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Characterization of anti-hapten antibodies generated in vitro by channel catfish peripheral blood lymphocytes
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The Scientific & Social Program: Vth ISDCI Congress
A15 CHARACTERIZATION OF ANTI-HAFFEN ANTIBODIES GENERATED [_NVITRO BY CHANNEL CATFISH PERIPHERAL BLOOD LYMPHOCYrES. *Frederik W. van Ginkel, Norman W. Miller, Craig J. Lobb and L. William Clem. Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216.
Secondaryin vitro stimulation of channel catfish peripheral blood lymphocytes with haptenated T-dependent antigen (TNP-KLH) eficited large numbers of hapten-specitlc Ab-producing cells and relatively high levels (10-.96.~ g/ml) of TN/'-specific Ab in the culture medium. These in vitro generated Abs were compared to_m wvo generated Abs from the serum of the same f'Lshwith respect to covalent structure, affmity and isotypic composition of heavy and light chains. SDS-PAGE analysis under both reducing and non-reducing conditions revealed that the in vitro Abs were structurally similar to the serum Abs. Similarly, in vitro pulsed-labeled Abs also exhibited the 8 band profile characteristic of channel catfish serum Abs when run under non-reducing denaturing conditions. Scatchard analysis of equilibrium dialysis data revealed that the alTmities of the culture- and serum-derived Abs were quite similar, Le., exhibited association constants o f - 2.0 X 10e M 1 . However, it was routinely observed that thein vitro generated Abs exhibited somewhat fewer binding sites per molecule than those derived from serum. The use of murine monoclonal Abs specific for different isotypes of channel catfish heavy and fight chains demonstrated that the isotypic composition of the culture- and serum-derived fish anti-TNP Abs were similar; exceptions occurred with cultures producing lower levels of Abs. These results strongly suggest that channel catfish in vitro Ab responses closely reflect what normally occurs in vivo. [Supported in part by NIH grant # 5-R37-AI-19530]
B1 ARE PRIMITIVE CYTOKINES INVOLVED IN I N V E R T E B R A T E HOST DEFENSE? "Gregory Beck and Gall S. Habicht, Department of Pathology, SUNY at Stony Brook, Stony
Brook, NY 11794-8691, USA, (Tel. 516-444-3030; Fax 516-444-3424). Cytokines are polypeptide m e d i a t o r s released by cells involved in vertebrate host defenses that are used to c o m m u n i c a t e with similar or different cells. Interleukin 1 (IL-1) and t u m o r necrosis factor (TNF) are major i m m u n o r e g u l a t o r y cytokines with m a n y host defense related properties. The crucial need for and importance of cytokines to vertebrates suggests to us that they are p r o t e i n s that have developed and evolved with the host defense s y s t e m s of all animals. Since the host defense s y s t e m s of invertebrates are diverse, we are Interested In studying cytokine-like mediators that can be found t h r o u g h o u t the animal kingdom. We have isolated and c h a r a c t e r i z e d IL-1 and TNF from several invertebrate phyla u s i n g v e r t e b r a t e assay systems. Invertebrate IL-l s h a r e s m a n y biological activities with vertebrate IL-1 w h e n a s s a y e d in several v e r t e b r a t e systems. TNF from i n v e r t e b r a t e s h a s b e e n Identified and characterized by using the L 929 cytotoxicity assay which is routinely employed for detecting vertebrate TNF. The advantage of using a vertebrate system for the characterization of the f u n c t i o n a l p r o p e r t i e s of i n v e r t e b r a t e c y t o k i n e s is that it e m p h a s i z e s the e v o l u t i o n a r y continuity of the i m m u n e / i n f l a m m a t o r y response. On the other hand, these a s s a y s y s t e m s m a y m i s s potentially u n i q u e functions of the invertebrate mediators. For this reason, we have used invertebrate assay systems as well as those utilizing vertebrate ceils for s t u d y i n g c y t o k i n e activity. In i n v e r t e b r a t e s y s t e m s , i n v e r t e b r a t e IL-1 s t i m u l a t e s c o e l o m o c y t e phagocytosis and proliferation. By studying defense m e c h a n i s m s of i n v e r t e b r a t e s and their regulatory cytokines, we may be able to unravel the complex web of Interactions a m o n g cells and factors of vertebrate i m m u n e responses. This will help to d e m o n s t r a t e conclusively the universitalily of cytokme regulation of host defenses. Since phagocytosis is as old as animal life itself, it is likely that tile cytokine a r m a m e n t a r i u m of the phagocyte has an ancient origin as well. (Supported by NSF Grant DCB 8810448)
The Scientific & Social Program: Vth ISDCI Congress
A15 CHARACTERIZATION OF ANTI-HAFFEN ANTIBODIES GENERATED [_NVITRO BY CHANNEL CATFISH PERIPHERAL BLOOD LYMPHOCYrES. *Frederik W. van Ginkel, Norman W. Miller, Craig J. Lobb and L. William Clem. Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216.
