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1.
Clin Microbiol Infect ; 15(5): 422-6, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19489925

RESUMO

Neurological manifestations of Lyme disease (or neuroborreliosis) occur variably and while it is clear that Borrelia burgdorferi can invade the nervous system, how it does so is not well understood. Pathogen penetration through the blood brain barrier (BBB) is often influenced by calcium signaling in the endothelial cells triggered by extracellular host-pathogen interactions. We examined the traversal of B. burgdorferi across the human BBB using in vitro model systems constructed of human brain microvascular endothelial cells (HBMEC) grown on Costar Transwell inserts. Pretreatment of the cell monolayers with BAPTA-AM (an intracellular calcium chelator) or phospholipase C (PLC) inhibitor U73122 inhibited B. burgdorferi transmigration. By 5 h, BAPTA-AM significantly inhibited (82-99%; p <0.017) spirochete traversal of HBMEC compared to DMSO controls. Spirochete traversal was almost totally blocked (> or =99%; p <0.017) after pretreatment with the PLC-beta inhibitor U73122 as a result of barrier tightening based on electric cell-substrate impedance sensing (ECIS). The data suggest a role for calcium signaling in CNS spirochete invasion through endothelial cell barriers.


Assuntos
Borrelia burgdorferi/patogenicidade , Encéfalo/microbiologia , Sinalização do Cálcio , Células Endoteliais/microbiologia , Linhagem Celular , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Humanos , Pirrolidinonas/farmacologia
2.
Int J Parasitol ; 36(5): 601-5, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16600247

RESUMO

The manifestations of Lyme disease, caused by Ixodes spp. tick-transmitted Borrelia burgdorferi, range from skin infection to bloodstream invasion into the heart, joints and nervous system. The febrile infection human granulocytic anaplasmosis is caused by a neutrophilic rickettsia called Anaplasma phagocytophilum, also transmitted by Ixodes ticks. Previous studies suggest that co-infection with A. phagocytophilum contributes to increased spirochetal loads and severity of Lyme disease. However, a common link between these tick-transmitted pathogens is dissemination into blood or tissues through blood vessels. Preliminary studies show that B. burgdorferi binds and passes through endothelial barriers in part mediated by host matrix metalloproteases. Since neutrophils infected by A. phagocytophilum are activated to release bioactive metalloproteases and chemokines, we examined the enhanced B. burgdorferi transmigration through vascular barriers with co-infection in vitro. To test whether endothelial transmigration is enhanced with co-infection, B. burgdorferi and A. phagocytophilum-infected neutrophils were co-incubated with EA.hy926 cells (HUVEC-derived) and human brain microvascular endothelial cells in Transwell cultures. Transmigration of B. burgdorferi through endothelial cell barriers was determined and endothelial barrier integrity was measured by transendothelial electrical resistivity. More B. burgdorferi crossed both human BMEC and EA.hy926 cells in the presence of A. phagocytophilum-infected neutrophils than with uninfected neutrophils without affecting endothelial cell integrity. Such a mechanism may contribute to increased blood and tissue spirochete loads.


Assuntos
Anaplasma phagocytophilum/fisiologia , Borrelia burgdorferi/fisiologia , Ehrlichiose/complicações , Doença de Lyme/complicações , Neutrófilos/microbiologia , Barreira Hematoencefálica/microbiologia , Células Cultivadas , Técnicas de Cocultura , Ehrlichiose/microbiologia , Células Endoteliais/microbiologia , Endotélio Vascular/microbiologia , Humanos , Doença de Lyme/microbiologia , Neutrófilos/fisiologia
3.
Med Hypotheses ; 59(1): 1-10, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12160674

RESUMO

It is proposed that peripheral blood fibrocytes will be a new and important player in the pathogenesis of Lyme disease. Peripheral blood fibrocytes are a circulating leukocyte subpopulation that: (a) express collagen; (b) are an abundant source of cytokines, chemoattractants and growth factors; and (c) are able to recruit and activate naive T-cells and memory T-cells. We predict that peripheral blood fibrocytes will represent a new and important antigen-presenting cell which will play an important role in directing the immune response from the pathogenic Th1 to the protective Th2 response cell in Borrelia infections.


