1 AND LEUKOCYTE SURFACE CR1 IN PATIENTS WITH GLOMERULONEPHRITIS B.Sivasankar, Sadia Ayub, S.C. Tiwari*, L.M. Srivastava and Nibhriti Das. Dept. of Biochemistry, *Dept. of Nephrology, All India Institute of Medical Sciences, New Delhi, India.
In view of the therapeutic role of recombinant soluble Complement Receptor1 (sCRl)it is aimed to study the expression and role of plasma sCR1 in health, and in patients suffering from glomerulonephritis. The parameters were plasma sCRl levels , levels of CR1 in culture supernatants and surface of leukocytes subjected to Interferon gamma (IFN-y ) and Interleukin-4 (IL-4) treatment. Plasma and culture supernatant sCRl levels were monitored by an indigeneous sandwich ELISA and
leukocyte surface CR1 expression was followed by flowcytometry. sCRl levels were found higher in patients (106.4Oi24.34 ng/ml ) as compared to the healthy individuals (44.68*12.5 nglml). GMSCF/IL-4, and GMCSFIIL-4/ IFN-y increased the expression of CR1 both on the surface of leukocytes and inculutre supernates. Whereas GMCSF/ IFN-7 down regulated the same. These results suggest an upregulation of sCR1 in the patients which may be under the influence of prevailing cytokines. In depth studies on the genetic expression of leukocyte CRI and cytokines by RT-PCR, its correlation with disease activity and plasma sCR1 levels are in progress.
085- ~EXPRESSION OF COMPLEMENT REGULATORY PROTEINS IN IDIOPATHIC FOCAL SEGMENTAL GLoMERuLOSCLEROSIS. Meen&& Arora, Re&kshi Arora, N. Das lud L.M Srivaatava AU India Institute of Medical Sciences, New Delhi 110029, INDIA Focal SegmentaI Glome~IosclemsIs (FSGS) is a hetemgenous gmup_o~~~i~$e&~ morphology, cIinicaI CharadcrisedbylloIlInvoIvemem of immunecomplexesinthe~& however, invoIvement and association of imn~10108icaI &ctors [email protected] assesses the expr&on of CRl, DAF and CD59 on the er@mcytes of FSGS @ents using flow cytometry to unders&d their role in the pa&genesis of FSGS. Si@icantIy reducedoqnessionofCRl,D~audCDS9wasd~uvedcm the e@uucy&s of FSGS patients. Expression of CRl, DAF and CD59 on kidney biop&s was also assessed ll6ix@ immmmfIuoresccnce technique. CR1 expression was either decnased or lost in segmentIy s&msed &memIus of FSGS whfzeaanonnaIgIomeruIishowuIhighdegreeofvariabii WlliChCOUldlKltbe-
Witllthepresenceor~of
[email protected] of DAF and CD59 was observed on the kidney biopsies of FSGS patients. A ditrerential expression of CR1 found within thetissuemayberegulatedbymauylocnIfacto~incI~ cytokincs which are Im9wn inducers of complement reaptor synthesis or lass of CR1 by pmteolytic cIeavage. Some gIomemIi of FSGS showing MCNS like expmssion indicate that FSGS repnsent a stage in evolution of a group of patients with MCNS and is a subset ofMCNS.
