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1.
J Infect Dis ; 227(11): 1322-1332, 2023 05 29.
Article in English | MEDLINE | ID: mdl-36571148

ABSTRACT

BACKGROUND: A drawback in the treatment of chronic Chagas disease (American trypanosomiasis) is the long time required to achieve complete loss of serological reactivity, the standard for determining treatment efficacy. METHODS: Antibody-secreting cells and memory B cells specific for Trypanosoma cruzi and their degree of differentiation were evaluated in adult and pediatric study participants with chronic Chagas disease before and after etiological treatment. RESULTS: T. cruzi-specific antibody-secreting cells disappeared from the circulation in benznidazole or nifurtimox-treated participants with declining parasite-specific antibody levels after treatment, whereas B cells in most participants with unaltered antibody levels were low before treatment and did not change after treatment. The timing of the decay in parasite-specific antibody-secreting B cells was similar to that in parasite-specific antibodies, as measured by a Luminex-based assay, but preceded the decay in antibody levels detected by conventional serology. The phenotype of total B cells returned to a noninfection profile after successful treatment. CONCLUSIONS: T. cruzi-specific antibodies in the circulation of chronically T. cruzi-infected study participants likely derive from both antigen-driven plasmablasts, which disappear after successful treatment, and long-lived plasma cells, which persist and account for the low frequency and long course to complete seronegative conversion in successfully treated participants.


Subject(s)
Chagas Disease , Nitroimidazoles , Trypanocidal Agents , Trypanosoma cruzi , Humans , Trypanosoma cruzi/genetics , Chagas Disease/drug therapy , Nitroimidazoles/therapeutic use , Treatment Outcome , B-Lymphocytes , Nifurtimox/therapeutic use , Persistent Infection , Trypanocidal Agents/therapeutic use , Chronic Disease
2.
J Antimicrob Chemother ; 68(2): 424-37, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23104493

ABSTRACT

OBJECTIVES: Even though the use of combined drugs has been proved to be effective in other chronic infections, assessment of combined treatment of antiparasitic drugs in human Chagas' disease has not been performed. Herein, a pilot study was conducted to evaluate the tolerance and side effects of a sequential combined treatment of two antiparasitic drugs, allopurinol and benznidazole, in the chronic phase of Trypanosoma cruzi infection. PATIENTS AND METHODS: Changes in total and T. cruzi-specific T and B cells were monitored during a median follow-up of 36 months. Allopurinol was administered for 3 months (600 mg/day) followed by 30 days of benznidazole (5 mg/kg/day) in 11 T. cruzi-infected subjects. RESULTS: The combined sequential treatment of allopurinol and benznidazole was well tolerated. The levels of T. cruzi-specific antibodies significantly decreased after sequential combined treatment, as determined by conventional serology and by a multiplex assay using recombinant proteins. The frequency of T. cruzi-specific interferon-γ-producing T cells significantly increased after allopurinol treatment and decreased to background levels following benznidazole administration in a substantial proportion of subjects evaluated. The levels of total naive (CD45RA + CCR7 + CD62L+) CD4 + and CD8 + T cells were restored after allopurinol administration and maintained after completion of the combined drug protocol, along with a decrease in T cell activation in total peripheral CD4 + and CD8 + T cells. CONCLUSIONS: This pilot study shows that the combination of allopurinol and benznidazole induces significant modifications in T and B cell responses indicative of a reduction in parasite burden, and sustains the feasibility of administration of two antiparasitic drugs in the chronic phase of Chagas' disease.


Subject(s)
Allopurinol/administration & dosage , Antiprotozoal Agents/administration & dosage , Chagas Disease/drug therapy , Nitroimidazoles/administration & dosage , Adult , Allopurinol/adverse effects , Antiprotozoal Agents/adverse effects , B-Lymphocytes/immunology , Chronic Disease , Drug Therapy, Combination/adverse effects , Drug Therapy, Combination/methods , Female , Follow-Up Studies , Humans , Male , Middle Aged , Nitroimidazoles/adverse effects , Pilot Projects , T-Lymphocytes/immunology , Treatment Outcome , Trypanosoma cruzi/immunology
3.
Parasitology ; 132(Pt 6): 867-82, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16700961

