Ribosomal DNA ITS-1 intergenic spacer polymorphism in triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.

Spatial patterns or trees in the Picinguara coastal sandy forest (state of Sao-Paulo, Brazil)

The species of the sandy plains forests (forests of the ''restingas'') have not yet had their spatial patterns studied as aids to the understanding of the diversity found in the different physiognomies along the Brazilian coast. In this paper a 10 x 10 m quadrat framework laid in a hectare of a tree dominant forest in the sandy plains of the Picinguaba area of the Serra do Mar State Park (municipality of Ubatuba, state of São Paulo, Brazil) was used to assess the spatial pattern of distribution for the ten most important species : Pera glabrata, Euterpe edulis, Eugenia brasiliensis, Alchornea triplinervea, Guatteria australis, Myrcia racemosa, Jacaranda semiserrata, Guarea macrophylla, Euplassa cantareirae and Nectandra oppositifolia. The spatial patterns were inferred through the calculations of their T-Square Index (C) and Dispersal Distance Index (I). P. glabrata shows a random pattern, E. edulis aggregate, E. brasiliensis, A. triplinervia, G. australis, E. cantareirae and N. oppositifolia with a tendency between aggregate and uniform and, M. racemosa, J. semiserrata and G. macrophylla between aggregate and random. Although the indexes are dependent of the sample size and of the technique adjustments, the relationship of the pattern with the environmental factors is shown by clustering methods. The results give confirmation of how the spatial patterns bring associations between populations and shape of the vegetation physiognomy.

Study of pulmonary experimental paracoccidioidomycosis by analysis of bronchoalveolar lavage cells: Resistant vs. susceptible mice

Adult Swiss (susceptible) and BALB/c (non-susceptible) mice were inoculated by the intravenous route with 1 x 10(6) yeast cells of Paracoccidioides brasiliensis, strain 18. Immunologic parameters, histopathology and features of the bronchoalveolar lavage (BAL) were evaluated at week 2, 4, 8 and 16 post-infection. The pulmonary infection was progressive in Swiss mice and regressive in BALB/c mice. The numbers of total cells, lymphocytes and polymorphonuclear neutrophils increased in BAL, as well as the percentages of giant cells, and CD4 and CD8 positive cells. The ultrastructural study of BAL cells revealed a predominance of macrophages and a frequency of 13.2% of type II pneumocytes. As the infection progressed, the number of fungal cells and spreading macrophages, as well as the stimulated release of H2O2 by macrophages, increased. The animals exhibited an exacerbation of the humoral immune response and a depression of cellular immunity during the infection. There was a good correlation between the intensity and the pattern of the pulmonary histopathology and the cellular findings in the BAL. The present model reproduces some anatomoclinical patterns of the human disease and shows that BAL may be a useful tool in monitoring the pulmonary infection caused by P. brasiliensis.

Ocorrência e flutuação populacional de ácaros associados a seringais vizinhos de fragmentos de cerrado

The basic knowledge of the seasonal occurrence of mites can supply data for elaboration of programs of ecological management to be implanted with success in the future. The objective of this study was to determine the species richness and the seasonality of mites present in two areas of rubber tree crops neighboring to native areas in Itiquira, MT. Along one year, 25 quantitative samplings were accomplished in rubber tree crop neighboring to two fragments of Cerrado (Cerradao and Mata Riparia). There were registered 199,380 mites, of 48 species, belonging to 15 families. of those, 13 species are phytophagous, 18 predators and 17 mycophagous or of unknown alimentary habit. Three phytophagous species represented more than 97% of the mites collected: Phyllocoptruta seringueirae Feres (80.8%), Tenuipalpus heveae Baker (12.7%) and Calacarus heveae Feres (3.6%). Among the predators, the most abundant species were Agistemus sp., Scirula sp. and Euseius concordis (Chant). Twenty-eight species were common to both crops. The families that had the largest number of species collected in the neighboring area to Cerradao were Tydeidae (7), Tarsonemidae (6), Eriophyidae and Phytoseiidae (4), and in the area close to Mata Riparia, Tydeidae (9) and Phytoseiidae (8). The presence of vegetation near the crop should explain the great number of species of mites classified as accidental found in this study. The largest abundances and species richness occurred in the end of the rainy season and beginning of the dry season.

Cytotaxonomic analysis of Brazilian species of the genus Amazona (Psittacidae, Aves) and confirmation of the genus Salvatoria (Ribeiro, 1920)

The karyotypes of 12 species of Psittacidae of the genus Amazona were studied: A. aestiva, A. amazonica, A. brasiliensis, A. autumnalis, A. farinosa, A, festiva, A. kawalli, A. ochrocephala, A. pretrei, A. rhodocorytha, A. vinacea and A. xanthops. The metaphases were obtained using a short term culture of leather pulp. Eleven of the twelve analyzed species were karyotypically homogeneous, with only a few divergences in chromosomes 2 and 3. The species A. xanthops showed large karyotypic differences compared to the genus Amazona. Consequently, the genus Salvatoria (Ribeiro, Rev. Mus. Paul. 12: 1-82, 1920) was confirmed, and A. xanthops renamed Salvatoria xanthops. The study showed the chromosomic conservation of the genus Amazona and the need for further taxonomic studies of the karyotype of the Psittacidae.

