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M4 11:00 Sequential activation and deactivation of antimicrobial killing mechanisms of cytokine activated goldfish macrophages
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Abstracts of the 7th Congress of the ISDCI: Session M
M3 lo:45
CLONING TRANSFORMING GROWTH FACTOR-B (TGF-/3) FROM HYBRID STRIPED BASS (Afurone saxatilis x M chrysops) Craig A. Harms’, Suzanne Kennedy-Stoskopf’, Fred J. Fuller’, William A. Home: Wayne A.F. Tompkins’ Departments of ‘Microbiology, Pathology and Parasitology, and *Anatomy, Physiology and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina.
The study of fish immunology is hampered by a paucity of specific reagents, including those for detecting cytokines. Reverse transcription-quantitative competitive polymerase chain reaction (RT-qcPCR) with primers derived from conserved regions of known cytokine sequences can be used to quantify cytokine mRNA in diverse species for which specific reagents are not available. Consensus primers derived from mammal cytokine sequences were used for RT-PCR of hybrid striped bass anterior kidney mononuclear cell mRNA. Amplification products were sequenced to determine specificity. A 416 bp RT-PCR product was cloned into a pGEM-T vector and transfected into JM109 cells. Sequencing of the plasmid insert revealed significant homology to TGF-/3 precursors. Rapid amplification of cDNA ends (RACE) produced an overlapping 580 bp 3’ RACE product that contains coding sequences for the entire mature active protein. This segment translates to a 112 amino acid polypeptide with 70% identity with rat TGF-/3,, and 79% identity with rainbow trout TGF-0. Acquisition of the 5’ RACE product is being pursued to allow PCR amplification and isolation of a full-length TGF-/3 clone. Subsequent aims are 1) measure TGF-fl mRNA from fish by RT-qcPCR and characterize tissue distribution; 2) demonstrate changes in TGF-/3 mRNA expression in response to immunomodulating agents; and 3) determine applicability of RT-qcPCR to quantify TGF-/3 mRNA in multiple fish species. M4 11:OO
SEQUENTIAL ACTIVATION AND DEACTIVATION OF ANTIMICROBIAL KILLING MECHANISMS OF CYTOKINE ACTIVATED GOLDFISH MACROPHAGES N-F.’ and M. Belosevic”z. Departments of Biological Sciences’ and Medical Microbiologyand Immunology*. University of Alberta, Edmonton, Alberta, Canada. T6G 2E9.
Macrophages are pivitol cells in mediating cellular immune mechanisms in vertebrates. These normally quiescent cells can be transformed into potent killer cells via stimulation with cytokines and/or foreign molecules; a process known as macrophage activation. Goldfish macrophages activated with macrophage activating factor (MAF) upregulate respiratory burst mediated cytotoxicity as early as 1 hr after stimulation and have maximal responses after 6-12 hr of stimulation. This killing response is subsequently “deactivated”, such that respiratory burst responses of activated macrophages are similar to the basal burst levels of unactivated macrophages by 24-48 hrs. The deactivation of primed respiratory burst responses corresponds to the production of reactive nitrogen intermediates by activated macrophages, but is not a direct result of its production. Thus, it would appear that fish macrophages undergo sequential activation and deactivation of cytokine inducible antimicrobial mechanisms.
Abstracts of the 7th Congress of the ISDCI: Session M
M3 lo:45
CLONING TRANSFORMING GROWTH FACTOR-B (TGF-/3) FROM HYBRID STRIPED BASS (Afurone saxatilis x M chrysops) Craig A. Harms’, Suzanne Kennedy-Stoskopf’, Fred J. Fuller’, William A. Home: Wayne A.F. Tompkins’ Departments of ‘Microbiology, Pathology and Parasitology, and *Anatomy, Physiology and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina.
The study of fish immunology is hampered by a paucity of specific reagents, including those for detecting cytokines. Reverse transcription-quantitative competitive polymerase chain reaction (RT-qcPCR) with primers derived from conserved regions of known cytokine sequences can be used to quantify cytokine mRNA in diverse species for which specific reagents are not available. Consensus primers derived from mammal cytokine sequences were used for RT-PCR of hybrid striped bass anterior kidney mononuclear cell mRNA. Amplification products were sequenced to determine specificity. A 416 bp RT-PCR product was cloned into a pGEM-T vector and transfected into JM109 cells. Sequencing of the plasmid insert revealed significant homology to TGF-/3 precursors. Rapid amplification of cDNA ends (RACE) produced an overlapping 580 bp 3’ RACE product that contains coding sequences for the entire mature active protein. This segment translates to a 112 amino acid polypeptide with 70% identity with rat TGF-/3,, and 79% identity with rainbow trout TGF-0. Acquisition of the 5’ RACE product is being pursued to allow PCR amplification and isolation of a full-length TGF-/3 clone. Subsequent aims are 1) measure TGF-fl mRNA from fish by RT-qcPCR and characterize tissue distribution; 2) demonstrate changes in TGF-/3 mRNA expression in response to immunomodulating agents; and 3) determine applicability of RT-qcPCR to quantify TGF-/3 mRNA in multiple fish species. M4 11:OO
SEQUENTIAL ACTIVATION AND DEACTIVATION OF ANTIMICROBIAL KILLING MECHANISMS OF CYTOKINE ACTIVATED GOLDFISH MACROPHAGES N-F.’ and M. Belosevic”z. Departments of Biological Sciences’ and Medical Microbiologyand Immunology*. University of Alberta, Edmonton, Alberta, Canada. T6G 2E9.
Macrophages are pivitol cells in mediating cellular immune mechanisms in vertebrates. These normally quiescent cells can be transformed into potent killer cells via stimulation with cytokines and/or foreign molecules; a process known as macrophage activation. Goldfish macrophages activated with macrophage activating factor (MAF) upregulate respiratory burst mediated cytotoxicity as early as 1 hr after stimulation and have maximal responses after 6-12 hr of stimulation. This killing response is subsequently “deactivated”, such that respiratory burst responses of activated macrophages are similar to the basal burst levels of unactivated macrophages by 24-48 hrs. The deactivation of primed respiratory burst responses corresponds to the production of reactive nitrogen intermediates by activated macrophages, but is not a direct result of its production. Thus, it would appear that fish macrophages undergo sequential activation and deactivation of cytokine inducible antimicrobial mechanisms.