Avaliação quantitativa e qualitativa das alterações no tecido esplênico de camundongos esquistossomóticos e dislipidêmicos

Neste estudo, investigamos a organização qualitativa e quantitativa do tecido esplênico na fase aguda (nona semana) da infecção pelo Schistosoma mansoni em camundongos Swiss alimentados com uma dieta hipercolesterolêmica (29% lipídios) ou uma ração padrão (12% de lipídios). O volume do baço foi medido pelo método de Scherle. Os cortes histológicos (5m) foram corados com hematoxilina-eosina, Lennerts Giemsa e Picrosirius. O tecido foi avaliado por histopatologia, morfometria e estereologia. A análise estatística foi realizada utilizando-se o programa Graph Pad Instat. Nossos resultados demonstraram que tanto a dieta quanto a infecção determinam perturbações em compartimentos esplênicos. Camundongos infectados, independentemente da dieta, tiveram aumento do baço (P=0,004) e arquitetura esplênica desorganizada quando comparado aos não infectados. O compartimento de polpa branca foi reduzida, enquanto a polpa vermelha e centro germinativo foram aumentadas (P<0,01). Camundongos infectados alimentados com alto teor de gordura apresentaram maior diâmetro de polpa branca (P=0,013) do que os não infectados. O exame microscópico do baço de camundongos não infectados alimentados com ração hipercolesterolêmica indicou que a polpa branca foi reduzida e indistinta. Além disso, camundongos infectados mostraram infiltrados celulares caracterizados por células polimorfonucleares, com mitose linfocítica intensa e células de Mott. Hemossiderina tende a ser em menor grau em camundongos infectados em comparação com os controles não infectados. A polpa vermelha mostrou um aumento significativo (P=0,008) no número médio de megacariócitos em comparação com camundongos não infectados. Camundongos alimentados com dieta padrão mostraram granuloma exsudativo-produtivo em torno de ovos do S. mansoni distribuídas apenas escassamente na polpa vermelha, enquanto que uma resposta do tecido caracterizada por uma infiltração de células em camundongos alimentados com alto teor de gordura foi encontrado. Os resultados do estudo sugerem que a ingestão de dieta rica em gorduras e a infecção contribuíram para a desorganização esplênica

Estudo estrutural e funcional das proteínas ligadoras de ácidos graxos (FABP- Fatty Acid Binding Proteins) de Fasciola hepatica

As proteínas ligadoras de ácidos graxos (Fatty Acid Binding Proteins, FABPs) de parasitos têm um papel importante no processo de infecção por estes organismos. Por este motivo, estas proteínas são antígenos candidatos para vacina contra a infecção por Schistosoma mansoni e Fasciola hepatica. No presente trabalho foram caracterizadas FABPs de F. hepatica e comparadas com a proteína Sm14 de S. mansoni, a FABP de parasito melhor caracterizada, mediante análise de sequências e estruturas modeladas. Também foram clonadas, expressas e purificadas as FABPs tipo 1 e tipo 3 de F. hepatica. Os resultados do presente estudo indicam que a FABP tipo 3 de F. hepatica é relacionada estrutural, imunológica e funcionalmente com a Sm14, um candidato vacinal amplamente estudado. Devido à importância da Sm14 como alvo para o desenvolvimento de vacina para a esquistossomose, as características apresentadas pela FhFABP3 de F. hepatica apontam esta proteína como um candidato importante também para o desenvolvimento de uma vacina contra a fasciolose

Alterações no tecido esplênico de camundongos esquistossomóticos com desnutrição calórica e protéica induzida no período de lactação

