Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome

Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the α5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.     

Trypanosoma cruzi: Induction of benznidazole resistance in vivo and its modulation by in vitro culturing and mice infection

Through a continuous in vivo drug pressure protocol, using mice as experimental model, we induced benznidazole resistance in Trypanosoma cruzi stocks. Full resistance was obtained for four out of five T. cruzi stocks analyzed. However, the number of benznidazole doses (40–180), as well as the time (4–18 months) necessary to induce resistance varied among the different T. cruzi stocks. The resistance phenotype remained stable after T. cruzi stocks has been maintained by 12 passages in mice (six months) and in acellular culture for the same time. However, the maintenance of resistant parasite for 12 months in acellular culture induces a reduction in its level of benznidazole resistance, while no alteration was detected in parasite maintained for the same time in mice. The data showed the stability of the resistance acquired by drug pressure, but suggest the possibility of reversible changes in the resistance levels after maintenance for long time in acellular culture.     

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS–PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K_i = 3.29 nM) and human cathepsin L (K_i = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.     

Nanoemulsions of sulfonamide carbonic anhydrase inhibitors strongly inhibit the growth of Trypanosoma cruzi

Sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors targeting the α-class enzyme from the protozoan pathogen Trypanosoma cruzi, responsible of Chagas disease, were recently reported. Although many such derivatives showed low nanomolar activity in vitro, they were inefficient anti-T. cruzi agents in vivo. Here, we show that by formulating such sulfonamides as nanoemulsions in clove (Eugenia caryophyllus) oil, highly efficient anti-protozoan effects are observed against two different strains of T. cruzi. These effects are probably due to an enhanced permeation of the enzyme inhibitor through the nanoemulsion formulation, interfering in this way with the life cycle of the pathogen either by inhibiting pH regulation or carboxylating reactions in which bicarbonate/CO₂ are involved. This type of formulation of sulfonamides with T. cruzi CA inhibitory effects may lead to novel therapeutic approaches against this orphan disease.     

Glycoinositolphospholipids from Trypanosomatids Subvert Nitric Oxide Production in Rhodnius prolixus Salivary Glands

Background

Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands.

Methodology/Principal Findings

Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities.

Conclusions/Significance

Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.     

Trypanosoma cruzi: Correlation of resistance and susceptibility in infected inbred mice with the in vivo primary antibody response to sheep red blood cells

Nonspecific immune responses during the course of murine Trypanosoma cruzi infection were examined in mouse strains genetically resistant or susceptible to the Brazil strain of T. cruzi. Spleen cells from infected susceptible (C3H) or resistant [C57 B1/10 and FI (C3H × C57)]mice at various points during the course of infection exhibited a reduced response to concanavalin A and lipopolysaccharide in vitro. Since this reduced response occurred in both susceptible and resistant mice, it was not predictive of resistance or susceptibility in vivo. We next examined the kinetics of in vivo primary antibody response to sheep red blood cells (SRBC) in infected C3H and C57 mice. C3H mice exhibited inhibition of the direct plaque-forming cell assay (d-PFC) which persisted until death. In contrast C57 mice exhibited no inhibition of the response at Day 5 and subsequently a markedly augmented response was observed. Other strains of mice were similarly investigated: all the susceptible mice examined (A/J, BALB/c) showed inhibition or depression of the primary antibody response and resistant mice [B10Br, C57B1/10, SJL, F1 (C3H × C57)]demonstrated either no inhibition or considerable augmentation of this response. CS7 mice resistant to the Brazil strain were susceptible to the Tulahuén strain. The mice in this latter group exhibited a markedly significant inhibition of the in vivo primary antibody response to SRBC. Culture forms of the Brazil strain protected C3H mice from a virulent challenge. This immunization resulted in a markedly augmented antibody response. The data reported herein are consistent with the notion that inhibition of the primary antibody response to SRBC correlates with susceptibility whereas no inhibition or, indeed, augmentation of the response correlates with natural as well as acquired resistance.     

Trypanosoma cruzi infection disturbs mitochondrial membrane potential and ROS production rate in cardiomyocytes

In this study, we investigated the role of Trypanosoma cruzi invasion and inflammatory processes in reactive oxygen species (ROS) production in a mouse atrial cardiomyocyte line (HL-1) and primary adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with T. cruzi (Tc) trypomastigotes, Tc lysate (TcTL), or Tc secreted proteins (TcSP) for 0–72 h, and ROS were measured by amplex red assay. Cardiomyocytes infected by T. cruzi (but not those incubated with TcTL or TcSP) exhibited a linear increase in ROS production for 2–48 h postinfection (max 18-fold increase), which was further enhanced by recombinant cytokines (IL-1β, TNF-α, and IFN-γ). We observed no increase in NADPH oxidase, xanthine oxidase, or myeloperoxidase activity, and specific inhibitors of these enzymes did not block the increased rate of ROS production in infected cardiomyocytes. Instead, the mitochondrial membrane potential was perturbed and resulted in inefficient electron transport chain (ETC) activity and enhanced electron leakage and ROS formation in infected cardiomyocytes. HL-1 rho (ρ) cardiomyocytes lacked a functional ETC and exhibited no increase in ROS formation in response to T. cruzi. Together, these results demonstrate that invasion by T. cruzi and an inflammatory milieu affect mitochondrial integrity and contribute to electron transport chain inefficiency and ROS production in cardiomyocytes.     

