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.

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.

Purification and ultrastructural localization of surface glycoproteins of Taenia solium (Cestoda) cysticerci.

A glycoprotein-enriched fraction was obtained by Concanavalin A-Sepharose 4B affinity chromatography from a crude extract of T. solium cysticerci. The six most prominent glycoproteins with molecular sizes of 180, 103, 96, 68, 55 and 45 kDa were purified by electro-elution from polyacrylamide gel slices. Ultrastructural localization assays using hyperimmune rabbit sera to each glycoprotein, demonstrated their presence on the tegumentary surface of the bladder wall of T. solium cysticerci. Similar studies showed that the 180 kDa glycoprotein is also present on the surface of the T. solium and T. saginata adult worms, as well as in T. saginata, T. pisiformis and T. crassiceps cysticerci. The 55 kDa glycoprotein, which is one of the most abundant on the cyst surface, was found to correspond to the heavy chain of pig IgG by Western blotting.

Intraocular cysticercosis.

Two intravitreal Taenia cysts were removed intact by pars plana vitrectomy from a 59-year-old woman who had never left the continental United States. The intraocular course of the cysts progressed from an initial chorioretinal location, accompanied by an intense focal inflammatory reaction, to free floating in the vitreous cavity within two months; thereafter, there was only a low-grade vitritis for an additional five months until removal. Light and electron microscopic studies suggested Cysticercus cellulosae as the infecting agent, although mature hooklets were absent. Local pork products were considered to be the source of the infection. Preretinal fibrosis and posterior subcapsular vacuoles were the final residua and did not progress after removal of the cysts. Although uncommon in the United States, cysticercosis should be considered in cases of focal necrotizing chorioretinitis.

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.

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.

Twinning in metacestodes of Taenia solium.

Two cysticerci containing 2 scolices were found among several thousand Taenia solium metacestodes dissected from swine. Microscopic study of tissue sections revealed that both worms were equally well developed in 1 bladder worm, whereas 1 member of the other pair was incompletely formed.

Fine structure of a racemose cysticercus from human brain.

The surface of a racemose cysticercus from the human brain was studied by scanning electron microscopy and the tegument of the cyst by transmission electron microscopy. The surface of the larva is covered by microvilli of uniform shape and size. The glycocalyx of the microvilli bears knotlike areas positive for acid mucopolysaccharides. Microvilli are interconnected by a fine, electron-dense network. The tegumental and subtegumental tissues vary in thickness from one region to the next, and the tissues below the vesicular layer are scattered irregularly, in a seemingly disordered manner.

Cysticerci (Cestoda: Taeniidae) from white-tailed deer, Odocoileus virginianus, in southern Florida.

Metacestodes (cysticerci) of Taenia omissa Lühe, 1910, and Taenia hydatigena Pallas, 1776, were found in 9 and 1 of 124 white-tailed deer, respectively, in southern Florida in 1984-1986. Intensities of T. omissa varied from 1 to 15 (mean = 4.6); only 1 cysticercus of T. hydatigena was collected. No significant difference in the prevalences of T. omissa according to sex, age, or locality was observed.

Scanning electron microscopic observations of Cysticercus fasciolaris (=Taenia taeniaeformis) after treatment of mice with mebendazole.

The time-related topographical changes in mature cysticerci of Taenia taeniaformis induced after medication of infected mice with 250 ppm of mebendazole are described. The changes included the gradual disappearance of microtriches and progressive degeneration of the tegment resulting in an irregular surface with grooves, holes, and craterlike structures. Host cells adhered to the altered areas and the number of these cells increased when more severe changes became apparent. Finally the necrotized cysticerci, which lost their tegument completely, were almost entirely covered with adhesive host cells. A difference in the time sequence of the reported changes occurred between the scolex, the pseudoproglottids, and the bladder. This difference in susceptibility towards the drug between the three parts of the parasite in relation to the morphology of their microtrichous covering is discussed.

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.

Morphological changes in cysticerci of Taenia taeniaeformis after mebendazole treatment.

The progressive micromorphological changes in Taenia taeniaeformis cysticerci, induced by a single parenteral treatment of the infected mice with mebendazole, are described. The time-related alterations concerned the tegument and tegumental cells and were successively: disappearance of microtubules, accumulation of secretory substances in the Golgi areas, decrease in number to complete loss of microtriches, "ballooning" of all tegumental cells with subsequent burst, vacuolization and degeneration of the tegument, and finally necrosis of the pseudoproglottids. Similar but less pronounced injuries were seen in the scolices, although microtubules disappeared as early as in the pseudoproglottids. Microtubules from the host tissues remained intact. The meaning of the apparent primary interference of mebendazole with the microtubular system in relation to the subsequently observed death of the cysticercoids is discussed.

Transhydrogenase activities in the mitochondria of Taenia crassiceps (Zeder, 1800) cysticerci.