Dialysable leukocyte extracts modify the course of experimental paracoccidioidomycosis in the Syrian hamster

The effect of dialysable leukocyte extracts (DLE) obtained from hamsters immunized with Paracoccidioides brasiliensis (immune DLE) and from non-immunized hamsters (non-immune DLE) was studied in hamsters inoculated with P. brasiliensis by the intratesticular route. Treatment with immune or non-immune DLE was started during the third week of infection and was repeated at 7, 11, 15 and 19 weeks. A group of untreated infected animals was used as control. Animals were submitted to the delayed hypersensitivity skin test to P. brasiliensis antigen (PbAg) in vivo and assayed in vitro by the macrophage migration inhibition test in the presence of Phytohemagglutinin (PHA) and PbAg and by immunodiffusion for specific antibody. The animals were sacrificed at 4, 8, 12, 16 and 20 weeks. The morphology and extension of the lesions were studied at the inoculation site, and in lymph nodes, lungs, liver, spleen and kidneys. In contrast to the controls, animals treated with both DLEs maintained a positive cell-mediated immune response throughout the experiment and developed less extensive infection with a significantly lower number of fungi in the lesions. The results suggest that immune and non-immune DLE preparations modified the evolution of experimental paracoccidioidomycosis with equal efficiency. This similarity may be explained by the immunoregulatory activities of both extracts.

SEXUAL DISTINCTION BETWEEN 5TH INSTAR NYMPHS OF 6 SPECIES OF TRIATOMINAE (HEMIPTERA, REDUVIIDAE)

The aim of this paper is to present the analysis of sexual morphological differences observed in 5th instar nymphs of the following species: Panstrongylus megistus; Rhodnius neglectus; Triatoma brasiliensis; T. infestans; T. matogrossensis and T. tibiamaculata. Male and female nymphs were examined and photographed with a Scanning Electron Microscope. The 9th segment dimensions of dorsal and ventral faces were determined through a Profile Projector. Results and statistical analysis showed significant differences: the 9th sternite is significantly broader in male than in female nymphs, while in five species, tergites in female nymphs are broad and in male are narrow.

Experimental paracoccidioidomycosis in hamsters (Mesocricetus auratus): Gestational interactions

Paracoccidioides brasiliensis is a dimorphic fungus presenting specific steroid hormone receptors, both in the yeast and mycelial forms and estrogen inhibits the transition from mycelium to yeast. In the acute phase, the disease occurs with equal frequency in both sexes but in adults, females are spared. Placental fungal infection has been reported, but references to fetal infection have not been confirmed. We used 78 Syrian female hamsters divided into 3 groups: GI consisted of 30 infected mated females, GII of 20 infected unmated females and GIII of 28 uninfected mated females. Animals of group I were mated 4 weeks after infection and half of them were submitted to cesarean section on day 15 after successful mating; the other half was maintained and submitted to cesarean section and sacrificed 14 weeks after infection. Half of the animals of group II were sacrificed seven weeks and the other half 14 weeks after infection. Uninfected animals of group III were treated the same as the animals of group I. The animals were infected with strain 18 of P. brasiliensis by the intracardiac route. We evaluated the disease by the volume of granulomas in different organs, number of fungi in liver and spleen and the immunologic responses [ELISA, Double Immunodifusion (DID), Delayed Hypersensitivity Skin Test (DHT) and Macrophage Migration Inhibition (MMI)]. We studied the infection through the gestation by evaluation of the abortions, morphologic and clinic examinations of the fetuses. Our results showed that the infection did not transfer to the fetus through the placenta, but the number of abortions was larger among infected females. The newborns of GI females were smaller, weighed less and showed little vitality. The disease was more severe and disseminated in infected mated females, especially in the second sacrifice 14 weeks after inoculation, when the total volume of granulomas in them (56.3 mm) was much greater than in the infected unmated females (12 mm).

Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient, monocytes activated with IFN-gamma (1 000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover,,patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H2O2 in vitro. Unlike the results obtained with Ph 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN-gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All fights reserved.

POLYSEROSITIS IN A PATIENT WITH ACUTE PARACOCCIDIOIDOMYCOSIS AND HEPATOSPLENIC SCHISTOSOMIASIS

A severe case of juvenile paracoccidioidomycosis (PCM), manifested as cholestatic jaundice, lymphnode enlargement and an unusual form of polyserositis, associated with portal hypertension secondary to schistosomiasis, as well as bacteremias caused by E. coli and S. aureus and post-transfusional hepatitis C is reported. Temporary unresponsiveness of in vivo and in vitro cellular immune responses to P. brasiliensis were registered. The authors discuss the possible interference of either agent in the host immune response, thus explaining the severity of PCM in the present case.