Apesar de significativos avanços obtidos no estudo da esquistossomose mansônica, as relações existentes entre esquistossomose e má-nutrição ainda não se acham completamente esclarecidas. Sendo a fase de lactação um período de vida de extrema importância para o indivíduo, alterações metabólicas na gestante podem afetar diretamente o desenvolvimento do feto sugerindo uma programação (imprinting) no metabolismo deste indivíduo em resposta adaptativa aos fatores ambientais encontrados em períodos iniciais de desenvolvimento. Este trabalho teve como objetivo avaliar as características do baço na fase aguda da infecção esquistossomótica de camundongos programados metabolicamente por restrição calórica e restrição protéica. Os baços dos animais eutanasiados na 9 semana de infecção foram submetidos a cortes histológicos (5m) e corados com hematoxilina-eosina. Foi realizada avaliação histopatológica, análise morfométrica e estereologia. A análise estatística foi realizada utilizando-se o programa Graph Pad Instat. Foi observada desorganização estrutural da polpa branca e da polpa vermelha nos grupos programados, independente da presença de infecção. Animais infectados apresentaram hiperplasia e hipertrofia da polpa branca e maior quantidade de pigmentos dispersos no tecido esplênico, bem como a presença de eosinófilos no interior de estruturas vasculares. A polpa branca dos grupos infectados tanto de restrição calórica quanto de restrição protéica apresentaram medidas morfométricas maiores quando comparados aos grupos não infectados. Os resultados estereológicos mostraram que o grupo de restrição calórica infectado apresentou menor densidade de volume de polpa vermelha, enquanto não houve diferenças significativas na densidade de volume de polpa branca. Megacariócitos foram vistos em maior quantidade nos grupos infectados, com ênfase no grupo de restrição protéica. Estes dados sugerem que a programação pela desnutrição materna na lactação e a infecção esquistossomótica provocam desorganização do tecido esplênico.

Organization of the musculature of schistosome cercariae

Phalloidin-fluorescein isothiocyanate staining of filamentous actin was used to identify muscle systems within the cercariae of Schistosoma mansoni. Examination of labeled cercariae by confocal scanning laser microscopy revealed distinct organizational levels of myofiber arrangements within the body wall, anterior cone, acetabulum, and esophagus. The body wall throughout showed a typical latticelike arrangement of outer circular and inner longitudinal myofibers, with an additional innermost layer of diagonal fibers in the anterior portion of the body. Circular and longitudinal fibers were also evident in the anterior organ and esophagus and. to some extent, the ventral acetabulum. Most striking was the striation of the cercarial tail musculature.

The schistosome excretory system: a key to regulation of metabolism, drug excretion and host interaction

There is a gulf between the enormous information content of the various genome projects and the understanding of the life of the parasite in the host. In vitro studies with adult Schistosoma mansoni using several substrates suggest that the excretory system contains both P-glycoproteins and multiresistance proteins. If both these families of protein were active in vivo, they could regulate parasite metabolism and be responsible for the excretion of drugs. During skin penetration, membrane-impermeant molecules of a wide range of molecular weights can be taken into the cercaria and schistosomulum through the nephridiopore, through the surface membrane or through both. We speculate that this uptake process might stimulate novel signalling pathways involved in growth and development.

Discovery of multiple neuropeptide families in the phylum Platyhelminthes

Available evidence shows that short amidated neuropeptides are widespread and have important functions within the nervous systems of all flatworms (phylum Platyhelminthes) examined, and could therefore represent a starting point for new lead drug compounds with which to combat parasitic helminth infections. However, only a handful of these peptides have been characterised, the rigorous exploration of the flatworm peptide signalling repertoire having been hindered by the dearth of flatworm genomic data. Through searches of both expressed sequence tags and genomic resources using the basic local alignment search tool (BLAST), we describe 96 neuropeptides on 60 precursors from 10 flatworm species. Most of these (51 predicted peptides on 14 precursors) are novel and are apparently restricted to flatworms; the remainder comprise nine recognised peptide families including FMRFamide-like (FLPs), neuropeptide F (NPF)-like, myomodulin-like, buccalin-like and neuropeptide FF (NPFF)-like peptides; notably, the latter have only previously been reported in vertebrates. Selected peptides were localised immunocytochemically to the Schistosoma mansoni nervous system. We also describe several novel flatworm NPFs with structural features characteristic of the vertebrate neuropeptide Y (NPY) superfamily, previously unreported characteristics which support the common ancestry of flatworm NPFs with the NPY-superfamily. Our dataset provides a springboard for investigation of the functional biology and therapeutic potential of neuropeptides in flatworms, simultaneously launching flatworm neurobiology into the post-genomic era. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Schistosome I/Lamides - A new family of bioactive helminth neuropeptides

