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1.
Lancet Infect Dis ; 18(9): 969-982, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30061051

RESUMO

BACKGROUND: Pfs25H-EPA is a protein-protein conjugate transmission-blocking vaccine against Plasmodium falciparum that is safe and induces functional antibodies in malaria-naive individuals. In this field trial, we assessed Pfs25H-EPA/Alhydrogel for safety and functional immunogenicity in Malian adults. METHODS: This double-blind, randomised, comparator-controlled, dose-escalation trial in Bancoumana, Mali, was done in two staggered phases, an initial pilot safety assessment and a subsequent main phase. Healthy village residents aged 18-45 years were eligible if they had normal laboratory results (including HIV, hepatitis B, hepatitis C tests) and had not received a previous malaria vaccine or recent immunosuppressive drugs, vaccines, or blood products. Participants in the pilot safety cohort and the main cohort were assigned (1:1) by block randomisation to a study vaccine group. Participants in the pilot safety cohort received two doses of Pfs25H-EPA/Alhydrogel 16 µg or Euvax B (comparator vaccine), and participants in the main cohort received Pfs25H-EPA/Alhydrogel 47 µg or comparator vaccine (Euvax B for the first, second, and third vaccinations and Menactra for the fourth vaccination). Participants and investigators were masked to group assignment, and randomisation codes in sealed envelopes held by a site pharmacist. Vials with study drug for injection were covered by opaque tape and labelled with a study identification number. Group assignments were unmasked at final study visit. The primary outcomes were safety and tolerability for all vaccinees. The secondary outcome measure was immunogenicity 14 days after vaccination in the per-protocol population, as confirmed by the presence of antibodies against Pfs25H measured by ELISA IgG and antibody functionality assessed by standard membrane feeding assays and by direct skin feeding assays. This trial is registered with ClinicalTrials.gov, number NCT01867463. FINDINGS: Between May 15, and Jun 16, 2013, 230 individuals were screened for eligibility. 20 individuals were enrolled in the pilot safety cohort; ten participants were assigned to receive Pfs25H-EPA/Alhydrogel 16 µg, and ten participants were assigned to receive comparator vaccine. 100 individuals were enrolled in the main cohort; 50 participants were assigned to receive Pfs25H-EPA/Alhydrogel 47 µg, and 50 participants were assigned to receive comparator vaccine. Compared with comparator vaccinees, Pfs25H vaccinees had more solicited adverse events (137 events vs 86 events; p=0·022) and treatment-related adverse events (191 events vs 126 events, p=0·034), but the number of other adverse events did not differ between study vaccine groups (792 vs 683). Pfs25H antibody titres increased with each dose, with a peak geometric mean of 422·3 ELISA units (95% CI 290-615) after the fourth dose, but decreased relatively rapidly thereafter, with a half-life of 42 days for anti-Pfs25H and 59 days for anti-EPA (median ratio of titres at day 600 to peak, 0·19 for anti-Pfs25H vs 0·29 for anti-EPA; p=0·009). Serum transmission-reducing activity was greater for Pfs25H than for comparator vaccine after the fourth vaccine dose (p<0·001) but not after the third dose (p=0·09). Repeated direct skin feeds were well tolerated, but the number of participants who infected at least one mosquito did not differ between Pfs25H and comparator vaccinees after the fourth dose (p=1, conditional exact). INTERPRETATION: Pfs25H-EPA/Alhydrogel was well tolerated and induced significant serum activity by standard membrane feeding assays but transmission blocking activity was not confirmed by weekly direct skin feed. This activity required four doses, and titres decreased rapidly after the fourth dose. Alternative antigens or combinations should be assessed to improve activity. FUNDING: Division of Intramural Research, National Institute of Allergy and Infectious Diseases.


