RESUMO
Developing a preclinical canine model that predicts outcomes for hematopoietic cell transplantation in humans requires a model that mimics the degree of matching between human donor and recipient major histocompatibility complex (MHC) genes. The polymorphic class I and class II genes in mammals are typically located in a single chromosome as part of the MHC complex. However, a divergent class I gene in dogs, designated dog leukocyte antigen-79 (DLA-79), is located on chromosome 18 while other MHC genes are on chromosome 12. This gene is not taken into account while DLA matching for transplantation. Though divergent, this gene shares significant similarity in sequence and exon-intron architecture with other class I genes, and is transcribed. Little is known about the polymorphisms of DLA-79 and their potential role in transplantation. This study was aimed at exploring the reason for high rate of rejection seen in DLA-matched dogs given reduced intensity conditioning, in particular, the possibility that DLA-79 allele mismatches may be the cause. We found that about 82% of 407 dogs typed were homozygous for a single, reference allele. Owing to the high prevalence of a single allele, 87 of the 108 dogs (â¼80%) transplanted were matched for DLA-79 with their donor. In conclusion, we have developed an efficient method to type alleles of a divergent MHC gene in dogs and identified two new alleles. We did not find any statistical correlation between DLA-79 allele disparity and graft rejection or graft-versus-host disease, among our transplant dogs.
Assuntos
Rejeição de Enxerto/veterinária , Doença Enxerto-Hospedeiro/veterinária , Antígenos de Histocompatibilidade Classe I/imunologia , Teste de Histocompatibilidade/veterinária , Alelos , Animais , Cromossomos de Mamíferos/imunologia , Cães , Éxons , Expressão Gênica , Rejeição de Enxerto/imunologia , Doença Enxerto-Hospedeiro/imunologia , Histocompatibilidade , Antígenos de Histocompatibilidade Classe I/classificação , Antígenos de Histocompatibilidade Classe I/genética , Homozigoto , Íntrons , Leucócitos/imunologia , Leucócitos/metabolismo , Tipagem Molecular/métodos , Filogenia , Polimorfismo GenéticoRESUMO
BACKGROUND: Preclinical studies of peripheral blood mononuclear cell (PBMC) transplantation conducted in a well-established canine hematopoietic cell transplantation (HCT) model have been successfully translated to human patients over the past 5 decades. OBJECTIVE: We retrospectively investigated the safety and feasibility of PBMC apheresis in the canine model of HCT by analyzing apheresis parameters, cell yields, and the impacts of donor-related and apheresis-related variables on collection yields and donor stability. ANIMALS: One hundred and twenty dogs that underwent PBMC aphereses were evaluated. METHODS: Aphereses were performed with a COBE Spectra blood separator and a central dual-lumen catheter, with or without recombinant canine granulocyte colony-stimulating factor (rcG-CSF) stem cell mobilization. RESULTS: Aphereses from dogs not given rcG-CSF yielded an average volume of 280 +/- 42 mL containing an average of 15,086 +/- 9,834 leukocytes/mL. Aphereses from dogs given rcG-CSF yielded an average volume of 261 +/- 55 mL containing an average of 39,711 +/- 24,488 leukocytes/mL. Higher pre-apheresis white blood cell (WBC) counts correlated with higher apheresis WBC yields (R=0.50, P<.0001). The correlations of collection time, inlet volume, and collection flow rate on WBC yields were statistically significant but only weak to moderate in magnitude (R=0.34, P=.0001; R=0.38, P=.0006; R=0.26, P=.002, respectively) as were the correlations of collection time and inlet volume on collection volumes (R=0.30, P=.002; R=0.42, P<.0001, respectively). All dogs recovered promptly after PBMC aphereses and catheter removal, without complications. CONCLUSIONS AND CLINICAL IMPORTANCE: These data may be useful for translating PBMC apheresis technology to the field of veterinary oncology for the treatment of dogs with hematologic malignancies.
