Biology and Ultrastructure (BI - 081/090)
BI-81
Interaction of chronic chagasic patients' SERA with cardiac adenosine receptor
Farias de Oliveira S, Almeida NA, dos Santos Costa PC, Pedrosa RC*, Campos de Carvalho AC*, Masuda MO
Instituto de Biofísica Carlos Chagas Filho *Hospital Universitário Clementino Fraga Filho, UFRJ, Rio de Janeiro, RJ, Brasil
It has been shown that sera from chronic chagasic patients (CrCh) induce alterations in cardiac electrogenesis by activation of muscarinic and b-adrenergic receptors. In a previous report we demonstrated that sera from 13 out of 25 CrCh patients were able to induce bradycardia and atrioventricular conduction block (AVB) in the presence of atropine, a muscarinc antagonist. In the present work we investigate the for interaction of these sera with the adenosine receptor in isolated rabbit heart since adenosine is known to depress AV conduction and pacemaker activity.
ECG was continuously monitored in isolated rabbit hearts during perfusion with Tyrode (Ty) solution (in mmol/L NaCl 2.7, glucose 9, NaHCO3 18, KCl 2.7, NaH2PO4 1.8, MgCl2 0.5, CaCl2 2.7) equilibrated with a mixture of 95% O2 and 5% CO2, at 35± 0.5oC. The experimental protocol consisted of 30 min control perfusion with Ty containing DCPCX (10-6 M) , a antagonist of A1 receptors, followed by another 30 min perfusion with Ty containing DCPCX + serum (diluted 1:100 v:v) and a washout period of 30 min with Ty containing DCPCX. Nine out of 11 non-muscarinic sera tested had their ability to induce bradycardia blocked by DCPCX. In addition 2 out of 7 sera that induce AV block had the effect reversed by the A1 receptor blocker.
We conclude that sera from a fraction of CrCh patients are able to depress the electrogenesis in the isolated rabbit heart through activation of A1 receptors.
Financial support by Finep, CNPq, Pronex.
BI-82
INTERACTION OF PHYTOMONAS SP. WITH AEDES ALBOPICTUS CELL CULTURE
Miguens FC, da Cunha M, Gomes RA, Keller DG, De Souza W*
Laboratório de Biologia Celular e Tecidual, CBB/UENF, Av. Alberto Lamego 2000, 28015-620 Campos dos Goytacazes, RJ *Laboratório Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, RJ, Brasil
Phytomonas can be defined as promastigote parasites that lack arginase, do not have opithomastigote stages, and infecte plants and insects. They have been known as parasites of laticiferous plants, except cassava, and pathogenic for several palm trees. Trypanosomatids of the genus Phytomonas have been reported in 158 species of 9 plant families. Several species of phytophagous insects, mainly Heteroptera, have been imputed as vectors of these trypanosomatids. Phytomonas davidi, cultured from laticiferous Euphorbiaceae, and Phytomonas serpens, isolated from Solanaceae, had been cultured in different culture medium. However, ATCC CCL 126 cells from Aedes albopictus in Mitsuhashi and Maramorosch insect medium supplemented with 10% fetal bovine serum has been used as an alternative approach to isolate Phytomonas staheli . In this study, we report our preliminary results of the interaction of P. davidi and P. staheli with Ae. albopictus cell line grown in MM medium supplemented with 10% fetal bovine serum. P. davidi were obtained from laticifers tubes of Chamaesyce thymifolia and inoculated in cell culture. After 30 min, 1, 2 and 6 hr samples were washed in PBS. Then, samples were fixed with 2.5% glutaraldehyde + 2% formaldehyde in 0.1M phosphate buffer and observed by light or electron microscopy. Similar experiments were carried out with coconut juice containing P. staheli. The washed material was also fixed and observed. During the whole period of interaction, Phytomonas were alive and presented high motility. A small number of protozoa was found adhered to Ae. albopictus cells. Most of them, were adhered by the flagellum. We never found flagellates in cell cytoplasm or in endocytic vacuoles. A large number of trypanosomatids could be found free in culture medium. None of them present alterations in their ultrastructure. In conclusion, we believe that a monolayer of Ae. albopictus grown in MM medium can be a good substrate to isolate Phytomonas.
