RESUMO
Fifty-eight consecutive children with high-risk malignancies were treated with CY, and targeted topotecan followed by autologous hematopoietic cell transplantation (AHCT) in a phase I/II Institutional Review Board-approved study. Twelve participants enrolled in phase I; 5 received dose level 1 of topotecan 3 mg/m(2) per day, with subsequent doses targeted to total systemic exposure of 100±20 ng h/mL and CY 750 mg/m(2) per day. Seven participants received dose level 2. CY dose escalation to 1 g/m(2) per day was considered excessively toxic; one died from irreversible veno-occlusive disease and two experienced reversible hepatotoxicity. These adverse events halted further dose escalation. A total of 46 participants were enrolled in phase II; results are on the 51 participants who received therapy at dose level 1, the maximum tolerated dose. Diagnoses included neuroblastoma (26), sarcoma (9), lymphoma (8), brain tumors (5), Wilms (2) and retinoblastoma (1). Twenty participants (39.3%) were in î¶CR1 at enrollment; median age was 5.1 years. Most common non-hematological grade III-IV toxicity was gastrointestinal (n=37). Neutrophil and platelet engraftment occurred at a median of 15 and 24 days, respectively. Twenty-six (51%) participants remain alive at a median of 6.4 years after AHCT. CY 3.75 g/m(2), and targeted topotecan followed by AHCT are feasible and produce acceptable toxicity in children with high-risk malignancies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/métodos , Neoplasias/tratamento farmacológico , Neoplasias/cirurgia , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Criança , Pré-Escolar , Terapia Combinada , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Humanos , Fatores de Risco , Taxa de Sobrevida , Inibidores da Topoisomerase I/administração & dosagem , Inibidores da Topoisomerase I/efeitos adversos , Topotecan/administração & dosagem , Topotecan/efeitos adversos , Transplante AutólogoRESUMO
Immunostimulating complexes (ISCOMs) are unique multimolecular structures formed by encapsulating antigens, lipids and triterpene saponins and are one of the most successful antigen delivery systems for microbial antigens. In the current study, both the route of administration and the antigen concentration of ISCOMs, containing Eimeria tenella antigens and saponins from native plants, were evaluated in their ability to stimulate humoral immunity and to protect chickens against a challenge infection with E. tenella. Broiler chickens were immunized with ISCOM preparations containing E. tenella antigens and the purified saponins Gg6, Ah6 and Gp7 isolated from Glycyrrhiza glabra, Aesculus hippocastanum and Gipsophila paniculata, respectively. The effects of the route of administration, dose of antigen and type of saponin used for construction of ISCOMs were evaluated for ability to stimulate serum IgG and IgM and to protect chickens against a homologous challenge. A single intranasal immunization was the most effective route for administering ISCOMs although the in ovo route was also quite effective. Dose titration experiments demonstrated efficacy after single immunization with various ISCOM doses but maximum effects were observed when ISCOMs contain 5-10mug antigen. Immunization of birds by any of the three routes with E. tenella antigens alone or antigens mixed with alum hydroxide adjuvant resulted in lower serum antibody and reduced protection to challenge relative to immunization with ISCOMs. Overall the results of this study confirm that significant immunostimulation and protection to challenge are achieved by immunization of chickens with ISCOMs containing purified saponins and native E. tenella antigens and suggest that ISCOMs may be successfully used to develop a safe and effective vaccine for prevention of avian coccidiosis.
