Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

Background: Schistosoma mansoni is the major causative agent of schistosomiasis. The parasite takes advantage of host signals to complete its development in the human body. Tumor necrosis factor-alpha (TNF-alpha) is a human cytokine involved in skin inflammatory responses, and although its effect on the adult parasite's metabolism and egg-laying process has been previously described, a comprehensive assessment of the TNF-alpha pathway and its downstream molecular effects is lacking. Methodology/Principal Findings: In the present work we describe a possible TNF-alpha receptor (TNFR) homolog gene in S. mansoni (SmTNFR). SmTNFR encodes a complete receptor sequence composed of 599 amino acids, and contains four cysteine-rich domains as described for TNFR members. Real-time RT-PCR experiments revealed that SmTNFR highest expression level is in cercariae, 3.5 (+/- 0.7) times higher than in adult worms. Downstream members of the known human TNF-alpha pathway were identified by an in silico analysis, revealing a possible TNF-alpha signaling pathway in the parasite. In order to simulate parasite's exposure to human cytokine during penetration of the skin, schistosomula were exposed to human TNF-alpha just 3 h after cercariae-to-schistosomula in vitro transformation, and large-scale gene expression measurements were performed with microarrays. A total of 548 genes with significantly altered expression were detected, when compared to control parasites. In addition, treatment of adult worms with TNF-alpha caused a significantly altered expression of 1857 genes. Interestingly, the set of genes altered in adults is different from that of schistosomula, with 58 genes in common, representing 3% of altered genes in adults and 11% in 3 h-old early schistosomula. Conclusions/Significance: We describe the possible molecular elements and targets involved in human TNF-alpha effect on S. mansoni, highlighting the mechanism by which recently transformed schistosomula may sense and respond to this host mediator at the site of cercarial penetration into the skin.

Fruit flies (Diptera, Tephritidae) and their associations with native host plants in a remnant area of the highly endangered Atlantic Rain Forest in the State of Espirito Santo, Brazil

The results presented in this paper refer to a host survey, lasting approximately three and a half years (February 2003-july 2006), undertaken in the Vale do Rio Doce Natural Reserve, a remnant area of the highly endangered Atlantic Rain Forest located in Linhares County, State of Espirito Santo, Brazil. A total of 330 fruit samples were collected from native plants, representing 248 species and 51 plant families. Myrtaceae was the most diverse family with 54 sampled species. Twenty-eight plant species, from ten families, are hosts of ten Anastrepha species and of Ceratitis capitata (Wiedemann). Among 33 associations between host plants and fruit flies, 20 constitute new records, including the records of host plants for A. fumipennis Lima and A. nascimentoi Zucchi. The findings were discussed in the light of their implications for rain forest conservation efforts and the study of evolutionary relationships between fruit flies and their hosts.

Counterregulation of Th2 immunity by interleukin 12 reduces host defenses against Strongyloides venezuelensis infection

The aim of this study was to investigate the role of interleukin 12 (IL-12) during Strongyloides venezuelensis infection. IL-12(-/-) and wildtype C57BL/6 mice were subcutaneously infected with 1500 larvae of S. venezuelensis. On days 7, 14, and 21 post-infection, we determined eosinophil and mononuclear cell numbers in the blood and broncoalveolar lavage fluid (BALF), Th2 cytokine secretion in the lung parenchyma, and serum antibody levels. The numbers of eggs in the feces and worm parasites in the duodena were also quantified. The eosinophil and mononuclear cell counts and the concentrations of IL-3, IL-5, IL-10, IL-13, and IgG1 and IgE antibodies increased significantly in infected IL-12(-/-) and wild-type mice as compared with uninfected controls. However, the number of eosinophils and mononuclear cells in the blood and BALF and the Th2 cytokine levels in the lungs of infected IL-12-/- mice were greater than in infected wild-type C57BL/6 mice. In addition, serum IgE and IgG1 levels were also significantly enhanced in the infected mice lacking IL-12. Meanwhile, parasite burden and fecal egg counts were significantly decreased in infected IL-12-/- mice. Together, our results showed that the absence of IL-12 upregulates the Th2 immune response, which is important for control of S. venezuelensis infection. (C) 2009 Elsevier Masson SAS. All rights reserved.

