The effect of adjuvant and microbial challenge on the expression of antimicrobial polypeptides in channel catfish (Ictalurus punctatus).

While antimicrobial polypeptides (AMPPs) are increasingly recognized as one of the most important components of innate immunity, there is very little information in vertebrates that documents their upregulation to levels that are microbicidal in vivo. Here we demonstrate that intraperitoneal injection of either Freund's complete adjuvant (FCA) or live Tetrahymena pyriformis (a parasitic ciliate) upregulated AMPP expression in channel catfish skin. FCA induced significant upregulation of total antibacterial activity, anti-Edwardsiella ictaluri activity (the fraction of antibacterial activity active against E. ictaluri), and HLP-1 (the major AMPP in channel catfish skin). Tetrahymena induced a similar upregulation, except that HLP-1 was not significantly induced and the response appeared to be more transient than FCA immunostimulation. AMPP levels were increased up to five-fold higher than resting levels and levels expressed were well within concentrations known to be inhibitory to many important pathogens in vitro. These results provide encouragement that AMPP upregulation may be a promising tool in aquaculture for enhancing the resistance of fish to disease.

[Modeling of interaction between Yersinia pestis and Tetrahymena pyriformis in experimental ecosystems].

Modeling of interaction Yersinia pestis-Tetrahymena pyriformis in artificial soil ecosystem (ASE) containing soil of burrows of main carrier from Gorno-Altayski natural plague reservoir, as well as in physiological solution (PS) and in Hottinger broth (HB). Optimal proportion of bacterial and protozoa cells was possible to obtain and depended from virulence of Y. pestis and environmental conditions. In ASE at 18-22 degrees C association was the most stable under the microbial burden of 100 microbial cells (m.c.) per infusorian. Resistance of plague agent to phagocytosis by T. pyriformis was determined by strain's virulence. Avirulent strain Y. pestis [cyrillic letter: see text]-2377 was rapidly eliminated by protozoan in HB, PS and in ASE under the burden of 10 m.c per infusorian. Y. pestis [cyrillic letter: see text]-3443 with selective virulence compared with [cyrillic letter: see text]-2377 preserved in association longer in any tested medium. Highly virulent Y. pestis [cyrillic letter: see text]-3448 was the most resistant to phagocytosis by T. pyriformis.

[Experimental evaluation of interaction between Yersinia pestis and soil infusoria and possibility of prolonged preservation of bacteria in the protozoan oocysts]. / Eksperimental'naia otsenka vzaimodeistvii Yersinia pestis EV s pochvennymi infuzoriiami i vozmozhnosti dlitel'nogo sokhraneniia bakterii v tsistakh prosteishikh.

The character and outcome of interactions between Y. pestis (vaccine strain and soil infusoria Tetrahymena pyriformis (axenic culture) were under experimental study. The parallel use of the bacteriological method and PCR test systems made it possible to follow the dynamics of Y. pestis cells (strain EV) with different plasmid profiles in their interaction with infusoria, as well as their passage into the protozoa cysts. The study revealed the complete utilization of Y. pestis cells lacking virulence factors by infusoria. The presence of plasmids of virulence influenced only the duration of complete bacterial phagocytosis. A drop in the temperature of cultivation to 2 degrees C induced the mass and rapid encystment of infusoria. In the PCR analysis specific DNA fragments of Y. pestis cells, preserved in the latent (uncultivable) state, were detected in the cysts of protozoa within the period of up to 14 months, while the results of bacteriological studies were negative. The data thus obtained are discussed with regard to the possible mechanisms of the existence and prolonged reservation of Y. pestis in the soils of natural foci with participation of protozoa.

Chemotactic selection with insulin, di-iodotyrosine and histamine alters the phagocytotic responsiveness of Tetrahymena.

