Cysticidal activity of extracts and isolated compounds from Teloxys graveolens: In vitro and in vivo studies.

In the search of new alternatives for neurocysticercosis treatment, the cysticidal activity of organic extracts of Teloxys graveolens was evaluated. The in vitro activity of hexane, ethyl acetate and methanol extracts against Taenia crassiceps cysts was tested and the selectivity index relative to human fibroblasts was determined. Subsequently, the in vivo efficacy of the methanolic extract at doses of 200 and 500 mg/kg in the murine cysticercosis model was evaluated. The ultrastructural effects in vitro and in vivo of the methanolic extract were also investigated using scanning electron microscopy. Additionally, a bioassay-guided fractionation for the isolation of the cysticidal components was performed. Our in vitro findings revealed that all extracts exhibited good cysticidal activity with EC50 values from 44.8 to 67.1 µg/mL. Although the ethyl acetate and methanolic extracts displayed low cytotoxicity, the methanolic extract was the most selective. The methanolic extract also showed in vivo efficacy which was similar to that obtained with ABZ. Significant alterations were found on the germinal layer of the cysts, with a high accumulation of granules of glycogen and vacuoles. The bioguided fractionation of methanolic extract led to the isolation of three flavonoids: chrysin, pinocembrin and pinostrobin; among them, pinocembrin was the compound that displayed cysticidal activity. This is the first study which reveals that T. graveolens could be a potential source for cysticidal and non-toxic compounds.

[In vitro effect of the S3Pvac vaccine against cysticercosis in human mononucleate cells]. / Efecto in vitro de la vacuna S3Pvac contra cisticercosis en celulas mononucleares humanas.

INTRODUCTION: Neurocysticercosis (NCC) is a parasitic infection caused by the establishment of Taenia solium cysticerci in the central nervous system. The larval stage of the parasite also affects the pig, which is the essential intermediate host for transmission. For this reason, many researchers have focused on identifying protective antigens to prevent swine cysticercosis and interrupt the transmission. These include S3Pvac vaccine antigens. Vaccine is constituted by three protective synthetic peptides: KETc1, KETc12 and GK1. AIM. To evaluate the effect of the vaccine peptides KETc1, KETc12 and GK1 in mononuclear cells of patients with neuro-cysticercosis and healthy individuals. SUBJECTS AND METHODS: Comparative, prospective, transverse study. We studied the proliferation and cytokine profile induced by the three peptides in mononuclear cells from three patients with active NCC, 16 patients by calcified NCC and 16 healthy subjects. RESULTS: KETc1 induces low levels of proliferation in cells from patients with active and controlled NCC, both in lymphocytes and in monocytes. KETc12 and GK-1 induce positive proliferation levels of monocytes in healthy subjects. CONCLUSIONS: KETc1 peptide could be used as an adjuvant in the treatment of patients with active NCC, as induced a Th2 response also GK1 peptide as stimulator of monocyte/macrophage in immunizations with other proteins.

TITLE: Efecto in vitro de la vacuna S3Pvac contra cisticercosis en celulas mononucleares humanas.Introduccion. La neurocisticercosis (NCC) es una infeccion parasitaria generada por el establecimiento de cisticercos de Taenia solium en el sistema nervioso central. La fase larvaria del parasito tambien afecta al cerdo, que es el huesped intermediario indispensable para la transmision. Por tal motivo, muchos investigadores se han enfocado en identificar antigenos protectores para prevenir la cisticercosis porcina e interrumpir la transmision. Entre ellos figuran los antigenos de la vacuna S3Pvac, constituida por tres peptidos protectores: KETc1, KETc12 y GK1. Objetivo. Evaluar el efecto de los peptidos vacunales KETc1, KETc12 y GK1 en celulas mononucleares de pacientes con NCC e individuos sanos. Sujetos y metodos. Estudio comparativo, prospectivo y transversal. Se analizo la proliferacion y el perfil de citocinas inducidos por los tres peptidos en celulas mononucleares de tres pacientes con NCC activa, 16 pacientes con NCC calcificada y 16 sujetos sanos. Resultados. KETc1 induce bajos niveles de proliferacion en las celulas de los pacientes con NCC activa y controlada, tanto en linfocitos como en monocitos. KETc12 y GK-1 inducen niveles positivos de proliferacion de monocitos en sujetos sanos. Conclusiones. El peptido KETc1 podria usarse como coadyuvante en el tratamiento de los pacientes con NCC activa, ya que indujo una respuesta Th2; y el peptido GK1, como estimulador del monocito/macrofago en inmunizaciones con otras proteinas.