Assuntos
Galinhas , Coccidiose/veterinária , Eimeria tenella/imunologia , ISCOMs/farmacologia , Imunização/veterinária , Doenças das Aves Domésticas/parasitologia , Administração Intranasal , Animais , Antígenos de Protozoários/imunologia , Coccidiose/imunologia , Coccidiose/parasitologia , Ensaio de Imunoadsorção Enzimática/veterinária , ISCOMs/administração & dosagem , Imunização/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Contagem de Ovos de Parasitas/veterinária , Doenças das Aves Domésticas/imunologia , Saponinas/imunologiaRESUMO
The killer cell Ig-like receptor (KIR) expression repertoire may offer valuable information for hematopoietic SCT (HSCT). We designed a quantitative KIR RNAtype assay and used it to determine KIR gene expression in healthy donors and patients before HSCT. The specificity of the assay was ensured by specific primers and by electrophoretic distinction of PCR products of unique length. In 87 healthy donors, the KIR repertoire was broadly distributed (32 categories of profiles). There was an overall trend toward inverse correlation of KIR expression level and donor age. Age affected mainly the activating KIR families. Leukemia patients showed lower KIR expression before transplantation than healthy donors. Stem cell mobilization caused a transient increase of KIR expression. We conclude that KIR expression differs quantitatively with age and primary disease and is transiently altered by stem cell recruitment and selection.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células Matadoras Naturais/imunologia , Receptores KIR/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Leucemia/imunologia , Masculino , Receptores KIR/biossíntese , Doadores de TecidosRESUMO
Serpins are serine protease inhibitors that are widely distributed in metazoans but have not been previously characterized in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed and characterized. Random screening of an E.acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame (ORF) consisting of 1,245 nts that encode a peptide 413 amino acids in length with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues in length. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55 kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 kDa and 48 kDa in sporozoites (SZ) and merozoites (MZ) respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa which may correspond to secreted serpin. By immuno-staining the serpin was located in granules distributed throughout both the SZ and MZ but granules appeared to be concentrated in the parasite's anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis the endogenous serpin in MZ extracts did not form the expected high molecular weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes.
Assuntos
Eimeria/química , Serpinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Western Blotting , Galinhas , Clonagem Molecular , Sequência Conservada , DNA Complementar/química , Eimeria/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Dados de Sequência Molecular , RNA de Protozoário/genética , Coelhos , Alinhamento de Sequência , Serpinas/química , Serpinas/genéticaRESUMO
Immunostimulating complexes (ISCOMs) are unique, multimolecular structures formed by encapsulating antigens, lipids, and triterpene saponins of plant origin, and are an effective delivery system for various kinds of antigens. The uses of ISCOMs formulated with saponins from plants collected in Kazakhstan, with antigens from the poultry coccidian parasite Eimeria tenella, were evaluated for their potential use in developing a vaccine for control of avian coccidiosis. Saponins isolated from the plants Aesculus hippocastanum and Glycyrrhiza glabra were partially purified by HPLC. The saponin fractions obtained from HPLC were evaluated for toxicity in chickens and chicken embryos. The HPLC saponin fractions with the least toxicity, compared to a commercial saponin Quil A, were used to assemble ISCOMs. When chicks were immunized with ISCOMs prepared with saponins from Kazakhstan plants and E. tenella antigens, and then challenged with E. tenella oocysts, significant protection was conveyed compared to immunization with antigen alone. The results of this study indicate that ISCOMs formulated with saponins isolated from plants indigenous to Kazakhstan are an effective antigen delivery system which may be successfully used, with low toxicity, for preparation of highly immunogenic coccidia vaccine.
Assuntos
Adjuvantes Imunológicos/normas , Antígenos de Protozoários/imunologia , Eimeria tenella/imunologia , ISCOMs/imunologia , Vacinas Protozoárias/administração & dosagem , Saponinas/imunologia , Adjuvantes Imunológicos/química , Aesculus/química , Animais , Galinhas , Cromatografia Líquida de Alta Pressão , Coccidiose/prevenção & controle , Coccidiose/veterinária , Glycyrrhiza/química , ISCOMs/química , Doenças das Aves Domésticas/parasitologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas Protozoárias/imunologia , Vacinas Protozoárias/normas , Saponinas/análise , Saponinas/toxicidadeRESUMO
BACKGROUND: We sought to develop a method for the clinical large-scale depletion of alphabeta T lymphocytes from mobilized peripheral stem cells, which would allow the allogeneic transplantation of a graft enriched for stem cells, natural killer (NK) cells and gammadelta T lymphocytes. METHODS: Therefore, we obtained mononuclear cells from either mobilized or non-mobilized healthy adult volunteer donors and incubated the cells with a biotinylated anti-alphabeta T-cell Ab and subsequently with an anti-biotin Ab conjugated with magnetic microbeads. The depletion was then performed using a CliniMACS device. RESULTS: The median T-cell depletion was 3.9 log (range 3.5-4.1 log). The recovery of the gammadelta and NK cells was 92% and 80%, respectively. The recovery of CD34+ stem cells from the mobilized donors was 66%. DISCUSSION: This method had no negative influence on the in vitro colony formation of stem cells, and transplantation of alphabeta-depleted cells into NOD-SCID IL-2 common gamma chain knockout (NOD-scid IL2r (null)) mice resulted in a rapid engraftment of human myeloid and lymphoid cells. This method will allow large-scale depletion of alphabeta T cells from mobilized peripheral blood in clinical trials.