ABSTRACT

The Trypanosoma cruzi cyclophilin gene family comprises 15 paralogues whose nominal masses vary from 19 to 110 kDa, namely TcCyP19, TcCyP20, TcCyP21, TcCyP22, TcCyP24, TcCyP25, TcCyP26, TcCyP28, TcCyP29, TcCyP30, TcCyP34, TcCyP35, TcCyP40, TcCyP42 and TcCyP110. Under the conditions used, only some of the T. cruzi cyclophilin paralogue products could be isolated by affinity chromatography. The 15 paralogues were aligned with 495 cyclophilins from diverse organisms. Analyses of clusters formed by the T. cruzi cyclophilins with others encoded in various genomes revealed that 8 of them (TcCyP19, TcCyP21, TcCyP22, TcCyP24, TcCyP35, TcCyP40, TcCyP42 and TcCyP110) have orthologues in many different genomes whereas the other 7 display less-defined patterns of their sequence attributes and their classification to a specific group of cyclophilin's orthologues remains uncertain. Seven epimastigote cDNA clones encoding cyclophilin isoforms were further studied. These genes were found dispersed throughout the genome of the parasite. Amastigote and trypomastigote mRNAs encoding these 7 genes were also detected. We isolated 4 cyclosporin A-binding proteins in T. cruzi epimastigote extracts, which were identified by mass spectrometry as TcCyP19, TcCyP22, TcCyP28 and TcCyP40. Cyclosporin A-binding to these cyclophilins might be of importance to the mechanism of action of Cyclosporin A and its non-immunosuppressive analogues, whose trypanocidal effects were previously reported, and therefore, of potential interest in the chemotherapy of Chagas' disease.


Subject(s)
Cyclophilins/genetics , Cyclosporine/metabolism , Gene Expression/physiology , Protozoan Proteins/genetics , Trypanosoma cruzi/genetics , Amino Acid Sequence , Animals , Chromatography, Affinity/veterinary , Cyclophilins/chemistry , Cyclophilins/classification , DNA Primers/chemistry , Gene Order , Genome/genetics , Humans , Life Cycle Stages/genetics , Molecular Sequence Data , Protozoan Proteins/chemistry , Protozoan Proteins/classification , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sequence Alignment/veterinary , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/veterinary , Trypanosoma cruzi/chemistry
4.
Science ; 309(5733): 473-6, 2005 Jul 15.
Article in English | MEDLINE | ID: mdl-16020736

ABSTRACT

To complement the sequencing of the three kinetoplastid genomes reported in this issue, we have undertaken a whole-organism, proteomic analysis of the four life-cycle stages of Trypanosoma cruzi. Peptides mapping to 2784 proteins in 1168 protein groups from the annotated T. cruzi genome were identified across the four life-cycle stages. Protein products were identified from >1000 genes annotated as "hypothetical" in the sequenced genome, including members of a newly defined gene family annotated as mucin-associated surface proteins. The four parasite stages appear to use distinct energy sources, including histidine for stages present in the insect vectors and fatty acids by intracellular amastigotes.


Subject(s)
Proteome , Protozoan Proteins/analysis , Trypanosoma cruzi/chemistry , Trypanosoma cruzi/growth & development , Adaptation, Physiological , Animals , Antigens, Protozoan/analysis , Chromatography, Liquid , Computational Biology , Databases, Genetic , Energy Metabolism , Enzymes/genetics , Enzymes/metabolism , Genes, Protozoan , Genome, Protozoan , Glycoproteins/analysis , Glycoproteins/genetics , Histidine/metabolism , Life Cycle Stages , Mass Spectrometry , Membrane Proteins/analysis , Membrane Proteins/genetics , Mucins/analysis , Multigene Family , Neuraminidase/analysis , Neuraminidase/genetics , Peptides/analysis , Protein Transport , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Trypanosoma cruzi/genetics , Trypanosoma cruzi/metabolism
5.
Curr Opin Immunol ; 13(4): 395-402, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11498294

ABSTRACT

The past year has brought great progress in the genome-sequencing efforts on a large number of protozoan and metazoan parasites. Whereas many of these projects are in their initial stages, at least one (for Plasmodium falciparum) is nearing completion. The information released to date has been most revealing with respect to immune evasion mechanisms.


Subject(s)
Genomics , Parasites/genetics , Animals , Antigenic Variation , Chromosomes/genetics , Databases, Factual , Expressed Sequence Tags , Genomics/trends , Parasites/immunology , Plasmodium falciparum/genetics , Symbiosis/genetics , Symbiosis/immunology , Vaccines/isolation & purification
6.
Int J Parasitol ; 31(5-6): 550-4, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11334941

ABSTRACT

Two primary hypotheses are proposed to account for pathogenesis in chronic Trypanosoma cruzi infections: that the persistence of T. cruzi at specific sites in the infected host results in chronic inflammatory reactivity and that T. cruzi infection induces immune responses which are targetted at self tissues. The data supporting parasite persistence as the primary cause of disease in T. cruzi infection have been recently reviewed and the reader is referred to this review for extensive documentation of most of the arguments outlined herein. This manuscript will briefly reiterate the main points of this previous review, adding additional data that have been presented since its publication. Then, philosophical and practical arguments on why Chagas disease should be investigated and treated as a parasitic infection and not as an autoimmune disease are presented. This is admittedly an 'opinion piece' and not a balanced review of the literature on Chagas disease. There are substantial data other than those reviewed here, which have been presented in support of the autoimmunity hypothesis. It is left to others to review that body of literature.


