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Activation of complement in miniature pigs undergoing cardiopulmonary bypass
61 A C T I V A T I O N OF C O M P L E M E N T IN M I N I A T U R E PIGS U N D E R G O I N G C A R D I O P U L M O N A R Y BYPASS W a g n e r R~, mlaba~ V~, ~ern9 J~ IRes. Inst. C a r d i o v a s c u l a r Surgery, Brno 2Inst. M i c r o b i o l . , Czech Acad. Sci., Prague C a r d i o p u l m o n a r y bypass (CPB) r e p r e s e n t s an a r t e f i c i a l e x t r a c o r p o r a l b l o o d c i r c u i t leading to u n p h y s i o l o g i c a l c o n t a c t of b l o o d w i t h d i f f e r e n t types of p l a s t i c m a t e r i a l and g a s / f l u i d interface. This t r i g g e r s m a n y humoral and c e l l u l a r i n f l a m m a t o r y events which may be d e t r i m e n t a l and may lead to severe p o s t p e r f u s i o n c o m p l i c a t i o n s . W i t h the aim to study the role of i n d i v i d u a l i n f l a m m a t o ry c o m p o n e n t s in the u n d e s i r e d sequelae of CPB, m i n i a t u r e pigs were c h o s e n as a m o d e l to study the u n d e r l y i n g m e c h a n i s m s and to m o n i t o r the course of p o s t p e r f u s i o n syndrom. In this study we tried to p a r t i a l l y e l i m i n a te the i n f l a m m a t o r y effect of CPB u s i n g p l a s m a / p l a t e l e t s e p a r a t o r H a e m o n e t i c s combined w i t h l e u c o c y t e removal filter b e f o r e the start of CPB. The results were c o m p a r e d w i t h a group of pigs w i t h s t a n d a r d CPB using Shiley o x y g e n a t o r only. In b o t h two groups u n d e r g o i n g c a r d i o s u r g e r y a drop of c l a s s i c al and a l t e r n a t i v e p a t h w a y was o b s e r v e d w h i c h was m u c h more p r o n o u n c e d in pigs of the former group. This could be only partially e x p l a i n e d by the d i l u t i o n of b l o o d w h i c h follows the s e p a r a t i o n of p l a s m a in the separator. The a c t i v a t i o n of c o m p l e m e n t could be d e m o n s t r a t e d by t w o - d i m e n s i o n a l immunoe l e c t r o p h o r e s i s and by m o l e c u l a r t i t r a t i o n of s e l e c t e d c o m p o n e n t s of complement.
CLONING AND SEQUENCING OF A cDNA CODING FOR BOVINE CONGLUTININ N. Wakamiya~),Y. Suzuki 2), Y.P. Yin 2) and T. Kurimura ~). ~ Osaka University, Osaka and 2)Osaka Prefectural Institute of Public Health, Osaka, Japan. Conglutinin is a bovine serum protein which mediates the agglutination of erythrocytes reacting with antibody and complement and was first described as a vertebrate lectin. The biological activity of bovine conglutinin is known to be mediated by binding of conglutinin to the carbohydrate moiety on the et chain of i C 3 b . Previous studies showed that bovine conglutinin directly inhibited the infectivity and hemagglutinating activity of influenza A viruses of the H1 and I-/3 subtypes by its binding to a carbohydrate site at the HA. Here, we try to isolate a cDNA clone encoding bovine conglutinin to obtain further information on its biological function, cDNA clones encoding the bovine conglutinin were isolated from a bovine liver cDNA library using a specific probe obtained from the PCR product. These cDNAs carry an insert of 1113 bp coding for a protein of 371 amino acid residues with a signal peptide of 20 residues. Southern blot analysis of total bovine genomic DNA indicated that there is only one copy of the gene encoding bovine conglutinin. Northern blot analysis of bovine tissues showed that conglutinin mRNA of about 1.5 kb is expressed in the liver and also slightly in the lung. The deduced amino acid sequence of cDNA shares considerable sequence similarity with those of c-lectins, especially bovine SP-D. These results suggest that conglutinin is produced in the liver and SP-D in the lung.