Chemotactic selection is a method by which populations of cells exposed to ligands can be isolated and subsequently cultivated. We used Tetrahymena pyriformis GL cultures selected by chemotactic selection to insulin (10 nM), histamine (0.1 nM) and di-iodotyrosine (T2, 10 nM) to study the phagocytotic capacity under the induction of selector hormones. Our results show a long-lasting link between chemotactically selected cultures and phagocytotic activity. Cells selected to histamine produced the highest phagocytotic activity upon a second exposure to the selector hormone. T2 selection was also strongly effective, however, the phagocytosis stimulation was not specific to the hormone given later. Insulin selected sub-populations had different phagocytotic responses to the control substance itself, whereas histamine selected sub-populations seem to be heterogeneous in the phagocytotic response to histamine. For insulin, the increased endocytotic or metabolic activity was demonstrated by the lack of non-phagocytotic cells. These experiments call attention to the evolutionary role of selection in the later developing receptor-hormone relationship.

Use of sequence-specific antisense oligodeoxynucleotides to determine the protozoan parasite antigen recognized by nonspecific cytotoxic cells.

The antigen on the protozoan parasite Tetrahymena pyriformis recognized by catfish nonspecific cytotoxic cells (NCC) is a 46- to 48-kDa protein referred to as NKTag. The complete cDNA-derived amino acid sequence of NKTag has been obtained. The antigenic determinant of NKTag corresponding to the NCC binding site has been determined with synthetic peptides in target cell competition experiments. To more directly characterize the mechanism of parasite:effector cell interaction, we applied NKTag sequence-specific antisense oligodeoxynucleotides to Tetrahymena in vitro. NKTag mRNA translation by Tetrahymena was blocked by specific antisense (AS) oligodeoxynucleotides. 5'-3' sense (S) oligodeoxynucleotide sequences were synthesized corresponding to the first 17 N-terminal amino acids of NKTag (in addition to -2 untranslated codons plus the start codon). Complimentary AS oligodeoxynucleotides were likewise synthesized. To determine the optimum in vitro conditions for AS treatment, we tested parasites at various phases of their growth cycle for the effects of a single AS treatment. At 9 h post-AS treatment (during the linear phase of the growth curve), maximum reduction in membrane expression of NKTag was observed. Eighty-five percent of Tetrahymena were positive for expression of NKTag at 0 time post-AS treatment versus 13% positive at 9 h. Membrane expression of AS-treated parasites returned to normal levels by 24 h post-treatment. In cold target inhibition experiments, the reduced NKTag expression by Tetrahymena at 9 h AS treatment was confirmed by observing a complete inability (compared with S-treated parasites) to compete with IM-9 cells for binding with NCC. These data demonstrated a unique experimental in vitro system to define the antigen determinant on target cells responsible for recognition by cytotoxic effector cells that participate in innate immune responses.

Non-specific activation of mouse peritoneal macrophages by a freshwater ciliate, Tetrahymena pyriformis.

Toxoplasma-killing activities of mouse peritoneal macrophages activated by the extracts of Tetrahymena pyriformis (Korean and Chinese strains) were evaluated, and the active protein fractions from both strains were partially characterized by a method including chromatographies and SDS-PAGE. The first peak in Korean strain and the second peak in Chinese strain of T. pyriformis obtained by DEAE-Sephadex A-50 chromatography were most effective in the activation of macrophages to kill Toxoplasma gondii tachyzoites in vitro. Subsequent fractionations of obtained peak fractions were performed on a Sephadex G-200 gel. The first peaks fractionated from both strains of T. pyriformis had the highest toxoplasmacidal activities, and when subjected to the SDS-PAGE, one prominent band was visualized for each of the strains showing the same molecular weight of ca. 52.6 kDa. This active protein is suggested to be related to non-specific activation of mouse peritoneal macrophages.

Evidence for antigen recognition by nonspecific cytotoxic cells: initiation of 3H-thymidine uptake following stimulation by a protozoan parasite and homologous cognate synthetic peptide.

