Reflection imaging of China ink-perfused brain vasculature using confocal laser-scanning microscopy after clarification of brain tissue by the Spalteholz method.

Confocal laser-scanning microscopy is a useful tool for visualizing neurons and glia in transparent preparations of brain tissue from laboratory animals. Currently, imaging capillaries and venules in transparent brain tissues requires the use of fluorescent proteins. Here, we show that vessels can be imaged by confocal laser-scanning microscopy in transparent cortical, hippocampal and cerebellar preparations after clarification of China ink-injected specimens by the Spalteholz method. This method may be suitable for global, three-dimensional, quantitative analyses of vessels, including stereological estimations of total volume and length and of surface area of vessels, which constitute indirect approaches to investigate angiogenesis.

An historical perspective on how advances in microscopic imaging contributed to understanding the Leishmania Spp. and Trypanosoma cruzi host-parasite relationship.

The literature has identified complex aspects of intracellular host-parasite relationships, which require systematic, nonreductionist approaches and spatial/temporal information. Increasing and integrating temporal and spatial dimensions in host cell imaging have contributed to elucidating several conceptual gaps in the biology of intracellular parasites. To access and investigate complex and emergent dynamic events, it is mandatory to follow them in the context of living cells and organs, constructing scientific images with integrated high quality spatiotemporal data. This review discusses examples of how advances in microscopy have challenged established conceptual models of the intracellular life cycles of Leishmania spp. and Trypanosoma cruzi protozoan parasites.

The challenge of Chagas' disease: has the human pathogen, Trypanosoma cruzi, learned how to modulate signaling events to subvert host cells?

Chagas' disease, caused by Trypanosoma cruzi, is an urgent and highly prevalent danger that is endemic to Latin America, and which the research community continues to ignore. Each year, Chagas' disease kills more people in Latin America compared to any other parasite-borne disease, including malaria. In addition, between 15 and 18 million people worldwide are afflicted with this potentially lethal disease. Despite these devastating numbers, less than 0.5% of worldwide research and development for neglected diseases was aimed at Chagas' disease. The aim of this review is to draw the attention of biotechnologists to the intriguing parasite that causes Chagas' disease, which is T. cruzi. Additionally, we would also like to convince the community that basic science research can have a profound impact on the diagnosis and treatment of Chagas' disease. In this review, we introduce distinct features of T. cruzi such as its complex life cycle (e.g. the potentially infective extracellular amastigote form), its genome and genomics, as well as proteomic analysis of this parasite. Notably, the PIK pathway has been widely acknowledged as an excellent target for drug discovery to combat this pathogen. Furthermore we also describe how the identification and characterization of PIK genes can aid in neutralizing Trypanosoma infections.

Microbicidal property of B1 cell derived mononuclear phagocyte.

A mononuclear phagocyte derived from B1b cells (B1CDP) has been described. As these cells migrate from the peritoneal cavity to non-specific inflammatory lesion sites and are highly phagocytic via Fc and mannose receptors, their microbicidal ability of these cells was investigated using the Coxiella burnetii cell infection model in vitro. In this report, the pattern of infection and C. burnetii phase II survival in B1CDP phagosomes was compared with the pattern of infection of peritoneal macrophages from Xid mice (PMphi) and bone marrow derived macrophages (BMMphi). Infection was assessed by determining the large parasitophorous vacuole formation, the relative focus forming units and the quantification of DAPI (4',6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy. When compared to macrophages, B1CDP are more permissive to the bacterial infection and less effective to kill them. Further, results suggest that IL-10 secreted by B1 cells are involved in their susceptibility to infection by C. burnetti, since B1CDP from IL-10 KO mice are more competent to control C. burnetii infection than cells from wild type mice. These data contribute further to characterize B1CDP as a novel mononuclear phagocyte.

Testing of four Leishmania vaccine candidates in a mouse model of infection with Leishmania (Viannia) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World.