NADPH/NAD and NADH/NAD transhydrogenase activities have been demonstrated in the mitochondrial fraction of Taenia crassiceps cysticerci. These activities seem to result from the activities of two different enzyme systems. Both transhydrogenase activities exhibited a high heat resistence and they were completely abolished only by the temperatures higher than 100 degrees C. The activity of NADH/NAD transhydrogenase was rather high (116 nmol.min-1.mg-1 protein), but it was found to exhibit a low affinity to NADH (K'M = 1.43.10(-4) M). The physiological significance of NADH/NAD transhydrogenase is discussed.

Fine structure of cells and tissues of Cysticercus tenuicollis aged 13 and 16 days.

A study has been made on the fine structure of three postembryonic phases of C. tenuicollis. We observed the differentiation of the villus-like microtriches without a distinct point. A lysis of host cells was caused by a secretion released from vesicles and droplets in the microthrix border. We distinguished 5 types of little-specialized cells during the initial phase of bladder development. The smallest of these cells were persisting oncospheral germ cells. The differentiation of cells terminated with the differentiation of the excretory system in a 16 day-old larva.

Purification and properties of cytoplasmic malate dehydrogenase from Taenia crassiceps (Zeder, 1800) cysticerci.

Malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) from the cytoplasm of Taenia crassiceps cysticerci was purified and the basic kinetic parameters of this enzyme were determined. The pH optimum range of enzyme reaction was found to be very wide: 8.8-11.0 for malate oxidation and 6.0-8.5 for oxaloacetate reduction. KM values for oxaloacetate, malate, NAD, and NADH were 7.8.10(-5) M, 1.4.10(-4) M, 1.2.10(-4) M, and 6.10(-5) M, respectively. Malate dehydrogenase activity was inhibited by malate excess. Molecular weight of malate dehydrogenase was 70,800. A comparison of the data obtained with those from other organisms including vertebrates showed that the cytoplasmic malate dehydrogenase from T. crassiceps is almost identical with the enzymes from other sources in its kinetic and regulatory properties, as well as in its molecular weight.

Cysticercus bovis: pinocytosis in the Cysticercus tegument.

The authors have found that pinocytosis occurs in the tegument of C. bovis from the fourth week after infection. Electron-lucid bladders surrounded by plasma membrane were encountered in the distal cytoplasm. The pinocytosis occurred in form of micropinocytotic bladders only in the bladder tegument but not in the scolex. The bladders appeared first in the superficial part of the distal cytoplasm. During the following periods of development of the larva they were dispersed in the whole distal cytoplasm and were found even in the processes of subtegumental cells and inside these cells near the heterolysosomes.

Ultrastructure of the bladder tegument of Cysticercus bovis in various stages of its development.

Early developmental stages of C. bovis possess microvilli on the tegument. The differentiation of microtriches occurs at the time of scolex formation. The distal cytoplasm contains rod-shaped bodies and vesicles of various sizes. Both organelles originate from subtegumental cells. During the development of the bladder wall, three types of cells differing in their structure and organization of granular endoplasmic reticulum are present in the subtegumental layer. After formation of the scolex the cells in which the glycogen is formed sink into the region of parenchyma. The distal cytoplasm contains sensory endings of only one type, which do not penetrate on the surface of the tegument and remain under the plasmatic membrane covering the distal cytoplasm.

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.

Methyl 5(6)-(alpha-hydroxyphenyl methyl) benzimidazole-2-carbamate and cysticercosis: chemotherapeutic and electron microscopic studies.

Methyl 5(6)-(alpha-hydroxyphenyl methyl) benzimidazole-2-carbamate, a major metabolite of mebendazole was evaluated against Cysticercus fasciolaria (larval form of Taenia taeniaeformis) in rats. The metabolite was assessed in various doses. A regimen of 50 mg/kg x 10 (ip), given one day apart, was found to be most effective and killed all the mature cysticerci. On developing cysts, the treatment was initiated in two schedules; 5 days prior to (d-5 to d-1) and 5 days after (d + 6 to d + 10) administration of T. taeniaeformis eggs to rats. The later protocol with 100 mg/kg x 5 dose (ip) resulted in 95% inhibition in the establishment of cysticerci. Activity of mebendazole against mature cysts was parallel to metabolite whereas against developing cysts, it was inferior. The time related topographical changes that occurred in mature C. fasciolaris after treatment with metabolite (50 mg/kg x 10, ip, one day apart) were observed by scanning electron microscopy. There was loss of contractivity, gradual disappearance of microtriches and progressive degeneration of tegument. Similar changes were noticed with mebendazole. The possession of better efficacy and higher safety range [Indian J Exp. Biol, 25 (1987) 871], suggests that the metabolite can be a potential anthelmintic for man and animals.