Assuntos
Doença de Lyme/sangue , Linfócitos T/imunologia , Borrelia burgdorferi/fisiologia , Humanos , Doença de Lyme/imunologia , Linfócitos T/microbiologia
4.
J Parasitol ; 87(5): 1064-70, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11695366

RESUMO

Tritrichomonas foetus is a common, sexually transmitted, protozoan parasite of cattle. It has an essential requirement for iron, which it obtains from host lactoferrin. However, specific lactoferrin-binding protein receptors have not yet been identified in T. foetus. To differentiate specific and nonspecific binding of lactoferrin, lactoferrin affinity chromatography and Western blotting was used to identify metabolically or surface-labeled T. foetus lactoferrin-binding proteins. Bovine lactoferrin was shown to bind more efficiently than human lactoferrin, and each of these bound much better than bovine transferrin. This is relevant because T. foetus is both species-specific and only infects the mucosal surface of the reproductive tract, which has little transferrin. Whereas the majority of lactoferrin binding was specific, competitive inhibition studies showed that nonspecific, charge-related binding of lactoferrin to T. foetus may also be involved. In the presence of bovine cervical mucus, binding of lactoferrin to T. foetus was diminished, suggesting that mucus has an effect on lactoferrin binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surface biotinylated proteins affinity-purified on lactoferrin-Sepharose showed biotinylated bands at Mr values of 22, 49, 55, 72, and 155 kDa. Because lactoferrin-binding proteins may be susceptible to digestion by T. foetus extracellular cysteine proteinases, it is suspected that the 155-kDa protein is the specific lactoferrin-binding protein and that the lower-Mr lactoferrin-binding molecules may be fragmentation products that contain the lactoferrin-binding site; however, other interpretations are clearly feasible. It is possible that there may be multiple proteins or multimers of the same protein. In summary, the data showed that binding of lactoferrin to T. foetus may be regulated by an interplay of specific receptor interactions as well as by hydrophobic and charge-related interactions.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte/metabolismo , Doenças dos Bovinos/parasitologia , Lactoferrina/metabolismo , Infecções Protozoárias em Animais , Proteínas de Protozoários/metabolismo , Tritrichomonas foetus/metabolismo , Animais , Ligação Competitiva , Western Blotting , Bovinos , Muco do Colo Uterino/metabolismo , Muco do Colo Uterino/parasitologia , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Ligação Proteica , Infecções por Protozoários/parasitologia
5.
Vet Immunol Immunopathol ; 78(3-4): 325-39, 2001 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-11292533

RESUMO

The membrane-associated form of the variable surface glycoprotein (mfVSG) from African trypanosomes is a potent macrophage activator capable of inducing production of tumor necrosis factor alpha (TNFalpha) in both bovine and murine models. Using a bovine model, we have re-investigated the hypothesis that the diacylglycerol moiety of the glycosylphosphatodylinositol (GPI) anchor is involved in macrophage activation and might be the actual parasite toxin. The anchor of the variable surface glycoprotein (VSG) was labeled with (3)H-myristic acid and VSG purified in its membrane-associated form. The dimyristylglycerol moiety of the anchor was released by phospholipase C cleavage. Integrity of the anchor and efficiency of cleavage was verified by autoradiography and methanol:hexane extraction. For analysis of biological function, bovine monocytes were used which had been incubated with bovine interferon gamma (primed) or with culture medium (unprimed). The VSG purified in its membrane-associated form was found to stimulate both primed and unprimed cells to secrete TNFalpha. The same preparation from which the dimyristylglycerol moiety had been cleaved was no longer able to stimulate unprimed cells but could still stimulate primed cells. Our data indicate that the presence of the dimyristylglycerol is not an absolute requirement for induction of TNFalpha production but can substitute for the interferon gamma priming. Therefore, we favor the hypothesis that stimulation of macrophages to secrete TNFalpha by the mfVSG is mediated by an as yet unknown trigger moiety and is facilitated by the dimyristylglycerol anchor.