Assuntos
Depleção Linfocítica/métodos , Transplante de Células-Tronco de Sangue Periférico , Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T , Adulto , Animais , Mobilização de Células-Tronco Hematopoéticas/métodos , Humanos , Separação Imunomagnética/métodos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Células-Tronco de Sangue Periférico/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Transplante HomólogoRESUMO
A number of complex processes are involved in Eimeria spp. survival, including control of sporulation, intracellular invasion, evasion of host immune responses, successful reproduction, and nutrition. Proteases have been implicated in many of these processes, but the occurrence and functions of serine proteases have not been characterized. Bioinformatic analysis suggests that the Eimeria tenella genome contains several serine proteases that lack homology to trypsin. Using RT-PCR, a gene encoding a subtilisin-like and a rhomboid protease-like serine protease was shown to be developmentally regulated, both being poorly expressed in sporozoites (SZ) and merozoites (MZ). Casein substrate gel electrophoresis of oocyst extracts during sporulation demonstrated bands of proteolytic activity with relative molecular weights (Mr) of 18, 25, and 45 kDa that were eliminated by coincubation with serine protease inhibitors. A protease with Mr of 25 kDa was purified from extracts of unsporulated oocysts by a combination of affinity and anion exchange chromatography. Extracts of SZ contained only a single band of inhibitor-sensitive proteolytic activity at 25 kDa, while the pattern of proteases from extracts of MZ was similar to that of oocysts except for the occurrence of a 90 kDa protease, resistant to protease inhibitors. Excretory-secretory products (ESP) from MZ contained AEBSF (4-[2-Aminoethyl] benzenesulphonyl fluoride)-sensitive protease activity with a specific activity about 10 times greater than that observed in MZ extracts. No protease activity was observed in the ESP from SZ. Pretreatment of SZ with AEBSF significantly reduced SZ invasion and the release of the microneme protein, MIC2. The current results suggest that serine proteases are present in all the developmental stages examined.
Assuntos
Eimeria tenella/enzimologia , Serina Endopeptidases/metabolismo , Animais , Western Blotting , Caseínas/metabolismo , Linhagem Celular , Galinhas , Cromatografia de Afinidade , DNA de Protozoário/análise , Eimeria tenella/genética , Eimeria tenella/crescimento & desenvolvimento , Eimeria tenella/fisiologia , Eletroforese em Gel de Poliacrilamida , Oocistos/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Esporos de Protozoários/fisiologia , Subtilisina/genética , Subtilisina/metabolismoRESUMO
A 21-year-old white male with relapsed acute lymphoblastic leukemia (ALL) developed an invasive Zygomycosis infection 3 weeks after beginning re-induction chemotherapy. Because of the high risk of fatal recurrence of the fungal infection, neither long-term maintenance chemotherapy nor allogeneic hematopoietic stem cell transplant (HSCT) was considered appropriate. Because his ALL blasts expressed CD34 but lacked CD133, he received a CD133 selected autologous graft following high-dose consolidation chemotherapy. The patient survives in remission 19 months after HSCT.
