[Show abstract][Hide abstract] ABSTRACT: Obesity is a rising problem in cats. It is a risk factor for several diseases and has been linked to impaired immunity. The goal of this study was to determine the effect of body composition and effects of diet on immune function in cats. Twenty-eight short-term obese and 12 lean cats with equal gender distribution were evenly and randomly divided into two groups which were either fed a diet containing saturated (SFA) or long-chain n-3 polyunsaturated fatty acids (3-PUFA) for a period of 6 months prior to testing. Blood was collected by venipuncture from the jugular vein. Blood samples were analyzed in a double blind fashion. A complete blood count was performed and lymphocyte distribution was examined by flow cytometric analysis with specific fluorescein-conjugated subset markers. Immune function was measured as follows: the proliferative activity of different cellular fractions was tested with polyclonal mitogens such as lipopolysaccharide (LPS), phytohaemagglutinin (PHA), phorbol 12-myristate 13-acetate (PMA), Ca ionophore, and concanavalin A. Innate immune functions assessed were phagocytosis and natural killer cell (NK) cytotoxicity. A similar immune innate and adaptive immune response was elicited regardless of diet or body condition. However, there was no correlation between body condition, diet, and any of the quantitative and qualitative functional responses of the immune system. We conclude that short-term obesity and the fatty acid composition of the diet do not alter immune responses in cats.
Veterinary Immunology and Immunopathology 04/2008; 122(1-2):146-52. · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cellular apoptosis susceptibility (CAS) gene is a homologue of the chromosome segregation gene (CSE) in yeast, involved in multiple cellular mechanisms associated with cell proliferation as well as cell death. CAS is highly expressed in proliferating cells but at a lower level in quiescent cells and tissues. Therefore it appears that CAS may play an important role in cancer development. We have previously identified CAS in tilapia non-specific cytotoxic cells (NCC) with a cross-reacting monoclonal antibody. Its expression was up-regulated in NCC in response to apoptosis regulatory factors. In the present report, the molecular cloning and expression of CAS in NCC is described, suggesting the importance of this protein in regulation of teleost immune functions. Furthermore, CAS expression is proposed as one of the mechanisms of regulation of activation induced programmed cell death (AIPCD) in these cytotoxic cells. As CAS expression is ubiquitous, we expect that these studies will help identify proliferating cells protected from apoptosis in additional tissues.
Fish & Shellfish Immunology 05/2006; 20(4):647-55. · 2.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Granzymes are members of the serine protease family and major components of cytotoxic granules of professional killer cells. Multiple granzymes have been identified from human and rodents with different substrate specificities. Although the significance of granzymes A and B in cell-mediated cytotoxicity has been extensively investigated, recent reports suggest that other granzymes may have either equal or greater importance in mediating cell death. Studies on the evolution of these closely related proteases were hindered by the lack of sequence and biochemical information of granzymes from "lower vertebrates." Here we report the generation of a catalytically active recombinant granzyme identified in the cytotoxic cells of an ectothermic vertebrate. Fully active, soluble recombinant catfish granzyme-1 (CFGR-1) was generated using a yeast-based expression system. In vitro enzyme kinetic assays using various thiobenzyl ester substrates verified its tryptase activity in full agreement with previous observations by sequence comparison and molecular modeling. The tryptase activity that was secreted from catfish NCC during an in vitro cytotoxicity assay strongly correlated with the cytotoxicity induced by these cells. Evidence for additional granzymes with different substrate specificities in NCC was obtained by analysis of the protease activity of supernatants collected from in vitro cytotoxicity assays. Searches of the catfish EST database further confirmed the presence of teleost granzymes with different substrate specificities. Granzyme activity measurements suggested a predominance of chymase and tryptase activities in NCC. Further proof that the granule exocytosis pathway is one of the cytotoxic mechanisms in NCC was provided by the expression of granule components perforin, granulysin and serglycin detected by RT-PCR analysis. These results demonstrate the evidence for a parallel evolution of effector molecules of cell-mediated cytotoxicity in teleosts.
[Show abstract][Hide abstract] ABSTRACT: We have identified the gene coding for a novel serine protease with close similarities to mammalian granzymes from nonspecific cytotoxic cells of a teleost fish Oreochromis niloticus. The genomic organization of tilapia granzyme-1 (TLGR-1) has the signature five-exon-four-intron structure shared by all granzymes and similar hematopoietic Ser proteases. Molecular modeling studies suggested a granzyme-like structure for this protein with four disulfide linkages and two additional Cys residues. The expression of this gene is found to be restricted to cytotoxic cell populations with a low level of constitutive expression when compared to similar granzymes in other teleost species. High levels of transcriptional activation of TLGR-1 with different stimuli suggested that this gene is highly induced during immune reactions. Triplet residues around the active site Ser of TLGR, which determines the primary substrate specificity of granzymes, differ significantly from that of other granzymes. Recombinant TLGR-1 was expressed in the mature and proenzyme forms using pPICZ-alpha vector in the Pichia pastoris expression system. Recombinant TLGR-1 was used to determine the primary substrate specificity of this protease using various synthetic thiobenzyl ester substrates. In vitro enzyme kinetics assays suggested a preference for residues with bulky side chains at the P1 site, indicating a chymase-like activity for this protease. These results indicate the presence of novel granzymes in cytotoxic cells from ectothermic vertebrates.