Supported by CNPq, Fenorte, Finep and Pronex 0885.
BI-83
JG STRAIN OF TRYPANOSOMA CRUZI FAIL TO INDUCE CARDIAC SYMPATHETIC DENERVATION IN YOUNG RATS
Silva GC, Camargos ERS, Chiari E, Machado CRS
Departamentos de Morfologia e Parasitologia, Instituto de Ciências Biológicas, UFMG, 31270-901 Belo Horizonte, MG, Brasil
Previous studies have demonstrated that several strains or clones of Trypanosoma cruzi are able to induce severe (Y, ABC, CL-Brener) or moderate (Col 1.7G2) acute myocarditis in rats. In all animals cardiac sympathetic denervation was also observed. Now we test the JG strain that was recently isolated from a patient with chagasic megaesophagus. Previous study had demonstrated that in BALB/c mice this strain showed tropism for the heart muscle, causing prominent myocarditis. Holtzman rats aged 27-29 days were inoculated with 1, 000 or 10,000. Trypomastigotes/50 g of body weight, ip) and sacrificed 21, 35 and 45 days after inoculation for histological (heart, esophagus and intestine) and histochemical (heart) studies. Parasitemic curves showed few circulating trypomastigotes (maximum of 14 or 80 parasites/5 µl, respectively), no parasites being found after day 29. Histological analysis showed absence of parasitism and inflammatory processes in the esophagus and rectum of all animals at different periods of the acute phase. In the heart only discrete and diffuse inflammation was observed in ventricles and right auricular appendages (days 35 and 45 days) in accordance with a low myocardial parasitism. The histochemical method for visualizing the sympathetic nerves (glyoxylic acid-induced fluorescence) showed that the density of noradrenergic nerve terminals in T. cruzi-infected rats was similar to that of controls. From the five T. cruzi strains or clones already tested in rats, the JG strain was the first to fail in inducing moderate or severe myocarditis. The myocarditis induced by the other T. cruzi isolates (inoculum = 10,000) was always accompanied by sympathetic denervation. Because of these results, we studied the effect of JG strain in BALB/c mice (1,000 trypomastigotes/ 50 g of body weight). In this animal patent parasitemia was found till day 100 after inoculation, the highest values being found at 23 and 29 days (mean value of 1,200 and 1,300 trypomastigotes/5 µl, respectively). At day 36 of infection all mice presented moderate denervation in the auricular appendages coexistent with moderate to intense inflammatory process.
Supported by CNPq and Pronex-1996.
BI-84
LECTIN-BINDING SITES IN FIBROBLASTS FROM PRIMARY CULTURES DURING ITS INTERACTION WITH LEISHMANIA AMAZONENSIS PROMASTIGOTE FORMS
Côrte-Real S, Soeiro MN, Moreno MLV, Airano RC, Almeida DS, Meirelles MNL
Laboratório de Ultra-estrutura Celular, Departamento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Av. Brasil 4365, 21945-900 Rio de Janeiro, RJ, Brasil
Lectins are useful tools for the detection and localization of carbohydrate residues. The mechanisms that govern the invasion of parasites of the genus Leishmania in fibroblasts are yet poorly studied. This work analyses the presence of Con A and RCA binding sites at the fibroblasts surface during the early events of Leishmania amazonensis promastigotes invasion.
Primary cultures of fibroblast were obtained from skin (SF) and skeletal muscle (SMF) by enzymatic treatments and were infected for 2h with L. amazonensis promastigote forms. For the detection of mannosyl and galactosyl residues, infected and non-infected cultures were incubated with ConA-FITC or RCA 120- TRITC (50mg/ml) during 60 mim/4°C, respectively. Afterwards, the cultures were fixed with 2% paraformaldehyde for 5min/4°C, and further incubated with DAPI for DNA detection. As controls, competition assays were performed incubating the cells with specific sugars.