Host and parasite apoptosis following<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis> infection in in vitro and in vivo models

The mechanism of cell death which occurs during Chagas' cardiopathy is disputed. To address this issue we analyzed the molecular pathways implicated in the death of cardiomyocytes during T. cruzi invasion and found that they undergo apoptosis during both in vitro and in vivo infections. However, the death rates and onset were related to the parasite stocks belonging to different biodemes, which can be correlated to the different histological inflammation findings that have already been reported. Our in vitro data provide additional support for this hypothesis since higher levels and earlier apoptosis induction were noted during the interaction with the Dm28c (type I) as compared to the Y and CL stocks (type II). Modifications of the surface carbohydrates of the infected cardiomyocytes were observed and these molecular events may be acting as "eat me" tags for their final engulfment by macrophages and/or other non-professional phagocytes. The analysis of other host cell types showed that the in vitro infection of fibroblasts did not result in host apoptosis even when a highly infective stock was used. Conversely, infected macrophages undergo apoptosis but at a higher degree than cardiomyocytes. Apoptotic intracellular parasites were observed to varied extents depending on the T. cruzi stock, which was related to the parasite invasion and proliferation. In summary, our results show that during T. cruzi infection, the extent of apoptosis varies according to the host cell type and the parasite stocks. The apoptosis of both host and T. cruzi can contribute to the silent spreading and persistence of the parasite without triggering an exacerbated inflammatory response.The present study was supported by grants from the Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), the Conselho Nacional Desenvolvimento Científico e Tecnológico (CNPq), INSERM and PAPESIII/FIOCRUZ. Support by the "INSERM-FIOCRUZ convention" is also acknowledged.     

Growth-Inhibition Drug Test with Trypanosoma cruzi Culture Forms*

SYNOPSIS. A 48-hr drug screening test is described which evaluates inhibition of exponential growth of T. cruzi culture forms by electronic cell count. About 80% of drugs active in vivo produced a > 50% growth inhibition, whereas among, compounds inactive in vivo, only 19.6% induced such inhibition. Advantages of this test are low cost, rapid results, small amounts of drugs needed, and feasibility without animal facilities. Comparative studies showed that culture forms are not suitable for screening additives to prevènt transmission of T. cruzi by banked blood.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Structural variation in the glycoinositolphospholipids of different strains ofTrypanosoma cruzi

The structures of the glycoinositolphospholipids (GIPLs) from five strains of the protozoan parasiteTrypanosoma cruzi have been determined. Two series of structures were identified, all but one containing the same Man₄(AEP)GlcN-Ins-PO₄ core. Series 1 oligosaccharides are substituted at the third mannose distal to inositol (Man 3) by ethanolamine-phosphate or 2-aminoethylphosphonic acid, as are some glycosyl-phosphatidylinositol-protein anchors ofT. cruzi. The core can be further substituted by terminal (1–3)-linked -galactofuranose units. In contrast, Series 2 oligosaccharides do not have additional phosphorus-containing groups attached to Man 3, the latter being substituted instead by a single side chain unit of -galactofuranose. Series 1 oligosaccharides are present in all strains (G, G-645, Tulahuen CL, and Y) whereas Series 2 structures are present mainly in CL and Y strains. The lipid moiety in the GIPLs from the G, G-645 and Tulahuen strains is predominantly ceramide, as reported for the Y strain, whilst that from the CL strain is a mixture of ceramide and alkylacylglycerol species. The lipid moiety of the GIPLs, and probably also the phosphoinositol-oligosaccharide structures may play an important immunomodulatory role in infection byT. cruzi.Abbreviations GIPL glycoinositolphospholipid - LPPG lipopeptidophosphoglycan - GPI glycosylphosphatidylinositol - AEP 2-aminoethylphosphonic acid - PI phosphoinositol - GC gas-liquid chromatography - MS mass spectrometry - FAB fast atom bombardment - NMR nuclear magnetic resonance - DQF-COSY double quantum-filtered correlation spectroscopy - TOCSY total correlation spectroscopy - ROESY rotating frame nuclear Overhauser enhancement spectroscopy - EtNP ethanolaminephosphate - HMQC heteronuclear multiple quantum coherence - Man mannose - Galf galactofuranose - GlcN glucosamine - Ins inositol - InsP inositolphosphate - Man 3 third mannose distal to inositol - NOE nuclear Overhauser effect - [M+H]⁺ protonated molecule - [M–H]^– deprotonated molecule - RMM relative molecular mass (monoisotopic)     

