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P Buscher - One of the best experts on this subject based on the ideXlab platform.
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recombinant rotat 1 2 Variable Surface Glycoprotein as antigen for diagnosis of trypanosoma evansi in dromedary camels
International Journal for Parasitology, 2005Co-Authors: Veerle Lejon, Phelix A O Majiwa, Filip Claes, D Verloo, M Maina, T Urakawa, P BuscherAbstract:The transcript encoding a predominant Trypanosoma evansi Variable Surface Glycoprotein RoTat 1.2 was cloned and expressed as a recombinant protein in Spodoptera frugiperda and Trichoplusia ni (insect) cells. Its potential as an antigen for specific detection of antibody in serum of dromedary camels affected by surra, was evaluated. In ELISA, the reactivity of the recombinant RoTat 1.2 VSG was similar to that of native RoTat 1.2 VSG. An indirect agglutination reagent was therefore prepared by coupling the recombinant RoTat 1.2 VSG onto latex particles. The performance of the latex agglutination test was evaluated on camel sera, and compared with the performance of CATT/T. evansi and LATEX/T. evansi tests, using the immune trypanolysis assay with T. evansi RoTat 1.2 as a reference test. The relative sensitivity and specificity of the latex coated with recombinant RoTat 1.2 VSG, using a 1:4 serum dilution, were respectively, 89.3 and 99.1%. No differences were observed between the performance of latex coated with recombinant RoTat 1.2 VSG and LATEX/T. evansi or CATT/T. evansi. Here, we describe the successful use of the recombinant RoTat 1.2 VSG for detection of specific antibodies induced by T. evansi infections.
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Variable Surface Glycoprotein rotat 1 2 pcr as a specific diagnostic tool for the detection of trypanosoma evansi infections
Kinetoplastid Biology and Disease, 2004Co-Authors: Filip Claes, Phelix A O Majiwa, Magda Radwanska, Toyo Urakawa, Bruno Goddeeris, P BuscherAbstract:Background: Based on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR). Results: This PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml. Conclusion: PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum.
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the expression of rotat 1 2 Variable Surface Glycoprotein vsg in trypanosoma evansi and t equiperdum
Veterinary Parasitology, 2003Co-Authors: Filip Claes, Phelix A O Majiwa, Theo Baltz, Bruno Goddeeris, D Verloo, D T De Waal, P BuscherAbstract:Abstract In order to define whether the Variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.
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general expression of rotat 1 2 Variable antigen type in trypanosoma evansi isolates from different origin
Veterinary Parasitology, 2001Co-Authors: D Verloo, E Magnus, P BuscherAbstract:Abstract The Variable Surface Glycoprotein of Trypanosoma evansi RoTat 1.2 Variable antigen type (VAT) is used as an antigen in different antibody detection assays for T. evansi. To obtain more information on the predominant character of RoTat 1.2 and its diagnostic potential in antibody detection tests, we checked its expression in 10 different T. evansi stocks and clones from different parts of the world. Cryostabilates were injected into mice and the trypanosomes of the first peak parasitaemia were screened for the presence of RoTat 1.2 by VAT specific immunofluorescence. To monitor the appearance of RoTat 1.2 specific antibodies during infection, rabbits were infected and serologically tested at different time intervals with VAT specific immune trypanolysis, CATT/T. evansi, LATEX/T. evansi and ELISA/T. evansi. Test results confirm the predominant character of RoTat 1.2.
Phelix A O Majiwa - One of the best experts on this subject based on the ideXlab platform.
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recombinant rotat 1 2 Variable Surface Glycoprotein as antigen for diagnosis of trypanosoma evansi in dromedary camels
International Journal for Parasitology, 2005Co-Authors: Veerle Lejon, Phelix A O Majiwa, Filip Claes, D Verloo, M Maina, T Urakawa, P BuscherAbstract:The transcript encoding a predominant Trypanosoma evansi Variable Surface Glycoprotein RoTat 1.2 was cloned and expressed as a recombinant protein in Spodoptera frugiperda and Trichoplusia ni (insect) cells. Its potential as an antigen for specific detection of antibody in serum of dromedary camels affected by surra, was evaluated. In ELISA, the reactivity of the recombinant RoTat 1.2 VSG was similar to that of native RoTat 1.2 VSG. An indirect agglutination reagent was therefore prepared by coupling the recombinant RoTat 1.2 VSG onto latex particles. The performance of the latex agglutination test was evaluated on camel sera, and compared with the performance of CATT/T. evansi and LATEX/T. evansi tests, using the immune trypanolysis assay with T. evansi RoTat 1.2 as a reference test. The relative sensitivity and specificity of the latex coated with recombinant RoTat 1.2 VSG, using a 1:4 serum dilution, were respectively, 89.3 and 99.1%. No differences were observed between the performance of latex coated with recombinant RoTat 1.2 VSG and LATEX/T. evansi or CATT/T. evansi. Here, we describe the successful use of the recombinant RoTat 1.2 VSG for detection of specific antibodies induced by T. evansi infections.