Mannosyl residues were seen over the surface of fibroblasts from both non-infected SF and SMF using both cultures, no considerable difference was noticed on mannosyl labeling due to promastigotes infection. On the other hand, attached parasites displayed a stronger signal than to both host cells. The parasite surface labeling displayed a discontinous labeling similar to patches. The flagellar pocket and also the posterior end of the parasite displayed a higher fluorescence intensity.
The expression of galactosyl residues seemed to be different between non-infected SF and SMF, being more intense in the latter cells. In infected SMF, RCA 120 binding sites seem to be decreased while no significant alteration was detected in infected SF cells. Extracellular parasites were devoid of RCA 120 labeling.
Our previous data showed that Con A binding sites participate in the attachment of promastigotes to fibroblasts. Here we demonstrate that after 2 hr of interaction, the mannose content of fibroblasts seems to be the same as in non-infected cells. Our preliminary studies suggest that the down-modulation of the galactosyl residues is directly related to the stage of parasite-SMF interaction. Other studies are underway to better clarify the events related with the whole process of promastigotes invasion into fibroblasts.
Supported by CNPq, and Fiocruz.
BI-85
LEISHMANIA (VIANNIA) BRAZILIENSIS EXPERIMENTAL INFECTIONS IN THE ASIAN RHESUS MONKEYS (MACACA MULATTA)
Teva A, Oliveira-Neto MP, Amaral VF, Silva AJ, Pereira MS, Carvalho-Paes LE, Coutinho SG*, Pirmez C**, Ferreira V***, Grimaldi Jr G
Departamento de Imunologia, HEC *Departamento de Protozoologia **DBBM, Instituto Oswaldo Cruz ***Departamento de Primatologia, Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
Nonhuman primates are emerging as invaluable tools employed in studying human diseases. As nonhuman primate host responses to Leishmania are very similar to those observed in humans, primate models could provide an indication of the potential success and/or limitations for human vaccine against leishmaniasis. This will be specially important in the case of L.. braziliensis complex parasites where the monkey models studied to date are susceptible to infection and the murine model is limited.
In this study, laboratory-bred and _reared adult rhesus macaques of both sexes were infected using L .(V.) braziliensis isolates originating either from localized cutaneous (strain MHOM/BR/97/SIS) [group A; N=5] or mucosal (strain MHOM/BR/95/OSC) [group B; N= 5) leishmaniasis patients from Rio de Janeiro. All the monkeys inoculated intradermally in the upper eyelid area with 10(7) virulent promastigotes of either parasite strain developed skin lesions at the site of inoculation. Variation in the level of susceptibility between individual monkeys was observed, but earlier infection onset and larger size of ulcerating nodules was found in group A. An erythematous-papular nature, were first visible at 1-2 wk p.i. Lesion development progressed rapidly and all lesions ulcerated (after 3 to 6 weeks p.i.) and was subsequently followed by regression and healing. Some monkeys developed satellite lesions peripheral to the resolving primary nodule (N=2) or metastases in the extremities (N=2). Most of the primary lesions had disappeared from 8 of the infected monkeys by 18 weeks, whereas satellite/metastatic lesions persisted at this time.
Level of IgG antibodies to promastigote antigens rose during active infection and then declined. Parasite-specific lymphoproliferative responses of PBL from monkeys were negative iat the initiation of infection, but positive reactions (SI = 16) developed as early as 6 weeks p.i. (mean SI values were not significantly different between groups) and subsequently continued to increase, peaking at 27 weeks p.i. (SI = 28) after which levels persisted (B) or declined (A) beyond selfe-cure. In addition, strong leishmanin skin test (LST reaction size = 10-30 mm) positivity to homologous antigen was detected in 7 (70%) animals, during active infection and following self-cure. There was no conspicous correlation between the LST positivity reaction sizes and the level of antigen-stimulated cell responses. Further studies are in progress to (a) determine whether the presence of proliferative (and/or IL-2, IFN-g responses) and a skin test response are sufficient indicators of self-healing and (b) for testing if these responses elicit substancial resistance to a subsequent homologous challenge with virulent parasites.