Assuntos
Antimaláricos/imunologia , Antimaláricos/toxicidade , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/toxicidade , Malária Falciparum/tratamento farmacológico , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/toxicidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimaláricos/uso terapêutico , Método Duplo-Cego , Feminino , Humanos , Vacinas Antimaláricas/uso terapêutico , Malária Falciparum/epidemiologia , Masculino , Mali/epidemiologia , Pessoa de Meia-Idade , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/uso terapêutico
2.
Genes Cells ; 22(9): 825-835, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28776895

RESUMO

Raw horsemeat has the potential to induce food poisoning which often presents with diarrheal symptoms. A sample of horsemeat was found to be infected with Sarcocystis fayeri, and a 15-kDa protein isolated from the cysts of S. fayeri was found to clearly show its diarrhea-inducing activity. A nested polymerase chain reaction was used to clone the cDNA of the 15-kDa protein. The deduced amino acid sequence showed homology to actin-depolymerizing factor (ADF). A recombinant 15-kDa protein depolymerized prepolymerized actins in a test tube. The 15-kDa protein possessed conserved amino acid sequences of ADF of Toxoplasma gondii and Eimeria tenella. These characteristics indicate that the 15-kDa protein of S. fayeri belongs to the ADF/cofilin protein family. The recombinant 15-kDa protein evoked fluid accumulation in the looped ileum, resulting in diarrhea, but it did not kill the cultured fibroblast cells, macrophages or intestinal mucosal cells. In addition, the culture supernatant of the macrophages treated with the recombinant 15-kDa protein killed the fibroblast L929 cells. This fact indicates that ADF of S. fayeri induced cytotoxic substances, such as tumor necrosis factor-α, according to the published reports. Although further experiments are needed now to elucidate the enterotoxic mechanism of S. fayeri's ADF, our findings may offer new insight into research on parasites and parasite-instigated food poisoning.


Assuntos
Fatores de Despolimerização de Actina/toxicidade , Diarreia/parasitologia , Proteínas de Protozoários/toxicidade , Sarcocystis/patogenicidade , Toxinas Biológicas/toxicidade , Fatores de Despolimerização de Actina/química , Animais , Linhagem Celular , Sequência Conservada , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Domínios Proteicos , Proteínas de Protozoários/química , Coelhos , Toxinas Biológicas/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
3.
Regul Toxicol Pharmacol ; 89: 13-19, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28709686

RESUMO

Interferon alpha as the one of FDA recommended drugs for Hepatitis B virus (HBV) infection has many side effects. Targeting IFNα to the liver may be a strategy to increase its efficacy locally and may increase efficacy of IFNα-based therapy of HBV infection. We have prepared a novel liver-targeting fusion interferon (IFN-CSP) combining IFN α2b with plasmodium region I peptide and have revealed it may be an excellent candidate as a liver-targeting anti-HBV agent. In this study, we investigated the genotoxic and teratogenic effects of IFN-CSP. The genotoxicity of IFN-CSP was evaluated by using a standard battery of tests (bacterial reverse mutation assay, mouse bone marrow micronucleus assay, and mouse sperm malformation assay). The results showed that IFN-CSP did not increase the number of revertant colonies in the plates of four strains, had no marked effect on the incidence of mouse bone marrow micronucleus and did not affect sperm deformity proportion at doses up to 8.8 × 108IU/kg, which was 1128.2 folds of the maximum' clinical equivalent dosage. Meanwhile, for teratogenicity test of IFN-CSP in female SD rats at the dosage of 6.3 × 107 IU/kg, no toxicological signs were observed. These results indicated that IFN-CSP has no genotoxicity and teratogenicity under the testing conditions.


Assuntos
Vírus da Hepatite B/efeitos dos fármacos , Interferon-alfa/toxicidade , Fígado/efeitos dos fármacos , Proteínas de Protozoários/toxicidade , Testes de Toxicidade/métodos , Animais , Feminino , Testes de Mutagenicidade/métodos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/toxicidade
4.
Malar J ; 15: 186, 2016 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-27036417

RESUMO

BACKGROUND: Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). METHODS: The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. RESULTS: During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. CONCLUSION: These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.


Assuntos
Proteínas de Membrana Transportadoras/toxicidade , Isoformas de Proteínas/toxicidade , Proteínas de Protozoários/toxicidade , Proteínas Recombinantes/toxicidade , Saccharomyces cerevisiae/fisiologia , Vacúolos/fisiologia , Cloroquina/metabolismo , Malásia , Proteínas de Membrana Transportadoras/genética , Isoformas de Proteínas/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética
5.
Glycobiology ; 25(11): 1142-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26224786