Here we report the identification of a new family of helminth neuropeptides with members in both nematodes and flatworms, and include preliminary cell biological and functional characterisation of one of the peptides from the trematode parasite of humans, Schistosoma mansoni. Bioinformatics and Rapid Amplification of cDNA Ends (RACE)-PCR were used to identify the completes. mansoni neuropeptide precursor gene Sm-npp-1, which encodes three pentapeptides bearing the motif (A/G)FVR(I/L).NH2. Similar peptides were identified in three other flatworm species and in 15 nematode species. Quantitative PCR (qPCR) and immunocytochemical (ICC) analyses showed that Sm-npp-1 is constitutively expressed in larval and adult worms. ICC and confocal microscopy were employed to localise one of the schistosome NPP-1 peptides (GFVRIamide) in adult worms and schistosomules; antibodies labelled a pair of neurones in the cerebral ganglia that extend posteriorly along the main nerve cords. GFVRIamide displayed no detectable co-localisation with FMRFamide-like peptides (FLPs), nor was it detectable in muscle innervation. Exogenously applied peptide had a significant inhibitory effect on the mobility of whole adult worm pairs at 10(-5) M (n = 9). Finally, we explored Sm-npp-1 function in schistosomules using RNA interference (RNAi); we successfully achieved specific knockdown of the Sm-npp-1 transcript (54.46 +/- 10.41% knockdown, n = 3), but did not detect any clear, aberrant mobility or morphological phenotypes. NPP-1-like peptides are a new family of helminth peptides with a cell-specific expression pattern distinct from FLPs and a modulatory effect on schistosome muscular activity. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Helminth Cysteine Proteases Inhibit TRIF-dependent Activation of Macrophages via Degradation of TLR3

Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCL1) and Schistosoma mansoni (SmCB1) protect mice from the lethal effects of lipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor alpha, and interleukin-12, from macrophages. The proteases specifically block the MyD88-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR) 4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation of TLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development of Th1 responses.

Observations on the mechanism of eggshell formation in the liver fluke, Fasciola hepatica

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1 x 10(-5) M) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1 x 10(-6) M), a sodium ionophore and ammonium chloride (NH4Cl) (5 x 10(-2) M), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate (DDC, 1 x 10(-3) M) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

COMPARATIVE ANALYSES OF THE NEUROPEPTIDE-F (NPF)-RELATED AND FMRFAMIDE-RELATED PEPTIDE (FARP)-IMMUNOREACTIVITIES IN FASCIOLA-HEPATICA AND SCHISTOSOMA SPP

Immunochemical techniques were used to determine the distribution, chemical characteristics and relative abundance of immunoreactivity (IR) to two native platyhelminth neuropeptides, neuropeptide F (NPF) (Moniezia expansa) and the FMRFamide-related peptide (FaRP), GNFFRFamide, in the trematodes, Fasciola hepatica and Schistosoma mansoni; the larger S. margrebowiei was used in the chemical analysis. Extensive immunostaining for the two peptides was demonstrated throughout the nervous systems of both F. hepatica and S. mansoni, with strong IR also in the innervation of muscular structures, including those associated with the egg-forming apparatus. The patterns of immunostaining were similar to those previously described for the vertebrate neuropeptide Y superfamily of peptides and for FMRFamide. Ultrastructurally, gold labelling of NPF- and GNFFRFamide-IRs was localized exclusively to the contents of secretory vesicles in the axons and somatic cytoplasm of neurones. Double-labelling experiments showed an apparent homogeneity of antigenic sites, in all probability due to the demonstrated cross-reactivity of the FaRP antiserum with NPF. Radioimmunoassay of acid-ethanol extracts of the worms detected 8.3 pmol/g and 4.7 pmol/g equivalents of NPF- and FMRFamide-IRs, respectively, for F. hepatica, and corresponding values of 4.9 pmol/g and 4.3 pmol/g equivalents for S. margrebowiei. Gel-permeation chromatography resolved IR to both peptides in discrete peaks and these eluted in similar positions to synthetic NPF (M. expansa) and GNFFRFamide, respectively.

LOCALIZATION OF DIPLOPTERA-PUNCTATA ALLATOSTATIN-LIKE IMMUNOREACTIVITY IN HELMINTHS - AN IMMUNOCYTOCHEMICAL STUDY

The nervous systems of helminths are predominantly peptidergic in nature, although it is likely that the full range of regulatory peptides used by these organisms has yet to be elucidated. Attempts to identify novel helminth neuropeptides are being made using immunocytochemistry with antisera raised against peptides isolated originally from insects. One of these antisera was raised against allatostatin III, a peptide isolated originally from the cockroach, Diploptera punctata, and a member of a family of related peptides found in insects. Allatostatin immunoreactivity was found throughout the nervous systems of Mesocestoides corti tetrathyridia, and adult Moniezia expansa, Diclidophora merlangi, Fasciola hepatica, Schistosoma mansoni, Ascaris suum and Panagrellus redivivus. Immunostaining was observed in the nerve cords and anterior ganglia of all the helminths. It was also apparent in the subtegumental nerves and around the reproductive apparatus of the flatworms, in neurones in the pharynx of D. merlangi, F. hepatica, A. suum and P. redivivus, and in fibres innervating the anterior sense organs in the nematodes. Immunostaining in all species was both reproducible and specific in that it could be abolished by pre-absorption of the antiserum with allatostatins I-IV. These results suggest that molecules related to the D. punctata allatostatins are important components in the nervous systems of a number of helminth parasites, and a free-living nematode. Their distribution within the nervous system suggests they function as neurotransmitters/ neuromodulators with roles in locomotion, feeding, reproduction and sensory perception.