Release of glycoprotein (GP1) from the tegumental surface of Taenia solium by phospholipase C from Clostridium perfringens suggests a novel protein-anchor to membranes.

In order to explore how molecules are linked to the membrane surface in larval Taenia solium, whole cysticerci were incubated in the presence of phospholipase C from Clostridium perfringens (PLC). Released material was collected and analyzed in polyacrylamide gels with sodium dodecyl sulfate. Two major bands with apparent molecular weights of 180 and 43 kDa were observed. Western blot of released material and localization assays in cysticerci tissue sections using antibodies against five known surface glycoproteins of T. solium cysticerci indicated that only one, previously called GP1, was released. Similar localization studies using the lectins wheat-germ-agglutinin and Concanavalin A showed that N-acetyl-D-glucosamine, N-acetylneuraminic, sialic acid, alphamethyl-D-mannoside, D-manose/glucose, and N-acetyl-D-glucosamine residues are abundantly present on the surface. On the other hand, we find that treatment with PLC releases molecules from the surface; they do not reveal Cross Reacting Determinant (CRD), suggesting a novel anchor to the membrane for the glycoprotein GP1.

Cysteine proteinase inhibitors in murine cysticercosis.

We explored the prophylactic efficacies of two novel protease inhibitors in murine cysticercosis. Our results demonstrated a 95% and 80% reduction in parasite burden for mice injected with Z-LLL-FMK and Z-LLY-FMK, respectively. Further studies are merited on the role of cysteine proteinase inhibitors in treatment of cysticercosis.

Metabolite pattern of Cysticercus cellulosae metacestode from different predilection sites of swine using proton magnetic resonance spectroscopy.

Cysticercosis due to Taenia solium is one of the most common public health problems in various regions of the world. We have performed prolon magnetic resonance spectroscopy (1H MRS) experiments of the fluid aspirated from cysticerci excised from skeletal muscle (n = 16) and brain (n = 9) of infected swine to compare the metabolite pattern of cysticerci in different predilection sites. Perchloric acid extract of cysticercus cysts excised from skeletal muscles (n = 16) was also prepared to ascertain water-soluble, low molecular weight metabolites using 1H MRS. Absolute quantification and statistical analysis of different metabolites was done to look for any significant differences in different locations of cysts. The metabolite pattern of cysticerci was found to be similar in the various predilection sites. Metabolites observed were leucine, valine, alanine, lysine, glycine, lipid contents, lactate, glutamate, acetate, succinate, creatine, choline, and glucose. Concentration of creatine in cysticercus fluid of cysts removed from the muscle was found to be significantly higher (p = 0.001) than the cysts located in the brain. We conclude that the metabolite pattern in the cysticerci is not influenced by the surrounding tissue location; however concentration of certain metabolites may depend upon the tissue location.

Prediction of viability of porcine neurocysticercosis with proton magnetic resonance spectroscopy: correlation with histopathology.

Neurocysticercosis (NCC) is the most frequent parasitic disease of central nervous system. In our earlier study, we had observed creatine [(creatine + phosphocreatine); (tCr)] on ex vivo proton MR spectroscopy (1H MRS) in some of the cysticercus cyst fluid samples obtained from swine's brain parenchyma. In current study, swine brains of freshly slaughtered animals naturally infected with NCC were subjected to ex vivo magnetic resonance (MR) imaging on a 1.5Tesla MR system. Cysticercus cysts (n = 12) were removed from these brains and were labeled depending upon presence or absence of edema around cysts as observed on imaging. Cysticercus cyst fluid (100 microl) was subjected to different 1H MRS experiments and results were compared with histopathological examinations to look for any relationship between tCr and parameters like quantification of musculature, and cellular infiltration in wall of the parasite. Histopathology of cyst wall was categorized into two groups based on cellular characteristics and the amount of musculature. Grade I cysts (n = 5) with no or minimal inflammation and large amount of musculature showed tCr on 1H MRS. However, grade II cysts (n = 7) with profuse inflammation and less amount of musculature in the cyst wall lacked tCr. Higher amount of musculature in grade I cysts was associated with higher concentration of tCr in the cyst fluid (r2 = 0.93, P = 0.007). Creatine appears to be a marker of innocuous and viable NCC.

Experimental studies on physiological and morphological aspects of Cysticercus cellulosae in pigs.