RESUMO

Chagas' disease is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Since the description of Chagas'disease in 1909 extensive research has identified important events in the disease in order to understand the biochemical mechanism that modulates T. cruzi-host cell interactions and the ability of the parasite to ensure its survival in the infected host. Exactly 30 years ago, we presented evidence for the first time of a trans-sialidase activity in T. cruzi (T. cruzi-TS). This enzyme transfers sialic acid from the host glycoconjugates to the terminal ß-galactopyranosyl residues of mucin-like molecules on the parasite's cell surface. Thenceforth, many articles have provided convincing data showing that T. cruzi-TS is able to govern relevant mechanisms involved in the parasite's survival in the mammalian host, such as invasion, escape from the phagolysosomal vacuole, differentiation, down-modulation of host immune responses, among others. The aim of this review is to cover the history of the discovery of T. cruzi-TS, as well as some well-documented biological effects encompassed by this parasite's virulence factor, an enzyme with potential attributes to become a drug target against Chagas disease.


Assuntos
Doença de Chagas/parasitologia , Glicoproteínas/toxicidade , Neuraminidase/toxicidade , Proteínas de Protozoários/toxicidade , Trypanosoma cruzi/patogenicidade , Fatores de Virulência/toxicidade , Animais , Doença de Chagas/imunologia , Glicoproteínas/imunologia , Humanos , Neuraminidase/imunologia , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/imunologia , Fatores de Virulência/imunologia
6.
Biomed Res Int ; 2015: 796456, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26075262

RESUMO

Carbon nanotubes (CNTs) are used as carriers in medicine due to their ability to be functionalized with chemical substances. However, cytotoxicity analysis is required prior to use for in vivo models. The aim of this study was to evaluate the cytotoxic effect of CNTs functionalized with a 46 kDa surface protein from Entamoeba histolytica (P46-CNTs) on J774A macrophages. With this purpose, CNTs were synthesized by spray pyrolysis and purified (P-CNTs) using sonication for 48 h. A 46 kDa protein, with a 4.6-5.4 pI range, was isolated from E. histolytica HM1:IMSS strain trophozoites using an OFFGEL system. The P-CNTs were functionalized with the purified 46 kDa protein, classified according to their degree of functionalization, and characterized by Raman and Infrared spectroscopy. In vitro cytotoxicity was evaluated by MTT, apoptosis, and morphological assays. The results demonstrated that P46-CNTs exhibited cytotoxicity dependent upon the functionalized grade. Contrary to what was expected, P46-CNTs with a high grade of functionalization were more toxic to J774 macrophages than P46-CNTs with a low grade of functionalization, than P-CNTs, and had a similar level of toxicity as UP-CNT. This suggests that the nature of the functionalized protein plays a key role in the cytotoxicity of these nanoparticles.


Assuntos
Apoptose/efeitos dos fármacos , Entamoeba histolytica/química , Macrófagos/metabolismo , Nanotubos de Carbono/efeitos adversos , Proteínas de Protozoários/toxicidade , Animais , Linhagem Celular , Macrófagos/patologia , Camundongos , Nanotubos de Carbono/química , Proteínas de Protozoários/química , Proteínas de Protozoários/farmacologia
7.
Graefes Arch Clin Exp Ophthalmol ; 253(1): 65-75, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25161076

RESUMO

BACKGROUND: Proteolytic enzymes secreted by trophozoites (amoebic secretome) are suggested as the main virulence factor involved in the severity of Acanthamoeba keratitis. The degradation profile of the main glycoprotein components of anterior and posterior portions of the cornea and the cytopathic effect of secretomes on endothelial cells by contact-independent mechanism were evaluated. METHODS: Trophozoites were isolated primarily from corneal tissue samples (n = 11) and extracellular proteins were collected from axenic cell culture supernatants. The molecular weights of proteolytic enzymes were estimated by zymography. Enzymatic cleavage of laminin and fibronectin substrates by amoebic secretome was investigated and cluster analysis was applied to the proteolysis profiles. Primary cultures of endothelial cells were used in both qualitative and quantitative assays of cytophatogenicity. RESULTS: Differential patterns of proteolysis were observed among the Acanthamoeba secretomes that were analysed. The uniformity of laminin degradation contrasted with the diversity of the proteolysis profiles observed in the fibronectin substrate. Acanthamoeba secretome extracted from four clinical isolates was shown to be toxic when in contact with the endothelial cell monolayer (p < 0.01). Induction of apoptosis and membrane permeability, at different percentual values, were suggested as the main mechanisms that could induce endothelial cell death when in contact with amoebic secretome. CONCLUSIONS: Our results provide evidence that virulence factors secreted by Acanthamoeba trophozoites can be related to an increased pathogenicity pattern by an independent contact-trophozoite mechanism, through induction of endothelial cell death by apoptosis at a higher percentage than providing the lack of cell viability by the membrane-associated pore-forming toxin activity.