A new social parasite in the ant genus Ectatomma F. Smith (Hymenoptera, Formicidae, Ectatomminae)

Ectatomma parasiticum Feitosa & Fresneau, a new ant species socially parasitic on Ectatomma tuberculatum (Olivier), is described from gynes discovered in Apazapan, state of Veracruz, Mexico, and reared in the laboratory. Ectatomma parasiticum is the first social parasite described in the Ectatomminae. This species can be distinguished from its host by morphological and behavioral features characteristic of the inquilines known in other ant subfamilies including reduced size, thickened petiole, and agonistic interactions with host species.

THE ENDOPARASITE PILOSTYLES ULEI (APODANTHACEAE - CUCURBITALES) INFLUENCES WOOD STRUCTURE IN THREE HOST SPECIES OF MIMOSA

Pilostyles species (Apodanthaceae) are endoparasites in stems of the plant family Fabaceae. The body comprises masses of parenchyma in the host bark and cortex, with sinkers, comprising groups of twisted tracheal elements surrounded by parenchyma that enter the secondary xylem of the host plant. Here we report for the first time the effects of Pilostyles parasitism on host secondary xylem. We obtained healthy and parasitized stems from Mimosa foliolosa, M. maguirei and M. setosa and compared vessel element length, fiber length, vessel diameter and vessel frequency, measured through digital imaging. Also, tree height and girth were compared between healthy and parasitized M. setosa. When parasitized, plant size, vessel diameter, vessel element length and fiber length are all less than in healthy plants. Also, vessel frequency is greater and vessels are narrower in parasitized stems. These responses to parasitism are similar to those observed in stressed plants. Thus, hosts respond to the parasite by changing its wood micromorphology in favour of increased hydraulic safety.

Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca(2+) levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca(2+) increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca(2+) levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca(2+) in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca(2+)](c). Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca(2+)](c) in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.

Melatonin and IP(3)-induced Ca(2+) Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Variety matters: adaptive genetic diversity and parasite load in two mouse opossums from the Brazilian Atlantic forest

The adaptive potential of a species to a changing environment and in disease defence is primarily based on genetic variation. Immune genes, such as genes of the major histocompatibility complex (MHC), may thereby be of particular importance. In marsupials, however, there is very little knowledge about natural levels and functional importance of MHC polymorphism, despite their key role in the mammalian evolution. In a previous study, we discovered remarkable differences in the MHC class II diversity between two species of mouse opossums (Gracilinanus microtarsus, Marmosops incanus) from the Brazilian Atlantic forest, which is one of the most endangered hotspots for biodiversity conservation. Since the main forces in generating MHC diversity are assumed to be pathogens, we investigated in this study gastrointestinal parasite burden and functional associations between the individual MHC constitution and parasite load. We tested two contrasting scenarios, which might explain differences in MHC diversity between species. We predicted that a species with low MHC diversity would either be under relaxed selection pressure by low parasite diversity (`Evolutionary equilibrium` scenario), or there was a recent loss in MHC diversity leading to a lack of resistance alleles and increased parasite burden (`Unbalanced situation` scenario). In both species it became apparent that the MHC class II is functionally important in defence against gastrointestinal helminths, which was shown here for the first time in marsupials. On the population level, parasite diversity did not markedly differ between the two host species. However, we did observe considerable differences in the individual parasite load (parasite prevalence and infection intensity): while M. incanus revealed low MHC DAB diversity and high parasite load, G. microtarsus showed a tenfold higher population wide MHC DAB diversity and lower parasite burden. These results support the second scenario of an unbalanced situation.