Assuntos
Diglicerídeos/farmacologia , Monócitos/efeitos dos fármacos , Glicoproteínas Variantes de Superfície de Trypanosoma/farmacologia , Animais , Bovinos , Células Cultivadas , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Glicosilfosfatidilinositóis/farmacologia , Monócitos/metabolismo , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/química
7.
Mol Med ; 5(1): 46-54, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10072447

RESUMO

BACKGROUND: Borrelia Burgdorferi has a predilection for collagenous tissue and can interact with fibronectin and cellular collagens. While the molecular mechanisms of how B. burgdorferi targets connective tissues and causes arthritis are not understood, the spirochetes can bind to a number of different cell types, including fibroblasts. A novel circulating fibroblast-like cell called the peripheral blood fibrocyte has recently been described. Fibrocytes express collagen types I and III as well as fibronectin. Besides playing a role in wound healing, fibrocytes have the potential to target to connective tissue and the functional capacity to recruit, activate, and present antigen to CD4(+) T cells. MATERIALS AND METHODS: Rhesus monkey fibrocytes were isolated and characterized by flow cytometry. B. burgdorferi were incubated with human or monkey fibrocyte cultures in vitro and the cellular interactions analyzed by light and electron microscopy. The two strains of B. burgdorferi studied included JD1, which is highly pathogenic for monkeys, and M297, which lacks the cell surface OspA and OspB proteins. RESULTS: In this study, we demonstrate that B. burgdorferi binds to both human and monkey (rhesus) fibrocytes in vitro. This process does not require OspA or OspB. In addition, the spirochetes are not phagocytosed but are taken into deep recesses of the cell membrane, a process that may protect them from the immune system. CONCLUSIONS: This interaction between B. burgdorferi and peripheral blood fibrocytes provides a potential explanation for the targeting of spirochetes to joint connective tissue and may contribute to the inflammatory process in Lyme arthritis.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/microbiologia , Grupo Borrelia Burgdorferi/imunologia , Grupo Borrelia Burgdorferi/patogenicidade , Tecido Conjuntivo/imunologia , Tecido Conjuntivo/microbiologia , Animais , Células Apresentadoras de Antígenos/ultraestrutura , Células Sanguíneas/imunologia , Células Sanguíneas/microbiologia , Células Sanguíneas/ultraestrutura , Fibroblastos/imunologia , Fibroblastos/microbiologia , Fibroblastos/ultraestrutura , Humanos , Técnicas In Vitro , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Leucócitos Mononucleares/ultraestrutura , Doença de Lyme/etiologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Macaca mulatta , Microscopia Eletrônica , Fagocitose
8.
Novartis Found Symp ; 226: 199-211; discussion 211-4, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10645547

RESUMO

The malaria parasite extensively modifies the host erythrocyte. Many of these modifications are mediated by proteins exported from the parasite and targeted to specific locations within the infected erythrocyte. However, little is known about how the parasite targets proteins to specific locations beyond its own plasma membrane. Treatment of infected erythrocytes with brefeldin A results in the accumulation of many exported Plasmodium proteins into a compartment distinct from the ER. Proteins destined for the host erythrocyte membrane, the parasitophorous vacuole or inclusions within the erythrocyte cytoplasm accumulate in this novel compartment, and co-localization studies indicate that there is a single compartment per parasite. Exported proteins only accumulate in this novel compartment if brefeldin A treatment is concurrent with their synthesis. This novel compartment is probably a membrane-bound organelle located at the parasite periphery, and may be the first step in an alternative secretory pathway that specializes in the export of proteins into the host cell. Such an alternative secretory pathway raises questions about how exported proteins are differentially targeted to this novel organelle versus the ER and the fate of exported proteins after this novel organelle.


Assuntos
Eritrócitos/parasitologia , Malária/sangue , Organelas/metabolismo , Plasmodium/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo , Retículo Endoplasmático/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas de Protozoários/metabolismo , Transdução de Sinais
9.
J Eukaryot Microbiol ; 45(5): 528-34, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9783454