We evaluated whether four recombinant antigens previously used for vaccination against experimental infection with Leishmania (Leishmania) major could also induce protective immunity against a challenge with Leishmania (Viannia) braziliensis, the species responsible for 90% of the 28,712 annual cases of cutaneous and mucocutaneous leishmaniasis recorded in Brazil during the year of 2004. Initially, we isolated the homolog genes encoding four L. (V.) braziliensis antigens: (i) homologue of receptor for activated C kinase, (ii) thiol-specific antioxidant, (iii) Leishmania elongation and initiation factor, and (iv) L. (L.) major stress-inducible protein 1. At the deduced amino acid level, all four open reading frames had a high degree of identity with the previously described genes of L. (L.) major being expressed on promastigotes and amastigotes of L. (V.) braziliensis. These genes were inserted into the vector pcDNA3 or expressed as bacterial recombinant proteins. After immunization with recombinant plasmids or proteins, BALB/c mice generated specific antibody or cell-mediated immune responses (gamma interferon production). After an intradermal challenge with L. (V.) braziliensis infective promastigotes, no significant reduction on the lesions was detected. We conclude that the protective immunity afforded by these four vaccine candidates against experimental cutaneous leishmaniasis caused by L. (L.) major could not be reproduced against a challenge with L. (V.) braziliensis. Although negative, we consider our results important since they suggest that studies aimed at the development of an effective vaccine against L. (V.) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World, should be redirected toward distinct antigens or different vaccination strategies.

Characterization of a Trypanosoma cruzi antigen with homology to intracellular mammalian lectins.

Two cDNAs, isolated from a Trypanosoma cruzi amastigote library immunoscreened with sera from patients with Chagas disease, encode proteins with sequence homology to eukaryotic components of the cellular sorting and recycling machinery. These proteins, denominated TcAGL, present an N-terminal lectin domain and a C-terminal region containing repetitive amino acids and a poly-glutamine tract. They are products of polymorphic alleles of a single copy gene constitutively expressed during the parasite life cycle. Polyclonal antibodies obtained from mice immunized with the recombinant antigen recognize proteins with apparent molecular weight ranging from 95 to 120 kDa in cell lysates from all three life stages and in various strains of the parasite. Sera from Chagas disease patients recognize the recombinant antigen in ELISA and immunoprecipitation assays but not in Western blot assays under denaturing conditions. Consistent with its proposed role in the glycoprotein secreting pathway, immunofluorescence analyses and expression of a green fluorescent protein-tagged TcAGL protein indicate a sub-cellular localization in the vicinity of the flagellar pocket membrane and the Golgi complex of the parasite.

The distribution of motor proteins in the muscles and flame cells of the Schistosoma mansoni miracidium and primary sporocyst.

Schistosoma mansoni eggs, miracidia and primary sporocysts were labelled with phalloidin-rhodamine to visualize filamentous actin structures. Analysis of these forms by confocal fluorescence microscopy revealed the presence of previously well-defined circular and longitudinal muscle layers. Besides these muscular layers that sustain and provide motility to these parasite forms, we found in these 3 consecutive developmental stages of the parasite previously unidentified actin-rich tubular structures. In the 3 forms, 4 actin-rich tubules could be observed by optical sectioning underneath the well-developed muscle layers. The tubules appear in pairs, transversal to the length of the parasite, and located towards the extremities. By using an anti-flame cell specific antibody we confirmed that the tubules co-localize with flame cells and also determined that the tubule core is filled with microtubules. The additional presence of myosin in these tubules strongly suggests that they are contractile structures.

Human antibody responses of patients living in endemic areas for schistosomiasis to the tegumental protein Sm29 identified through genomic studies.

Surface proteins of schistosomes are exposed to host tissues and thus present as potential candidate molecules for the development of new intervention strategies. Herein, we have identified a new tegumental protein of Schistosoma mansoni, termed Sm29. In silico analysis revealed a signal peptide, three glycosylation sites and a transmembrane region on Sm29 amino acid sequence. Sm29 transcription in mammalian developmental stages cDNA libraries of S. mansoni was verified by PCR using specific primers for Sm29 nucleotide sequence and it revealed the presence of transcripts in schistosomula and adult worm stages of the parasite. Sm29 (40-169) fragment was produced in Escherichia coli and purified by affinity chromatography to be used in the immunological assays. Confocal microscopy confirmed bioinformatic studies, revealing that Sm29 is a membrane-bound protein localized on the tegument of S. mansoni adult worm. ELISA was performed using rSm29 protein to investigate the antibody isotype profile to Sm29 in sera of patients living in endemic areas for schistosomiasis. IgG1 and IgG3 subclass antibodies to rSm29 were predominant in sera of individuals naturally resistant to infection and resistant to re-infection whereas low levels of IgM, IgA or IgE were measured. Since, IgG1 and IgG3 are involved in parasite killing and in protective immunity the findings reported here suggest the use of Sm29 as a potential candidate vaccine against schistosomiasis.