Assuntos
Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/enzimologia , Fibronectinas/metabolismo , Laminina/metabolismo , Proteínas de Protozoários/toxicidade , Serina Proteases/toxicidade , Tubulina (Proteína)/metabolismo , Acanthamoeba castellanii/isolamento & purificação , Acanthamoeba castellanii/patogenicidade , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Córnea/parasitologia , Eletroforese em Gel de Poliacrilamida , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteólise , Fatores de Virulência
8.
Appl Microbiol Biotechnol ; 98(10): 4347-53, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24676751

RESUMO

Some amoeboid protozoans are facultative or obligate parasites in humans and bear an enormous cytotoxic potential that can result in severe destruction of host tissues and fatal diseases. Pathogenic amoebae produce soluble pore-forming polypeptides that bind to prokaryotic and eukaryotic target cell membranes and generate pores upon insertion and oligomerization. This review summerizes the current knowledge of such small protein toxins from amoebae, compares them with related proteins from other species, focuses on their three-dimensional structures, and gives insights into divergent activation mechanisms. The potential use of pore-forming toxins in biotechnology will be briefly outlined.


Assuntos
Amoeba/química , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Proteínas de Protozoários/toxicidade , Animais , Biotecnologia/métodos , Humanos , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/química , Conformação Proteica , Proteínas de Protozoários/química
9.
Vaccine ; 26(48): 6143-50, 2008 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-18804135

RESUMO

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, PfAMA1 ectodomain (amino acid 25-545, FVO strain) was produced in Pichia pastoris by 35L scale fed batch fermentation under current Good Manufacturing Practice (cGMP). Fermentation was followed by a three-step chromatographic purification procedure resulting in a yield of 5.8g of purified protein. As judged by size exclusion chromatography, the cGMP-product comprised >95% PfAMA1 monomer, the remainder being predominantly PfAMA1 dimer. In SDS-PAGE two main bands of 68 and 70kDa and some minor bands were evident. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident; less than 15% of the protein had this internal cleavage. By mass-spectrometric analysis, all bands analyzed in overloaded SDS-PAGE gels comprised PfAMA1 derived products. The protein was quantitatively bound by immobilized 4G2, a monoclonal antibody reactive with a reduction sensitive conformational determinant. The lyophilized product was stable for over 1 year. Immunopotency did not diminish, and storage did not lead to alterations in the behaviour of the protein upon formulation with adjuvants selected for Phase I clinical evaluation. These formulations also showed no pharmacotoxicity in rabbits. The final product conformed to preset criteria and was judged suitable for use in human clinical trials.


Assuntos
Antígenos de Protozoários/biossíntese , Indústria Farmacêutica/normas , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/normas , Proteínas de Membrana/biossíntese , Proteínas de Membrana/normas , Pichia/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/normas , Adjuvantes Imunológicos , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/toxicidade , Western Blotting , Clonagem Molecular , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Fermentação , Liofilização , Cobaias , Vacinas Antimaláricas/toxicidade , Masculino , Espectrometria de Massas , Proteínas de Membrana/toxicidade , Camundongos , Dados de Sequência Molecular , Pichia/metabolismo , Plasmodium falciparum/imunologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/toxicidade , Controle de Qualidade , Coelhos , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/normas , Vacinas Sintéticas/toxicidade
10.
PLoS One ; 3(8): e2940, 2008 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-18698359