Secondaryin vitro stimulation of channel catfish peripheral blood lymphocytes with haptenated T-dependent antigen (TNP-KLH) eficited large numbers of hapten-specitlc Ab-producing cells and relatively high levels (10-.96.~ g/ml) of TN/'-specific Ab in the culture medium. These in vitro generated Abs were compared to_m wvo generated Abs from the serum of the same f'Lshwith respect to covalent structure, affmity and isotypic composition of heavy and light chains. SDS-PAGE analysis under both reducing and non-reducing conditions revealed that the in vitro Abs were structurally similar to the serum Abs. Similarly, in vitro pulsed-labeled Abs also exhibited the 8 band profile characteristic of channel catfish serum Abs when run under non-reducing denaturing conditions. Scatchard analysis of equilibrium dialysis data revealed that the alTmities of the culture- and serum-derived Abs were quite similar, Le., exhibited association constants o f - 2.0 X 10e M 1 . However, it was routinely observed that thein vitro generated Abs exhibited somewhat fewer binding sites per molecule than those derived from serum. The use of murine monoclonal Abs specific for different isotypes of channel catfish heavy and fight chains demonstrated that the isotypic composition of the culture- and serum-derived fish anti-TNP Abs were similar; exceptions occurred with cultures producing lower levels of Abs. These results strongly suggest that channel catfish in vitro Ab responses closely reflect what normally occurs in vivo. [Supported in part by NIH grant # 5-R37-AI-19530]
B1 ARE PRIMITIVE CYTOKINES INVOLVED IN I N V E R T E B R A T E HOST DEFENSE? "Gregory Beck and Gall S. Habicht, Department of Pathology, SUNY at Stony Brook, Stony
Brook, NY 11794-8691, USA, (Tel. 516-444-3030; Fax 516-444-3424). Cytokines are polypeptide m e d i a t o r s released by cells involved in vertebrate host defenses that are used to c o m m u n i c a t e with similar or different cells. Interleukin 1 (IL-1) and t u m o r necrosis factor (TNF) are major i m m u n o r e g u l a t o r y cytokines with m a n y host defense related properties. The crucial need for and importance of cytokines to vertebrates suggests to us that they are p r o t e i n s that have developed and evolved with the host defense s y s t e m s of all animals. Since the host defense s y s t e m s of invertebrates are diverse, we are Interested In studying cytokine-like mediators that can be found t h r o u g h o u t the animal kingdom. We have isolated and c h a r a c t e r i z e d IL-1 and TNF from several invertebrate phyla u s i n g v e r t e b r a t e assay systems. Invertebrate IL-l s h a r e s m a n y biological activities with vertebrate IL-1 w h e n a s s a y e d in several v e r t e b r a t e systems. TNF from i n v e r t e b r a t e s h a s b e e n Identified and characterized by using the L 929 cytotoxicity assay which is routinely employed for detecting vertebrate TNF. The advantage of using a vertebrate system for the characterization of the f u n c t i o n a l p r o p e r t i e s of i n v e r t e b r a t e c y t o k i n e s is that it e m p h a s i z e s the e v o l u t i o n a r y continuity of the i m m u n e / i n f l a m m a t o r y response. On the other hand, these a s s a y s y s t e m s m a y m i s s potentially u n i q u e functions of the invertebrate mediators. For this reason, we have used invertebrate assay systems as well as those utilizing vertebrate ceils for s t u d y i n g c y t o k i n e activity. In i n v e r t e b r a t e s y s t e m s , i n v e r t e b r a t e IL-1 s t i m u l a t e s c o e l o m o c y t e phagocytosis and proliferation. By studying defense m e c h a n i s m s of i n v e r t e b r a t e s and their regulatory cytokines, we may be able to unravel the complex web of Interactions a m o n g cells and factors of vertebrate i m m u n e responses. This will help to d e m o n s t r a t e conclusively the universitalily of cytokme regulation of host defenses. Since phagocytosis is as old as animal life itself, it is likely that tile cytokine a r m a m e n t a r i u m of the phagocyte has an ancient origin as well. (Supported by NSF Grant DCB 8810448)