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A C Frasch

Publications and source records attributed to A C Frasch.

At least 73 records · Page 4Linked to original sources

Bloodstream Trypanosoma cruzi parasites from mice simultaneously express antigens that are markers of acute and chronic human Chagas disease.

Several recombinant Trypanosoma cruzi proteins previously isolated were used as antigens to analyse antibody specificities present in sera from human infections. Some parasite proteins such as SAPA (Shed Acute Phase Antigen) are antigenic early after infection. Others, like antigens 1 and 30, are antigenic mainly during the chronic phase of the infection. To understand why different proteins are antigenic at different periods of infection, specificities of antibodies present in the sera of infected mice were compared with the antigens expressed by parasites collected directly from blood. Parasites collected during the acute parasitaemia peak expressed not only antigen SAPA, but also antigens 1 and 30. However, only antibodies against SAPA were frequently observed during the early period and also in the chronic phase of murine infection. Long-lasting antibodies against SAPA were detected regardless of the mouse and parasite strains used. Furthermore, all 8 recombinant clones detected in a T. cruzi expression library with pooled sera from acutely infected mice were homologous to the SAPA gene. These results show that even though parasites from the acute parasitaemia peak in mice may express simultaneously several proteins known to be antigenic, only antibodies against SAPA were consistently detected.

Acute Disease↗

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop.

A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.

Animals↗

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is antigenic in human infections.

Antibodies against the major cysteine proteinase (cruzipain) from Trypanosoma cruzi were detected in human sera obtained from patients with chronic Chagas' disease. Not only the intact 60-kDa enzyme but also its 25-kDa self-proteolysis fragment are antigenic in vivo. Although T. cruzi antigens 13 and 36 also reacted with the apparently monospecific antiproteinase serum, the antigenicity of cruzipain to human patients is genuine, since its reactivity was not modified by the adsorption of human sera with the recombinant proteins 13 and 36.

Animals↗

Assay for detection of Trypanosoma cruzi antibodies in human sera based on reaction with synthetic peptides.

Synthetic peptides modelled according to the amino acid sequences derived from the repeated domains of five Trypanosoma cruzi antigens were used in an immunoradiometric assay to detect antibodies appearing after natural human infections. An enzyme-linked immunosorbent assay and an indirect immunofluorescence assay performed with a complex antigenic mixture from parasites were used as controls. The results indicate that the synthetic peptides were recognized by a large proportion of serum samples collected from 34 patients with Chagas' disease in Chile and point to their possible use in diagnosis.

Adolescent↗

Diagnosis of Chagas disease using recombinant DNA technology.

In this article, Alberto Frasch and Maria Reyes discuss the development of new tools for the serodiagnosis of Chagas disease (caused by Trypanosoma cruzi) and describe recombinant antigens that have proved to be of great diagnostic potential in distinguishing different stages of the disease. The work is important because different treatment strategies are needed for acute and chronic infection.

Journal Article↗

Fetal IgG specificities against Trypanosoma cruzi antigens in infected newborns.

A panel of Trypanosoma cruzi antigens produced by recombinant DNA techniques was used to analyze the IgM and IgG specificities present in sera from 22 mothers with chronic Chagas disease and their newborn infants. Ten of the newborns were congenitally infected and the other 12 children were healthy. While in most cases IgG specificities in the newborns mirrored those of their mothers, congenitally infected newborns had, in addition, IgG specificities that were undetectable in their mothers. The new IgG specificities observed most frequently were against a shed acute-phase antigen (SAPA), and less frequently, against other nine different parasite antigens. Thus, SAPA is able to identify new fetal IgGs because antibodies against this antigen are generated during the acute phase of the infection and not in their chronically infected mothers. Sera from congenital cases also had IgMs against several parasite antigens, but again, SAPA was the most frequently detected. Neither IgMs nor new IgG specificities were detected in healthy children born to mothers with Chagas disease. We conclude that individual antigens can be used to detect new IgG specificities present in the cord blood from infected newborns. Furthermore, detection of IgMs and new fetal IgGs with recombinant antigens may be used to sort out congenitally infected infants from uninfected ones, a method that might be applied to other infectious diseases.