[Show abstract][Hide abstract] ABSTRACT: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are the main killer cell populations of the immune system. The mechanisms by which these cells recognize target cells vary considerably, while the effector molecules used to facilitate target cell death are highly conserved. The main pathways utilized by killer cells consist of granule exocytosis and those mediated by members of the TNF superfamily. Nonspecific cytotoxic cells (NCC) are the first identified cytotoxic cell population in teleosts. We have previously demonstrated the expression of granzymes and Fas ligand in these cells. This is the first report of the expression of tumor necrosis factor-alpha in these killer cells. A cDNA coding for TNF was cloned and sequenced from NCC purified from Nile tilapia (Oreochromis niloticus). Factors regulating the transcriptional modulation of TNF in these cells were identified by RT-PCR analysis. The mature form of tilapia TNF was expressed as a recombinant protein and biological activities were analyzed. Using a cross-reacting anti-TNF polyclonal antibody, analysis of TNF expression suggested that tilapia NCC constitutively express the membrane-bound as well as secreted forms of TNF. Recombinant tilapia TNF effectively induced cytotoxicity in the mammalian cell line WEHI, although to a lesser extent compared to the murine TNF. Treatment with recombinant TNF protected NCC from activation-induced cell death. Recombinant tilapia TNF was also effective in upregulation of granzyme transcription in tilapia NCC. These data suggest that teleost TNF may play a role in diverse effector functions of cytotoxic cells from ectotherms, similar to the biological functions described for mammalian TNF.
[Show abstract][Hide abstract] ABSTRACT: The present study was designed to examine the binding and signalling effects of single base and CpG dinucleotide phosphodiester (Po) oligodeoxynucleotides (ODN) on the human natural killer (NK)-like cell line (YT-INDY). Single base Po ODN composed of 20-mers of guanosine (dG20), adenosine (dA20), cytosine (dC20) or thymidine (dT20) as well as 'conventional' Po CpG ODN were examined for their ability to bind and activate YT-INDY cells. Binding by dG20 and CpG ODN to YT-INDY cells was saturable and specific. dG20 binding was competitively inhibited by homologous dG20 and heterologous CpG ODN but not by dC20 and dA20. Two different YT-INDY membrane proteins (18 and 29 kDa) were identified by ligand (Southwestern) blotting with biotinylated dG20 and CpG. The specificity of the ODN-binding protein(s) was further confirmed by ODN depletion experiments using a teleost recombinant protein orthologue [nonspecific cytotoxic cells (NCC) cationic antimicrobial protein-1 (ncamp-1)] known to bind CpG and dG20. Cell proliferation and activation studies showed that dG20 and CpG treatment of YT-INDY cells induced cellular DNA synthesis (i.e. G1 to S-phase conversion). This signalling function was accompanied in dG20-treated cells by proliferation 10 h posttreatment. Both dG20 and CpG ODN binding induced a calcium flux in YT-INDY cells within seconds of treatment. These experiments demonstrated that Po single base dG20 and CpG ODN bind to a (potential) new class of cell-surface proteins that mediate the activation of YT-INDY cells.
Scandinavian Journal of Immunology 11/2005; 62(4):361-70. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nonspecific cytotoxic cells (NCC) are the first identified and most extensively studied killer cell population in teleosts. NCC kill a wide variety of target cells including tumor cells, virally transformed cells and protozoan parasites. The present study identified a novel evolutionarily conserved oligodeoxynucleotide (ODN) binding membrane protein expressed by channel catfish (Ictalurus punctatus) NCC. Peptide fingerprinting analysis of the ODN binding protein (referred to as NCC cationic anti-microbial protein-1/ncamp-1) identified a peptide that was used to design degenerate primers. A catfish NCC cDNA library was used as template with these primers and the PCR-amplified product was sequenced. The translated sequence contained 203 amino acids (molecular mass of 22,064.63 Da) with characteristic lysine rich regions and a pI=pH 10.75. Sequence comparisons of this protein indicated similarity to zebrafish (51.2%) histone family member 1-X and (to a lesser extent) to trout H1. A search of EST databases confirmed that ncamp-1 is also expressed in various tissues of channel catfish as well as zebrafish. Inspection for signature repeats in ncamp-1 and comparisons with histone-like peptides from different species indicated the presence of multiple lysine based motifs composed of AKKA or PKK repeats. The novel protein was cloned, expressed in E. coli and the recombinant was used to generate rabbit anti-serum. The recombinant ncamp-1 bound GpC and CpG ODNs and was detected with homologous anti-ncamp-1 polyclonal antibodies. Western blots of NCC membranes using anti-ncamp-1 serum detected a 29 kDa protein. Binding competition experiments demonstrated that anti-ncamp-1 antibodies and GpC bound to the same protein on NCC. Two different truncated forms of ncamp-1 as well as the full-length recombinant protein exhibited anti-microbial activity. The present study demonstrated the expression by NCC of a new membrane protein that may participate in the recognition of bacterial DNA and as such participate in innate anti-microbial immune responses in teleosts.