Cross-Reactive Polysaccharides from Trypanosoma cruzi and Fungi (Especially Dactylium dendroides)1

ABSTRACT. Cross-reactivity between fungal and Trypanosoma cruzi polysaccharides, owing to common residues of β-D-galactofuranose, β-D-galactopyranose, and α-D-mannopyranose, was demonstrated by using a) rabbit immune sera against T. cruzi epimastigotes and b) sera from patients with Chagas’disease. Several chagasic (Ch) sera precipitated partly purified galactomannans from Aspergillus fumigatus and from T. cruzi epimastigotes and also the galactoglucomannan from Dactylium dendroides. Reaction of one Ch serum with T. cruzi galactomannan (GM) was completely inhibited by synthetic β-D-Galf-(1 → 3)-Me α-D-Manp, and that of another Ch serum with a purified D. dendroides galactoglucomannan (GGM) was partly inhibited by (1 → 6)-linked (81%) or by (1 - 3)-linked (33%) β-D-Galf-Me α-D-Manp. The β-D-Galf-(1 → 3)-α-D-Manp epitope was present in both T. cruzi and D. dendroides polysaccharides. Rabbit anti-T. cruzi antisera precipitated A. fumigatus GM, T. cruzi antigenic extracts containing the lipopeptidophosphoglycan (LPPG), T. cruzi alkali-extracted GM, a synthetic GM, and D. dendroides GGM. Weak reactivities were obtained for a Torulopsis lactis-condensi GM containing β-D-Galp terminal residues and for baker's yeast mannan with α-D-Manp-(1 - 3)-α-D-Manp-(1- → 2)-α-D-Manp-(1 → 2) side chains. An anti-LPPG rabbit serum precipitated D. dendroides GGM—a reaction inhibited (82%) by β-D-Galf-(1 → 3)-Me α-D-Manp and, less efficiently, by a (1 → 5)-linked β-D-Galf-tetrasaccharide. Sera from mice immunized with D. dendroides whole cells reacted with CL-strain trypomastigotes as shown a) by indirect immunofluorescence, b) by a Staphylococcus adherence test, but were not lytic. Mice immunized with D. dendroides were not protected against a challenge with virulent T. cruzi trypomastigotes.     

Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes

Immunofluorescence studies of normal and Trypanosoma cruzi-infected primary cultures of heart muscle cells were performed to gather information about the arrangement of myofibrillar components during the intracellular life cycle of this parasite. By using a panel of monoclonal antibodies against various myofibrillar proteins, a progressive disruption and loss of contractile proteins (such myosin and actin) of the host cell was detected during infection. The host cell formed a loose network of myofibrillar proteins around the parasites. Breakdown of the myofibrils occurred in regions where the parasites were present, and heavily infected cells showed myofibrillar proteins at their periphery. In parallel, we investigated the effect of T. cruzi infection on intracellular calcium levels by using a Ca²⁺ fluorescent indicator (confocal microscopy). Infected cardiomyocytes displayed a marked impairment in contractility, and calcium influxes became irregular and less intense when compared with those of non-infected cells. Our results demonstrate that T. cruzi infection dramatically affects calcium fluxes and causes myofibrillar breakdown disturbing cardiomyocyte contractility.Financial support through grants and scholarships from the Brazilian funding agencies FAPESP, CNPq, and CAPES is gratefully acknowledged.     

Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host

Current knowledge of the biochemistry of Trypanosoma cruzi has led to the development of new drugs and the understanding of their mode of action. Some trypanocidal drugs such as nifurtimox and benznidazole act through free radical generation during their metabolism. T. cruzi is very susceptible to the cell damage induced by these metabolites because enzymes scavenging free radicals are absent or have very low activities in the parasite. Another potential target is the biosynthetic pathway of glutathione and trypanothione, the low molecular weight thiol found exclusively in trypanosomatids. These thiols scavenge free radicals and participate in the conjugation and detoxication of numerous drugs. Inhibition of this key pathway could render the parasite much more susceptible to the toxic action of drugs such as nifurtimox and benznidazole without affecting the host significantly. Other drugs such as allopurinol and purine analogs inhibit purine transport in T. cruzi, which cannot synthesize purines de novo. Nitroimidazole derivatives such as itraconazole inhibit sterol metabolism. The parasite's respiratory chain is another potential therapeutic target because of its many differences with the host enzyme complexes. The pharmacological modulation of the host's immune response against T. cruzi infection as a possible chemotherapeutic target is discussed. A large set of chemicals of plant origin and a few animal metabolites active against T. cruzi are enumerated and their likely modes of action are briefly discussed.     