H U M A N C5 D E F I C I E N C Y : A N O N S E N S E M U T A T I O N IN T H E FIRST C O D O N OF T H E I - C H A I N X. Wang~, D.T. Fleischer~, G. Peter~, W.T. Whitehead1, D.L HavilandI, R.A. Wetsel ~ 1Washingtou University School of Medicine, St. Louis, MO USA 2Brown University, Providence, RI USA Several cases of hereditary homozygous C5 deficiency (C5D) have been discovered, but the molecular genetic defect causing C5 protein deficiency is not known in any C5D kindred. Lack of RFLPs, restricted tissue expression, gene size (80 kb), and gene complexity (41 exons) have precluded many protein and molecular genetic studies of C5 deficiency. We are presently examining the molecular basis of C5 deficiency in a homozygous C5D Black-American female. By ELISA with a sensitivity of 5 ng/ml, no C5 protein was detected in the semm of this C5D individual. To determine the molecular genetic basis of the deficiency, a genomic cosmid library was constructed using DNA from the patient's white blood cells. C5 clones were isolated and the C5D structural gene was mapped using four overlapping cosmid clones. The C5D genomic inserts were purified, self-ligated, randomly sheared by sonication, and fragments of 600bp were cloned into Ml3mpl8. M13 subclones that contained exons were identified by C5 cDNA hybridization and sequenced. Approximately 50% of the C5D structural gene has been characterized by this method thus far. One mutation has been discovered during this analysis that would cause C5 protein deficiency. A nonsense mutation (CAG to TAG) was found encoding the first amino acid of the C5 [~-chain(Gln~ to Stop). This finding indicates that the molecular genetic defect causing C5 protein deficiency in this patient can be partly explained by a nonsense mutation in one of the C5 alleles. Current PCR studies will determine if this mutation is present in both C5 alleles of this patient as well as in C5 genes of other CSD kindred.
A N A L Y S I S O F C5a R E C E P T O R ANTIBODY.
BY M O N O C L O N A L
H. W a t a n a b e , M. Kuraya, R. K a s u k a w a , T. F u j i t a , H. Yanagisawa, and M. Yanagisawa Dept. of Internal Medicine II, and Biochemistry, F u k u s h i m a Medical College, F u k u s h i m a , 960-12 Japan, Dept. of M o l e c u l a r G e n e t i c s , The U n i v e r s i t y of Texas, Dallas, Texas 75235-9050, USA. We prepared mouse monoclonal a n t i b o d y to h u m a n C5a r e c e p t o r (C5aR) by f u s i n g s p l e e n c e l l s from mice immunized with Ltk'cell trasfected by r e c o m b i n a n t h u m a n C 5 a R (Ltk-/C5aR) to the m o u s e m y e l o m a line P3U1. On i m m u n o b l o t t i n g a n a l y s i s , we detected the 44 kDa band on cell lysates of Ltk-/C5aR. T h i s m o n o c l o n a l a n t i b o d y (4C8), b e l o n g i n g to I g M K, c o m p l e t e l y i n h i b i t e d b i n d i n g of 125I-C5a to its r e c e p t o r on Ltk'/C5aR and PMN when c r o s s l i n k e d by a n t i - m o u s e IgM a n t i b o d y . F u r t h e r m o r e , 4C8 p a r t i a l l y b l o c k e d a [Ca2+]i i n c r e a s e in Ltk-/C5aR and PMN s t i m u l a t e d by C5a. T h e r e f o r e , we c o n c l u d e that 4C8 is s p e c i f i c for C5aR. U s i n g this m o n o c l o n a l a n t i b o d y , we a n a l y z e d the e x p r e s s i o n of C5aR on l e u k o c y t e s by flow c y t o m e t r y . We found that C5aR was expressed on PMN, m o n o c y t e s and p l a t e l e t s , but not on l y m p h o c y t e s . W h e n U937 was s t i m u l a t e d by d b - c A M P , the e x p r e s s i o n of C5aR was i n d u c e d in a dose d e p e n d e n t m a n n e r . B a s e d on t h e s e results, it is likely that 4C8 is useful for analysis of the expression of C5aR under the conditions of some i m m u n o l o g i c a l and i n f l a m m a t o r y d i s e a s e s .
CLONING AND SEQUENCING OF A cDNA CODING FOR BOVINE CONGLUTININ N. Wakamiya~),Y. Suzuki 2), Y.P. Yin 2) and T. Kurimura ~). ~ Osaka University, Osaka and 2)Osaka Prefectural Institute of Public Health, Osaka, Japan. Conglutinin is a bovine serum protein which mediates the agglutination of erythrocytes reacting with antibody and complement and was first described as a vertebrate lectin. The biological activity of bovine conglutinin is known to be mediated by binding of conglutinin to the carbohydrate moiety on the et chain of i C 3 b . Previous studies showed that bovine conglutinin directly inhibited the infectivity and hemagglutinating activity of influenza A viruses of the H1 and I-/3 subtypes by its binding to a carbohydrate site at the HA. Here, we try to isolate a cDNA clone encoding bovine conglutinin to obtain further information on its biological function, cDNA clones encoding the bovine conglutinin were isolated from a bovine liver cDNA library using a specific probe obtained from the PCR product. These cDNAs carry an insert of 1113 bp coding for a protein of 371 amino acid residues with a signal peptide of 20 residues. Southern blot analysis of total bovine genomic DNA indicated that there is only one copy of the gene encoding bovine conglutinin. Northern blot analysis of bovine tissues showed that conglutinin mRNA of about 1.5 kb is expressed in the liver and also slightly in the lung. The deduced amino acid sequence of cDNA shares considerable sequence similarity with those of c-lectins, especially bovine SP-D. These results suggest that conglutinin is produced in the liver and SP-D in the lung.