Catfish nonspecific cytotoxic cells bind to and lyse certain protozoan parasites and tumor cells. Target cell binding is facilitated by recognition of (minimally) one antigenic determinant. Binding to this determinant initiates multiple signalling pathways in NCC including protooncogene kinase phosphorylation, regulation of phosphatase activity and increased membrane receptor expression. In the present study, highly purified NCC were activated in vitro with the protozoan parasite Tetrahymena pyriformis, with a multiple antigenic peptide (MAP) composed of the cognate antigenic determinant of this parasite (i.e. natural killer target antigen/NKTag) and NCC were activated with a monoclonal antibody specific for the NCC receptor which binds NKTag. NCC were purified by Percoll density gradients and negative selection by panning (2x) over anti-sIg specific mab 9E1. In 5 day proliferation experiments, treatment of NCC with immobilized Tetrahymena initiated a significant increase in uptake of tritiated thymidine. This appeared to be a primary response in that NCC from in vivo parasite primed catfish did not have secondary-like proliferation responses. Stimulation of NCC with immobilized synthetic peptides composed of the cognate antigenic determinant of this parasite (i.e. MAP) also caused significant increased uptake of tritiated thymidine. An indication that NCC recognize a specific antigenic determinant was that sMAP (i.e. peptides composed of the same amino acids as MAP but in a scrambled sequence) failed to increase incorporation. Similar to the MAP results, mab 5C6 binding to NCC also caused increased thymidine uptake. To determine if an IL-2 cosignal was required to achieve optimum activation responses by NCC, different concentrations of human recombinant IL-2 (rHuIL-2) were tested individually or as costimulants. Co-treatment of NCC with rHuIL-2 and any of the three stimuli (parasite, MAP, mab 5C6) did not produce increased proliferation of NCC. These studies demonstrated that NCC specifically recognize an antigenic determinant on protozoan parasites and binding to this antigen produces an activation signal that may have important consequences for elicitation of innate immunity.

The third member of the Tetrahymena CCT subunit gene family, TpCCT alpha, encodes a component of the hetero-oligomeric chaperonin complex.

The sequence of a third member of the Tetrahymena pyriformis chaperonin CCT ('chaperonin containing TCP1') subunit gene family is presented. This gene, designated TpCCT alpha, is the orthologue of the mouse chaperonin gene TCP1/CCT alpha. To characterize the CCT complex in this ciliate, we have produced polyclonal antibodies against synthetic peptides based on C-terminal sequences deduced from the primary sequences of the TpCCT alpha, TpCCT gamma and TpCCT eta subunits. We have also used polyclonal antibodies produced against recombinant yeast CCT alpha and CCT beta subunits. Using these antibodies, we show that Tetrahymena cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits. Three of these were assigned to specific TpCCT genes, whereas a fourth was recognized by the polyclonal antibody against yeast CCT beta, suggesting that this gene is also present in the ciliate. The CCT complex also contains other unidentified proteins that were recognized by the polyclonal antibody UM-1, raised against the putative ATP binding domain of the chaperonin proteins. TpCCT alpha gene expression was shown in exponentially growing cells and cells regenerating their cilia for different periods to have a similar pattern to the previously identified genes TpCCT gamma and TpCCT eta, and also to tubulin genes.

Molecular characterization of a protozoan parasite target antigen recognized by nonspecific cytotoxic cells.

The target cell antigen(s) on tumor cells and on protozoan parasites recognized by NK and nonspecific cytotoxic cells (NCC) has not yet been specifically identified. NCC may be the teleost equivalent of NK cells and IL-2-activated NK cells. A ligand recognized by NCC has been identified. It is expressed on both protozoan parasites and mammalian tumor target cells. In the present study, a protozoan parasite antigen (NK target antigen/NKTag/p46) was purified from Tetrahymena pyriformis and the entire amino acid sequence was deduced from cDNA. Soluble and purified NKTag inhibited NCC lysis of human and mouse transformed target cells. Homology comparisons using Swissprot database revealed that NKTag is a novel protein. Molecular weight computation of the deduced sequence demonstrated that NKTag is a 48.17-kDa protein containing 422 amino acids with relatively high percentages of tyrosine and serine residues. Expression of NKTag on various mammalian tumor target cells, normal tissue, and T. pyriformis was determined using anti-multiple antigenic peptide (MAP) monoclonal antibody (mab) 22A12 [generated against an N-terminal 20-mer (aa 61-80) of p46]. This mab bound to tissue-cultured and tumor cells (YAC-1, IM-9, NC-37, MOLT-4, and U937) with low levels of binding to fish, mouse, and equine cells. Studies were also done to determine if purified and iodinated NKTag bound specifically to NCC. Binding was saturable and specific. These data provide evidence that NCC recognize a target cell ligand which is found on both protozoan and tumor cells. This may provide an explanation as to how NCC (including activated NK cells) recognize a vast array of targets in the absence of haplotype recognition and in spite of a diverse species of origin.