Human autoantibodies to diacyl-phosphatidylethanolamine recognize a specific set of discrete cytoplasmic domains.

The aim of this study was to characterize a novel human autoantibody-autoantigen system represented as cytoplasmic discrete speckles (CDS) in indirect immunofluorescence (IIF). A distinct CDS IIF pattern represented by 3-20 discrete speckles dispersed throughout the cytoplasm was identified among other cytoplasmic speckled IIF patterns. The cytoplasmic domains labelled by human anti-CDS-1 antibodies did not co-localize with endosome/lysosome markers EEA1 and LAMP-2, but showed partial co-localization with glycine-tryptophan bodies (GWB). CDS-1 sera did not react with several cellular extracts in immunoblotting and did not immunoprecipitate recombinant GW182 or EEA1 proteins. The typical CDS-1 IIF labelling pattern was abolished after delipidation of HEp-2 cells. Moreover, CDS-1 sera reacted strongly with a lipid component co-migrating with phosphatidylethanolamine (PE) in high performance thin-layer chromatography (HPTLC)-immunostaining of HEp-2 cell total lipid extracts. The CDS-1 major molecular targets were established by electrospray ionization-mass spectrometry (ESI-MS), HPTLC-immunostaining and chemiluminescent enzyme-linked immunosorbent assay as diacyl-PE species, containing preferentially a cis-C18 : 1 fatty acid chain at C-2 of the glycerol moiety, namely 1,2-cis-C18 : 1-PE and 1-C16 : 0-2-cis-C18 : 1-PE. The clinical association of CDS-1 sera included a variety of systemic and organ-specific autoimmune diseases but they were also observed in patients with no evidence of autoimmune disease.

Survival of Trypanosoma cruzi metacyclic trypomastigotes within Coxiella burnetii vacuoles: differentiation and replication within an acidic milieu.

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics. Infective stages of Trypanosoma cruzi, the causative agent of Chagas' disease, actively invade a wide variety of cells, a process followed by lysosomal recruitment. Recently, we have investigated and characterized early events that occur in Vero cells persistently colonized with C. burnetii when doubly infected with T. cruzi trypomastigote forms. Kinetic studies of trypomastigote transfer indicated that parasitophorous vacuoles (PV) of metacyclic trypomastigotes are rapidly and efficiently fused to C. burnetii vacuoles. Based on these observations we have investigated the behavior of metacyclic trypomastigotes within C. burnetii vacuoles beyond 12 h of co-infection inside Vero cells. Using indirect immunofluorescence with MAb against different developmental stages, it was possible to follow the T. cruzi differentiation process within C. burnetii vacuoles after up to 96 h post-invasion. We observed that metacyclic trypomastigotes began to differentiate after 12 h of infection, and 24 h later amastigotes were the prevailing forms within C. burnetii vacuoles. T. cruzi amastigote replication within C. burnetii vacuoles was confirmed using video and time-lapse confocal microscopy and around 36 h of co-infection, cytokinesis took about 70 min to occur. After 72 h, we observed that amastigote forms seemed to escape from C. burnetii vacuoles. Labeling of amastigotes within C. burnetii vacuoles using a polyclonal antibody to C9 complement protein suggested that TcTOX (T. cruzi hemolysin) could play a role in parasite escape from C. burnetii. We concluded that T. cruzi has an outstanding adaptation capability and can survive within a hostile milieu such as C. burnetii vacuoles.

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells.

Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells

Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

Extracellular matrix alterations in experimental murine Leishmania (L.) amazonensis infection.