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Variable Surface Glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansiinfections
Kinetoplastid Biology and Disease, 2004Co-Authors: Filip Claes, Phelix A O Majiwa, Magda Radwanska, Toyo Urakawa, Bruno Goddeeris, Philip BüscherAbstract:Background Based on the recently sequenced gene coding for the Trypanosoma evansi ( T. evansi ) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR). Results This PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei , T. b. gambiense , T. b. rhodesiense , T. congolense , T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml. Conclusion PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum .
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Variable Surface Glycoprotein rotat 1 2 pcr as a specific diagnostic tool for the detection of trypanosoma evansi infections
Kinetoplastid Biology and Disease, 2004Co-Authors: Filip Claes, Phelix A O Majiwa, Magda Radwanska, Toyo Urakawa, Bruno Goddeeris, P BuscherAbstract:Background: Based on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR). Results: This PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml. Conclusion: PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum.
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the expression of rotat 1 2 Variable Surface Glycoprotein vsg in trypanosoma evansi and t equiperdum
Veterinary Parasitology, 2003Co-Authors: Filip Claes, Phelix A O Majiwa, Theo Baltz, Bruno Goddeeris, D Verloo, D T De Waal, P BuscherAbstract:Abstract In order to define whether the Variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.
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metacyclic form specific Variable Surface Glycoprotein encoding genes of trypanosoma nannomonas congolense
Gene, 1992Co-Authors: Yuki Eshita, Wallace R Fish, T Urakawa, Hiroyuki Hirumi, Phelix A O MajiwaAbstract:Abstract A complementary DNA expression library in phage λgt11 was synthesized using MRNA from in vitro-produced metacyclic forms of a clone of Trypanosoma (Nannomonas) congolense. The unamplified library was screened with antiserum from a goat immune to infection with metacyclic (m)-forms of T. congolense ILRAD Nannomonas antigen repertoire 2(ILNaR2). Of the 100 antiserum-reactive phage clones identified, 22 were analyzed further: 21 of the clones contained overlapping portions of a single transcript, while one other contained a different transcript. Northern blot analyses indicated that the sequences contained in the clones were transcribed only by m-forms of ILNaR2. Immunological and sequence analyses indicated that the two different cloned sequences encode m-form-specific Variable Surface Glycoproteins.
D J L Williams - One of the best experts on this subject based on the ideXlab platform.
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the role of anti Variable Surface Glycoprotein antibody responses in bovine trypanotolerance
Parasite Immunology, 1996Co-Authors: D J L Williams, Katherine A Taylor, J Newson, B Gichuki, Jan NaessensAbstract:: It has been reported that some breeds of cattle such as the N'Dama mount a more effective antibody response to the Variable Surface Glycoprotein coat of trypanosomes and that this may contribute to their ability to control the infection. Thus we have investigated antibody responses to Surface exposed epitopes of the Variable Surface Glycoprotein in Trypanosoma congolense-infected N'Dama (trypanotolerant) and Boran (susceptible) cattle. Similar titres and isotypes were found in both N'Damas and Borans indicating that trypanotolerance is not associated with superior antibody-mediated destruction of trypanosomes. However, significant differences in antibody responses to cryptic VSG epitopes and non-trypanosome antigens were identified. Trypanosusceptible Boran cattle had low IgG1 responses to cryptic epitopes but high IgM responses to non-trypanosome antigens such as beta-galactosidase. In contrast the N'Dama cattle had significantly higher IgG1 responses to cryptic VSG epitopes and negligible responses to beta-galactosidase. These results indicate differences in the induction of anti-trypanosome immune responses between trypanotolerant and susceptible cattle infected with T. congolense.
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Mechanisms for the elimination of potentially lytic complement-fixing Variable Surface Glycoprotein antibody-complexes inTrypanosoma brucei
Parasitology Research, 1994Co-Authors: D. C. W. Russo, D J L Williams, D. J. GrabAbstract:Live antibody-coated Trypanosoma brucei parasites remove Variable Surface Glycoprotein (VSG)-antibody complexes from their Surface upon warming to 37° 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 climination of complement-fixing VSG-antibody complexes.
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mechanisms for the elimination of potentially lytic complement fixing Variable Surface Glycoprotein antibody complexes in trypanosoma brucei
Parasitology Research, 1994Co-Authors: D. C. W. Russo, D J L Williams, D. J. GrabAbstract:Live antibody-coatedTrypanosoma brucei parasites remove Variable Surface Glycoprotein (VSG)-antibody complexes from their Surface upon warming to 37° 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 climination of complement-fixing VSG-antibody complexes.