Supported by grants from Fiocruz, Faperj and CNPq (Brazil), and WHO (Geneve).
BI-86
LEISHMANIA AMAZONENSIS AND L. BRAZILIENSIS PROMASTIGOTES CAN RESIDE INTRACELLULARLY IN HUMAN MACROPHAGES AND RESPOND TO IFN-b AND TGF-b TREATMENT
Silva MP, Barral A, Barral-Netto M, Van Weyenbergh J
LIMI, Centro de Pesquisas Gonçalo Moniz , Fiocruz, Salvador, BA, Brasil
Leishmania are protozoan parasites that are transmitted in the promastigote form by a phlebotomine vector to the human host, where they differentiate into the amastigote form, preferentially residing in cells from the macrophage lineage. Leishmania promastigotes are considered to exist exclusively extracellularly and amastigotes intracellularly, although both forms can be grown axenically. To our knowledge, intracellular promastigotes have never been documented for any of the Leishmania species. When infecting human macrophages (derived from peripheral blood of healthy donors) with stationary phase L. amazonensis promastigotes, and examining by ordinary light microscopy, we readily observed large phagosomes with vividly moving intact promastigotes in a small number of cells (<0.1 %), whereas with L. braziliensis fewer and smaller phagosomes containing promastigotes were observed. The number of cells displaying visible promastigotes increased with time and reached a plateau between 48 to 72 hr, although live intracellular promastigotes could be observed for up to five days following infection. The presence of intact intracellular promastigotes was confirmed and quantified on cytospin preparations stained with hematoxylin/eosin. Macrophages infected with L. amazonensis were found to contain 5 to 10 intact promastigotes per phagosome in 10-20% of the cells. Treatment with IFN-b or TGF-b increased the number of intracellular promastigotes with ± 50%, similar to what we previously demonstrated for normal amastigote parasite burden. Conversely, IFN-g treatment diminished the number of intracellular promastigotes, but increased the size of promastigote-containing phagosomes, rendering them more easily visible by light microscopy. However, IFN-b, IFN-g or TGF-b treatment did not alter the promastigote/amastigote ratios (1/20 approximately), indicating that intracellular promastigotes behave similarly to amastigotes in response to these cytokines. Interestingly, intracellular promastigotes were easily detected when macrophages were cultured in medium containing human serum and virtually undetectable in cultures supplemented with fetal bovine serum, suggesting a stimulatory or inhibitory factor in human or bovine serum, respectively, that seems to act specificically upon the promastigote form of Leishmania.
Supported by Pronex, CNPq and CADCT.
BI-87
LEPTOMONAS SP. ISOLATED FROM THE SANDFLY LUTZOMYIA AYROZAI (DIPTERA: PSYCHODIDAE)
Sousa MA, Barrett TV***, Naiff RD***, Branco DCB, Sá-Xavier C, Santos SM, Cysne L*, Brandão A**
Coleção de Tripanosomatídeos, Laboratório de Transmissores de Hematozoários, Departamento de Entomologia *Departamento de Protozoologia **Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil ***I.N.P da Amazônia, Caixa Postal 478, 69011-970 Manaus, AM, Brasil
Trypanosomatids belonging to the genera Leishmania, Endotrypanum and Trypanosoma have been isolated on several occasions from phlebotomines, but few reports exist on presumed monoxenous parasites isolated from these hosts. In the present work, we characterized two isolates (IM 3943 e IM 3944) obtained from Lutzomyia ayrozai captured in August 1993 in Porto Urucu (State of Amazonas, Brazil) with a view to determine their generic identification. Both isolates presented promastigotes in axenic cultures and were unable to infect hamsters. Then, they were cloned in a flow cytometer and a clone from each one (IM 3943-c and IM 3944-c) was studied with regard to the capacity of multiplication within macrophages in vitro, growth, morphological differentiation and division process in the Yaeger's LIT medium at 27.3oC (from 48-144 hr). A morphometric analysis of the predominant stage found in these cultures was carried out as well. The kDNA minicircle size of both isolates was estimated and compared with those from two reference strains of Endotrypanum and with data published on Leishmania species.