Assuntos
Antígenos CD/análise , Glicoproteínas/análise , Peptídeos/análise , Transplante de Células-Tronco de Sangue Periférico , Leucemia-Linfoma Linfoblástico de Células Precursoras/cirurgia , Terapia de Salvação , Antígeno AC133 , Anfotericina B/uso terapêutico , Antifúngicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Asparaginase/administração & dosagem , Caspofungina , Criança , Terapia Combinada , Contraindicações , Dexametasona/administração & dosagem , Quimioterapia Combinada , Equinocandinas , Humanos , Separação Imunomagnética , Lipopeptídeos , Masculino , Mercaptopurina/administração & dosagem , Metotrexato/administração & dosagem , Peptídeos Cíclicos/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Pirimidinas/uso terapêutico , Recidiva , Indução de Remissão , Transplante Autólogo , Transplante Homólogo , Triazóis/uso terapêutico , Vincristina/administração & dosagem , Voriconazol , Zigomicose/complicações , Zigomicose/tratamento farmacológicoRESUMO
The developmental expression of the antigen SO7, which has been previously shown to protect chickens against infection by several Eimeria species, was investigated. Using RT-PCR, mRNA for SO7 was found to be restricted primarily to unsporulated oocysts (0 hr). Western blot (WB) analysis with an antibody to recombinant SO7 (rbSO7) revealed expression of the protein from 6 to 72 hr (fully sporulated) of sporulation and in sporozoites (SZ). SO7 was absent in host-derived second-stage merozoites (MZ) and was present in culture-derived first-stage MZ but at a level of only 25% of that exhibited by SZ. During invasion of Madin-Darby bovine kidney (MDBK) cells by SZ in vitro, the level of SO7 within cells, as determined by WB analysis, remained relatively constant until 48 hr of development and then decreased by about 40% at the next time point (72 hr). The SO7 secreted into the culture media during in vitro development increased to a relative maximum at 48 hr and then decreased to about 20% of maximum at 72 hr. Immunostaining with anti-rbSO7 indicates that SO7 is highly concentrated in both refractile bodies (RB) of SZ, with some limited distribution in the apical complex. Anti-rbSO7 intensively stained the intracellular parasites and the first-stage schizonts during in vitro development of E. tenella in MDBK cells. Upon release from the schizonts, the first-stage merozoites stained with 1 or 2 bright spots typically at each end. The results suggest that SO7 is closely associated with the SZ RB and is developmentally regulated but may not play a direct role in cellular invasion.
Assuntos
Antígenos de Protozoários/metabolismo , Eimeria tenella/crescimento & desenvolvimento , Regulação da Expressão Gênica , Rim/parasitologia , Organelas/metabolismo , Esporozoítos/ultraestrutura , Animais , Antígenos de Protozoários/genética , Linhagem Celular , Eimeria tenella/genética , Eimeria tenella/imunologia , Eimeria tenella/metabolismo , Eimeria tenella/ultraestrutura , Interações Hospedeiro-Parasita , Rim/citologia , Estágios do Ciclo de Vida , Organelas/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esporozoítos/crescimento & desenvolvimento , Esporozoítos/metabolismoRESUMO
Our previous investigation demonstrated the expression in Eimeria tenella sporulated oocysts of an aminopeptidase (AP) with strong homology to AP N. To further understand the role of proteases during development, we investigated the molecular and biochemical properties of E. tenella AP. Greater than 95% AP activity was present in a soluble extract during sporulation of oocysts with highest activity in fully sporulated oocysts. The AP activity was inhibited by the AP inhibitors bestatin and 1,6-phenanthroline, but not by serine protease inhibitors. The AP had specificity for synthetic endopeptidase substrates that contain arginine, alanine, or glycine at the N terminus. Partial purification of the enzyme yielded a major protein band with an Mr of about 106 kDa and an isoelectric point (Ip) of 5.1. Reverse transcription-polymerase chain reaction indicated that the gene for AP is expressed during sporulation, but expression is absent or greatly reduced in the sporozoites and merozoites. On the basis of the deduced gene structure, the predicted Mr is 110 kDa with a pI of 5.59. Database search indicates that the E. tenella AP shares significant homology with the AP from Apicomplexan taxa: Toxoplasma gondii, Cryptosporidium parvum, and Cryptosporidium hominis. Together, these results confirm the presence of a cytosolic AP related to AP N, which is expressed and active during sporulation of E. tenella oocysts.