PEPTIDES RELATED TO THE DIPLOPTERA-PUNCTATA ALLATOSTATINS IN NONARTHROPOD INVERTEBRATES - AN IMMUNOCYTOCHEMICAL SURVEY

The allatostatins are a family of peptides isolated originally from the cockroach, Diploptera punctata. Related peptides have been identified in Periplaneta americana and the blowfly, Calliphora vomitoria. These peptides have been shown to be potent inhibitors of juvenile hormone synthesis in these species. A peptide inhibitor of juvenile hormone biosynthesis has also been isolated from the moth, Manduca sexta; however, this peptide has no structural homology with the D. punctata-type allatostatins. Investigations of the phylogeny of the D. punctata allatostatin peptide family have been started by examining a number of nonarthropod invertebrates for the presence of allatostatin-like molecules using immunocytochemistry with antisera directed against the conserved C-terminal region of this family. Allatostatin-like immunoreactivity (ALIR) was demonstrated in the nervous systems of Hydra oligactis (Hydrozoa), Moniezia expansa (Cestoda), Schistosoma mansoni (Trematoda), Artioposthia triangulata (Turbellaria), Ascaris suum (Nematoda), Lumbricus terrestris (Oligochaeta), Limax pseudoflavus (Gastropoda), and Eledone cirrhosa (Cephalopoda). ALIR could not be demonstrated in Ciona intestinalis (Ascidiacea). These results suggest that molecules related to the allatostatins may play an important role in nervous system function in many invertebrates as well as in insects and that they also have an ancient evolutionary lineage. (C) 1994 Wiley-Liss, Inc.

A novel calmodulin-like protein from the liver fluke, Fasciola hepatica

An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.

Aflatoxin Exposure May Contribute to Chronic Hepatomegaly in Kenyan School Children

BACKGROUND: Presentation with a firm type of chronic hepatomegaly of multifactorial etiology is common among school-age children in sub-Saharan Africa.

OBJECTIVE: Aflatoxin is a liver toxin and carcinogen contaminating staple maize food. In this study we examined its role in chronic hepatomegaly.

METHODS: Plasma samples collected in 2002 and again in 2004 from 218 children attending two schools in neighboring villages were assayed for aflatoxin exposure using the aflatoxin-albumin adduct (AF-alb) biomarker. Data were previously examined for associations among hepatomegaly, malaria, and schistosomiasis.

RESULTS: AF-alb levels were high in children from both schools, but the geometric mean (95% confidence interval) in year 2002 was significantly higher in Matangini [206.5 (175.5, 243.0) pg/mg albumin] than in Yumbuni [73.2 (61.6, 87.0) pg/mg; p < 0.001]. AF-alb levels also were higher in children with firm hepatomegaly [176.6 (129.6, 240.7) pg/mg] than in normal children [79.9 (49.6, 128.7) pg/mg; p = 0.029]. After adjusting for Schistosoma mansoni and Plasmodium infection, we estimated a significant 43% increase in the prevalence of hepatomegaly/hepatosplenomegaly for every natural-log-unit increase in AF-alb. In 2004, AF-alb levels were markedly higher than in 2002 [539.7 (463.3, 628.7) vs. 114.5 (99.7, 131.4) pg/mg; p < 0.001] but with no significant difference between the villages or between hepatomegaly and normal groups [539.7 (436.7, 666.9) vs. 512.6 (297.3, 883.8) pg/mg], possibly because acute exposures during an aflatoxicosis outbreak in 2004 may have masked any potential underlying relationship.

CONCLUSIONS: Exposure to aflatoxin was associated with childhood chronic hepatomegaly in 2002. These preliminary data suggest an additional health risk that may be related to aflatoxin exposure in children, a hypothesis that merits further testing.

Cathelicidin-like Helminth Defence Molecules (HDMs):Absence of Cytotoxic, Anti-microbial and Anti-protozoan Activities Imply a Specific Adaptation to Immune Modulation

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects.