Intestinally implanted Nippostrongylus brasiliensis adult worms decrease serum paraoxonase-1 activity in rats

We have previously reported a significant decrease in serum PON1 activity after Nippostrongylus brasiliensis infection in Wistar rats in association with the inflammatory response mounted against the parasite in the migratory phase of infection. However, the roles of intestinal phase and the associated oxidative stress during N. brasiliensis infection on PON1 activity have not yet been elucidated. In the present study, we observed a significant reduction in serum paraoxonase and arylesterase activity on days 6 and 9 post-implantation with N. brasiliensis adult worms in the absence of a significant increase in various serum pro-inflammatory cytokines. In addition, provision of the antioxidant butylated hydroxyanisole (BHA) to adult worm-implanted rats did not ameliorate the reduction in PON1 activity. Due to the prolonged intestinal phase of gastrointestinal nematode infections, alterations in PON1 activity during this phase need to be further examined to elucidate the mechanism of alteration in PON1 activity.     

Nippostrongylus brasiliensis: effect of dexamethasone upon anthelmintic efficacy

A study has been made to determine the chemotherapeutic susceptibility of natural and “steroid sustained” Nippostrongylus brasiliensis infections in rats. Dexamethasone in the drinking water (2.5 × 10^?4 mg/ml) aided establishment of greater parasite populations but restricted growth of young rats. Comparison trials with thiabendazole, cambendazole, and parbendazole, as well as 10 candidate drugs, showed that the parasiticidal activity of these materials was not significantly altered between the 10-day-old naturally maintained and the 17-day-old steroid-treated parasitic infections. As expected, 17-day-old naturally maintained parasite populations were highly susceptible to the anthelmintic action of these materials. In one instance, the toxic response elicited by a candidate drug was apparently potentiated by dexamethasone.     

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen. International Journal for Parasitology4: 173–176. Third stage larvae of N. brasiliensis were frozen over liquid nitrogen and after storage for 7 days were thawed rapidly and inoculated subcutaneously into rats. When ensheathed larvae were used, none survived freezing as judged by motility and infectivity trials. Separate vials of exsheathed larvae survived freezing in proportions ranging from 10 to 64 per cent. Female worms, derived from frozen exsheathed larvae, had a normal complement of eggs in the uterus and both male and female worms had a normal histological appearance. Exsheathed larvae frozen in the presence of 10 per cent dimethylsulphoxide had the same survival rate as those frozen without the addition of cryoprotectant. The addition of 10 per cent glycerol adversely effected the survival of frozen exsheathed larvae.     

Nippostrongylus brasiliensis: Mast cell kinetics at small intestinal sites in infected rats

Mast cell kinetics during infection with the nematode Nippostrongylus brasiliensis were studied at various sites in the small bowel of rats and in heterotopically transplanted isografts of foetal small intestine placed under the kidney capsule. Infection produced an increase in the number of mast cells not only in the proximal jejunum, where most of the worms are located, but also in the distal ileum and in isografts of small intestine. However, globule leucocyte infiltration of the gut epithelium was confined to the proximal small intestine and did not occur in the distal ileum or isografts. These results show that the mast cell increase in the small bowel of N. brasiliensis-infected rats is a property of the whole organ, and is not restricted to sites of worm infection; but that in contrast, globule leucocyte infiltration of the epithelium is dependent upon the presence of worms within the bowel lumen.     

Paracoccidoides brasiliensis 30 kDa Adhesin: Identification as a 14-3-3 Protein,Cloning and Subcellular Localization in Infection Models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.     

Macrophage Interaction with Paracoccidioides brasiliensis Yeast Cells Modulates Fungal Metabolism and Generates a Response to Oxidative Stress

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.     

Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42’s expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host–pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.     

An assessment of Schinopsis brasiliensis Engler (Anacardiacea) for dendroclimatological applications in the tropical Cerrado and Chaco forests,Bolivia

Given the scarcity of instrumental climatic data in the South American tropics, it is valuable to explore the dendrochronological potential of the numerous tree species growing in the region. In this paper, we assessed for the first time the dendrochronological characteristics of Schinopsis brasiliensis, an arboreal species from the dry-tropical Cerrado and Chaco forests in Bolivia and adjacent countries. Similar to most woody species in the Cerrado and Chaco regions, growth rings of S. brasiliensis are delimited by the presence of thin but continuous lines of marginal parenchyma. Based on 22 samples from 15 trees, we present the first ring-width chronology for this species covering the period 1812–2011 (200 years). Additionally, a 106-year floating chronology from S. brasiliensis was developed using cores from four columns from the church of San Miguel, Santa Cruz, built in the period 1720–1740. Standard dendrochronological statistics indicate an important common signal in the radial growth of S. brasiliensis. The comparison of variations in regional climate and ring widths shows that tree growth is directly related to spring-summer rainfall and inversely related to temperature. Following the winter dry season, rainfall in late spring and early summer increases soil water supply, which activates tree growth. In contrast, above-average temperatures during the same period increase evapotranspiration, intensify the water deficit and reduce radial growth. The dependence of S. brasiliensis growth on water supply is evidence of its dendrochronological potential for reconstructing past precipitation variations in the extensive tropical Cerrado and Chaco forest formations in South America. Using wood from historical buildings opens the possibility of extending the chronologies of S. brasiliensis over the past 400–500 years.     

IgE formation in the rat following infection with Nippostrongylus brasiliensis: I. Proliferation and differentiation of IgE-bearing cells

Sprague-Dawley rats were infected with Nippostrongylus brasiliensis larvae, and IgE formation was studied. Before infection, the serum IgE level was less than 0.4 μg/ml. The IgE level began to increase from the 10th day of infection, reached its maximum (50–100 μg/ml) at the 14th day and gradually declined. Reinfection of the rats resulted in an increase of the serum IgE level within 7 days. The IgE antibody response to N. brasiliensis antigens did not parallel the increase of IgE synthesis. In most animals, the antibody became detectable in the serum at the 21st day when the total IgE level already began to decrease. The animals showed a secondary IgE antibody response upon reinfection. Both mesenteric lymph nodes and spleen cell suspensions were examined for the presence of IgE-bearing cells (IgE-B cells) and IgE-forming cells by fluorescent antibody technique. The IgE-bearing lymphocytes became detectable in the mesenteric lymph nodes and spleen at the 8th day of infection. The proportion of the IgE-B cells in nonadherent cell population gradually increased and reached maximum at the 14th day; about 20% of immunoglobulin (Ig)-bearing cells in the mesenteric lymph nodes and 10% of Ig-bearing cells in spleen bore IgE on their surface. Evidence was obtained that these lymphocytes synthesized IgE. The IgE-forming cells were detected in both mesenteric lymph nodes and spleen of the infected animals. The number of IgE-forming cells was greater in the mesenteric lymph nodes than in spleen, indicating that the regional lymph nodes are the major source of serum IgE in the N. brasiliensis-infected animals.     

Canatoxin-, concanavalin A- and canavalin-cross-reactive materials during maturation of Canavalia brasiliensis (Mart.) seeds

The distribution of three cross-reactive materials (CRMs), a toxic protein analogous to canatoxin, CNTX-CRM, a lectin analogous to concanavalin A, Con A-CRM, and a major storage protein, canavalin-CRM, was investigated during successive stages of maturation of Canavalia brasiliensis Mart. seeds. The data obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunological analyses indicated that these proteins share extensive homology with the analogous proteins found in Canavalia ensiformis seeds. The changes in CNTX-CRM and Con A-CRM levels throughout the maturation process were assayed by rocket immunoelectrophoresis. Synthesis of Con A-CRM was detectable at 30 days post-anthesis (DPA) while its hemagglutinating activity appeared only at 35 DPA. The CNTX-CRM was detected as a biologically active protein from 30 DPA onwards. The behavior of CNTX-CRM during maturation of C. brasiliensis seeds was quite distinct from that of Con A-CRM, pointing to different biological roles of these proteins in the seed.     

Nippostrongylus brasiliensis: effects of immunity on the pre-intestinal and intestinal larval stages of the parasite

Nippostrongylus brasiliensis: effects of immunity on the pre-intestinal and intestinal larval stages of the parasite. International journal for Parasitology4: 183–191. Migration of the pre-intestinal larval stages of N. brasiliensis was studied in rats undergoing either primary or challenge infections. In rats undergoing a primary infection, more than 67 percent of larvae successfully migrated from the skin to the oesophagus by 70 h after infection, and subsequently over 90 per cent of these larvae became established in the small intestine as sexually mature adults. In immune rats undergoing a second infection, 46 per cent of larvae completed migration to the oesophagus by 70 h and of these, only 1·6 per cent became established in the intestine to produce eggs. These inhibitory effects on the pre-intestinal and intestinal larval stages were even more pronounced in immune rats undergoing a third or fourth infection and in addition, there was a prolonged sojourn and substantial retention of larvae in their lungs. There was no evidence that the immune response had an adverse effect on oesophageal fourth stags larvae as these organisms (obtained from immune donors) were able to establish and develop to maturity when transferred per os to normal animals.Syngeneic transfer of immune mesenteric lymph node cells to normal recipients, caused expulsion of parasites from the intestine but failed to effect migration of pre-intestinal larval stages. The implications of these findings are discussed in the context of current knowledge of the mechanisms of immunity to helminths.     

Detection of Antibodies Against Paracoccidioides brasiliensis in Free-Range Domestic Pigs (Sus scrofa)

Paracoccidioidomycosis, caused by the thermodimorphic fungus Paracoccidioides brasiliensis, is a human systemic mycosis prevalent in Latin America. Paracoccidioidomycosis affects mainly male rural workers, causing granulomatous lesions in several organs such as the lungs, liver and spleen. The participation of other animal species in the fungus epidemiology is not well understood. The objective of this study was to evaluate the infection of free-range domestic pigs by P. brasiliensis. Serum samples from 106 pigs were analyzed by ELISA and the immunodiffusion test, using P. brasiliensis gp43 and exoantigen as antigens, respectively. The overall positivity to gp43 in ELISA was 37.7 %, although no reactivity was observed in the immunodiffusion test and nor was P. brasiliensis detected in tissue samples (spleen, lung, liver and lymph nodes) from slaughtered animals submitted to culture, histopathological examination and PCR analysis. Five pigs seronegative to gp43 were exposed to natural infection by P. brasiliensis, and all animals seroconverted 3 months after exposure. The results suggest that free-range pigs are frequently infected with P. brasiliensis but are resistant to disease development. This is the first report of paracoccidioidomycosis in pigs.     