Subject(s)
Chagas Disease/parasitology , Animals , Autoimmunity/immunology , Chagas Disease/immunology , Humans , Trypanosoma cruzi/immunology
7.
J Immunol ; 166(7): 4596-603, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11254717

ABSTRACT

Infection with Trypanosoma cruzi results in the development of both type 1 and type 2 patterns of cytokine responses during acute and chronic stages of infection. To investigate the role of Th1 and Th2 subsets of CD4(+) T cells in determining the outcome of T. cruzi infection in mice, we have developed T. cruzi clones that express OVA and have used OVA-specific TCR-transgenic T cells to generate OVA-specific Th1 and Th2 cells. BALB/c mice receiving 10(7) OVA-specific Th1 cells and then challenged with OVA-expressing T. cruzi G-OVA.GPI showed significantly lower parasitemia and increased survival in comparison to mice that received no cells. In contrast, recipients of OVA-specific Th2 cells developed higher parasitemias, exhibited higher tissue parasitism and inflammation, and had higher mortality than recipients of Th1 cells after infection with T. cruzi G-OVA.GPI. Mice receiving a mixture of both Th1 and Th2 OVA-specific cells also were not protected from lethal challenge. The protective effect of the OVA-specific Th1 cells was OVA dependent as shown by the fact that transfer of OVA-specific Th1 or Th2 cells failed to alter the course of infection or disease in mice challenged with wild-type T. cruzi. Immunohistochemical analysis of OVA-specific Th1 and Th2 cells at 4, 15, and 30 days postinfection revealed the persistence and expansion of these cells in mice challenged with T. cruzi G-OVA.GPI but not in mice infected with wild-type T. cruzi. We conclude that transfer of Ag-specific Th1 cells but not Th2 cells protect mice from a lethal infection with T. cruzi.


Subject(s)
Chagas Disease/immunology , Chagas Disease/prevention & control , Epitopes, T-Lymphocyte/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Trypanosoma cruzi/immunology , Adoptive Transfer , Amino Acid Sequence , Animals , Cell Division/genetics , Cell Division/immunology , Cells, Cultured , Chagas Disease/mortality , Chagas Disease/pathology , Glycosylphosphatidylinositols/administration & dosage , Glycosylphosphatidylinositols/genetics , Glycosylphosphatidylinositols/immunology , Lymphocyte Activation/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Transgenic , Molecular Sequence Data , Ovalbumin/administration & dosage , Ovalbumin/biosynthesis , Ovalbumin/genetics , Ovalbumin/immunology , Plasmids/administration & dosage , Plasmids/immunology , Th1 Cells/parasitology , Th1 Cells/transplantation , Th2 Cells/parasitology , Th2 Cells/transplantation , Trypanosoma cruzi/genetics
8.
J Immunol ; 165(3): 1520-5, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10903759

ABSTRACT

Although Th1-type responses tend to be associated with resistance to Trypanosoma cruzi infection, mixed Th1 and Th2 cytokine responses are generally observed in both resistant and susceptible mice. To help clarify the role of type 1 and type 2 cytokine responses in immunity to T. cruzi, mice with induced deficiencies in the Stat4 or Stat6 genes were infected with T. cruzi. As expected, Stat4-/- mice deficient in type 1 cytokine responses were highly susceptible to infection, exhibiting increased parasitemia levels relative to wild-type mice and 100% mortality. In contrast, parasitemia levels and survival in Stat6-deficient mice were not different from wild type. The type 1 and type 2 cytokine bias of Stat6- and Stat4-deficient mice, respectively, was confirmed by in situ immunocytochemical analysis of cytokine-producing cells in the tissues of infected mice and by subclass analysis of anti-T. cruzi serum Abs. Notably, both Stat4- and Stat6-deficient mice produced substantial amounts of anti-T. cruzi Abs. Tissues from chronically infected Stat6-deficient mice had little to no evidence of inflammation in the heart and skeletal muscle in contrast to wild-type mice, which exhibited substantial inflammation. In situ PCR analysis of these tissues provided evidence of the persistence of T. cruzi in wild-type mice, but no evidence of parasite persistence in Stat6-deficient mice. These data suggest that type 1 T cells are required for the development of immune control to T. cruzi, but that type 2 T cells contribute to parasite persistence and increased severity of disease.