Infestin 1R, an intestinal subtilisin inhibitor from Triatoma infestans able to impair mammalian cell invasion by Trypanosoma cruzi

Infestins are Kazal-type serine protease inhibitors described in the midgut of Triatoma infestans, Chagas disease vector. Of all infestins, only infestin 1R (INF1R) does not control host blood coagulation, due to its inhibitory specificity for chymotrypsin-like proteases. We further investigated the effect of INF1R on cell infection by Trypanosoma cruzi. The importance of INF1R reactive site to inhibit T. cruzi cell invasion was confirmed using 1RSFTI, a synthetic cyclic peptide containing the inhibitor reactive site region hybridized to the Sunflower Trypsin Inhibitor-1 (SFTI-1). Our results suggest that INF1R efficiently inhibited parasite cell invasion. For the first time, a serine protease inhibitor, derived from T. infestans, was shown to impair cell invasion by T. cruzi, representing possible new target in parasite cell invasion.     

Notch receptor expression in Trypanosoma cruzi‐infected human umbilical vein endothelial cells treated with benznidazole or simvastatin revealed by microarray analysis

Chagas disease is a vector‐borne disease caused by the protozoan parasite Trypanosoma cruzi. Current therapy involves benznidazole. Benznidazole and other drugs can modify gene expression patterns, improving the response to the inflammatory influx induced by T. cruzi and decreasing the endothelial activation or immune cell recruitment, among other effects. Here, we performed a microarray analysis of human umbilical vein endothelial cells (HUVECs) treated with benznidazole and the anti‐inflammatory drugs acetylsalicylic acid or simvastatin and infected with T. cruzi. Parasitic infection produces differential expression of a set of genes in HUVECs treated with benznidazole alone or a combination with simvastatin or acetylsalicylic acid. The differentially expressed genes were involved in inflammation, adhesion, cardiac function, and remodeling. Notch1 and high mobility group B1 were genes of interest in this analysis due to their importance in placental development, cardiac development, and inflammation. Quantitative polymerase chain reaction confirmation of these two genes indicated that both are upregulated in the presence of benznidazole.     

PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE₂ involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE₂ serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE₂ release per cell by CD8+; values of PGE₂ release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE₂ (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE₂ could play a role in resistance in mice infected with K98.     

Cytotoxic, immunosuppressive and trypanocidal activities of agrocybin, a polyacetylene produced by Agrocybe perfecta (Basidiomycota)

Summary The ethyl acetate extract from the culture of the fungus Agrocybe perfecta (Rick) Singer was selected for further study in a screening of Brazilian basidiomycetes for bioactivity. The extract showed significant activity against the recombinant enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, lymphocyte proliferation in human peripheral mononuclear cells (PBMC) stimulated with phytohemaglutinin (PHA), and the human cancer cell lines UACC-62 (melanoma), MCF-7 (mammary), and TK-10 (kidney). The chromatographic fractionation of the extract was monitored by the above bioassays and showed that agrocybin was the active component. Agrocybin, a known polyacetylene amide, showed an IC₅₀ of 2 μM in the TryR assay but killed only 60% of the trypomastigote form of T. cruzi in infected murine blood even at 680 μM. This weaker activity could be due to the low temperature used to mimic banked blood or as a consequence of its inactivation by blood, already reported in the literature. On the other hand, it inhibited the proliferation of PBMC by 50% at 3.4 μM and the growth of the cancer cell lines at concentrations between 9 and 24.5 μM. Measurements of DNA fragmentation using flow cytometry suggest that agrocybin promotes cell death via apoptosis.     

Inhibition of mammalian host cell infection by insect-derived, metacyclic forms of Trypanosoma cruzi in the presence of human or rabbit anti-T. cruzi antibodies

The presence of serum from chronic chagasic patients or rabbits immunized with killed epimastigote forms of Trypanosoma cruzi inhibited infection of rat heart myoblasts by insect-vector (Triatoma infestans)-derived, metacyclic forms of Trypanosoma cruzi. The effect was produced even after diluting the chagasic serum to non-agglutinating levels and was evidenced by marked reductions in both the percentage of infected myoblasts and the number of parasites per 100 cells. Human IgG or IgM purified from chronic chagasic serum and serum from rabbits immunized with killed T. cruzi epimastigotes also reduced both parameters. While previous work has shown that immunological destruction of invasive forms of T. cruzi may underlie the protective effects of the humoral immune response against this parasite, the present in vitro results suggest that specific anti- T. cruzi antibodies could also contribute to protection via inhibition of host cell infection by the vectortransmissible form of the parasite.