Assuntos
Cães/cirurgia , Transplante de Células-Tronco Hematopoéticas/veterinária , Leucaférese/veterinária , Leucócitos Mononucleares/citologia , Distribuição por Idade , Animais , Contagem de Células Sanguíneas/veterinária , Peso Corporal/fisiologia , Cães/sangue , Feminino , Fator Estimulador de Colônias de Granulócitos/farmacologia , Leucaférese/métodos , Masculino , Estudos Retrospectivos , Caracteres SexuaisRESUMO
Major histocompatibility complex (MHC) genes in mammals include highly polymorphic class I and class II genes that are critical for donor-recipient matching for transplantation. Dogs have served as an effective, directly translatable model for stem/progenitor cell transplantation. Previous analyses of MHC class I genes in dogs point to a single highly polymorphic gene, dog leukocyte antigen (DLA)-88, as an important factor in the success or failure of hematopoietic stem cell transplants. Fifty-nine DLA-88 alleles have been identified and reported so far. Here, we extend this list by presenting 13 novel DLA-88 alleles found in domestic dogs.
Assuntos
Alelos , Cães/genética , Antígenos de Histocompatibilidade Classe I/genética , Polimorfismo Genético , Animais , Cães/imunologia , Técnicas de Genotipagem , Antígenos de Histocompatibilidade Classe I/imunologiaRESUMO
A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V(L) and V(H) regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 A of a CDR. Synthesis of the V(L) and V(H) regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMV(P+E). We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile when compared with the murine monoclonal antibody (muNR-LU-10). The huNR-LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft. Humanization of the chNR-LU-13 antibody is expected to eliminate an undesired human antimouse antibody response, allowing for repeated i.v. administration into humans.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacocinética , Antígenos de Neoplasias/imunologia , Células CHO , Moléculas de Adesão Celular/imunologia , Cricetinae , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Molécula de Adesão da Célula Epitelial , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Células Tumorais CultivadasRESUMO
Streptavidin provides an effective receptor for biotinylated tumoricidal molecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this bacterial protein to patients repeatedly, so as to maximize the antitumor effect without eliciting an immune response. Therefore, we attempted to reduce the antigenicity of streptavidin by mutating surface residues capable of forming high energy ionic or hydrophobic interactions. A crystallographic image of streptavidin was examined to identify residues with solvent-exposed side chains and residues critical to streptavidin's structure or function, and to define loops. Mutations were incorporated cumulatively into the protein sequence. Mutants were screened for tetramer formation, biotin dissociation, and reduced immunoreactivity with pooled patient sera. Patient antisera recognized one minor continuous epitope with binding locus at residue E101 and one major discontinuous epitope involving amino acid residues E51 and Y83. Mutation of residues E51, Y83, R53, and E116 reduced reactivity with patient sera to <10% that of streptavidin, but these mutations were no less antigenic in rabbits. Mutant 37, with 10 amino acid substitutions, was only 20% as antigenic as streptavidin. Rabbits immunized with either streptavidin or mutant 37 failed to recognize the alternative antigen. Biotin dissociated from mutant 37 four to five times faster than from streptavidin. Residues were identified with previously undescribed impact on biotin binding and protein folding. Thus, substitution of charged, aromatic, or large hydrophobic residues on the surface of streptavidin with smaller neutral residues reduced the molecule's ability to elicit an immune response in rabbits.
Assuntos
Formação de Anticorpos/imunologia , Proteínas de Transporte/imunologia , Epitopos/imunologia , Estreptavidina/genética , Estreptavidina/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/genética , Antígenos/genética , Antígenos/imunologia , Proteínas de Transporte/química , Epitopos/química , Humanos , Mutagênese Sítio-Dirigida/genética , Dobramento de Proteína , Coelhos , Estreptavidina/químicaRESUMO
The mechanisms of lysis of nonspecific cytotoxic cells (NCC) from the channel catfish (Ictalurus punctatus) were studied by determining the effects of various inhibitors of cellular metabolism on cytolysis of NC-37 human lymphoma target cells. Inhibition of NCC-mediated lysis by dinitrophenol (DNP) and sodium azide (NaN3) indicated a requirement for cellular energy metabolism. Cytochalasin B, an inhibitor of microfilaments, and monensin, an inhibitor of cellular secretion, both prevented lysis by NCC. Three microtubule inhibitors, vinblastine sulfate, colchicine, and demecolcine, all inhibited target cell lysis. Two divalent cation chelators, EDTA and EGTA, blocked NCC activity. Elimination of both Ca2+ and Mg2+ by EDTA prevented target cell binding and killing. Selective removal of Ca2+ by EGTA prevented killing but did not block target cell binding. These results indicated that nonspecific cytotoxicity in fish is an active process which requires cell movement and an intact secretory apparatus. The mechanisms of cytolysis by NCC were found (except for the requirement of microtubules) to be analogous to those of mammalian NK cells. Combined with morphological studies, these data strongly suggest that a phylogenetic relationship exists between these effector cells.