Subject(s)
Chagas Disease/genetics , Chagas Disease/immunology , DNA-Binding Proteins/deficiency , Genetic Predisposition to Disease , Trans-Activators/deficiency , Animals , Antibodies, Protozoan/biosynthesis , Antibodies, Protozoan/blood , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chagas Disease/parasitology , Chagas Disease/pathology , Chronic Disease , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Dose-Response Relationship, Immunologic , Heart/parasitology , Immunity, Innate/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , Myocardium/immunology , Myocardium/pathology , STAT4 Transcription Factor , STAT6 Transcription Factor , Signal Transduction/genetics , Signal Transduction/immunology , Spleen/immunology , Spleen/parasitology , Spleen/pathology , Th2 Cells/immunology , Th2 Cells/metabolism , Trans-Activators/biosynthesis , Trans-Activators/genetics , Trypanosoma cruzi/immunology
9.
J Infect Dis ; 180(2): 480-6, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10395865

ABSTRACT

The protozoan parasite Trypanosoma cruzi infects up to 20 million people in Latin America, and the resulting disease (Chagas' disease) is a leading cause of heart disease and death in young adults in areas endemic for the parasite. The clinical symptoms of Chagas' disease have been attributed to autoimmune reactivity to antigens shared by the parasite and host muscle or neuronal tissue. In the present study, in situ polymerase chain reaction analysis was used in murine models of Chagas' disease to demonstrate an absolute correlation between the persistence of parasites and the presence of disease in muscle tissue. Clearance of parasites from tissues, presumably by immunologic mechanisms, correlated with the abatement of inflammatory responses and the resolution of disease. These data provide strong evidence for parasite persistence as a primary cause of Chagas' disease and argue for efforts to eliminate T. cruzi from the host as a means for prevention and treatment of Chagas' disease.


Subject(s)
Chagas Cardiomyopathy/parasitology , Chagas Disease/parasitology , Trypanosoma cruzi/isolation & purification , Trypanosoma cruzi/physiology , Animals , Chagas Cardiomyopathy/pathology , Chagas Disease/pathology , Chronic Disease , DNA, Kinetoplast/analysis , Female , Heart/parasitology , Heart Transplantation , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Muscle, Skeletal/parasitology , Myocardium/pathology , Polymerase Chain Reaction/methods , Tissue Distribution
10.
Rapid Commun Mass Spectrom ; 13(11): 1024-30, 1999.
Article in English | MEDLINE | ID: mdl-10368978

ABSTRACT

Cell-surface class I major histocompatibility complex (MHC) molecules present processed self- and nonself-peptides to thymus-derived (T) lymphocytes, allowing the intracellular compartment of cells to be sampled in order to detect infection. Since the class I MHC-peptide complex plays a critical role in cell-mediated immunity, it is important to obtain sequence information on the MHC-associated peptides unique to infected cells as a prelude to the development of vaccines. Here, we outline and test an alternative strategy for identifying the proteins that are processed through the MHC pathway. This new strategy eliminates the necessity of developing and maintaining cytotoxic T lymphocyte (CTL) lines for peptide identification. In this new approach genome sequences from the infecting agent are scanned for stretches of amino acids that match a particular MHC binding motif. Molecular masses from these putative MHC-binding peptide sequences are calculated and compared to those found for peptides isolated from pathogen-infected host cells using liquid chromatography/mass spectrometry (LC/MS). Peptides with masses matching those in the database are then analyzed by tandem mass spectrometry (MS/MS) to determine their identity. Using this approach we were able to confirm the processing and presentation of two Trypanosoma cruzi proteins by the MHC class I pathway. These data suggest that a rigorous approach employing two-dimensional separations in conjunction with MS/MS and bioinformatics is a feasible means of identifying pathogen gene products of immunological interest when a CTL assay is unavailable or unsuccessful.