H U M A N C5 D E F I C I E N C Y : A N O N S E N S E M U T A T I O N IN T H E FIRST C O D O N OF T H E I - C H A I N X. Wang~, D.T. Fleischer~, G. Peter~, W.T. Whitehead1, D.L HavilandI, R.A. Wetsel ~ 1Washingtou University School of Medicine, St. Louis, MO USA 2Brown University, Providence, RI USA Several cases of hereditary homozygous C5 deficiency (C5D) have been discovered, but the molecular genetic defect causing C5 protein deficiency is not known in any C5D kindred. Lack of RFLPs, restricted tissue expression, gene size (80 kb), and gene complexity (41 exons) have precluded many protein and molecular genetic studies of C5 deficiency. We are presently examining the molecular basis of C5 deficiency in a homozygous C5D Black-American female. By ELISA with a sensitivity of 5 ng/ml, no C5 protein was detected in the semm of this C5D individual. To determine the molecular genetic basis of the deficiency, a genomic cosmid library was constructed using DNA from the patient's white blood cells. C5 clones were isolated and the C5D structural gene was mapped using four overlapping cosmid clones. The C5D genomic inserts were purified, self-ligated, randomly sheared by sonication, and fragments of 600bp were cloned into Ml3mpl8. M13 subclones that contained exons were identified by C5 cDNA hybridization and sequenced. Approximately 50% of the C5D structural gene has been characterized by this method thus far. One mutation has been discovered during this analysis that would cause C5 protein deficiency. A nonsense mutation (CAG to TAG) was found encoding the first amino acid of the C5 [~-chain(Gln~ to Stop). This finding indicates that the molecular genetic defect causing C5 protein deficiency in this patient can be partly explained by a nonsense mutation in one of the C5 alleles. Current PCR studies will determine if this mutation is present in both C5 alleles of this patient as well as in C5 genes of other CSD kindred.
A N A L Y S I S O F C5a R E C E P T O R ANTIBODY.
BY M O N O C L O N A L
H. W a t a n a b e , M. Kuraya, R. K a s u k a w a , T. F u j i t a , H. Yanagisawa, and M. Yanagisawa Dept. of Internal Medicine II, and Biochemistry, F u k u s h i m a Medical College, F u k u s h i m a , 960-12 Japan, Dept. of M o l e c u l a r G e n e t i c s , The U n i v e r s i t y of Texas, Dallas, Texas 75235-9050, USA. We prepared mouse monoclonal a n t i b o d y to h u m a n C5a r e c e p t o r (C5aR) by f u s i n g s p l e e n c e l l s from mice immunized with Ltk'cell trasfected by r e c o m b i n a n t h u m a n C 5 a R (Ltk-/C5aR) to the m o u s e m y e l o m a line P3U1. On i m m u n o b l o t t i n g a n a l y s i s , we detected the 44 kDa band on cell lysates of Ltk-/C5aR. T h i s m o n o c l o n a l a n t i b o d y (4C8), b e l o n g i n g to I g M K, c o m p l e t e l y i n h i b i t e d b i n d i n g of 125I-C5a to its r e c e p t o r on Ltk'/C5aR and PMN when c r o s s l i n k e d by a n t i - m o u s e IgM a n t i b o d y . F u r t h e r m o r e , 4C8 p a r t i a l l y b l o c k e d a [Ca2+]i i n c r e a s e in Ltk-/C5aR and PMN s t i m u l a t e d by C5a. T h e r e f o r e , we c o n c l u d e that 4C8 is s p e c i f i c for C5aR. U s i n g this m o n o c l o n a l a n t i b o d y , we a n a l y z e d the e x p r e s s i o n of C5aR on l e u k o c y t e s by flow c y t o m e t r y . We found that C5aR was expressed on PMN, m o n o c y t e s and p l a t e l e t s , but not on l y m p h o c y t e s . W h e n U937 was s t i m u l a t e d by d b - c A M P , the e x p r e s s i o n of C5aR was i n d u c e d in a dose d e p e n d e n t m a n n e r . B a s e d on t h e s e results, it is likely that 4C8 is useful for analysis of the expression of C5aR under the conditions of some i m m u n o l o g i c a l and i n f l a m m a t o r y d i s e a s e s .