[Show abstract][Hide abstract] ABSTRACT: Granzymes are granule-associated serine proteases, which are important effector molecules in NK cell and CTL functions. The granzyme family poses a perplexing problem in phylogenetics due to the lack of nonmammalian sequence information. We now report the identification of a cDNA that codes for a granzyme homologue, channel catfish granzyme-1 (CFGR-1), from nonspecific cytotoxic cells (NCC) of a teleost. NCC are the first identified and extensively studied cytotoxic cell population in teleosts. Ictalurus punctatus (channel catfish) granzyme cDNA encodes a protein with approximately 50% similarity to granzymes A and K. Highly conserved catalytic triad residues of serine proteases and other motifs common to granzymes were also identified. Conserved amino acid sequences, structure-function data available for the serine protease family, and the crystal structure of human granzyme K supported a model of CFGR-1. It suggested an Arg/Lys primary substrate specificity that is shared with granzymes A and K. Furthermore, CFGR-1 has the four conserved disulfide bonds of granzymes A, K, and M. Phylogenetic analysis suggested that this molecule is a member of the granzyme family. Expression of CFGR-1 in NCC was confirmed by RT-PCR analysis. Presence of a granzyme-like molecule that might play an important role in the effector functions of NCC indicates that cell-mediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years.
Journal of Molecular Evolution 05/2004; 58(4):449-59. · 2.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to characterize the response of golden shiner fed one of eight purified diets with either 0 or 38 mg α-tocopherol (α-T) kg−1 and one of four levels of ascorbic acid (AA) (23, 43, 98, or 222 mg kg−1 diet) in a 2×4 factorial design. Growth, survival, signs of vitamin E deficiency, and immune and stress responses were monitored in groups of 30 golden shiners (mean average initial weight=0.79 g) fed the diets to apparent satiation twice daily in triplicate aquaria for 14–19 weeks. The average individual weight gain was not affected by dietary α-T concentrations at 14 weeks, but the survival of fish fed the α-T-unsupplemented diets was lower than that of fish fed the α-T-supplemented diets. After 10 weeks, fish fed the α-T-unsupplemented diets began to show vitamin-E-deficiency signs such as increased prevalence of fish with darkened skin. After 17 weeks the deficient fish had lower α-T concentrations in viscera, lower whole-body crude protein, total lipid, dry matter, hematocrit, hemoglobin, lymphocyte (%), alternative complement activity (ACH50), and survival after exposure to stressful water temperatures (36–37 °C) than those fed the α-T-supplemented diets. A sparing effect of vitamin E on vitamin C was evident. Elevated dietary vitamin C reduced the incidence and severity of vitamin E deficiency signs in a dose-dependent manner. The interactive effect of vitamins C and E on ACH50 activity and percentage of thrombocytes was also significant. Regardless of vitamin E levels, different vitamin C levels did not influence the α-T concentration in viscera and other interactions between vitamins C and E were not evident.
[Show abstract][Hide abstract] ABSTRACT: Golden shiners (Notemigonus crysoleucas) require a dietary source of ascorbic acid (AA) for growth or survival, depending on diet composition. However, no quantitative requirements of golden shiners for AA for growth, health or survival have been determined, and specific deficiency signs have not been observed. The purpose of this study was to determine the effects of different dietary levels of AA on the growth and health of golden shiners fed diets containing 0-218.5 mg AA/kg diet for 10-16 wk. Weight gain, survival and gross deformities were assessed at 10 wk. The remaining fish were fed the same diets from wk 11-16; hematology and alternative complement activity were then assessed and a subset of live fish from each tank was exposed to elevated temperature. Gross deformities appeared in fish fed 0 mg AA/kg diet at 9 wk. The 19.5 mg AA/kg diet was sufficient to prevent the deformities and optimize survival, whereas growth did not differ among treatments. Fish fed 40.3 mg of AA/kg diet had a higher survival rate than fish fed 0 or 19.5 mg AA/kg diet after exposure to elevated temperatures (34-35.5 degrees C). Alternative complement activity and visceral AA concentrations were greater in fish fed diets with 218.5 mg AA/kg than in all other groups. The results indicate that the dietary requirement of AA for golden shiners increases in response to heat stress, and that the alternative complement activity (one index of immune competence) was strongly enhanced in fish fed a diet with approximately 10 times the amount of AA required to prevent deficiency signs.
Journal of Nutrition 08/2003; 133(7):2281-6. · 4.20 Impact Factor