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directional movement of Variable Surface Glycoprotein antibody complexes in trypanosoma brucei
European Journal of Cell Biology, 1993Co-Authors: D. C. W. Russo, D. J. Grab, J D Lonsdaleeccles, M K Shaw, D J L WilliamsAbstract:: 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.
Filip Claes - One of the best experts on this subject based on the ideXlab platform.
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recombinant rotat 1 2 Variable Surface Glycoprotein as antigen for diagnosis of trypanosoma evansi in dromedary camels
International Journal for Parasitology, 2005Co-Authors: Veerle Lejon, Phelix A O Majiwa, Filip Claes, D Verloo, M Maina, T Urakawa, P BuscherAbstract:The transcript encoding a predominant Trypanosoma evansi Variable Surface Glycoprotein RoTat 1.2 was cloned and expressed as a recombinant protein in Spodoptera frugiperda and Trichoplusia ni (insect) cells. Its potential as an antigen for specific detection of antibody in serum of dromedary camels affected by surra, was evaluated. In ELISA, the reactivity of the recombinant RoTat 1.2 VSG was similar to that of native RoTat 1.2 VSG. An indirect agglutination reagent was therefore prepared by coupling the recombinant RoTat 1.2 VSG onto latex particles. The performance of the latex agglutination test was evaluated on camel sera, and compared with the performance of CATT/T. evansi and LATEX/T. evansi tests, using the immune trypanolysis assay with T. evansi RoTat 1.2 as a reference test. The relative sensitivity and specificity of the latex coated with recombinant RoTat 1.2 VSG, using a 1:4 serum dilution, were respectively, 89.3 and 99.1%. No differences were observed between the performance of latex coated with recombinant RoTat 1.2 VSG and LATEX/T. evansi or CATT/T. evansi. Here, we describe the successful use of the recombinant RoTat 1.2 VSG for detection of specific antibodies induced by T. evansi infections.
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Variable Surface Glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansiinfections
Kinetoplastid Biology and Disease, 2004Co-Authors: Filip Claes, Phelix A O Majiwa, Magda Radwanska, Toyo Urakawa, Bruno Goddeeris, Philip BüscherAbstract:Background Based on the recently sequenced gene coding for the Trypanosoma evansi ( T. evansi ) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR). Results This PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei , T. b. gambiense , T. b. rhodesiense , T. congolense , T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml. Conclusion PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum .
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Variable Surface Glycoprotein rotat 1 2 pcr as a specific diagnostic tool for the detection of trypanosoma evansi infections
Kinetoplastid Biology and Disease, 2004Co-Authors: Filip Claes, Phelix A O Majiwa, Magda Radwanska, Toyo Urakawa, Bruno Goddeeris, P BuscherAbstract:Background: Based on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR). Results: This PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml. Conclusion: PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum.
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the expression of rotat 1 2 Variable Surface Glycoprotein vsg in trypanosoma evansi and t equiperdum
Veterinary Parasitology, 2003Co-Authors: Filip Claes, Phelix A O Majiwa, Theo Baltz, Bruno Goddeeris, D Verloo, D T De Waal, P BuscherAbstract:Abstract In order to define whether the Variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.
D. J. Grab - One of the best experts on this subject based on the ideXlab platform.
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Mechanisms for the elimination of potentially lytic complement-fixing Variable Surface Glycoprotein antibody-complexes inTrypanosoma brucei
Parasitology Research, 1994Co-Authors: D. C. W. Russo, D J L Williams, D. J. GrabAbstract:Live antibody-coated Trypanosoma brucei parasites remove Variable Surface Glycoprotein (VSG)-antibody complexes from their Surface upon warming to 37° 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 climination of complement-fixing VSG-antibody complexes.
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mechanisms for the elimination of potentially lytic complement fixing Variable Surface Glycoprotein antibody complexes in trypanosoma brucei
Parasitology Research, 1994Co-Authors: D. C. W. Russo, D J L Williams, D. J. GrabAbstract:Live antibody-coatedTrypanosoma brucei parasites remove Variable Surface Glycoprotein (VSG)-antibody complexes from their Surface upon warming to 37° 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 climination of complement-fixing VSG-antibody complexes.
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directional movement of Variable Surface Glycoprotein antibody complexes in trypanosoma brucei
European Journal of Cell Biology, 1993Co-Authors: D. C. W. Russo, D. J. Grab, J D Lonsdaleeccles, M K Shaw, D J L WilliamsAbstract:: 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.