Subject(s)
Genes, MHC Class I/genetics , Peptides/chemistry , Animals , Chagas Disease/metabolism , Chromatography, High Pressure Liquid , Flow Injection Analysis , Mass Spectrometry , Mice , Molecular Weight , Myocardium/chemistry , Trypanosoma cruzi
11.
Parasitol Today ; 15(3): 94-9, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10322321

ABSTRACT

The question of the cause and the mechanisms of disease in chronic Trypanosoma cruzi infection continues to attract debate. Chagas disease, characterized by cardiomyopathy and/or megasyndrome involving the esophagus or colon, occurs in approximately 30% of individuals with chronic T. cruzi infections. Although the pathogenesis of Chagas disease is often attributed to autoimmune mechanisms, definitive proof of anti-self responses as the primary cause of disease in T. cruzi-infected hosts is lacking. Rick Tarleton and Lei Zhang here consider an alternative view that the primary cause of chronic Chagas disease is the failure of the host to clear the infection, resulting in infection-induced, immune-mediated tissue damage.


Subject(s)
Autoimmunity , Chagas Disease/etiology , Trypanosoma cruzi/physiology , Animals , Chagas Disease/immunology , Chagas Disease/parasitology , Humans , Mice
12.
Infect Immun ; 66(11): 5073-81, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9784506

ABSTRACT

DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2(b) and H-2(d) mice with a plasmid DNA encoding an N-terminally truncated TSA-1 lacking or containing the C-terminal nonapeptide tandem repeats, the antibody level, CTL response, and protection against challenge with T. cruzi were assessed. In H-2(b) mice, antiparasite antibodies were induced only by immunization with the DNA construct encoding TSA-1 containing the C-terminal repeats. However, both DNA constructs were efficient in eliciting long-lasting CTL responses against the protective H-2Kb-restricted TSA-1515-522 epitope. In H-2(d) mice, inoculation with either of the two TSA-1-expressing vectors effectively generated antiparasite antibodies and primed CTLs that lysed T. cruzi-infected cells in an antigen-specific, MHC class I-restricted, and CD8(+)-T-cell-dependent manner. When TSA-1 DNA-vaccinated animals were challenged with T. cruzi, 14 of 22 (64%) H-2(b) and 16 of 18 (89%) H-2(d) mice survived the infection. The ability to induce significant murine anti-T. cruzi protective immunity by immunization with plasmid DNA expressing TSA-1 provides the basis for the application of this technology in the design of optimal DNA multicomponent anti-T. cruzi vaccines which may ultimately be used for the prevention or treatment of Chagas' disease.


Subject(s)
Antigens, Protozoan/immunology , Antigens, Surface/immunology , Chagas Disease/prevention & control , Plasmids/immunology , Vaccines, DNA/immunology , Variant Surface Glycoproteins, Trypanosoma , Animals , Antibodies, Protozoan/biosynthesis , Antibody Specificity , Antigens, Protozoan/genetics , Antigens, Surface/genetics , CD8-Positive T-Lymphocytes/immunology , Chagas Disease/mortality , Female , Gene Expression/immunology , Histocompatibility Antigens Class I/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , T-Lymphocytes, Cytotoxic/immunology , Transfection/immunology , Trypanosoma cruzi/immunology
13.
Parasite Immunol ; 20(5): 207-16, 1998 May.
Article in English | MEDLINE | ID: mdl-9651921

ABSTRACT

The life cycle of the protozoan parasite Trypanosoma cruzi in mammalian hosts includes both non-dividing trypomastigote forms which circulate in the blood and replicating intracellular amastigotes which reside within the cytoplasm of a variety of host cells. In this study we have used mice with induced mutations in genes responsible for either antibody production or cytolytic T lymphocyte (CTL) function to examine the relative contributions of these effector mechanisms to control of T. cruzi. Mice deficient in the production of antibodies exhibited a delay in the rise in acute phase parasitaemia and an extended time to death relative to mice lacking CD8+ T cells. Nevertheless, B cell deficient mice eventually succumbed to the infection. Prior infection with an avirulent strain of T. cruzi failed to protect either CD8+ T cell-deficient mice or B cell deficient mice from challenge infection with virulent parasites. In contrast, mice with disruptions in the genes controlling perforin- or granzyme B-mediated cytolytic pathways had parasitaemia and mortality rates similar to wild-type mice and were protected from secondary infection by prior exposure to avirulent parasites. These results 1) confirm that antibody production, although secondary in importance to cellular responses, is nevertheless absolutely required and 2) perforin- or granzyme B-mediated lytic pathways are not required for control of T. cruzi infection.