Assuntos
Antimetabólitos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Peixes/imunologia , Animais , Citoesqueleto/efeitos dos fármacos , Ácido Egtázico/farmacologia , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Técnicas In Vitro , Células Matadoras Naturais/imunologia , Masculino , Mamíferos/imunologia , Microtúbulos/efeitos dos fármacos , Monensin/farmacologia , Especificidade da EspécieRESUMO
Nonspecific cytotoxic cells (NCC) obtained from the head (anterior) kidney of fish (Ictalurus punctatus) lyse human transformed B-cell targets. Lysis depended on direct cell-cell contact. Fish size, age, environmental holding temperatures, and lytic reaction conditions such as osmolality and optimum effector:target cell ratios were optimized. Experiments to characterize optimum kinetics demonstrated highly efficient killing after two hours incubation. This rapid cytolysis was further studied by determining NCC activity against appropriately labeled target cells after 30, 60, 90 and 120 minutes of cocultivation. At 160:1 (E:T) greater than 40% of the 5 hour percent specific release value was produced after 30 minutes. After 90 minutes, more than 90% of total percent specific release was observed. At least one mechanism of regulation of NCC killing was described. In the presence of normal (homologous or heterologous) catfish serum (CFS), essentially no NCC activity was observed. This suppression was reversible by preincubation in 10% fetal bovine serum (FBS). NCC "activation" by preincubation in 10% FBS was time-dependent (at least four hours was required to generate significant lysis). NCC activation could be reversed by treating potentially lytic cells with supernatants containing dissociated CFS. In addition, reversible activation could be demonstrated by treating potentially lytic effector cells with CFS to produce suppression. Regulation occurred at the effector cell level because treated target cells did not suppress NCC activity. These data demonstrate a population of nonspecific effector cytolytic cells that potentially represent a phylogenetic precursor to mammalian natural killer cells.
Assuntos
Citotoxicidade Imunológica , Peixes/imunologia , Imunidade Celular , Animais , Células Cultivadas , Feminino , Peixes/sangue , Humanos , Rim/imunologia , Células Matadoras Naturais/imunologia , Cinética , Masculino , FilogeniaRESUMO
Fish nonspecific cytotoxic cells (NCC) lyse various transformed human B-cells (NC-37, P3HR-1) and erythroblastoid cells (K562) as well as mouse YAC-1 and P815 cells. Highest NCC activity was found in the anterior (head) kidney, but spleen cells and peripheral blood leucocytes (PBL) also demonstrated cytolytic abilities. Lysis of chromium-51 labeled target cells occurred rapidly and optimum cytolysis developed at either 16 degrees C or 26 degrees C incubation temperatures. Preincubation at temperatures of 4 degrees C or 37 degrees C for 4 hours reduced NCC cytotoxicity. Although catfish (Ictalurus punctatus) are extensively outbred, interfish NCC activities did not significantly vary at the optimum E:T ratios (160). The NCC target antigen specificities were partially determined by cold target inhibition (CTI) studies. YAC-1 and K562 did not produce significant CTI, however. These studies demonstrated the presence of a highly active cytotoxic cell which is widely distributed in fish lymphoreticular tissue. NCC kill divergent kinds of transformed cell types, and the target cell specificity for human transformed B-cells is different from the NCC target cell antigens on other human (K562) and on mouse (YAC-1 and P815) cells.