These clones were unable to multiply within macrophages and grew well in LIT medium, reaching the maximum growth (3x107 cells/ml) within 72-96 hr. As seen in Giemsa-stained smears, slim promastigotes greatly predominated and were very similar in both clones. Paramastigotes also could be found, although at low percent (averages: 0.1% in IM 3943-c and 1.1% in IM 3944-c). Aflagellated cells were absent. No opisthomastigote was seen, then discarding the possibility of these clones belonging to the Herpetomonas genus. During the division process, in both clones, the nuclei were predominantly placed at different planes. However, in IM 3944-c, the majority of dividing cells (93%) presented the two kinetoplasts before the nuclei, while in IM 3943-c the kinetoplasts presented two main positions: (1) both being placed before the dividing nuclei (45% of the dividing cells) and (2) one kinetoplast placed before the nuclei, while the other was paranuclear (49%). The promastigotes of IM 3943-c and IM 3944-c were similar in size, having respectively 13.0 ±3.2 and 14.3 ±3.5 mm in length, 1.7 ±0.2 and 1.6 ±0.2 mm in width, free flagellum of 12.9 ±4.3 and 13.3 ±4.3 mm, and 1.5 mean nuclear index. The kDNA minicircle size of these clones was estimated at 2.3 kb, then being markedly higher than those of Endotrypanum and Leishmania spp. (0.8-0.9 kb). All these findings led us to consider these isolates as varieties of a same species, which do not belong to the Leishmania or Endotrypanum genera, otherwise having morphological features of a Leptomonas sp. The IM3943-c and IM3944-c cultures are deposited in the Trypanosomatid Collection of the Oswaldo Cruz Institute under the codes CT-IOC 100 and CT-IOC 058, respectively.
BI-88
LIPID TRAFFIC IN VERO CELLS INFECTED WITH TOXOPLASMA GONDII
Melo EJT, Oliveira AS, De Souza W
Laboratório de Biologia Celular e Tecidual, LBCT/CBB/UENF, Campos, RJ, Brasil
Toxoplasma gondii is an intracellular parasite widely distributed among the vertebrate animals. Tachyzoites of T. gondii multiply within parasitophorous vacuole. During its development and multiplication host cells structures change their position. In a previous study we have shown that intravacuolar parasites are able to capture metabolic of C6-NBD-ceramide. In the present study we explored this point further analyzing, using fluorescence microscopy, and the incorporation by the parasites of some labeled lipid analogues.
Vero Cells were cultured in Linbro tissue plates that contained a sterile coverslip (3-5 105/well) and maintained at 37°C overnight. The cultures were then infected with tachyzoites (RH strain). Cells were incubated in the presence of labeled lipids (C6-NBD-Ceramide; C5-sphingomieline, Br2-C5-ceramide, C6-NBD-sphingomieline) for 1-5 hr and then observed in a Zeiss Confocal Laser Scan Microscope. Fluorescence indicated of the presence of C5-SM and C6-NBD-SM was initially observed in the plasma membrane (1 hr incubation) and later in the cytoplasm of the host cell. No labeling of the parasites located within the parasitophorous vacuole was observed. However, extracellular parasites were labeled. Infected host cells incubated in the presence of Br2-C5-ceramide and C6-NBD-ceramide initially the perinuclear region was stained (1 hr incubation). After 5 hr incubation a intense labeling was observed in cytoplasm of host cell and intravacuolar tachyzoites. However, only the plasma membrane of intravacuolar parasites was stained with Br2-C5-ceramide. These observations suggest that there is a selective incorporation of host cell lipids in T. gondii containing parasitophorous vacuoles.