Assuntos
Aminopeptidases/isolamento & purificação , Eimeria tenella/enzimologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Galinhas , DNA Complementar/química , DNA de Protozoário/química , Eimeria tenella/genética , Eimeria tenella/fisiologia , Eletroforese em Gel de Poliacrilamida , Éxons/genética , Regulação Enzimológica da Expressão Gênica , Hidrólise , Íntrons/genética , Focalização Isoelétrica , Dados de Sequência Molecular , Peso Molecular , Alinhamento de Sequência , Esporos de Protozoários/fisiologia , Especificidade por SubstratoRESUMO
BACKGROUND: The presence of T and B cells in allogeneic grafts contributes to GvHD and to EBV-associated lymphoproliferative disease (LPD). Depletion of T and B cells from the graft decreases the risk of these complications. METHODS: T and B cells were depleted from mobilized peripheral stem cells from volunteer donors (n=5) using anti-CD3 and anti-CD19 Abs conjugated to magnetic microbeads, and the CliniMACS device. The function of the stem cells after depletion was evaluated using colony assays and non-obese diabetic (NOD)/SCID repopulating experiments. RESULTS: The mean mononuclear cell (MNC) count prior to T- and B-cell depletion was 2.19x10(10) (range 1.48-3.53). After depletion, the mean percentage of contaminating T cells was 0.02% (range 0.01-0.04%) with a mean log(10) depletion of 3.4 (range 3-3.8). The mean percentage of contaminating B cells was 0.1% (range 0.01-0.4%) with a mean log(10) depletion of 2.2 (range 1.4-3). The mean recovery of CD3- and CD19-negative MNCs after depletion was 70% (range 54-88%) and the mean recovery of CD34(+) stem cells was 69% (range 52-98%). The mean number of natural killer (NK) cells after T- and B-cell depletion was 5.2x10(8) (range 2-10x10(8)). In vitro colony assays and in vivo NOD/SCID repopulation assays showed no negative impact of this method on the function of the hematopoietic stem cells. DISCUSSION: Our results show that the CliniMACS system can be used to efficiently deplete PBSC of T and B cells simultaneously, without adverse effect on the graft.
Assuntos
Linfócitos B/imunologia , Depleção Linfocítica/métodos , Transplante de Células-Tronco de Sangue Periférico/métodos , Linfócitos T/imunologia , Animais , Antígenos CD19/análise , Antígenos CD34/análise , Complexo CD3/análise , Antígeno CD56/análise , Separação Celular/métodos , Doença Enxerto-Hospedeiro/prevenção & controle , Mobilização de Células-Tronco Hematopoéticas , Humanos , Células Matadoras Naturais/citologia , Camundongos , Transplante HomólogoRESUMO
To characterize the genes expressed by Eimeria tenella oocysts, the sequence of 499 expressed sequence tags (ESTs) was obtained from complementary DNA (cDNAs) enriched for transcripts expressed by unsporulated or sporulated oocysts. Of these, 225 clones were isolated from cDNA of sporulated oocysts and 274 from unsporulated oocysts. A total of 163 unique sequences were found, and the majority of these (64%) represent novel genes with no significant homology to the proteins in GenBank. Approximately half of the unique transcripts generated from sporulated oocysts are also expressed by sporozoites and merozoites, whereas the expression of most (79%) of the transcripts from unsporulated oocysts has not yet been detected at other stages of development. The expression of 4 transcripts obtained from the subtracted cDNAs was confirmed by quantitative reverse transcriptase-polymerase chain reaction. The results confirmed that these transcripts are in fact differentially expressed between sporulated and unsporulated oocysts.
Assuntos
Galinhas/parasitologia , Coccidiose/veterinária , Eimeria tenella/genética , Hibridização de Ácido Nucleico/métodos , Doenças das Aves Domésticas/parasitologia , Animais , Coccidiose/parasitologia , Sequência Consenso , DNA Complementar/química , DNA de Protozoário/química , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Biblioteca Gênica , Reação em Cadeia da Polimerase , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Transcrição GênicaRESUMO
A wide range of recombinant proteins from Eimeria species have been reported to offer some degree of protection against infection and disease, but the specific biological function of these proteins is largely unknown. Previous studies have demonstrated a 19-kDa protein of unknown function designated SZ-1 in sporozoites and merozoites of Eimeria acervulina that can be used to confer partial protection against coccidiosis. Reverse transcriptase-polymerase chain reaction indicated that the gene for SZ-1 is expressed by all the asexual stages of Eimeria tenella. Rabbit antisera to recombinant SZ-1 recognized an approximately 19-kDa protein from extracts of E. tenella sporozoites, merozoites, sporulated oocysts, and oocysts in various stages of sporulation. Immunofluorescence antibody staining indicated specific staining of E. tenella sporozoites and merozoites. Staining was most intense in the cytoplasm of the posterior end of the parasite. The primary amino acid sequence of the gene for E. tenella SZ-1 deduced from the E. tenella genome indicated a conserved domain for the actin-regulatory protein profilin. A conserved binding site for poly-L-proline (PLP), characteristic of profilin was also observed. SZ-1 was separated from soluble extract of E. tenella proteins by affinity chromatography using a PLP ligand, confirming the ability of SZ-1 to bind PLP. SZ-1 also partially inhibited the polymerization of actin. The current results are consistent with the classification of SZ-1 as a profilin-related protein.