RESUMO

The malarial parasite dramatically affects the structure and function of the erythrocyte membrane by exporting proteins that specifically interact with the host membrane. This report describes the complete sequence and some biochemical properties of a 93-kDa Plasmodium chabaudi chabaudi protein that interacts with the host erythrocyte membrane. Approximately 40% of the deduced protein sequence consists of tandem repeats of 14 amino acids that are rich in glutamic acid residues. Comparison of the repeat sequences from two different P. c. chabaudi strains derived from the same initial isolate revealed an exact duplication of 294 nucleotides suggesting a recent unequal crossing-over event. However, in spite of this potentially high level of intragenic recombination activity, the repeat sequences from P. c. adami are rather conserved suggesting structural or functional constraints on the protein and tandem repeats. The 93-kDa protein exists in an oligomeric form as revealed by gel filtration chromatography and non-denaturing gel electrophoresis. A predominantly alpha-helical predicted secondary structure and a discrepancy between the estimated molecular sizes determined from non-denaturing gel electrophoresis and gel filtration chromatography suggest that the protein is a long rod-shaped or fibrillar, protein. Attributes shared between the 93-kDa protein, some P. falciparum proteins with glutamate-rich tandem repeats, and cytoskeletal proteins suggest that these parasite proteins function as cytoskeletal proteins that possibly stabilize the erythrocyte membrane.


Assuntos
Eritrócitos/parasitologia , Plasmodium chabaudi/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Biblioteca Gênica , Genes de Protozoários , Malária/sangue , Camundongos , Dados de Sequência Molecular , Plasmodium chabaudi/genética , Proteínas de Protozoários/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica
10.
Biochim Biophys Acta ; 1407(2): 135-45, 1998 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-9685613

RESUMO

Recently, the term MSCRAMM (microbial surface components recognizing adhesive matrix molecules), has been introduced to describe microbial molecules that recognize extracellular matrix (ECM) [1]. Here we present evidence for the presence of fibronectin-binding molecules in Borrelia burgdorferi and several other Borrelia species. Immunofluorescence studies show that plasma fibronectin is bound uniformly over the cell surface of free swimming B. burgdorferi. In addition, the spirochetes are able to bind to plasma fibronectin-coated microwell plates, an interaction that is inhibited by anti-fibronectin antibody as well as exogenous plasma fibronectin. Taken together, the data suggest that fibronectin binds to the surface of the spirochete. On Western blot-like assays, B. burgdorferi and some B. afzelii strains express a major fibronectin-binding protein (Fn-BA) with an approximate molecular mass of 52 kDa. In addition, several other major Fn-BAs were found in B. hermsii (26, 31, 33, 39, 46, 54 and 58 kDa) and B. turicatae (39, 41, 45, 50, 56, 59 and 66 kDa). Preliminary evidence suggests that fibronectin (and Fn-BA) may play a role as a molecular bridge between the spirochete and other components of the extracellular matrix.


Assuntos
Adesinas Bacterianas , Grupo Borrelia Burgdorferi/fisiologia , Fibronectinas/metabolismo , Anticorpos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ligação Competitiva , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/química , Adesão Celular/fisiologia , Imunofluorescência , Ligação Proteica/fisiologia
11.
Electrophoresis ; 19(7): 1162-70, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9662179

RESUMO

In this paper we demonstrate the power of preparative free-flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free-flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time-dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conclusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket-derived membranes from other endosomal and lysosomal compartments of African trypanosomes.


Assuntos
Eletroforese/métodos , Endocitose , Organelas/química , Trypanosoma brucei brucei/química , Animais , Biomarcadores/análise , Fracionamento Celular , Peroxidase do Rábano Silvestre , Lisossomos/química , Ratos , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/fisiologia
13.
Biochem Biophys Res Commun ; 240(3): 540-4, 1997 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-9398600

RESUMO

Cysteine proteases have been identified in parasitic protozoa including the causative agent of Chagas' disease Trypanosoma cruzi. T. cruzi lysates subjected to substrate-containing SDS-polyacrylamide gel electrophoresis exhibit major bands of proteolytic activity in the 45-55 kDa molecular mass range (cruzipain activity). Paradoxically, addition of kininogen (a cystatin-like protease inhibitor) to the lysates before electrophoresis results in the appearance of additional bands of proteolytic activity in the 160-190 kDa molecular mass range. This inhibitor-activated protease activity depends upon the reaction conditions and exhibits novel properties. For example, a 24-48 hour preincubation at low temperature (-20 degrees C optimum) greatly enhances the proteolytic activity. The results suggest that a metastable complex forms between kininogen and a cryptic 30 kDa cysteine protease from T. cruzi and that this complex participates in the activation of proteolytic activity.