RESUMO

BACKGROUND: Apical Membrane Antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. This is the first reported use in humans of an investigational vaccine, AMA1-C1/Alhydrogel, with the novel adjuvant CPG 7909. METHODS: A phase 1 trial was conducted at the University of Rochester with 75 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine. Participants were sequentially enrolled and randomized within dose escalating cohorts to receive three vaccinations on days 0, 28 and 56 of either 20 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 15), 80 microg of AMA1-C1/Alhydrogel (n = 30), or 80 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 30). RESULTS: Local and systemic adverse events were significantly more likely to be of higher severity with the addition of CPG 7909. Anti-AMA1 immunoglobulin G (IgG) were detected by enzyme-linked immunosorbent assay (ELISA), and the immune sera of volunteers that received 20 microg or 80 microg of AMA1-C1/Alhydrogel+CPG 7909 had up to 14 fold significant increases in anti-AMA1 antibody concentration compared to 80 microg of AMA1-C1/Alhydrogel alone. The addition of CPG 7909 to the AMA1-C1/Alhydrogel vaccine in humans also elicited AMA1 specific immune IgG that significantly and dramatically increased the in vitro growth inhibition of homologous parasites to levels as high as 96% inhibition. CONCLUSION/SIGNIFICANCE: The safety profile of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine is acceptable, given the significant increase in immunogenicity observed. Further clinical development is ongoing. TRIAL REGISTRATION: ClinicalTrials.gov NCT00344539.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/toxicidade , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Adolescente , Adulto , Hidróxido de Alumínio/toxicidade , Animais , Antígenos de Protozoários/toxicidade , Humanos , Proteínas de Membrana/imunologia , Proteínas de Membrana/toxicidade , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/toxicidade , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/toxicidade , Segurança
11.
Int J Biochem Cell Biol ; 40(11): 2442-51, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18586550

RESUMO

Recently, we found that inhibition of putrescine synthesis by ornithine decarboxylase (ODC) significantly increased Trichomonas vaginalis adherence mediated by protein adhesins. Surprisingly and unexpectedly, trichomonal contact-dependent cytotoxicity was absent. Therefore, a role for polyamine depletion on regulation of T. vaginalis cytotoxicity mediated by the cysteine proteinase (CP) of 65-kDa, CP65, was investigated. We performed cytotoxicity and cell-binding assays followed by zymograms, as well as Western blot and indirect immunofluorescence assays using specific anti-CP65 antibodies to detect CP65. Trichomonads grown in the presence of the ODC inhibitor, 1-4-diamino-2-butanone (DAB) had lower levels of cytotoxicity that corresponded with diminished CP65 proteolytic activity when compared to untreated organisms handled identically. Likewise, semiquantitative and qRT-PCR as well as Western blot and immunofluorescence assays showed decreased amounts of tvcp65 mRNA and CP65 protein in DAB-treated parasites. These effects were reversed by addition of exogenous putrescine. These data show a direct link between polyamine metabolism and expression of the cytotoxic CP65 proteinase involved in trichomonal host cellular damage.


Assuntos
Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/toxicidade , Poliaminas/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/toxicidade , Tricomoníase , Trichomonas vaginalis/enzimologia , Animais , Cisteína Endopeptidases/genética , Regulação para Baixo , Humanos , Proteínas de Protozoários/genética , Tricomoníase/metabolismo , Tricomoníase/patologia , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/patogenicidade
12.
Microbes Infect ; 9(14-15): 1597-605, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18023389

RESUMO

Several cysteine proteinases (CPs) participate in the virulence of Trichomonas vaginalis. One of them is a 65kDa CP, CP65, involved in cytotoxicity. The aim of this work was to investigate the effect of iron on the trichomonal CP65-dependent cytotoxicity using parasites grown under distinct iron concentrations. Cytotoxicity and cell-binding assays, and zymograms were performed. At the highest iron concentration (250 microM), parasites exhibited the lowest levels of cytotoxicity and less CP65 proteolytic activity. Other cations in the culture medium did not affect the trichomonal CP65-dependent cytotoxicity as iron did. Another four trichomonad fresh isolates presented similar iron negative effect over cytotoxicity. Western blot and RT-PCR experiments also showed reduction in the amount of protein and transcript of CP65 in trichomonads grown under iron-rich conditions, as compared with parasites grown in normal and iron-depleted media. Indirect immunofluorescence using the anti-CP65 antibody showed that parasites grown in iron-rich medium expressed less CP65 than those grown in normal and iron-depleted media. Cytotoxicity inhibition experiments with the anti-CP65 antibody confirmed the iron negative effect over the CP65-dependent cytotoxicity. In conclusion, our data show that iron specifically down-regulates proteolytic activity, expression, and transcription of CP65, negatively affecting trichomonal cytotoxicity in vitro.