Three Small-Ear-Miniature, 3 Landrace-Small-Ear-Miniature, and one Douc-Yorkshire-Landrace pigs were inoculated orally with 100 000 eggs of Zhengzhou strain or 10 000 eggs of Harbin strain of Taenia solium. A total of 3739 cysticerci were recovered from 3 Small-Ear-Miniature and 3 Landrace-Small-Ear-Miniature pigs, giving an infection rate of 85.7% and a cysticercus recovery rate of 1.1%. The predilection sites of Cysticercus cellulosae in descending order were leg muscles, abdominal muscles, thoracic muscles, liver, head muscles, diaphragm, tongue, heart, trachea, and omentum/testes. Except 2 calcified cysticerci in the tongue, 2 in the heart, and 176 in the liver, the remaining cysticerci were all alive. The greatest number of cysticerci per 100 g of muscles or viscera was found in the head muscles, followed by the leg, diaphragm, heart, tongue, thoracic, abdominal, omentum, testes, and trachea. All cysticerci were evaginated in pig's bile after fluid was drawn out from cysticerci, whereas evagination occurred in only 83.2% of those without fluid drawing. In 364 evaginated cysticerci, the mean length and width of scolex, proglottid, and bladder, and diameter of rostellum and sucker were 826 x 747 microm, 5,370 x 1,734 microm, 2,885 x 3,002 microm, 155 microm, and 253 microm, respectively. In the protoscolex, the mean number of segments was 33. Each cysticercus had 2 rows of rostellar hooks on the scolex, and the mean length and width of inner and outer hooks were 151 x 18 microm and 117 x 14 microm, respectively. The number of paired hooks ranged from 10 to 18.

Formation of calcareous corpuscles in the lumen of excretory canals of Taenia solium cysticerci.

Platyhelminths, like many other organisms, are capable of producing mineral concretions. In cestodes these are referred to as calcareous corpuscles. Studies on these concretions in different cestodes both in vivo and in vitro have resulted in a number of hypotheses on their origin, formation, and structure. Calcareous corpuscles are believed to be of cellular origin, although the kind of cell involved and the mechanisms of mineralization remain under discussion. In the present paper we show that formation of calcareous corpuscles in cysticerci of Taenia solium is not of intracellular origin, as described for other cestodes, but occurs extracellularly in the lumen of protonephridial ducts in a way similar to that proposed for trematodes. This finding enhances the function of the protonephridial ducts, at least in the larvae of T. solium, to the roles formerly ascribed to the calcareous corpuscles.

Cytomorphological spectrum of cysticercosis--a review of 132 cases.

A retrospective analysis of fine needle aspirates of 132 cases of cysticercosis presenting as palpable nodule is presented. In 98 cases, larval parts, detached hooklets and scolex established the diagnosis; in another 24 cases, the background inflammatory pattern was helpful in suggesting the diagnosis of a parasitic lesion.

A method for the isolation of tegument syncytium mitochondria from Taenia crassiceps cysticerci and partial characterization of their aerobic metabolism.

Heterogeneous populations of mitochondria have been described in helminths. Mitochondria from different tissues have been isolated in adult organisms. However, in larvae, due to their small size, isolation from tissues has not been feasible. A method for the isolation of tegumental mitochondria from the larval stage of Taenia crassiceps is described. After solubilization of the plasma membrane with saponin, tegumental mitochondria were purified by a simple and rapid protocol of differential centrifugation, which allowed the retention of suitable quantities of well-preserved mitochondria, as judged by biochemical and ultrastructural parameters. Respiratory activity evoked by exogenous NADH was negligible, but its oxidation increased several-fold after sonication of intact mitochondria. Other substrates, e.g., succinate and malate-glutamate, were oxidized at high rate, leading to the formation of a H+ gradient across the inner mitochondrial membrane, which, in turn, supported oxidative phosphorylation. These results indicate that tegumental mitochondria carry out aerobic metabolism.

Demonstration of Taenia crassiceps cysteine proteinase activity in tegumentary lysosome-like vesicles.

Larval stages of Taenia species survive for prolonged periods in the tissues of their intermediate hosts. Other groups have demonstrated that host immunoglobulins are taken up by the cysticerci by adsorptive endocytosis, degraded, and the amino acids incorporated into parasite proteins. We have shown that a 43-kDa cysteine proteinase is the major parasite enzyme that degrades immunoglobulin in vitro. To localize this enzyme in situ, Taenia crassiceps cysticerci were incubated with the peptide substrate Z-Phe-Arg-methoxynaphthylamide. Free methoxynaphthylamide was coupled to p-rosanilin and osmium and visualized by transmission electron microscopy. Initial studies of cysticerci incubated without substrate confirmed the normal microanatomy and absence of significant host inflammation. In comparison to controls with no substrate, sections of cysticerci incubated with substrate revealed electron-dense deposits in round vesicles. The vesicles were found primarily within the tegumentary cytons and internuncial processes, a location similar to that described for vesicles associated with adsorptive endocytosis. There were proportionately more endocytotic vesicles and electron-dense vesicles in smaller cysticerci than larger ones. Formation of electron-dense deposits was inhibited by heat and partially inhibited by the cysteine proteinase inhibitor E-64. These data are consistent with localization of the cysteine proteinase activity to lysosome-like vesicles.