Activation of nonspecific cytotoxic cells with a multiple antigenic peptide: specificity and requirements for receptor crosslinkage.

Nonspecific cytotoxic cells (NCC) of teleost fish recognize a conserved antigenic determinant found on the protozoan Tetrahymena pyriformis and on many different tumor target cells. This determinant is located on a 46-kDa Tetrahymena protein referred to as natural killer target antigen (NKTag). The NKTag cognate sequence recognized by NCC is composed of seven to nine amino acids. In the present study, synthetic peptides of the cognate NKTag determinant were prepared as multiple antigenic peptides (MAP). Immobilized MAP activated NCC lysis of IM-9 target cells in the absence of antigen presenting cells or exogenous added cytokines. NCC binding to immobilized MAP produced two- to fivefold increased lysis of IM-9, U937, and HL-60 target cells compared to scrambled control MAP (composed of the same amino acids only in random sequence). NCC receptor crosslinkage was required for activation. Immobilized monomeric homologous cognate peptide did not activate increased NCC lysis of IM-9 target cells; however, NCC preincubated with soluble homologous monomer inhibited MAP activation of lysis. Ligand activation of NCC was antigen specific. Binding of the immobilized ligand with anti-MAP mab 22A12 prior to addition of NCC blocked activation. Mab 22A12 also inhibited NCC lysis of IM-9 target cells. A possible mechanism of NCC activation was determined. Binding of NCC to immobilized MAP produced significantly increased membrane expression of a putative receptor as defined by mab 5C6 binding. These studies demonstrate that activation of NCC by a target cell antigenic determinant depends on crosslinkage of an NCC "receptor" by polymeric repetitive sequences, a soluble or fixed monomer cannot activate but can inhibit activation, and MAP binding initiates increased expression of a putative NCC receptor protein.

Partial amino acid sequence of a novel protozoan parasite antigen that inhibits non-specific cytotoxic cell activity.

Monoclonal antibody (MoAb) 18C2, prepared against a human EBV transformed lymphoblastic cell line (NC-37) is specific for a target cell ligand recognized by fish NCC and by mammalian NK cells. MoAb 18C2 inhibits the lysis of a variety of transformed murine and human cells (e.g. NC-37, YAC-1, K562, etc.). This MoAb also recognizes a determinant on the fish protozoan parasite Tetrahymena pyriformis. In the present study, we used MoAb 18C2 to identify a target antigen in detergent lysates of T. pyriformis. MoAb 18C2 recognized a 46-50 kDa target antigen (NKTag) by Western blot analysis of both crude and ammonium sulphate (AS) fractionated (25-40% saturation) T. pyriformis lysates. AS fractionated or purified soluble NKTag inhibited NCC mediated lysis of IM-9 target cells in a dose dependent fashion. AS fractionated NKTag also inhibited NCC lysis of a variety of human and murine transformed targets (e.g. HL-60, MOLT-4, DAUDI, NC-37, U-937, YAC-1, EL-4). Inhibition was specific for NCC and inhibition could be removed by adsorption of AS fractionated NKTag with MoAb 18C2 hybridoma cells. NKTag was prepared for amino acid sequencing by preparative SDS PAGE of whole cell detergent (CHAPS) lysate followed by Western transfer to nitrocellulose. The MoAb 18C2 recognized NKTag was excised and submitted for microsequence analysis. Direct N-terminal analysis yielded a 12 residue sequence. Additional sequences, obtained from in situ trypsin digests of the NKTag on nitrocellulose yielded four additional peptides of 10, 13, 16 and 21 residues. None of the sequences examined had significant homology to known sequences (Swiss-Prot protein sequence database). These data indicate that MoAb 18C2 recognized a novel protein on T. pyriformis which may be involved in target cell recognition/lysis by NCC. Further, these data extend our previous observation that a common target determinant exists between higher and lower eukaryotic cells, and its expression may provide an explanation for the susceptibility of both protozoan parasites and transformed tumour cells to NK/NCC lysis.

The cellular proliferative response, humoral antibody response, and cross reactivity studies of Tetrahymena pyriformis with Ichthyophthirius multifiliis in juvenile carp (Cyprinus carpio L.).