Assuntos
Citotoxicidade Imunológica , Peixes/imunologia , Imunidade Celular , Animais , Linfócitos B , Linhagem Celular , Epitopos , Feminino , Humanos , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Neoplasias Experimentais , Especificidade da Espécie , Baço/imunologiaRESUMO
The morphology of nonspecific cytotoxic cells (NCC) was identified. NCC were purified by target cell conjugate formation and density gradient separation. NCC are monocyte-like. They have reniform nuclei and a low nucleus/cytoplasm ratio. Cytoplasmic granules were not seen after giemsa staining. Scanning electron microscopy demonstrated moderate surface villi and target cell attachment occurred via long membraneous filament-like surface projections extending to the target cell membranes. Transmission electron microscopy of effector:target cell conjugates revealed membrane contact areas without fusion or fragmentation. The nucleus of the NCC had accentuated peripheral chromation and a prominent Golgi apparatus; the cytoplasm contained osmiophilic granules. Michaelis-Menten and Lineweaver-Burk transformation of target cell binding revealed a Vmax of 11-15,000 and a Km of 40,000. The percentage of NCC bound to target cells was 16-18%. Results of these studies were combined with the conjugate experiment to obtain an estimated percentage of active NCC (5-7%). A maximum recycling capacity of .16-.30 indicated that once attachment by NCC to the target cell occurred (and a lethal signal delivered by an effector cell), either the NCC did not recycle or a long lag period was required to restore its cytotoxic capability.
Assuntos
Citotoxicidade Imunológica , Peixes/imunologia , Animais , Sítios de Ligação , Feminino , Peixes/anatomia & histologia , Imunidade Celular , Técnicas In Vitro , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/ultraestrutura , Masculino , Microscopia Eletrônica de VarreduraRESUMO
Nonspecific cytotoxic cells (NCC) from the catfish (Ictalurus punctatus) may comprise a population of cells that are responsible for cellular immunity in the fish. NCC kill a wide variety of transformed target cells, and previous studies have indicated that NCC share properties with mammalian natural killer cells. In the present study, many biophysical and biochemical properties of NCC were defined. NCC were nylon wool nonadherent and adherent. NCC activity was also enriched in plastic nonadherent cells. NCC were nonphagocytic (for carbonyl iron), and they did not bind to Sephadex G-10. Characterization of NCC by density gradient centrifugation indicated that they comprise a relatively homogenous population of cytolytic cells that band at 45.5% Percoll. Moderate to high doses (500-2500 R) of X-irradiation produced a stimulatory effect on NCC lysis of labeled target cells. Additional studies indicated that a soluble suppressor protein in catfish serum (CFS) regulated NCC activity. This S. aureus protein A binding component isolated from CFS suppressed NCC activity. Analysis by SDS-PAGE indicated that the soluble regulatory protein had properties similar to immunoglobulin. These data indicate that NCC share some biophysical properties with mammalian natural killer cells. In addition, NCC appear to be under partial cell regulation by a radiation sensitive suppressor cell and also by a soluble regulator serum immunoglobulin component.