Impairment of the inflammatory reaction on implanted Taenia solium metacestodes in mice by a T. solium RNA-peptide: a scanning electron microscopy study.

Inhibition of inflammation by a Taenia solium RNA-peptide (metacestode factor, MF) was studied by scanning electron microscopy (SEM). Viable (96%) T. solium metacestodes obtained from a naturally infected pig were dissected and implanted in treated and control mice, removed at 6 and 12 days postimplantation (p.i.), and studied by SEM. At day 6, metacestodes in control mice showed vigorous inflammation, whereas in mice treated with MF they were apparently intact with exiguous inflammation. Mice immunized with T. solium metacestode antigens showed a moderate inflammation; those treated with both MF and T. solium antigens presented scanty inflammation. At day 12, metacestodes presented copious inflammation and severe damage to the sucker tissues in mice immunized with T. solium; in mice treated with either MF or MF and T. solium antigens there was only discrete inflammation. These observations illustrate the central role of MF in the inhibition of the early events leading to the parasite's destruction by means of an inflammatory response.

[Establishment of Hymenolepis diminuta-animal model and morphology of cysticercoid].

AIM: To observe the morphological changes in the process of the development of Hymenolepis diminuta. METHODS: The life cycle of Hymenolepis diminuta was established between Rattus domesticus albus and Triboliun castaneum. The morphology of cysticercoid were observed microscopically, and the ultrastructure of the body surface of cysticercoid were observed by scanning electron microscopy. RESULTS: Three phases including the mature stage, the blister stage and the protective outer membrane-forming stage during the growing course of cysticercoid were observed microscopically. Under scanning electron microscope, lots of sieve-like micropores on the surface of mature cysticercoid were seen in the second week after infection. The blister phase was found in the third week and the outer membrane measuring about 45 microns in thickness were found surrounding the cysticercoid and vesicular surface, forming a smooth cyst wall in the fourth week. CONCLUSION: The life cycle of Hymenolepis diminuta has been established in the animal-model. The finding of the three phases during the growing course of cysticercoid is reported for the first time.

[Histological changes of Cysticercus cellulosae under the action of proteinase of Omphalia lapidescens in vitro].

AIM: For the purpose of finding an anthelmintic with high efficacy, low toxicity and low cost, especially from the Chinese traditional medicines. METHODS: Forty Cysticercus cellulosae were separately incubated with proteinase from artificially fermented Omphalia lapidescens for 2 h, 4 h and 8 h, and then examined for histological changes by light microscopy and compared with those of proteinase from natural dry Omphalia lapidescens after similar treatment. RESULTS: Cysticercus cellulosae were morphologically and structurally impaired after exposure to the action of proteinase of Omphalia lapidescens, and the degree of impairment was proportionally paralleled to the duration of treatment. CONCLUSION: The anti-cysticercus effect of proteinase of Omphalia lapidescens and the homogeneity of proteinases from artificially fermented and natural Omphalia lapidescens were confirmed.

Ultrastructural observation on spermatocytogenesis in Taeniid cestodes.

AIM: To study the spermatocytogenesis of taeniid cestodes at the ultrastructural leaves. METHODS: Transmission electron microscopy. RESULTS: The ultrastructural observation on spermatocytogenesis in Taenia solium, T. saginata and T. pisiformis were made by TEM. Two types of spermatogonia; type A and B, as well as the supporting cells surrounding the peripheral of spermatogonia are recognized. The type A spermatogonia are stem cells and the type B are mother cells which produce 16 primary spermatocytes by mitosis for 4 times with the cells unseparated. The primary spermatocytes are characterized by the ribosome masses in the cytoplasm. 32 secondary spermatocytes arranged in roselike were produced by reductive division of primary spermatocytes. The secondary spermatocytes become the spermatid quickly by short time development. CONCLUSION: The dividing mode of spermatogonia in Taeniid cestodes is mitosis with cells unseparated.

Ultrastructural observations on the formation and metabolism of calcareous corpuscles in Cysticercus cellulosae.