The immune response of 10-12-week-old juvenile carp to T. pyriformis [CCAP 1630/w, 1939 (w)] was investigated. The humoral antibody response following one and two intraperitoneal injections of whole live T. pyriformis (protein concentration of 25 micrograms/g body weight), separated by an interval of 4 weeks, was monitored over 12-16 weeks. Peak antibody titres were detected 6 weeks following antigen administration. Antibody titres were elevated following the second injection, and relatively high levels were still maintained by week 16. Proliferative responses measured by autoradiography following intraperitioneal injection of methyl 3H thymidine (1-2 microCi/g body weight) were recorded in the pronephros, opisthonephros, and spleen at weekly intervals for 4 weeks following each injection of T. pyriformis. Immunised fish had higher counts than nonimmunised controls, with the greatest counts occurring at 2 and/or 3 weeks following one injection of antigen and 2 weeks following two injections. Activity was recorded prior to peak antibody production with the pronephros and opisthonephros having greater proliferative responses than the spleen. Cross antigenicity studies to examine protection of carp against I. multifiliis following administration of T. pyriformis, showed little evidence in support of protection using this strain of T. pyriformis. Further, antisera raised in either carp, rabbits, or rats failed to show any cross antigenicity between these two species of protozoa.

[Effect of in vivo administration of Tetrahymena pyriformis on the in vitro toxoplasmacidal activity of mouse peritoneal macrophages].

Tetrahymena pyriformis is a free-living ciliate protozoan in the freshwater system. Experiments were carried out to determine whether intraperitoneal administration of T. pyriformis (GL strain) to mice activates macrophages to be able to kill Toxoplasma gondii tachyzoites in vitro. Mice were also injected intraperitoneally with several synthetic activators; dimethyldioctadecylammonium bromide (DDA), dextran sulfate, complete Freund's adjuvant (CFA) as well as Toxoplasma and Tetrahymena lysates in order to activate mouse peritoneal macrophages. One week after the administration of activators, peritoneal cells were harvested and the adherent macrophages were challenged with Toxoplasma tachyzoites. Macrophage monolayers were then fixed with absolute methanol after washing, and stained with Giemsa solution. The percentage of the adherent cells infected and total number of organisms per 100 macrophages were calculated to make toxoplasmacidal activity of macrophages according to the cultivation time. Peritoneal macrophages from mice administered with Tetrahymena exhibited significant protection against target parasites as compared with those treated with synthetic activators. Among non-biological synthetic activators, DDA was evaluated as an excellent activator.

Species-specific antibodies of Tetrahymena acid alpha-glucosidase.

1. Tetrahymena acid alpha-glucosidases A and B were purified from T. pyriformis W and T. thermophila 399, respectively. The acid alpha-glucosidases A and B were different in immunological properties and thermostability. 2. The acid alpha-glucosidases A and B reflected specific distribution between T. pyriformis and T. thermophila. 3. Type A and B of taurolipid showed a species-specific distribution pattern between T. pyriformis and T. thermophila.

Effect of glycosylation inhibitors on the binding of insulin and lectins and on hormonal imprinting in Tetrahymena.

Treatment of the unicellular Tetrahymena with inhibitors of glycosylation (glucosamine or 2-fluoro-2-deoxy-D-glucose) leads, with a few exceptions, to enhancement of the binding of FITC-labelled insulin and lectins (Con-A, Helix, Datura). The development of insulin-evoked imprinting (i.e., the enhancement of binding at subsequent contacts) is not prevented by the administration of glucosamine. Meanwhile the administration of fluoroglucose results, on its own, in enhanced binding. This increased binding capacity cannot be further enhanced by imprinting.

Salmon calcitonin-like immunoactivity in extracts of Tetrahymena pyriformis.

Acid extracts of Tetrahymena pyriformis, a ciliated protozoan grown in defined medium revealed the presence of materials with salmon-type calcitonin immunoactivity. These findings add support to earlier reports of the presence of materials closely resembling vertebrate peptide hormones in unicellular microbes.

Macrophage activation by Tetrahymena pyriformis. I. Active subcellular fractions of Tetrahymena.

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H2O2) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H2O2 release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.