Animals↗

Variable number of repeat units in genes encoding Trypanosoma cruzi antigens.

The genes encoding a protein antigenic during the acute phase (SAPA) and another one antigenic during the chronic phase (antigen 30) of human Chagas' disease were analyzed in 15 Trypanosoma cruzi isolates and clones collected in distant geographical regions. These two genes had tandem repeats which were present in all parasites tested. However, large differences in the length of restriction fragments were observed among isolates. This was readily explained by variations in the number of repeat units present in homologous genes. This result was confirmed after analysis of 3 members of the SAPA gene family. In the case of antigen 30, we propose that differences in the number of repeat units result in size differences in the corresponding RNAs and proteins, which explains the large size heterogeneity in otherwise homologous T. cruzi antigens.

Animals↗

Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease.

A Trypanosoma cruzi antigen which is shed into the culture medium by the trypomastigote stage of the parasite and detected in blood of acutely infected mice was cloned and characterized. We designate this antigen shed acute phase antigen (SAPA). Five protein bands with apparent molecular masses ranging from 160 to 200 kDa were detected by immunoblotting of plasma from infected mice and in supernatants of cultured trypomastigotes upon reaction with antibodies against SAPA. A serum obtained from a patient acutely infected with Chagas' disease revealed a similar set of polypeptides in supernatants of cultured trypomastigotes when tested by immunoblotting. SAPA seems thus to be a major shed protein during the acute period of the disease. Twenty-six of 28 sera from human acute cases of Chagas' disease tested reacted with SAPA. Conversely, only 8-10% of sera from chronic cases of the disease contained detectable levels of antibody against SAPA. Sera from rabbits infected with six different parasite strains all contained antibodies against SAPA. Antibodies against SAPA are detectable 15 days after the manifestation of acute Chagas' disease symptoms in humans and 15 days post-infection in sera from mice and rabbits. The nucleotide sequence of a genomic clone encoding the 3' end of the SAPA gene revealed the presence of 14 tandemly arranged 12-amino acid-long repeats. A 39-amino acid-long region that is very hydrophobic precedes the stop codon. Due to its early appearance it might be possible to design diagnostic assays which are based on SAPA for identification of recently infected cases of Chagas' disease.

Acute Disease↗

Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite.

Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite.

Amino Acid Sequence↗

Multiple Trypanosoma cruzi antigens containing tandemly repeated amino acid sequence motifs.

Chromosomal DNA from Trypanosoma cruzi, the agent of the American trypanosomiasis (Chagas' disease), was used for construction of a DNA library, employing the expression vector lambda gt11. Nine clones encoding different parasite antigens were isolated from this library by screening with an antiserum from a Chagasic patient. Nucleotide sequence analysis showed that seven out of the nine isolated clones code for antigens which contain tandemly repeated amino acid sequence motifs. Each of the seven antigens contains a unique repeat, ranging in length between 5 and 68 amino acids. The length of the repeats is highly conserved within each clone. Fusion proteins, expressed from two of the clones, reacted with a large proportion of sera collected from Chagasic patients in Argentina, Brazil and Chile. These clones appear thus to encode antigens which are shared between different strains of T. cruzi. Immunofluorescence experiments with live parasites showed that three of the antigens were detectable on the surface of trypanosomes.

Amino Acid Sequence↗

Antigenic determinants of Trypanosoma cruzi defined by cloning of parasite DNA.

A genomic DNA library from Trypanosoma cruzi, the agent of Chagas' disease, was constructed in the gt11 lambda vector and was screened with serum from a Chagasic patient. Out of 53 positive clones, 23 plaques were purified to homogeneity and 10 different groups were defined by cross-hybridization experiments and by reaction of antibodies selected with products from each recombinant clone. Native T. cruzi proteins of molecular mass ranging from 85 to larger than 205 kDa that share antigenic determinants with products of the recombinant clones were observed in Western blots of parasite extracts. Some of the native proteins were detected in the trypomastigote stage of the parasite, while others were present in epimastigotes as well. The latter result was confirmed for some recombinant clones by hybridization of the cloned DNA with Northern blots of parasite RNA. Clones from each group reacted differently with nine sera from rabbits infected with several T. cruzi strains as well as with eight sera from human patients. Clone 7 was detected by all rabbit sera but not by three human sera. Conversely, clones 1, 2 and 30 were detected by all human sera but failed to be detected by most rabbit sera. We conclude that several proteins from T. cruzi are antigenically active during infection and that some of them differ in their ability to generate antibodies in rabbit or human infections.