Subject(s)
Antibodies, Protozoan/immunology , CD8-Positive T-Lymphocytes/immunology , Chagas Disease/immunology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/immunology , Animals , Antibodies, Protozoan/biosynthesis , B-Lymphocytes/immunology , Chagas Disease/parasitology , Chagas Disease/pathology , Granzymes , Major Histocompatibility Complex/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Perforin , Pore Forming Cytotoxic Proteins , Serine Endopeptidases/genetics , Serine Endopeptidases/immunology , Trypanosoma cruzi/immunology , beta 2-Microglobulin/genetics , beta 2-Microglobulin/immunology
14.
J Immunol ; 160(4): 1817-23, 1998 Feb 15.
Article in English | MEDLINE | ID: mdl-9469442

ABSTRACT

Amastigotes of Trypanosoma cruzi express surface proteins that, when released into the host cell cytoplasm, are processed and presented on the surface of infected cells in the context of MHC class I molecules to be recognized by CD8+ CTL. To further understand the role of CTL in T. cruzi infection, we used the available MHC class I peptide binding motifs to identify potential CTL target epitopes in two recently described T. cruzi amastigote surface proteins, ASP-1 and ASP-2. The predicted amino acid sequences of ASP-1 and ASP-2 were screened for H-2b allele-specific class I peptide motifs, and four peptides (PA11, PA12, PA13, and PA14) and six peptides (PA5, PA6, PA7, PA8, PA9, and PA10) were synthesized from ASP-1 and ASP-2, respectively. The majority of the peptides bound to some degree to H-2b class I MHC molecules, and six of 10 of the peptides stimulated spleen cells from T. cruzi-infected mice to lyse target cells sensitized with the homologous peptides. Short term T cell lines specific for three of these peptides also lysed T. cruzi-infected target cells. These results demonstrate that ASP-1 and ASP-2 are targets of in vivo generated CTLs and that this CTL response induced by T. cruzi infection is parasite and peptide specific, MHC restricted, and CD8 dependent.


Subject(s)
Membrane Proteins/immunology , Neuraminidase/immunology , Protozoan Proteins/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/parasitology , Trypanosoma cruzi/growth & development , Trypanosoma cruzi/immunology , Animals , Antigen Presentation , Cytotoxicity, Immunologic , Female , H-2 Antigens/immunology , H-2 Antigens/metabolism , Mice , Mice, Inbred C57BL , Protein Binding/immunology , Protozoan Proteins/metabolism , Trypanosoma cruzi/metabolism
15.
Infect Immun ; 65(10): 4055-60, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9317007

ABSTRACT

Induction of a glycosylphosphatidylinositol (GPI) deficiency in Trypanosoma cruzi by the heterologous expression of Trypanosoma brucei GPI-phospholipase C (GPI-PLC) results in decreased expression of major surface proteins (N. Garg, R. L. Tarleton, and K. Mensa-Wilmot, J. Biol. Chem. 272:12482-12491, 1997). To further explore the consequences of a GPI deficiency on replication and differentiation of T. cruzi, the in vitro and in vivo behaviors of GPI-PLC-expressing T. cruzi were studied. In comparison to wild-type controls, GPI-deficient T. cruzi epimastigotes exhibited a slight decrease in overall growth potential in culture. In the stationary phase of in vitro growth, GPI-deficient epimastigotes readily converted to metacyclic trypomastigotes and efficiently infected mammalian cells. However, upon conversion to amastigote forms within these host cells, the GPI-deficient parasites exhibited a limited capacity to replicate and subsequently failed to differentiate into trypomastigotes. Mice infected with GPI-deficient parasites showed a substantially lower rate of mortality, decreased tissue parasite burden, and a moderate tissue inflammatory response in comparison to those of mice infected with wild-type parasites. The decreased virulence exhibited by GPI-deficient parasites suggests that inhibition of GPI biosynthesis is a feasible strategy for chemotherapy of infections by T. cruzi and possibly other intracellular protozoan parasites.


Subject(s)
Glycosylphosphatidylinositols/deficiency , Trypanosoma cruzi/growth & development , Trypanosoma cruzi/pathogenicity , Animals , Chagas Disease/parasitology , Female , Glycosylphosphatidylinositol Diacylglycerol-Lyase , Mice , Mice, Inbred C3H , Phosphatidylinositol Diacylglycerol-Lyase , Type C Phospholipases/genetics , Type C Phospholipases/metabolism , Virulence/genetics
16.
Mol Biochem Parasitol ; 88(1-2): 137-49, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9274875