Assuntos
Cisteína Endopeptidases/metabolismo , Cininogênios/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Gelatina/metabolismo , Cinética , Cininogênios/farmacologia , Peso Molecular , Temperatura
15.
FEMS Microbiol Lett ; 144(1): 39-45, 1996 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-8870250

RESUMO

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi, an invasive spirochete. B. burgdorferi has a predilection for collagenous tissue and one major clinical manifestation of the disease is arthritis. We have identified a collagenolytic activity in B. burgdorferi detergent lysates using iodinated gelatin as well as iodinated pepsinized human collagen types II and IV as protein substrates. In addition, we describe several proteolytic activities in B. burgdorferi with molecular masses greater than 200 kDa on sodium dodecyl sulfate polyacrylamide gels containing copolymerized gelatin. We propose that the collagenolytic activity of B. burgdorferi has a role in invasion, in the pathogenesis of Lyme arthritis, and perhaps also in other manifestations of Lyme borreliosis.


Assuntos
Grupo Borrelia Burgdorferi/metabolismo , Colágeno/metabolismo , Gelatina/metabolismo , Grupo Borrelia Burgdorferi/enzimologia , Grupo Borrelia Burgdorferi/patogenicidade , Gelatinases/metabolismo , Humanos , Colagenase Microbiana/análise , Especificidade por Substrato
16.
Vet Immunol Immunopathol ; 52(1-2): 65-76, 1996 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-8807777

RESUMO

A murine IgM monoclonal antibody (mAb), IL-A77, has been generated that recognises the bovine transferrin receptor (TfR) and will be a useful tool to measure the activation state of bovine lymphocytes and macrophages. The antigen is detected on immature erythroid cells and proliferating lymphocytes. It is undetectable on resting lymphocytes, but appears within 24 h after stimulation with concanavalin A (ConA) or pokeweed mitogen (PWM). Immune precipitations of lysates of both labeled activated lymphocytes and bone marrow erythroid cells showed that, similar to human TfR, the bovine receptor is a disulfide-bonded dimer of two identical chains of M(r) 97,000. A similar 97,000 M(r) protein was eluted from a column containing immobilised bovine transferrin (Tf) using conditions known to elute the human TfR, and this protein was recognised by mAb IL-A77, proving that it detected bovine TfR. Although the mAb inhibited binding of transferrin to its receptor, it did not block proliferation of Theileria parva-transformed or ConA-stimulated lymphocytes. When cells were metabolically labeled with 35S-methionine, a second 90,000-M(r) TfR band was detected in Theileria parva-transformed cells, but not in stimulated lymphocytes. This form of the TfR was not expressed on the cell surface. It may be an.


Assuntos
Anticorpos Monoclonais/química , Linfócitos B/citologia , Receptores da Transferrina/química , Receptores da Transferrina/imunologia , Theileria parva/fisiologia , Animais , Reações Antígeno-Anticorpo , Linfócitos B/imunologia , Bovinos , Divisão Celular/imunologia , Linhagem Celular Transformada , Feminino , Masculino
17.
Vet Parasitol ; 57(1-3): 189-203, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7597783

RESUMO

Current vaccines for bovine hemoparasites utilize live attenuated organisms or virulent organisms administered concurrently with antiparasitic drugs. Although such vaccines can be effective, for most hemoparasites the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selection of potentially protective antigens has traditionally made use of antibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predicts the ability of an antigen to confer protective immunity nor correlates with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immunogens should be used. Through the elaboration of cytokines, T helper 1-(Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an important role in protective immunity against bovine hemoparasites. CD4+ T cell clones specific for soluble or membrane antigens of either Theileria parva schizonts or Babesia bovis merozoites were therefore employed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatography, or a combination thereof, and fractions were tested for the ability to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. parva proteins of approximately 10 and 24 kDa. Antisera raised against the purified 24 kDa band reacted with a native schizont protein of approximately 30 kDa. Babesia bovis-specific Th cell clones tested against fractionated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of reactivity, and proteins separated by anion exchange revealed two patterns of reactivity when selected T cell clones were assayed for stimulation by antigenic fractions. Studies using a continuous-flow electrophoresis apparatus have indicated the feasibility of identifying T cell-stimulatory proteins from parasite membranes as well as from the cytosolic fraction of B. bovis merozoites. The Th cell clones reactive with these different hemoparasites expressed either unrestricted or Th1 cytokine profiles, and were generally characterized by the production of high levels of IFN-gamma. A comprehensive study of T cell and macrophage responses to defined parasite antigens will help elucidate the reasons for vaccine failure or success, and provide clues to the mechanisms of acquired immunity that are needed for vaccine development.