Animals↗

Trypanosoma cruzi isolates from Argentina and Chile grouped with the aid of DNA probes.

Fifty-two isolates and several clones from Trypanosoma cruzi, the agent of Chagas' disease, were analyzed using cloned minicircles or total kinetoplast DNA as probes. Isolates were obtained from triatomines, guinea pigs and infected humans in the Central and Northern regions of Argentina and the North of Chile. 35% of all the randomly selected isolates could be identified with one cloned minicircle probe. This widely distributed T. cruzi group was detected on both sides of the Andes mountain range (Argentina and Chile) in Triatoma infestans as well as in human infections. Most of the other isolates could be grouped with four kinetoplast DNAs as probes, but their geographical distribution seems to be restricted as compared with the one mentioned above. These results confirm the heterogeneity of T. cruzi subspecies in nature and the usefulness of DNA probes to group them.

Animals↗

Sequence diversity in the kinetoplast DNA minicircles of Trypanosoma cruzi.

Minicircles are the most abundant component of the mitochondrially located kinetoplast DNA in the members of the order Kinetoplastida. Minicircle sequences differ among most trypanosomatid species. To learn about the molecular mechanisms that give rise to this diversity, we sequenced a complete minicircle (pTckAWP-2) and two homologous but polymorphic minicircle fragments isolated from different Trypanosoma cruzi clones. Comparison of these sequences revealed 23 point mutations, 19 of which were transitions. A single base pair insertion was also detected in one of the two minicircle fragments sequenced. Analysis of pTckAWP-2 sequence showed the following features: the presence of four internal 118 base pairs conserved regions with 80% or higher homology; the fact that these four conserved regions also differed mainly by point mutations, although in this case a bias in favor of transversions was observed; the existence in each of these four regions of the highly conserved 13 bp sequence 5'GGGGTTGGTGTAA3', detected in all trypanosomatid minicircles, which is thought to be the origin of replication; and the presence of several direct and inverted repeat sequences of 8 base pairs or longer, scattered throughout the minicircle molecule. Comparison of the T. cruzi conserved minicircle region with that of other trypanosomatids showed a higher homology of T. cruzi with T. lewisi, another stercorarian trypanosome, than with African trypanosomes or Leishmania.

Animals↗

Trypanosoma cruzi: structure and transcription of kinetoplast DNA maxicircles of cloned stocks.

Restriction endonuclease mapping of the Trypanosoma cruzi kinetoplast DNA maxicircle was performed in nine cloned stocks using maxicircle probes from T. brucei. Analysis of the maxicircle 13-15-kbp encoding region allowed cloned stocks to be divided into three groups: A, B, and C. Parasites from groups A and B had 3% sequence divergence while parasites from group C showed 16-17% sequence divergence with regard to parasites from groups A and B. Cross-hybridization experiments demonstrated that the 23-25-kbp maxicircle divergent region was similar in sequence in group A and B, but different in group C parasites. The high homology of the T. cruzi and T. brucei encoding regions allowed the use of T. brucei probes to detect T. cruzi transcripts, whose overall picture did not differ for parasites from any of the nine assayed clones.

Animals↗

Damage of Trypanosoma cruzi deoxyribonucleic acid by nitroheterocyclic drugs.

Kinetoplast DNA (kDNA) from Trypanosoma cruzi epimastigotes pretreated with the trypanocidal drugs nifurtimox (10 or 100 microM) or benznidazole (38 or 380 microM) showed an increased number of strand-breaks, as revealed by (a) trapping of alkali-denatured kDNA by nitrocellulose filters and (b) electrophoresis in an alkaline 2% agarose gel. Similar damage was observed in nuclear DNA (nDNA), as detected by centrifugation in an alkaline-sucrose gradient. DNA damage was reversible since reincubation in fresh medium for 24 hr restored filtration and electrophoretic and sedimentation patterns to normal.

Animals↗