ABSTRACT

Amastigote surface proteins of Trypanosoma cruzi are likely targets of both humoral and cell-mediated immune responses, however, few such molecules have been well studied. In this study, we have used modified RACE (rapid amplification of cDNA ends) and SOE (gene splicing by overlap extension) polymerase chain reaction strategies to clone the gene for the previously described 83 kDa amastigote surface protein of T. cruzi. Of the several clones obtained, only one clone, clone 4, was found to encode the 20 amino acid sequence originally reported by Pan and McMahon-Pratt (J Immunol 1989;143:1001-1008). The identity of the cloned gene with the 83 kDa amastigote surface protein was further confirmed by the reactivity of polyclonal antisera against the purified 83 kDa protein with the gene product expressed in E. coli. Sequence analyses revealed that this amastigote surface protein (ASP-2) has two conserved aspartic acid box motifs and the highly conserved VTVxNVxLYNR motif characteristic of bacterial and viral sialidases and the type III module of fibronectin, respectively. ASP-2 thus joins ASP-1 as a member of the amastigote surface expressed family of sialidase-like molecules having strong homology with family 2 of the sialidase/trans-sialidase gene superfamily of T. cruzi.


Subject(s)
Genes, Protozoan , Neuraminidase/genetics , Protozoan Proteins/genetics , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Conserved Sequence , DNA Primers/genetics , DNA, Protozoan/genetics , Escherichia coli/genetics , Gene Expression Regulation, Developmental , Molecular Sequence Data , Molecular Weight , Neuraminidase/chemistry , Polymerase Chain Reaction , Protozoan Proteins/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Trypanosoma cruzi/growth & development
17.
Mol Biochem Parasitol ; 86(1): 1-11, 1997 May.
Article in English | MEDLINE | ID: mdl-9178263

ABSTRACT

An accumulating body of evidence suggests that T. cruzi-infected host cells are recognized and destroyed by class I major histocompatibility complex (MHC) restricted CD8+ T-cells thus contributing to immune control of the infection [1-6]. However, to date, only a few amastigote proteins which could be the target of this response have been described and gene sequence information is available only for the amastins [7]. In order to identify amastigote proteins which could contribute to immune detection of infected host cells, a panel of monoclonal antibodies specific for amastigote proteins was produced and screened. Three mAbs (IIIC4, VIIC1 and IIID4) were identified which recognized amastigote surface proteins of 78, 26 and 53 kDa, respectively. Screening of an amastigote cDNA expression library with mAb IIIC4 resulted in the isolation of a 2.8 Kb clone. pSI2. The derived amino acid sequence indicates that the pSI2 clone encodes an amastigote surface protein belonging to the T. cruzi trans-sialidase super-family. Based on its preferential expression in the amastigote stage we have named this protein amastigote surface protein-1 (ASP-1). ASP-1 contains the third and fourth Asp block motifs, SxDxGxTW and the fibronectin type III-like domain, VTVxNVxLYNR, thus placing it in family II of the T. cruzi trans-sialidases [8]. ASP-1 is the first trans-sialidase family member shown to be preferentially expressed in the amastigote stage of the T. cruzi life cycle. This expression of ASP-1 on parasites in infected cells and its apparent membrane attachment by a glycosylphosphatidylinositol (GP1)-anchor makes it a prime candidate to enter the class I MHC processing and presentation pathway.


Subject(s)
Genes, Protozoan , Membrane Proteins/biosynthesis , Multigene Family , Neuraminidase/biosynthesis , Neuraminidase/genetics , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/genetics , Amino Acid Sequence , Animals , Base Sequence , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/parasitology , Chagas Disease/immunology , DNA Primers , Histocompatibility Antigens Class I/immunology , Major Histocompatibility Complex , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Sequence Data , Neuraminidase/chemistry , Polymerase Chain Reaction , Protozoan Proteins/biosynthesis , RNA, Messenger/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Trypanosoma cruzi/immunology
18.
J Biol Chem ; 272(19): 12482-91, 1997 May 09.
Article in English | MEDLINE | ID: mdl-9139697

ABSTRACT

Glycosylphosphatidylinositols (GPIs) are membrane anchors for cell surface proteins of several major protozoan parasites of humans, including Trypanosoma cruzi, the causative agent of Chagas' disease. To investigate the general role of GPIs in T. cruzi, we generated GPI-deficient parasites by heterologous expression of T. brucei GPI-phospholipase C. Putative protein-GPI intermediates were depleted, causing the biochemical equivalent of a dominant-negative loss of function mutation in the GPI pathway. Cell surface expression of major GPI-anchored proteins was diminished in GPI-deficient T. cruzi. Four proteins that are normally GPI-anchored in T. cruzi exhibited different fates during the GPI shortage; Ssp-4 and p75 were secreted prematurely, while protease gp50/55 and p60 were degraded intracellularly. These observations demonstrate that secretion and intracellular degradation of GPI-anchored proteins may occur in the same genetic background during a GPI deficiency. We postulate that the interaction between a protein-GPI transamidase and the COOH-terminal GPI signal sequence plays a pivotal role in determining the fate of these proteins. At a nonpermissive GPI deficiency, T. cruzi amastigotes inside mammalian cells replicated their single kinetoplast but failed at mitosis. Hence, in these protozoans, GPIs appear to be essential for nuclear division, but not for mitochondrial duplication.