AIM: To study the formation and metabolism of calcareous corpuscles from Cysticercus cellulosae at the ultrastructure level. METHODS: Transmission electron microscopy. RESULTS: The developmental processes of calcareous corpuscles could be divided into two stages: the intracellular formation stage and the extracellular metabolic stage. The calcareous corpuscles were formed in a cell which we named calcareous corpuscle forming cell. At the early stage of the formation, the corpuscles appeared to be secretory granules in the cells. With the development of the corpuscles, they became bead-shaped and lamellae-like, then the calcareous corpuscle forming cell enlarged and the organellae degenerated. Finally the corpuscles gathered to form particle substances with black dense background, while the nucleus and organellae of the forming cell all disappeared. There were 1-3 or 10-20 calcareous corpuscles in a mature forming cell. Then, the corpuscles were released to the parenchymal tissues and gradually appeared to be concentric lamella or an empty cavity during the metabolic process. CONCLUSION: The calcareous corpuscles were formed in calcareous corpuscle forming cell and consumed in metabolic process in the parenchymal layer of Cysticercus cellulosae.

Observations on the effects of cysticercosis immune sera on cysticercosis by scanning electronmicroscopy.

Two antigens of Taenia solium cysticercus, cystic fluid antigen (CFA) and the culture medium antigen (CMA), were used respectively to immunize rabbits in order to obtain immunosera. The CMA immunoserum added to culture medium with cysticerci limited the activities of the bladder worms. By using the scanning electronmicroscope, we could observe particulate deposits on the surface of the scolices, suckers and necks of the worms. The CFA immunoserum group showed similar changes but the deposit was less than that on the worms in the former group and appeared mainly on the cystic wall. After adding complement to the two groups mentioned above, we found that the microcilia on the surface of the worms were swollen and were seriously damaged. The worms treated with praziquantel were damaged over large area of their surfaces and were affected deep into their tissues. The damaged parts of the worms were quite different between the two groups. CMA is secreted by the living worms and therefore the serum antibodies are more effective than CFA in anti-parasite activity.

[Electron microscopic observation on cysticercus cellulosae treated with mienangling in vitro].

OBJECTIVE: To observe the effect of Mienangling (MNL) in treating cysticercus cellulosae in vitro. METHODS: The changes of cysticercus cellulosae treated in vitro with the MNL extracts, praziquantel (PQT) and albendazole (ALB) were studied under the transmission and scanning electron microscopes. RESULTS: Under the scanning electron microscope, the authors can see that all of the cysticercus's scoleces did not grow in the MNL and PQT groups, cyst walls shrivelled, tegument's ectoplasts were damaged, microtriches's ultrastructures were not clear or exfoliated, some corpuscular matter densely was distributed in the surface; 46.7% of cysticercus cellulosaes in the ALB group had been excystated, the rostellar hooks of rostella developed normally, four sucker's surface wrer hollow, the necks were eroded, and grooves of sonite disappeared. Under the transmission electron microscope, we can see that the tegument, basement membrane, muscular layer and parenchyma in the groups of MNL and PQT were all clearly damaged, but the harmful effect of MNL to parenchyma's nerve cords is more serious than the PQT. The damage of all layers of the cysticercus cellulosae in the ALB group is smaller in degree than the MNL and the PQT. CONCLUSION: The MNL has marked damaging effect to cysticercus cellulosae.

Immunoperoxidase for the detection of antibodies in cerebrospinal fluid in neurocysticercosis: use of Cysticercus cellulosae and Cysticercus longicollis particles fixed on microscopy slides

A cepa ORF de Cysticercus longicollis (Cl) representa importante modelo para estudo de antigenos heterologos no imunodiagnostico da neurocisticercose (NC). Foi padronizada a tecnica de imunoperoxidase (IP) empregando suspensao antigenica particulada. Amostras de liquido cefalorraquiano (LCR) foram incubadas sobre o antigeno fixado em laminas de microscopia, o conjugado empregado foi anti-IgG-Peroxidase, a reacao enzimatica iniciou-se ao cobrirem-se as laminas com solucao cromogena (Diaminobenzidina/H2O2). Apos lavagens em agua destilada, a lamina foi corada com verde malaquita a 2 por cento em agua. De 21 LCR de pacientes com NC, 19 (90,5 por cento) foram reativos e 8 (100 por cento) LCR do grupo controle foram nao reativos. Os resultados do teste IP-Cl ensaiando 127 LCR de pacientes com suspeita de NC mostrou 89,7 por cento de concordancia com o teste ELISA empregando extrato salino de Cysticercus cellulosae (Cc) e 94,2 por cento de concordancia com o teste IP-Cc