Supported by Pronex, CNPq, Finep and Fenorte.
BI-89
MACROPHAGE IN VITRO INFECTION WITH LEISHMANIA (L.) AMAZONENSIS AND LEISHMANIA (V.) PANAMENSIS
Matta VLR, Gomes CMC*, Ura D.M, Laurenti MD, Corbett CEP
Laboratório de Patologia de Moléstias Infecciosas (LIM/50 HC-FMUSP), Departamento de Patologia, FMUSP, São Paulo, SP, Brasil *Universidade Federal do Maranhã, São Luís, MA, Brasil
We have been working with a strain of cutaneous leishmaniasis characterized by isoenzyme and hybridization as Leishmania (Viannia) panamensis, HSJD-1 strain. However this strain showed unexpected pattern of biological behaviour with ulceration and metastatic lesion in mice tail after six month of infection, in contrast with a local cutaneous lesion caused by panamensis species. A new characterization identified this strain as Leishmania (Leishmania) amazonensis by monoclonal antibodies (Dr JJ Shaw) and by hybridization (Dr S Uliana).
In order to study better the behaviour of this leishmania strain, we compared the BALB/c perioneal macrophages infection with L. (L.) amazonensis (HSJD-1) and with a WHO strain of L. (V.) panamensis (MHOM/PA/91/CIDEP002). The infection index were determined at 6 post infection.
The infection index post 6 hr with L. (L.) amazonensis were higher (69.12%) comparing to L. (V.) panamensis (32%).
The higher in vitro macrophage infection using HSJD-1 strain corroborates with the new characterization and the in vivo behaviour of the classical L. (L.) amazonensis infection in which we observed a higher hind footpad swelling than L. (V.) panamensis and characterized by intense vacuolated macrophage infiltrate with high amount of parasites.
Supported by LIM/50 HC-FMUSP and Fapesp.
BI-90
MAPPING OF HOST CELL RECOGNITION SITE OF TRYPANOSOMA CRUZI SURFACE MOLECULE GP82
Manque PM, Eichinger D*, Ruiz R, Araya JE, Juliano MA*, Juliano L*, Silveira JF, Yoshida N
Departamento de Microbiologia, Imunologia e Parasitologia *Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, R. Botucatu 862, São Paulo, SP, Brasil ** Department of Molecular and Medical Parasitology, New York University, New York, USA
Gp82, a surface glycoprotein expressed specifically in Trypanosoma cruzi metacyclic trypomastigotes, has been implicated in the process of mammalian cell invasion. The portion of gp82 that binds to target cells appears to be the central domain of the molecule, comprising aminoacids 224-357. In order to map within this central region the binding site for host cell receptor, we have: i) generated by site-directed mutagenesis a set of pGEX constructs containg the nucleotide sequence of gp82 with various deletions, ii) transformed bacteria with the various plasmid constructs to produce recombinant proteins iii) purified the mutated proteins and tested them for binding activity, capacity to inhibit host cell invasion and reactivity with monoclonal antibody 3F6, an antibody that inhibits parasite internalization. We observed that all mutated recombinant proteins, including the one containig the largest deletion (amino acid 257-321) bound to HeLa cells and was recognized by monoclonal antibody 3F6. Based on this result, we are producing additional truncated gp82 recombinant proteins with deletion spanning aminoacids 257-363 and 321-363. In parallel, we synthesized a set of 20-mer peptides with 10 overlapping amino acids corresponding to residues 314-363 (based on the G strain sequence). One peptide contained a stretch of 10 amino acids based on the sequence of the highly invasive CL strain and not shared by the poorly invasive G strain. These peptides were used for inhibition assay of HeLa cell invasion. The penetration of CL strain metacyclic forms were into HeLa cells was maximally inhibited (~80%) by the peptide containg the strain-specific sequence. This peptide failed to inhibit the internalization of G strain metacyclic trypomastigotes.
Supported by Fapesp and CNPq.
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