Assuntos
Citotoxicidade Imunológica , Peixes/imunologia , Animais , Adesão Celular , Separação Celular , Proteínas do Sistema Complemento , Citotoxicidade Imunológica/efeitos da radiação , Feminino , Peixes/anatomia & histologia , Imunoglobulinas , Técnicas In Vitro , Células Matadoras Naturais/imunologia , Masculino , Mamíferos/imunologia , Fagócitos/imunologiaRESUMO
The lethal hit stage in NK cell-mediated lysis requires a complex series of events involving the release of NKCF, subsequent binding of these factors to the target cell, and susceptibility of the target cell to lysis by NKCF. Binding of NKCF alone is not sufficient because a number of tumor cells are able to bind NKCF without being lysed, suggesting the need for an additional processing step active on susceptible target cells. In the present study, we show that the interaction with liposome-incorporated NKCF renders NK resistant target cells sensitive to NKCF-mediated lysis. These results suggest that NKCF may mediate their cytotoxic effects through internalization of these factors into the cytosol.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Proteínas/imunologia , Células Tumorais Cultivadas/imunologia , Animais , Fluoresceínas , Humanos , Fatores Matadores de Levedura , Lipossomos/administração & dosagem , Proteínas/administração & dosagemRESUMO
Channel catfish demonstrate a shift in the tissue distribution of nonspecific cytotoxic cells (NCC) when infected with the protozoan parasite, Ichthyophthirius multifiliis. NCC, isolated from head kidney (HK) tissues (hemopoietic organ) or peripheral blood leukocytes, were assessed for cytotoxic activity against NC-37 (a transformed mammalian cell line). NCC activity from HK tissue of moribund I. multifiliis-infected fish was depressed compared to HK-NCC activity in uninfected or I. multifillis-immune fish. The activity of NCC, isolated from the peripheral blood of moribund I. multifiliis-infected fish was significantly greater than the NCC activity in peripheral blood from either immune or uninfected fish. Chromium-51 release assays were combined with effector and target conjugate assays to determine killing capacity (Vmax) and affinity (Km) for target cells of peripheral blood NCC from moribund I. multifiliis-infected and uninfected fish. These experiments indicated that the peripheral blood from the moribund infected fish contained an increased percentage of active NCC with increased killing capacity and target cell affinity compared to peripheral blood NCC activity of uninfected fish.
Assuntos
Doenças dos Peixes/imunologia , Peixes/imunologia , Infecções Protozoárias em Animais , Animais , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Doenças dos Peixes/parasitologia , Peixes/parasitologia , Imunidade Celular , Masculino , Infecções por Protozoários/imunologiaRESUMO
Nonspecific cytotoxic cells (NCC) from fish (Ictalurus punctatus) were treated with different concentrations of retinolacetate and poly I:C. Both in vitro and in vivo experiments were conducted. Retinolacetate significantly increased NCC activity against chromium-51 labeled human B-cell lymphoma target cells (NC-37). Preincubation (treatment prior to adding the labeled target cells) of NCC for 4 to 8 h in 10(-3) to 10(-12) M concentration of retinolacetate produced significant increases in NCC activity compared to treatment during the killing assay only. Similar experiments with different concentrations of poly I:C had no NCC augmenting effects when tested by adding poly I:C either during preincubation periods or during the cytotoxicity assay. Retinolacetate probably produces positive modulation of cytotoxicity by increasing the killing effectiveness of individual NCC, rather than recruiting larger numbers of cytolytic cells. In vitro studies were also conducted by injecting catfish (i.p.) with 1 X, 3X and 5X the daily recommended vitamin A dosages and determining NCC activity after 24, 48 and 72 h treatment. The 1X dose significantly increased NCC activity at 72 h. This increase was not transient because NCC activity after 33-37 days' treatment was significantly higher than controls in the 1X, 2X and 3X groups. Intraperitoneal injections of fish with poly I:C produced no significant increases in NCC activity at 24 or 72 h post-inoculation.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Peixes/imunologia , Vitamina A/análogos & derivados , Animais , Linfoma de Burkitt/imunologia , Diterpenos , Feminino , Humanos , Técnicas In Vitro , Células Matadoras Naturais/imunologia , Masculino , Poli I-C/farmacologia , Ésteres de Retinil , Vitamina A/farmacologiaRESUMO
Development of preclinical dog models of solid organ and hematopoietic transplantation is critically dependent upon characterization of the polymorphic major histocompatibility complex class I and class II loci. While the class II alleles are easily typed as the polymorphic positions reside on a single exon, typing the class I locus is tedious. We have improved the class I typing method by designing improved primers and adopting alternative DNA amplification and cloning reagents that circumvent the use of radioactivity and the need for the single-stranded conformation polymorphism gels. The method is reliable in typing dogs for the class I dog leukocyte antigen (DLA)-88 locus, and through its use, we describe here two new alleles DLA-88*01101 and DLA-88*01201.