Effects of cyclooxygenase inhibitors on parasite burden, anemia and oxidative stress in murine Trypanosoma cruzi infection

Prostaglandins are known to be produced by macrophages when challenged with Trypanosoma cruzi, the etiological agent of Chagas` disease. It is not known whether these lipid mediators play a role in oxidative stress in host defenses against this important protozoan parasite. In this study, we demonstrated that inducible cyclooxygenase-mediated prostaglandin production is a key chemical mediator in the control of parasite burden and erythrocyte oxidative stress during T. cruzi infection in C57BL/6 and BALB/c mice, prototype hosts for the study of resistance and susceptibility in murine Chagas` disease. The results suggested the existence of at least two mechanisms of oxidative stress, dependent or independent with regard to the nitric oxide and cyclooxygenase pathway, where one or the other is more evident depending on the mouse strain.

5-Lipoxygenase plays a role in the control of parasite burden and contributes to oxidative damage of erythrocytes in murine Chagas` disease

Chagas` disease is accompanied by severe anemia and oxidative stress, which may contribute to mortality. In this study, we investigated the role of 5-lipoxygenase (5-LO) in the control of parasitism and anemia associated with oxidative damage of erythrocytes in experimental Trypanosoma cruzi infection. Wild-type C57BL/6, 129Sv mice treated or not with nordihydroguaiaretic acid (NDGA, 5-LO inhibitor), mice lacking the 5-LO enzyme gene (5-LO(-/-)) and inducible nitric oxide synthase gene (iNOS(-/-)) were infected with the Y strain of T cruzi. impairment of 5-LO resulted in increased numbers of trypomastigote forms in the blood and amastigote forms in the heart of infected mice. We assessed oxidative stress in erythrocytes by measuring oxygen uptake, induction time and chemiluminescence following treatment with tert-butyl hydroperoxide (TBH). Our results show that 5-LO metabolites increased lipid peroxidation levels in erythrocytes during the early phase of murine T cruzi infection. NDGA treatment reduced oxidative damage of erythrocytes in C57BL/6 T cruzi-infected mice but not in C57BL/6 iNOS-/- infected mice, showing that the action of NDGA is dependent on endogenous nitric oxide (NO). In addition, our results show that 5-LO metabolites do not participate directly in the development of anemia in infected mice. We conclude that 5-LO products may not only play a major role in controlling heart tissue parasitism, i.e., host resistance to acute infection with T cruzi in vivo, but in the event of an infection also play an important part in erythrocyte oxidative stress, an NO-dependent effect. (C) 2009 Elsevier B.V. All rights reserved.

Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T rangeli (rangelipain) closest to T cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T rangeli lineages associated with sympatric species of Rhodnius. (c) 2009 Elsevier B.V. All rights reserved.

Use of L-Proline and ATP Production by Trypanosoma cruzi Metacyclic Forms as Requirements for Host Cell Invasion

The process of host cell invasion by Trypanosoma cruzi depends on parasite energy. What source of energy is used for that event is not known. To address this and other questions related to T. cruzi energy requirements and cell invasion, we analyzed metacyclic trypomastigote forms of the phylogenetically distant CL and G strains. For both strains, the nutritional stress experienced by cells starved for 24, 36, or 48 h in phosphate-buffered saline reduced the ATP content and the ability of the parasite to invade HeLa cells proportionally to the starvation time. Inhibition of ATP production by treating parasites with rotenone plus antimycin A also diminished the infectivity. Nutrient depletion did not alter the expression of gp82, the surface molecule that mediates CL strain internalization, but increased the expression of gp90, the negative regulator of cell invasion, in the G strain. When L-proline was given to metacyclic forms starved for 36 h, the ATP levels were restored to those of nonstarved controls for both strains. Glucose had no such effect, although this carbohydrate and L-proline were transported in similar fashions. Recovery of infectivity promoted by L-proline treatment of starved parasites was restricted to the CL strain. The profile of restoration of ATP content and gp82-mediated invasion capacity by L-proline treatment of starved Y-strain parasites was similar to that of the CL strain, whereas the Dm28 and Dm30 strains, whose infectivity is downregulated by gp90, behaved like the G strain. L-Proline was also found to increase the ability of the CL strain to traverse a gastric mucin layer, a property important for the establishment of T. cruzi infection by the oral route. Efficient translocation of parasites through gastric mucin toward the target epithelial cells in the stomach mucosa is an essential requirement for subsequent cell invasion. By relying on these closely associated ATP-driven processes, the metacyclic trypomastigotes effectively accomplish their internalization.