Selenoprotein Expression in Macrophages Is Critical for Optimal Clearance of Parasitic Helminth Nippostrongylus brasiliensis

The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) toward an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J₂ (15d-PGJ₂) plays a key role. Here, we hypothesize that dietary selenium modulates macrophage polarization toward an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a selenium-adequate (0.08 ppm) diet significantly augmented intestinal AAM presence while decreasing adult worms and fecal egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diet. Further increase in dietary selenium to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the selenium-supplemented Trsp^fl/flCre^WT mice that express selenoproteins driven by tRNA^Sec (Trsp), whereas N. brasiliensis-infected Trsp^fl/flCre^LysM selenium-supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in selenium-adequate mice. This was rescued with 15d-PGJ₂, which partially recapitulated the effect of selenium supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARγ blocked the effect of selenium. These results suggest that optimal expression of selenoproteins and selenium-dependent production of COX-derived endogenous prostanoids, such as Δ¹²-PGJ₂ and 15d-PGJ₂, may regulate AAM activation to enhance anti-helminthic parasite responses.     

In-depth proteome analysis of the rubber particle of Hevea brasiliensis (para rubber tree)

The rubber particle is a special organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis. To better understand the biological functions of rubber particles and to identify the candidate rubber biosynthesis-related proteins, a comprehensive proteome analysis was performed on H. brasiliensis rubber particles using shotgun tandem mass spectrometry profiling approaches—resulting in a thorough report on the rubber particle proteins. A total of 186 rubber particle proteins were identified, with a range in relative molecular mass of 3.9–194.2 kDa and in isoelectric point values of 4.0–11.2. The rubber particle proteins were analysed for gene ontology and could be categorised into eight major groups according to their functions: including rubber biosynthesis, stress- or defence-related responses, protein processing and folding, signal transduction and cellular transport. In addition to well-known rubber biosynthesis-related proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and cis-prenyl transferase (CPT), many proteins were firstly identified to be on the rubber particles, including cyclophilin, phospholipase D, cytochrome P450, small GTP-binding protein, clathrin, eukaryotic translation initiation factor, annexin, ABC transporter, translationally controlled tumour protein, ubiquitin-conjugating enzymes, and several homologues of REF, SRPP and CPT. A procedure of multiple reaction monitoring was established for further protein validation. This comprehensive proteome data of rubber particles would facilitate investigation into molecular mechanisms of biogenesis, self-homeostasis and rubber biosynthesis of the rubber particle, and might serve as valuable biomarkers in molecular breeding studies of H. brasiliensis and other alternative rubber-producing species.     

Nippostrongylus brasiliensis: feeding activity in the mouse

Nippostrongylus brasiliensis incorporated the fluorescent dye, Rhodamine B, while feeding in vivo. Uptake of dye in both sexes of helminth increased linearly from 30 to 120 min after the hosts were given dye per os. Feeding by female N. brasiliensis significantly exceeded that of the male at 4 and 5 days postinfection. Feeding declined in older helminths of both sexes. The density of helminths had no effect on their incorporation of dye in vivo. Feeding in male- and female-only groups of worms was similar to that seen in populations of mixed sexes. Feeding by helminths decreased during the first 36 h of food deprivation in the host, but increased during subsequent fasting of the host. Both sexes of N. brasiliensis resumed feeding within 15 min after the fasted hosts were fed. Growth of N. brasiliensis increased linearly from 4 to 7 days postinfection, based on dry weight. Seven-day-old and older females were significantly heavier than males.     

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen

The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.     

A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.     

Immunity against trichostrongylus colubriformis infection in guinea-pigs and sheep: Some comparisons with Nippostrongylus brasiliensis infection in the rat

In Nippostrongylus brasiliensis infection in rats there is evidence that antibodies ‘damage’ the worms rendering them susceptible to other components of the host immune response. Some of the experiments from which this evidence was obtained were repeated in Trichostrongylus colubri-formis infections in guinea-pigs and sheep.Lesions similar to those described in ‘damaged’ N. brasiliensis were present in T. colubriformis, but unlike damaged N. brasiliensis, damaged T. colubriformis were not rapidly expelled from the intestine of either normal or adoptively immunized hosts. Other differences between the two infections included the failure of serum from T. colubriformis-immune donors either to regularly transfer immunity against infection to non-immune recipients or to endow additional immunity on recipients of lymphoid cells from immune donors.It is suggested that the results might reflect differences between the manner in which guinea-pigs and rats achieve immune expulsion of gastro-intestinal nematode parasites.