Assuntos
Alelos , Cães/genética , Cães/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Leucócitos/imunologia , Animais , Sequência de Bases , Éxons , Teste de Histocompatibilidade/veterinária , Íntrons , Dados de Sequência Molecular , Filogenia , Polimorfismo GenéticoRESUMO
Previous studies in our laboratory on the natural killer (NK) lytic mechanism demonstrated that following interaction of target cell with effector cell, the effector cell releases NK cytotoxic factors (NKCF) that can then bind to and lyse the target cell. This study investigates the mechanism by which the target cell signals the effector cell to release NKCF. Studies on other cell systems with secretory functions have indicated that receptor-induced transmembrane signaling leads to the metabolism of phosphatidylinositol and activation of protein kinase C (PKC) by increased cytosolic Ca++ and diacylglycerol (DAG). We tested the hypothesis that a similar sequence of activation events occurs in human NK cells by examining the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and the calcium ionophores A23187 and ionomycin in their ability to induce release of NKCF. The amount of NKCF released was determined in a 20-hr 51Cr release assay against an NK-sensitive target cell. A23187, ionomycin, or TPA alone did not induce release of NKCF. However, ionophores (200 mM) in conjunction with TPA (20 ng/ml) induced release of NKCF. Several properties of the induced NKCF by TPA and ionophores were concordant with those of the NK cell-mediated cytotoxicity (CMC) reaction. The kinetics of release were faster (less than 1 hr) than when either Con A or target cells were used to stimulate NKCF. Only NK-sensitive target cells were killed by NKCF. Pretreatment of effector cells with interferon enhanced release of NKCF from effector cells. Several lines of evidence suggested that the pathway of activation takes place through phosphatidyl inositol metabolism. Activation of PKC was indicated because TPA and A23187 enhanced protein phosphorylation in the LGL-enriched fraction. Experiments that made use of oleoyl acetyl glycerol, a synthetic DAG, showed release of NKCF in the absence of A23187 but was augmented by the ionophore. The above studies suggest that NKCF is released from NK effector cells within a period of time consistent with NK CMC, and the release of NKCF results either directly or indirectly from protein phosphorylation by PKC.
Assuntos
Citotoxicidade Imunológica , Imunidade Inata , Células Matadoras Naturais/fisiologia , Forbóis/farmacologia , Proteína Quinase C/fisiologia , Proteínas/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Cálcio/fisiologia , Fracionamento Celular , Diglicerídeos/farmacologia , Humanos , Interferons/farmacologia , Ionóforos/farmacologia , Fatores Matadores de Levedura , Fosfatidilinositóis/fisiologia , Taxa Secretória/efeitos dos fármacos , Fatores de TempoRESUMO
Nonspecific cytotoxic cells (NCC) obtained from channel catfish (Ictalurus punctatus) kill Tetrahymena pyriformis, an opportunistic parasite in fish. Based upon this fact, a new mechanism for nonspecific cellular anti-parasitic immunity in fish is proposed. Optimum in vitro conditions for NCC killing of deciliated T. pyriformis were first obtained. Lysis of T. pyriformis by NCC occurred by 10 hr of cocultivation of effector and target cells. During this time period, 50 to 60% cytotoxicity occurred. Fish anti-T. pyriformis serum enhanced NCC killing of T. pyriformis either by prolonging immobilization (after the cilia regeneration period) or by delaying cilia regeneration. Shared antigenic determinants between T. pyriformis, Ichthyophthirius multifiliis, and NC-37 target cells were demonstrated by binding-depletion experiments. For these studies, NCC were depleted from anterior kidney cells (the hemopoetic organ in fish) by preincubating formalin-treated T. pyriformis, I. multifiliis, or viable NC-37 target cells with NCC for 3 hr. Conjugates of effector and target cells were removed by overlaying on fetal bovine serum. Unconjugated fish anterior kidney cells were tested for cytotoxic activity against NC-37 or T. pyriformis target cells. Cold target inhibition experiments by using a 4-hr 51chromium cytotoxicity assay also demonstrated these shared antigenic determinants. Target-specific antisera, used to mediate the killing of T. pyriformis by NCC, were required only for immobilizing the targets, and did not function in an antibody-dependent cell-mediated (ADCC)-like mechanism. Scanning electron micrographs of NCC-T. pyriformis conjugates additionally demonstrated NCC binding to both cilia and cell surface determinants.