Assuntos
Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/toxicidade , Regulação para Baixo , Ferro/farmacologia , Trichomonas vaginalis/patogenicidade , Animais , Meios de Cultura , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Regulação da Expressão Gênica , Células HeLa , Humanos , Ferro/metabolismo , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/toxicidade , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/crescimento & desenvolvimento
13.
Int Immunol ; 18(10): 1487-97, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16943259

RESUMO

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) is a virulent molecule specific for tachyzoites of T. gondii. The expression of T.g.HSP70 rapidly increases just before death of the host, indicating that T.g.HSP70 functions as a danger signal during lethal acute T. gondii infection. In the present study, T.g.HSP70 was proven to be capable of inducing lethal anaphylactic reaction in T. gondii-infected wild-type (WT) mice. Anaphylactic reaction appeared within the first hour after intraperitoneal injection of T.g.HSP70 and was characterized by a series of consequent symptoms until death. T.g.HSP70-induced anaphylactic reaction was not observed in IFN-gamma knockout (GKO) mice, indicating the involvement of IFN-gamma in the reaction. The anaphylactic reaction was transferable to GKO mice by splenocytes but not serum from infected WT mice. Also, this reaction occurred in B cell-deficient mice, indicating that T.g.HSP70-induced anaphylactic reaction occurred through an Ig-independent pathway. The messenger RNA (mRNA) expression of IFN-gamma increased significantly in splenocytes from T. gondii-infected WT mice after T.g.HSP70 injection. Furthermore, the mRNA expression of platelet-activating factor (PAF) acetylhydrolase in WT, but not GKO mice, distinctly increased during the occurrence of T.g.HSP70-induced anaphylactic reaction, indicating the involvement of PAF in T.g.HSP70-induced anaphylactic reaction. Treatment with PAF receptor antagonist rescued WT mice from the anaphylactic reaction. These data demonstrated the involvement of IFN-gamma-dependent PAF activation in T.g.HSP70-induced anaphylactic reaction.


Assuntos
Anafilaxia/imunologia , Proteínas de Choque Térmico HSP70/toxicidade , Interferon gama/imunologia , Proteínas de Protozoários/toxicidade , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Anafilaxia/induzido quimicamente , Anafilaxia/tratamento farmacológico , Anafilaxia/etiologia , Anafilaxia/patologia , Animais , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/farmacologia , Interferon gama/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/imunologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/farmacologia , Compostos de Piridínio/farmacologia , Compostos de Piridínio/uso terapêutico , Toxoplasmose Animal/complicações , Toxoplasmose Animal/tratamento farmacológico , Toxoplasmose Animal/patologia
14.
Res Microbiol ; 154(2): 85-90, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12648722

RESUMO

Pfiesteria piscicida and Pfiesteria shumwayae are estuarine dinoflagellates thought to be responsible for massive fish deaths and associated human illnesses in the southeastern United States. These dinoflagellates are described as having a complex life cycle involving flagellated zoospores, cysts, and amoeboid stages. Although no Pfiesteria toxin has been identified, certain strains of these dinoflagellates are thought to produce a water-soluble toxin that can kill fish and cause human illness. Recent reports show no evidence for amoeboid stages and indicate that a much more simplified life cycle exists. In addition, researchers have shown that P. shumwayae only kills fish through direct contact that does not necessarily involve the production of one or more toxins. This review summarizes these and other recent findings with an emphasis on establishing basic facts regarding the toxicity and life history of Pfiesteria dinoflagellates.


Assuntos
Dinoflagelados , Doenças dos Peixes/parasitologia , Pfiesteria piscicida , Infecções Protozoárias em Animais/parasitologia , Animais , Dinoflagelados/crescimento & desenvolvimento , Dinoflagelados/metabolismo , Dinoflagelados/patogenicidade , Ecossistema , Peixes , Humanos , Estágios do Ciclo de Vida , Pfiesteria piscicida/crescimento & desenvolvimento , Pfiesteria piscicida/metabolismo , Pfiesteria piscicida/patogenicidade , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/toxicidade , Toxinas Biológicas/metabolismo , Toxinas Biológicas/toxicidade
15.
Exp Parasitol ; 102(3-4): 178-86, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12856314

RESUMO

In this study we have reported the detailed characterization of a 58 kDa excretory-secretory product (ESP) of Giardia lamblia. The method of purification has been simplified which has improved the purification fold as well as the yield of the ESP. The binding efficacy of disialoganglioside (GD2) to the purified ESP was found to be maximum among all other gangliosides used. The N-terminal sequence of the immunoreactive 29 kDa peptide obtained from partial tryptic digest of the ESP was found to be AD-FVPQVST. The IgG against the purified ESP (IgGES) showed cross-reactivity with the binding subunit of the commercially available cholera toxin and also with two protein bands of western cottonmouth moccasin snake toxin. The ESP could accumulate fluid in the intestine of sealed adult mice and also induce morphological changes in HEp-2 cells. The crude extract of G. lamblia trophozoites preincubated with Escherichia coli revealed 8-fold augmentation in the cytopathic activity on HEp-2 cells as compared to that of crude preparation from trophozoites only.