DELAYED-TYPE HYPERSENSITIVITY RESPONSE IN AN ISOGENIC MURINE MODEL OF PARACOCCIDIOIDOMYCOSIS

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265) Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses against P. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.

Composition and abundance of marine shrimps (Crustacea, Decapoda, Penaeidea) in Ubatuba bay, Ubatuba (SP), Brazil

This work aims to analyse the composition, abundance and distribution of the Penaeidea species which occur in the Ubatuba Bay (23 degrees 26'S and 45 degrees 02'W). The samples were monthly collected from October/1992 to September/1993. Each collect was composed of two parallel radials: the first (radial ''A'') was carried out in the mid region of the bay and the second one (radial ''B'') in the bay mouth. The trawls took one hour in a boat equipped with one otter-trawl (10 mm of mesh). The registered environmental factors were depth, bottom water temperature, granulometric composition and organic content of the sediment. After the trawls, the shrimp were separated from other marine organisms and counted. Eight species of shrimp were obtained: Xiphopenaeus kroyeri; Artemesia longinaris; Penaeus (L.) schmitti; P. (F.) brasiliensis; Trachypenaeus; constrictus; Sicyonia typica; S. dorsalis and Pleoticus muelleri. The most abundant species were X. kroyeri, A. longinaris, P. muelleri and T. constrictus. It was verified a very strong seasonality among the species. The X. kroyeri species occurred in both radials along the months but its abundance decreased from a November to March. Such fact is attributed to the temperature which reached a minimum value of 20 degrees C during this period. The species A. longinaris and P. muelleri were more frequent in the radial B which was caracterized by 14 +/- 1.3 m of depth, low organic content in the sediment (2.97%) and granulometric composition of medium sand. The distribution and abundance of these shrimps in the bay, beyond the hydrological features can be related to biotic factors as food availability, migration, inter and intra specific relations (competition, predation, etc.).

The use of glucan as immunostimulant in the treatment of paracoccidioidomycosis

A group of 10 patients, nine of them seriously infected with Paracoccidioides brasiliensis (G1), received glucan (beta-1,3 polyglucose) as an immunostimulant intravenously once a week for one month, followed by monthly doses (10 mg) over an ii-month period, together with a specific anti-fungal agent as an immunostimulant. A second group of eight moderately infected patients (G2) was treated with only the anti-fungal agent. Among the patients in G1, there was only one case of relapse compared with five in G2. Values for the erythrocyte sedimentation rate (ESR) showed a significant difference (P < 0.01) post-treatment in G1 patients, when compared with the pretreatment levels. There was also a significant reduction (P < 0.001) in the level of serum antibodies to P. brasiliensis in the G1 patients in post-treatment examinations. The phytohemagglutinin (PHA) skin test showed a positive reaction among the patients in G1 (P < 0.01) post-treatment and there was a tendency towards an increase in the number of CD4+ T lymphocytes in both groups after treatment. The serum level of tumor necrosis factor (TNF) proved to be significantly higher (P < 0.02) in the G1 patients during treatment. In the G1 patients, the correlation between ESR and TNF tended to be negative whereas that between ESR and serum antibodies was positive. The present results indicate that the patients who received glucan, in spite of being more seriously ill, had a stronger and more favorable response to therapy.

Native crystal structure of a nitric oxide-releasing lectin from the seeds of Canavalia maritima

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide. (C) 2005 Elsevier B.V. All rights reserved.

Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis

Patients with paracoccidioidomycosis (PCM) display a certain degree of immunecompromise characterized by lymphocyte hyporesponsiveness to the main Paracoccidioides brasiliensis antigen (gp43). To determine whether cytokines are involved in this state, we evaluated the secretion of IL-2, IL-10 and IFN-gamma by peripheral blood mononuclear cells (PBMC) from patients with the acute (AF) and chronic (CF) forms of PCM and from healthy, P. brasiliensis-sensitized subjects. gp43-stimulated PBMC from healthy subjects produced substantial amounts of IL-2, IFN-gamma and IL-10, whereas PBMC from AF and CF patients produced low levels of IL-2 and IFN-gamma but substantial amounts of IL-10, Phytohaemagglutinin-induced cytokine secretion was comparable among AF and CF patients and healthy subjects, suggesting integrity of non-specific cellular immune mechanisms in PCM. gp43-pulsed adherent cells, but not non-adherent cells, mere the main source of IL-10, Moreover, IL-2 and IFN-gamma secretion correlated inversely with the amount of specific antibodies produced by patients and healthy subjects. Our results suggest that the imbalance in cytokine production of patients with PCM plays a role in the gp43-hyporesponsiveness and the marked (non-protective) antibody production of these patients. (C) 2001 Academic Press.