Here we describe extracellular matrix alterations in footpad lesions and draining lymph nodes caused by Leishmania (L.) amazonensis in mouse strains with distinct susceptibilities to this parasite: BALB/c (susceptible), C57BL/6 (intermediate), and DBA/2 (resistant). Changes in ECM were observed mainly in BALB/c mice that, in general, presented tissue damage associated with high parasite burden. Under polarized light, Sirius Red revealed type I collagen that was predominant in the primary lesion in all strains studied at the early phase of infection, but gradually decreased and was replaced by abundant type III collagen fibres in chronic phase lesions. The presence of type III collagen seemed to provide support to inflammatory cells, mainly vacuolated and parasitized macrophages. Laminin expression was not altered during infection by L. (L.) amazonensis in any of the mouse strains studied. Furthermore, the decreased fibronectin expression, in all strains, in areas where amastigotes have been found, indicated that this decline was also not related to the genetic background.

Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi

In this study we have examined certain aspects of the process of cell invasion and parasitophorous vacuole escape by metacyclic trypomastigotes and extracellular amastigote forms of Trypanosoma cruzi (G strain). Using Vero (and HeLa) cells as targets, we detected differences in the kinetics of vacuole escape by the two forms. Alcalinization of intercellular pH influenced both invasion as well as the escape from the parasitophorous vacuole by metacyclic trypomastigotes, but not the escape kinetics of extracellular amastigotes. We used sialic acid mutants as target cells and observed that the deficiency of this molecule facilitated the escape of both infective forms. Hemolysin activity was only detected in extracellular amastigotes and neither form presented detectable transialidase activity. Invasion of extracellular amastigotes and trypomastigotes in Vero cells was affected in different ways by drugs that interfere with host cell Ca2+ mobilization. These results are in line with previous results that indicate that metacyclic trypomastigotes and extracellular amastigote forms utilize mechanisms with particular features to invade host cells and to escape from their parasitophorous vacuoles.

Central nervous system involvement in experimental infection with Leishmania (Leishmania) amazonensis.

We describe the pathologic alterations of the central nervous system (CNS) observed in experimental tegumentary leishmaniasis in BALB/c and Swiss mice. The mice were subcutaneously infected with 10(4) amastigotes of Leishmania (Leishmania) amazonensis. Animals were killed and brains were removed for histologic and immunocytochemical studies. Histologic examination showed that 66.6% of infected mice had a discrete hyperemia and inflammatory infiltrate in the meninges, composed of mononuclear cells and neutrophils with no detectable parasites. However, parasitized macrophages were detected in the cerebral parenchyma, as well as mast cells, lymphocytes, and polymorphonuclear cells. Necrosis in the cerebral parenchyma was also observed. Confocal fluorescence microscopy showed that CD8+ T lymphocytes are the major component of the inflammatory infiltrate in the CNS. In addition to these cells, CD4+, CD11b, and dendritic cells are present, in small numbers, in the inflammatory processes of the CNS. Thus, L. amazonensis is able to cross the blood-brain barrier and cause significant pathologic changes in the CNS.

T cell subpopulations in myocardial inflammatory infiltrates detected by confocal microscopy: dose dependence in mice treated with cyclophosphamide during acute Trypanosoma cruzi infection.

In this article, we have characterized cell subpopulations found in the hearts of mice presenting acute Chagas' disease by immunocytochemistry and subjected to different schedules of an immunosuppressive therapy with cyclophosphamide (CY). In this comparative study, CY treatment with different doses was carried out before or after infection with Trypanosoma cruzi Y strain trypomastigotes, enabling us to discriminate the parasitemic kinetics and inflammatory processes in the heart, 12 d after infection. Animals treated with 200 mg/kg of CY 2 d before infection presented high parasitaemia as well as heavy inflammation and low parasite loads in the heart. Mice treated 5 d after infection with the same dose, developed the same parasitaemic peak but were not able to control it. Their heart did not present inflammation, but a high number of parasites could be seen. Animals treated with five 3 mg/kg doses of CY every other day presented heavy inflammatory reaction and low parasitaemia. In this group, as well as the one treated before infection, immunocytochemistry studies have shown predominance of CD8(+) T cells in the myocardium. On the other hand, mice treated with 200 mg/kg of CY 5 d after infection, presented small amounts of CD4(+) T cells while no CD8(+) could be found. These results have confirmed the dose dependence influence of this drug on the T cell populations in the inflammatory infiltrates as well as the importance of the schedule employed.

Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi.

In this study we have examined certain aspects of the process of cell invasion and parasitophorous vacuole escape by metacyclic trypomastigotes and extracellular amastigote forms of Trypanosoma cruzi (G strain). Using Vero (and HeLa) cells as targets, we detected differences in the kinetics of vacuole escape by the two forms. Alcalinization of intercellular pH influenced both invasion as well as the escape from the parasitophorous vacuole by metacyclic trypomastigotes, but not the escape kinetics of extracellular amastigotes. We used sialic acid mutants as target cells and observed that the deficiency of this molecule facilitated the escape of both infective forms. Hemolysin activity was only detected in extracellular amastigotes and neither form presented detectable transialidase activity. Invasion of extracellular amastigotes and trypomastigotes in Vero cells was affected in different ways by drugs that interfere with host cell Ca2+ mobilization. These results are in line with previous results that indicate that metacyclic trypomastigotes and extracellular amastigote forms utilize mechanisms with particular features to invade host cells and to escape from their parasitophorous vacuoles.

Reactivity of MEST-1 (antigalactofuranose) with Trypanosoma cruzi glycosylinositol phosphorylceramides (GIPCs): immunolocalization of GIPCs in acidic vesicles of epimastigotes.

Using confocal microscopy, MEST-1-positive immunofluorescence was observed within various Trypanosoma cruzi forms, except in cell-derived trypomastigotes. Glycosylinositol phosphorylceramides were identified by thin-layer chromatography immunostaining as the antigens recognized by MEST-1 in these parasites. In epimastigotes, labeling of MEST-1 coincided with acidic vesicles, indicating an internal localization of these glycoconjugates.

Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation.

The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.

Comparative histopathology of endomyocardial biopsies in chagasic and non-chagasic heart transplant recipients.

BACKGROUND: Heart transplantation has been an option for the treatment of chagasic (C) cardiomyopathy despite difficulties concerning the control of rejection and reactivation. The parasite-host interaction under the influence of immunosuppressive therapy may affect the immunological response to the graft in a pattern different from that in non-chagasic (NC) patients. The aim of this study was to compare the major histopathological features in heart transplantation in C and NC patients. METHODS: We studied 293 endomyocardial biopsies from two groups of heart transplanted patients, including 18 C and 15 NC. Both groups had identical surgical and clinical procedure except immunosuppressive therapy was lower in C patients. The histopathological parameters evaluated were the Quilty effect, rejection, C myocarditis reactivation, fibrosis, hypertrophy, and ischemia. In addition, lymphocytic cellular infiltration of myocarditis due to rejection or reactivation was immunophenotyped in the biopsies of both groups with rejection grades 3 to 4, in biopsies with signs of reactivation, and in fragments of the receptor heart with chronic C myocarditis. A search for Trypanosoma cruzi was performed in all biopsies in the C group in which lymphocyte immunophenotyping was done. We used immunofluorescence and confocal microscopy. RESULTS: The Quilty effect was present in 23% of the biopsies, involving 69.7% of the patients without a significant difference between groups (p = 0.509). Rejection was frequently observed in biopsies with the Quilty effect and the effect often recurred in the same patient. Rejection grades 3 to 4 was more frequent in the C group (p = 0.023). There were 5 episodes of Chagas' disease reactivation with myocarditis in 2 cases. The mean numbers of CD8+ and CD4+ T cells, and the CD4+-to-CD8+ ratio were similar for rejection in both groups (p > 0.05), while the CD4+-to-CD8+ ratio was significantly lower in chronic C myocarditis compared to rejection in the C group (p = 0.043). There was no significant difference in ischemic damage or interstitial fibrosis in the groups but there was a higher frequency of hypertrophy in the NC group (p = 0.007). CONCLUSIONS: The histopathological features of heart transplantation in C patients did not differ from that in NC patients in regard to the Quilty effect, development of myocardial fibrosis and ischemia. However, the higher involvement of the C group for rejection grades 3 to 4 suggested higher susceptibility to this event. The similarity of the lymphocytic cellular composition for rejection in both groups indicates that C patients respond to immunological stimulus in a similar pattern as NC patients.