OS& COMPLEMENT FACTOR C3 IS NOT REQUIRED FOR FULL EXPRESSION OF LUPUS NEPHRITIS IN MRL/Ipr MICE. Hideharu Sekine, Harvey Colten, Gerard Gamier, Antonella Circolo, Gary Gilkeson. Medical University of South Carolina.CharIeston. SC 29425, Northwestern University Medical School, Chicago, IL 60611 and Washington University, St.Louis, MO 63198. Complement activation and tissue deposition of complement fragments appear to be involved in the pathogenesis of lupus nephritis. To define the role of complement component C3 in lupus nephritis, the C3 deficient genotype (C3-I-), derived by homologous recombination, was bred onto the lupus prone MRLlIpr background. We previously reported that after backcrossing the C3-/- genotype 4 times to MRL/lpr mice, there was slight demased proteinuria in the C3-/- mice, but no effect on survival or pathologic renal disease compared to C3+/+ mice. C3+/- mice had more severe disease than either of the other two groups. For this study, we further hackcrossed the C3-I- genotype onto the MRL/Ipr background an additional 5 generations and verified that al1 mice in al1 3 groups were H2m. Over 25 mice of each genotype were generated. As before, there was less proteinuria in the C3-/- group compared to the other two groups, although not statistically less. Serum anti-DNA antibody levels. spleen weights. and survival were similar in the tbrcc groups. In contrast to the previous study, there was no acceleration of disease in the C3+/- mice. These results are similar to findings in B6/lpr mice and suggest that activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice. Furthermore, the previous finding of enhanced disease in MRL/lpr C3+/- mice was not confumed in this study suggesting the prior enhanced disease was secondary to other gene(s) and not a direct result of the C3+/- genotype.
083- ACCELERATED NEPHROTOXIC NEPHRITIS: INCREASED SEVERITY OF DISEASE IN Clq DEFICIENT MICE M.G.Robson , H, T.Cook’, P Taylor, M.Botto, M.J.Walport and K.A. Davies, Division of Medicine and Dept Histopathology’, ICSM, Hammersmith Hospital, Du Cane Road, London, UK Complement deficiency in humans predisposes to severe SLE. We have studied the development of accelerated nephrotoxic nephritis in complement-deficient mice. Glomerulonephritis was induced by immunisation with 200pg rabbit IgG in Freund’s complete adjuvant i/p and 7 days later, i/v administration of nephrotoxic globulin (Img). Clq-/-, Bf/CZ-I- and Clq/Bf/CZ-/mice were studied. Animals (7- I2 per group) of mixed (129/SVxC57BL/6) genetic background were compared. Renal histology (day 4) showed mild proliferative changes in wildtype (WT), and Bf/C2/- animals, but severe glomerular thrombosis in Clq-/- and Clq/Bf/CZ-/- mice. Experiments were also performed in mice of C57BLi6 background (n=9 per group). At day 1I, mice had proliferative nephritis with significantly more neutrophils (median I per glomerular cross section compared to 0.14 (p< 0.05)), and macrophages (5.4 vs 2.7) in Clq-i- than WT mice (p< 0.05). In addition, by quantitative immunofluorescence, Clq-i- mice had more glomerular IgG2a (median 42 arbitrary fluoresence units v 19) and IgG3 (57 vs 31), but not IgG2b (70 v 86) than WT mice (~~0.05 for IgG2a and IgG3). Electron microscopy showed findings similar to human lupus nephritis with large subendothelial deposits in Clq-/- but not WT mice. These data suggest that Clq may protect from immunemediated renal damage, which is independent of alternative pathway activation. The results in C57BL/6 mice suggest that the increased disease in Clq-i- mice may reflect a failure to clear immune complexes, and the electron microsopy suggests important parallels with human lupus nephritis.
084-
EXPRESSION
OF PLASMA
SOLUBLE
CR1 AND LEUKOCYTE SURFACE CR1 IN PATIENTS WITH GLOMERULONEPHRITIS B.Sivasankar, Sadia Ayub, S.C. Tiwari*, L.M. Srivastava and Nibhriti Das. Dept. of Biochemistry, *Dept. of Nephrology, All India Institute of Medical Sciences, New Delhi, India.
In view of the therapeutic role of recombinant soluble Complement Receptor1 (sCRl)it is aimed to study the expression and role of plasma sCR1 in health, and in patients suffering from glomerulonephritis. The parameters were plasma sCRl levels , levels of CR1 in culture supernatants and surface of leukocytes subjected to Interferon gamma (IFN-y ) and Interleukin-4 (IL-4) treatment. Plasma and culture supernatant sCRl levels were monitored by an indigeneous sandwich ELISA and
leukocyte surface CR1 expression was followed by flowcytometry. sCRl levels were found higher in patients (106.4Oi24.34 ng/ml ) as compared to the healthy individuals (44.68*12.5 nglml). GMSCF/IL-4, and GMCSFIIL-4/ IFN-y increased the expression of CR1 both on the surface of leukocytes and inculutre supernates. Whereas GMCSF/ IFN-7 down regulated the same. These results suggest an upregulation of sCR1 in the patients which may be under the influence of prevailing cytokines. In depth studies on the genetic expression of leukocyte CRI and cytokines by RT-PCR, its correlation with disease activity and plasma sCR1 levels are in progress.