Assuntos
Giardia lamblia/metabolismo , Proteínas de Protozoários/isolamento & purificação , Toxinas Biológicas/isolamento & purificação , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia em Gel , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Gangliosídeos/metabolismo , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Glicoproteínas/toxicidade , Imunoglobulina G/imunologia , Intestino Delgado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/toxicidade , Coelhos , Toxinas Biológicas/química , Toxinas Biológicas/metabolismo , Toxinas Biológicas/toxicidade
17.
FEBS Lett ; 385(1-2): 96-100, 1996 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-8641476

RESUMO

Amoebapores are cytolytic peptides of Entamoeba histolytica which function by the formation of ion channels in target cell membranes. Three isoforms (amoebapore A, B, and C) exist in amoebic cytoplasmic granules. They are composed of 77 amino acid residues arranged in four alpha-helical domains. In order to analyze the structure-function relationships, 15 synthetic peptides of 24-25 residues were constructed based on the assumption that the third helix is the membrane-penetrating domain and on the previous finding that positively charged residues are significant for activity. Activity of these short versions of amoebapores was determined towards artificial and natural targets, such as liposomes, bacteria, erythrocytes and a human tumor cell line. It was found that some of the novel peptides were highly active and showed a broader activity spectrum compared to the parent molecules.


Assuntos
Bactérias/efeitos dos fármacos , Entamoeba histolytica/química , Canais Iônicos , Proteínas de Membrana/farmacologia , Proteínas de Membrana/toxicidade , Proteínas de Protozoários/farmacologia , Proteínas de Protozoários/toxicidade , Sequência de Aminoácidos , Animais , Bactérias/crescimento & desenvolvimento , Citotoxinas , Difusão , Eritrócitos , Humanos , Lipossomos , Proteínas de Membrana/síntese química , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Peptídeos/toxicidade , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/síntese química , Relação Estrutura-Atividade , Linfócitos T , Células Tumorais Cultivadas
18.
J Immunol ; 156(5): 1886-96, 1996 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8596041

RESUMO

In this study we demonstrate that glycosylphosphatidylinositol (GPI) of malaria parasite origin directly increases cell adhesion molecule expression in purified HUVECs in a dose- and time-dependent manner, resulting in a marked increase in parasite and leukocyte cytoadherence to these target cells. The structurally related glycolipids dipalmitoyl-phosphatidylinositol and iM4 glycoinositolphospholipid of Leishmania mexicana had no such activity. Malarial GPI exerts this effect by activation of an endogenous GPI-based signal transduction pathway in endothelial cells. GPI induces rapid onset tyrosine phosphorylation of multiple intracellular substrates within 1 min of addition to cells in a dose-dependent manner. This activity can be blocked by the protein tyrosine kinase-specific antagonist herbimycin A, genistein, and tyrphostin. These tyrosine kinase antagonists also inhibit GPI-mediated up-regulation of adhesion expression and parasite cytoadherence. GPI-induced up-regulation of adhesion expression and parasite cytoadherence can also be blocked by the NF kappa B/c-rel antagonist pyrrolidine-dithiocarbamate, suggesting the involvement of this family of transcription factors in GPI-induced adhesin expression. The direct activation of endothelial cells by GPI does not require the participation of TNF or IL-1. However, GPI is also responsible for the indirect pathway of increased adhesin expression mediated by TNF and IL-1 output from monocytes/macrophages. Total parasite extracts also up-regulate adhesin expression and parasite cytoadherence in HUVECs, and this activity is blocked by a neutralizing mAb to malaria GPI, suggesting that GPI is the dominant agent of parasite origin responsible for this activity. Thus, a parasite-derived GPI toxin activates vascular endothelial cells by tyrosine kinase-mediated signal transduction, leading to NF kappa B/c-rel activation and downstream expression of adhesins, events that may play a central role in the etiology of cerebral malaria.