Phylogenetic, morphological and behavioural analyses support host switching of Trypanosoma (Herpetosoma) lewisi from domestic rats to primates

We characterized four Brazilian trypanosomes isolated from domestic rats and three from captive nonhuman primates that were morphologically similar to T. lewisi, a considered non-pathogenic species restricted to rodents and transmitted by fleas, despite its potential pathogenicity for infants. These isolates were identified as T. lewisi by barcoding using V7V8 SSU rDNA sequences. In inferred phylogenetic trees, all isolates clustered tightly with reference T. lewisi and T. lewisi-like trypanosomes from Europe, Asia and Africa and despite their high sequence conservation formed a homogeneous clade separate from other species of the subgenus T. (Herpetosoma). With the aim of clearly resolving the relationships between the Brazilian isolates from domestic rats and primates, we compared sequences from more polymorphic ITS rDNA. Results corroborated that isolates from Brazilian rats and monkeys were indeed of the same species and quite close to T. lewisi isolates of humans and rats from different geographical regions. Morphology of the monkey isolates and their behaviour in culture and in experimentally infected rats were also compatible with T. lewisi. However, infection with T. lewisi is rare among monkeys. We have examined more than 200 free-ranging and 160 captive monkeys and found only three infected individuals among the monkeys held in captivity. The findings of this work suggest that proximity of monkeys and infected rats and their exposure to infected fleas may be responsible for the host switching of T. Iewisi from their natural rodent species to primates. This and previous studies reporting T. lewisi in humans suggest that this trypanosome can cause sporadic and opportunistic fleaborne infection in primates. (C) 2010 Elsevier B.V. All rights reserved.

In vivo infection by Trypanosoma cruzi: The conserved FLY domain of the gp85/trans-sialidase family potentiates host infection

Trypanosoma cruzi is a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas` disease. The infection of host cells by T. cruzi involves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1 mu g/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection by T. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7.6-fold), heart (3-fold) and small intestine (3.6-fold). Moreover, an intense inflammatory response and increment of CD4(+) T cells (1.7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4(+)CD25(+)FoxP3(+) T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitates in vivo infection by T. cruzi and concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas` disease.

Comparative study of intermaxillary relationships of manual and swallowing methods

This study compared the mandibular displacement from three methods of centric relation record using an anterior jig associated with (A) chin point guidance, (B) swallowing (control group) and (C) bimanual manipulation. Ten patients aged 25-39 years were selected if they met the following inclusion criteria: complete dentition (up to the second molars), Angle class I and absence of signs and symptoms of temporomandibular disorders and diagnostic casts showing stability in the maximum intercuspation (MI) position. Impressions of maxillary and mandibular arches were made with an irreversible hydrocolloid impression material. Master casts of each patient were obtained, mounted on a microscope table in MI as a reference position and 5 records of each method were made per patient. The mandibular casts were then repositioned with records interposed and new measurements were obtained. The difference between the two readings allowed measuring the displacement of the mandible in the anteroposterior and lateral axes. Data were analyzed statistically by ANOVA and Tukey's test at 5% significance level. There was no statistically significant differences (p>0.05) among the three methods for measuring lateral displacement (A=0.38 ± 0.26, B=0.32 ± 0.25 and C=0.32 ± 0.23). For the anteroposterior displacement (A=2.76 ± 1.43, B=2.46 ± 1.48 and C=2.97 ± 1.51), the swallowing method (B) differed significantly from the others (p<0.05), but no significant difference (p>0.05) was found between chin point guidance (A) and bimanual manipulation (C). In conclusion, the swallowing method produced smaller mandibular posterior displacement than the other methods.