Assuntos
Antígenos de Protozoários/química , Proteínas Contráteis/química , Eimeria tenella/imunologia , Proteínas dos Microfilamentos/química , Actinas/antagonistas & inibidores , Actinas/química , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Sequência de Bases , Western Blotting , Galinhas , Cromatografia de Afinidade , Coccidiose/parasitologia , Coccidiose/prevenção & controle , Coccidiose/veterinária , Proteínas Contráteis/genética , Proteínas Contráteis/imunologia , Eimeria tenella/genética , Eletroforese em Gel Bidimensional , Imunofluorescência , Expressão Gênica , Soros Imunes/imunologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/imunologia , Dados de Sequência Molecular , Peptídeos/metabolismo , Doenças das Aves Domésticas/parasitologia , Doenças das Aves Domésticas/prevenção & controle , Profilinas , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Changes in proteins during sporulation of Eimeria tenella oocysts were investigated. Unsporulated E. tenella oocysts collected from cecal tissue at 7 days postinoculation were sporulated in aerated media at 28 C for 0-48 hr. Gel analysis of soluble protein extracts prepared from oocysts from their respective time points indicated the presence of 2 prominent bands with relative molecular weight (Mr) in the range of 30 kDa and making up 20% of the total protein. These 2 bands, designated as major oocyst proteins (MOPs), were absent or barely detectable by 21 hr of sporulation. MOP bands were weakly reactive with glycoprotein stain but showed no mobility shift on deglycosylation. By gel analysis it was shown that the purified MOPs consisted of 2 bands of Mr 28.7 and 30.1 kDa. However, by matrix-assisted laser deabsorption-time of flight analysis it was shown that masses were about 17% lower. Internal sequence analysis of the 28.7-kDa protein generated 2 peptides of 17 and 14 amino acids in length, consistent with a recently described protein coded by the gam56 gene and expressed in E. maxima gametocytes. Rabbit antibodies made against MOPs were localized to outer portions of sporocysts before excystment and to the apical end of in vitro-derived sporozoites. These same antibodies were found to react with bands of Mr 101 and 65 kDa by Western blot but did not recognize MOPs in soluble or insoluble sporozoite extracts. The data suggest that the MOPs are derived from part of a gametocyte protein similar to that coded by gam56 and are processed during sporulation into sporocyst and sporozoite proteins. Alternatively, the binding of anti-MOP to 101- and 65-kDa proteins may result from alternatively spliced genes as the development of parasite proceeds.
Assuntos
Eimeria tenella/química , Proteínas de Protozoários/fisiologia , Sequência de Aminoácidos , Animais , Galinhas , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Coccidiose/parasitologia , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Feminino , Glicosilação , Immunoblotting , Dados de Sequência Molecular , Oocistos/química , Oocistos/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Coelhos , Solubilidade , Organismos Livres de Patógenos EspecíficosRESUMO
Chicks fed betaine supplemented diets and infected with Eimeria acervulina and Eimeria maxima had markedly higher levels of betaine in the duodenum and mid-gut than unsupplemented, infected chicks. Uninfected chicks fed betaine exhibited almost twice the levels of betaine in the gut as infected chicks. Plasma betaine levels were lower in E. maxima-infected chicks than in E. acervulina-or Eimeria tenella-infected chicks. Betaine supplementation reversed the decrease in weight gain in E. maxima- infected chicks but had no effect on the decrease in weight gains in E acervulina- and E. tenella-infected chicks. Coccidia-infected birds on normal diets regularly exhibit increases in plasma NO(2)(+)NO(3). This increase was abolished in E.tenella-infected birds on betaine supplement. Betaine feeding did not alter this effect in E. acervulina- and E. maxima-infected birds. Results indicate that betaine supplementation has a positive effect on gut betaine levels in birds infected with E. acervulina and E. maxima. In all treatment groups, infection lowered the levels of betaine.