Assuntos
Moléculas de Adesão Celular/biossíntese , Endotélio Vascular/metabolismo , Glicosilfosfatidilinositóis/toxicidade , Leucócitos Mononucleares/imunologia , Plasmodium falciparum/imunologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/imunologia , Regulação para Cima/imunologia , Animais , Adesão Celular/imunologia , Moléculas de Adesão Celular/efeitos dos fármacos , Selectina E/biossíntese , Selectina E/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/imunologia , Glicosilfosfatidilinositóis/imunologia , Glicosilfosfatidilinositóis/isolamento & purificação , Interações Hospedeiro-Parasita , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Fosforilação , Plasmodium falciparum/química , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/toxicidade , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/efeitos dos fármacos
19.
J Immunol ; 156(5): 1897-1907, 1996 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8596042

RESUMO

In this study, we demonstrate that glycosylphosphatidylinositol (GPI) is a major toxin of Plasmodium falciparum origin responsible for nitric oxide (NO) production in host cells. Purified malarial GPI is sufficient to induce NO release in a time- and dose-dependent manner in macrophages and vascular endothelial cells, and regulates inducible NO synthase expression in macrophages. GPI-induced NO production was blocked by the NO synthase-specific inhibitor L-N-monomethylarginine. GPI also synergizes with IFN-gamma in regulating NO production. The structurally related molecules dipalmitoylphosphatidylinositol and iM4 glycoinositolphospholipid from Leishmania mexicana had no such activity, and the latter antagonized IFN-gamma-induced NO output. GPI activates macrophages by initiating an early onset tyrosine kinase-mediated signaling process, similar to that induced by total parasite extracts. The tyrosine kinase antagonists tyrphostin and genistein inhibited the release of NO by parasite extracts and by GPI, alone or in combination with IFN-gamma, demonstrating the involvement of one or more tyrosine kinases in the signaling cascade. GPI-induced NO release was also blocked by the protein kinase C inhibitor calphostin C, demonstrating a role for protein kinase C in GPI-mediated cell signaling, and by pyrrolidine dithiocarbamate, indicating the involvement of the NF-kappa B/c-rel family of transcription factors in cell activation. A neutralizing mAb to malarial GPI inhibited NO production induced by GPI and total malarial parasite extracts in human vascular endothelial cells and murine macrophages, indicating that GPI is a necessary agent of parasite origin in parasite-induced NO output. Thus, in contrast to dipalmitoylphosphatidylinositol and glycoinositolphospholipids of Leishmania, malarial GPI initiates a protein tyrosine kinase- and protein kinase C-mediated signal transduction pathway, regulating inducible NO synthase expression with the participation of NF-kappa B/c-rel, which leads to macrophage and vascular endothelial cell activation and downstream production of NO. These events may play a role in the etiology of severe malaria.


Assuntos
Endotélio Vascular/enzimologia , Glicosilfosfatidilinositóis/toxicidade , Macrófagos/enzimologia , Óxido Nítrico Sintase/biossíntese , Plasmodium falciparum/metabolismo , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/farmacologia , Arginina/análogos & derivados , Arginina/farmacologia , Relação Dose-Resposta Imunológica , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Indução Enzimática/imunologia , Glicolipídeos/isolamento & purificação , Glicosilfosfatidilinositóis/imunologia , Glicosilfosfatidilinositóis/isolamento & purificação , Humanos , Leishmania mexicana/química , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , NG-Nitroarginina Metil Éster , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Plasmodium falciparum/química , Plasmodium falciparum/imunologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/toxicidade
20.
Mol Microbiol ; 14(5): 895-904, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7715451

RESUMO

Three peptides with pore-forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed-phase high-performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic alpha-helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore-forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram-positive bacteria. The amoebapores represent a distinct family of membrane-active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.


Assuntos
Entamoeba histolytica/química , Entamoeba histolytica/genética , Canais Iônicos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Bactérias/efeitos dos fármacos , Sequência de Bases , Membrana Celular/efeitos dos fármacos , Clonagem Molecular , Reações Cruzadas , Grânulos Citoplasmáticos/química , DNA de Protozoário/genética , Proteínas de Membrana/toxicidade , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas de Protozoários/toxicidade , Homologia de Sequência de Aminoácidos
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