Assuntos
Antígenos de Protozoários/imunologia , Babesia bovis/imunologia , Babesiose/imunologia , Doenças dos Bovinos , Vacinas Protozoárias , Linfócitos T Auxiliares-Indutores/imunologia , Theileria parva/imunologia , Theileriose/imunologia , Animais , Babesiose/prevenção & controle , Linfócitos T CD4-Positivos/imunologia , Bovinos , Células Clonais , Citocinas/biossíntese , Theileriose/prevenção & controle
18.
Biochem J ; 305 ( Pt 2): 549-56, 1995 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-7832773

RESUMO

African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition observed when this putative cysteine protease inhibitor was used under these conditions. The activation of parasite cysteine proteases by commonly accepted cysteine protease inhibitors is unexpected and may have important pathological repercussions.


Assuntos
Cisteína Endopeptidases/efeitos dos fármacos , Cininogênios/farmacologia , Trypanosomatina/enzimologia , Animais , Cumarínicos/metabolismo , Dipeptídeos/metabolismo , Relação Dose-Resposta a Droga , Corantes Fluorescentes/metabolismo , Hidrólise , Cininogênios/sangue , Cininogênios/isolamento & purificação , Leishmania/enzimologia , Trypanosoma/enzimologia
19.
Parasitol Res ; 80(6): 487-92, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7528915

RESUMO

Live antibody-coated Trypanosoma brucei parasites remove variable surface glycoprotein (VSG)-antibody complexes from their surface upon warming to 37 degrees C and evade antibody-activated complement lysis by both protein synthesis-dependent and protein synthesis-independent mechanisms. The protein synthesis-dependent process follows antibody-mediated trypanosome agglutination, whereas the protein synthesis-independent mechanism can occur in the absence of trypanosome agglutination. The latter process leads to a more rapid elimination of complement-fixing VSG-antibody complexes.


Assuntos
Anticorpos Antiprotozoários/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Ativação do Complemento , Trypanosoma brucei brucei/imunologia , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologia , Aglutinação , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Epitopos , Citometria de Fluxo , Imunofluorescência , Proteínas de Protozoários/biossíntese , Trypanosoma brucei brucei/citologia
20.
Eur J Cell Biol ; 62(2): 432-41, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7925498

RESUMO

Binding of antibody, antibody fragments (Fab and F(ab)2) and biotin molecules to variable surface glycoprotein (VSG) of Trypanosoma brucei was studied by both light microscopy and fluorescence activated cell sorter (FACS) analysis. Antibodies, antibody fragments and biotin molecules were distributed over the entire parasite surface after incubation at 0 degree C. Upon warming to 37 degrees C, surface bound Fab and F(ab)2 fragments showed different rates of clearance from the parasite surface. Clearance, which in both cases followed double exponential decay kinetics, resulted from a directional movement of VSG-bound antibody complexes from both the surface of the flagellum and the cell body towards the cellular site of active endocytosis, the flagellar pocket (FP), even in the absence of antibody-mediated crosslinking of VSG. Immunofluorescence on trypanosomes permeabilized after binding, clearance and internalization, indicated the location of small amounts of antibody intracellularly, between the nucleus and the flagellar pocket. However, if a cocktail of protease inhibitors was added to the medium, larger amounts of internalized antibody could be detected within vacuoles situated between the nucleus and the flagellar pocket. Movement of antibody-VSG complexes was reversibly inhibited at temperatures below 37 degrees C and by increasing the NaCl concentration in the medium to 200 mM.


Assuntos
Anticorpos Monoclonais/análise , Região Variável de Imunoglobulina/análise , Glicoproteínas de Membrana/análise , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Biotina , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Citometria de Fluxo , Imunofluorescência , Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Cloreto de Sódio/farmacologia , Temperatura , Trypanosoma brucei brucei/metabolismo
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