Subject(s)
Glycosylphosphatidylinositols/chemistry , Protozoan Proteins/chemistry , Animals , Cell Division , Cell Membrane/metabolism , Cell Nucleus/metabolism , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , Glycosylphosphatidylinositols/deficiency , Glycosylphosphatidylinositols/metabolism , Protozoan Proteins/metabolism , Trypanosoma cruzi
19.
Proc Natl Acad Sci U S A ; 94(8): 3932-7, 1997 Apr 15.
Article in English | MEDLINE | ID: mdl-9108082

ABSTRACT

Infection with the protozoan parasite Trypanosoma cruzi often results in chronic heart- and gut-associated disease known as Chagas disease. In this study we show that contrary to previous reports, neonatal hearts transplanted into mice chronically infected with T. cruzi do not exhibit signs of autoimmune-type rejection or any significant inflammatory response. In addition to an absence of inflammation, these syngeneic heart transplants survive for more than 1 year and are absolutely free of parasites as determined by in situ PCR analysis. However, if well-established transplanted hearts in chronically infected mice are directly injected with live parasites, a rapid and dramatic inflammatory response ensues that results in cessation of heart function. Likewise, transplanted hearts established in mice prior to systemic infection with T. cruzi or hearts transplanted into mice during the acute stage of T. cruzi infection become parasitized and develop inflammatory foci. In these cases where the transplanted hearts become parasitized, the ensuing inflammatory response is nearly identical to that observed in the native hearts of T. cruzi-infected mice in terms of cell types present and adhesion molecules and cytokines expressed. Importantly, this response is strikingly different from that observed in the allogeneic heart rejection. These results clearly document that parasitization of heart tissue is both necessary and sufficient for the induction of tissue damage in Chagas disease and strongly argue against a principal autoimmune etiology for this disease.


Subject(s)
Chagas Disease/therapy , Graft Rejection/immunology , Heart Transplantation/immunology , Trypanosoma cruzi/immunology , Animals , Antibodies, Protozoan/immunology , Antigens, Protozoan/immunology , Autoimmunity , Chagas Disease/immunology , Female , Mice , Mice, Inbred C3H , Mice, Inbred C57BL
20.
J Immunol ; 158(7): 3293-302, 1997 Apr 01.
Article in English | MEDLINE | ID: mdl-9120286

ABSTRACT

Class I MHC-restricted T cell responses have been shown to be critical for the development of immune resistance to Trypanosoma cruzi in mice. However, to date, no antigenic targets of this anti-parasite response have been characterized. We have analyzed the characteristics of potential T. cruzi CTL target molecules by expression of the model CTL target molecule chicken OVA in different cellular compartments of T. cruzi. OVA (amino acids 139-385) was expressed as a secretory, cytoplasmic, transmembrane, or glycosylphosphatidylinositol-anchored protein in T. cruzi transfectants. Host cells infected with T. cruzi transfectants that secreted or released OVA, but not those producing cytoplasmic or transmembrane forms of OVA, could process and present OVA peptide via the class I MHC pathway, as indicated by the stimulation of OVA-specific CD8+ T cell hybridomas and the cytolysis of host cells infected with OVA-secreting parasites by OVA-specific CTLs. In addition, infection of mice with OVA-secreting parasites elicited the production of OVA-specific CTLs. These studies demonstrate the ability to target proteins to specific cellular compartments in T. cruzi using either trypanosomal or mammalian signal sequences. Furthermore, these results suggest that proteins secreted or released by T. cruzi in infected cells are a major source of peptides for MHC class I presentation and for the generation of parasite-specific CTL.


Subject(s)
Antigen Presentation , H-2 Antigens/metabolism , Ovalbumin/immunology , Ovalbumin/metabolism , T-Lymphocytes, Cytotoxic/immunology , Trypanosoma cruzi/immunology , Trypanosoma cruzi/metabolism , Amino Acid Sequence , Animals , Biological Transport/immunology , Cell Line , Cytotoxicity, Immunologic , Genetic Vectors/immunology , H-2 Antigens/immunology , Host-Parasite Interactions , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Ovalbumin/genetics , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Transfection
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