085- ~EXPRESSION OF COMPLEMENT REGULATORY PROTEINS IN IDIOPATHIC FOCAL SEGMENTAL GLoMERuLOSCLEROSIS. Meen&& Arora, Re&kshi Arora, N. Das lud L.M Srivaatava AU India Institute of Medical Sciences, New Delhi 110029, INDIA Focal SegmentaI Glome~IosclemsIs (FSGS) is a hetemgenous gmup_o~~~i~$e&~ morphology, cIinicaI CharadcrisedbylloIlInvoIvemem of immunecomplexesinthe~& however, invoIvement and association of imn~10108icaI &ctors [email protected] assesses the expr&on of CRl, DAF and CD59 on the er@mcytes of FSGS @ents using flow cytometry to unders&d their role in the pa&genesis of FSGS. Si@icantIy reducedoqnessionofCRl,D~audCDS9wasd~uvedcm the e@uucy&s of FSGS patients. Expression of CRl, DAF and CD59 on kidney biop&s was also assessed ll6ix@ immmmfIuoresccnce technique. CR1 expression was either decnased or lost in segmentIy s&msed &memIus of FSGS whfzeaanonnaIgIomeruIishowuIhighdegreeofvariabii WlliChCOUldlKltbe-
Witllthepresenceor~of
[email protected] of DAF and CD59 was observed on the kidney biopsies of FSGS patients. A ditrerential expression of CR1 found within thetissuemayberegulatedbymauylocnIfacto~incI~ cytokincs which are Im9wn inducers of complement reaptor synthesis or lass of CR1 by pmteolytic cIeavage. Some gIomemIi of FSGS showing MCNS like expmssion indicate that FSGS repnsent a stage in evolution of a group of patients with MCNS and is a subset ofMCNS.
OS& COMPLEMENT FACTOR C3 IS NOT REQUIRED FOR FULL EXPRESSION OF LUPUS NEPHRITIS IN MRL/Ipr MICE. Hideharu Sekine, Harvey Colten, Gerard Gamier, Antonella Circolo, Gary Gilkeson. Medical University of South Carolina.CharIeston. SC 29425, Northwestern University Medical School, Chicago, IL 60611 and Washington University, St.Louis, MO 63198. Complement activation and tissue deposition of complement fragments appear to be involved in the pathogenesis of lupus nephritis. To define the role of complement component C3 in lupus nephritis, the C3 deficient genotype (C3-I-), derived by homologous recombination, was bred onto the lupus prone MRLlIpr background. We previously reported that after backcrossing the C3-/- genotype 4 times to MRL/lpr mice, there was slight demased proteinuria in the C3-/- mice, but no effect on survival or pathologic renal disease compared to C3+/+ mice. C3+/- mice had more severe disease than either of the other two groups. For this study, we further hackcrossed the C3-I- genotype onto the MRL/Ipr background an additional 5 generations and verified that al1 mice in al1 3 groups were H2m. Over 25 mice of each genotype were generated. As before, there was less proteinuria in the C3-/- group compared to the other two groups, although not statistically less. Serum anti-DNA antibody levels. spleen weights. and survival were similar in the tbrcc groups. In contrast to the previous study, there was no acceleration of disease in the C3+/- mice. These results are similar to findings in B6/lpr mice and suggest that activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice. Furthermore, the previous finding of enhanced disease in MRL/lpr C3+/- mice was not confumed in this study suggesting the prior enhanced disease was secondary to other gene(s) and not a direct result of the C3+/- genotype.