Development in vitro of the metacercaria of Bucephaloides gracilescens (Trematoda: Bucephalidae)

The development of Bucephaloides gracilescens metacercariae was studied using a range of cultivation conditions. The most rapid development occurred at 18°C in a medium containing NCTC 135 supplemented with 25% chicken serum, 25% hen egg yolk and 25% hen egg albumen, with a gas phase of air. Under these conditions, shell-protein synthesis was triggered by day 3 in culture; secondary oocytes were apparent in the ovary by day 10; and egg production began by day 14. Survival of worms in media containing chicken serum was twice as long as that achieved with either whiting or angler fish serum. The ingestion of yolk (feeding) appeared to be a necessary prerequisite to development and egg production. The presence of yolk in the culture medium greatly increased the amount of ³H-thymidine incorporated by the reproductive system of freshly excysted metacercariae but had little effect on the uptake and incorporation of tyrosine. The eggs produced in vitro failed to embryonale and were abnormal in appearance, being non-operculate with irregularly thickened shells.     

Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

Observations on morphology and hydrolytic enzyme histochemistry of excysted metacercariae of Himasthla rhigedana (Trematoda: Echinostomatidae)

The morphology of in vitro excysted metacercariae of Himasthia rhigedana was studied by light microscopy, enzyme histochemistry, and scanning electron microscopy (SEM). The body surface is covered with longitudinal rows of pointed spines which extend from the anterior end to just below the acetabulum. Histochemical methods for acid and alkaline phosphatases, β-glucoronidasc, leucine aminopeplidase, nonspecific esterase, acetylcholinesterase, butyrylcholincsterase, chymotrypsin-like protease, α-galactosidase and β-galactosidase were applied to the excysted metacercariae. Certain systems of the metacercaria containing these enzymes showed positive reactions to the appropriate methods and were selectively visualized. Reactions for alkaline phosphatase occur throughout most of the excretory system while acid phosphatase occurs in the gut, oral, and ventral suckers. Reactions for nonspecific esterases and cholinesterascs are found throughout the nervous system, in the gut, and in the oral and ventral suckers. The results of including specific inhibitors or activators in the incubation medium after preineubation m 10^?5m diethyl p-nitrophenyl phosphate (E-600) suggested that A-and C-typc esterases are present in the gut. Inclusion of 10^?4m eserine in the incubation medium abolished cholinesterase activity in the nervous system.     

Cultivation in vitro of Schistosomatium douthitti (trematoda: Schistosomatidae)

Cultivation in vitro of Schistosomatium douthitti (Trematoda : Schistosomatidae). International Journal for Parasitology12: 541–545. We grew S. douthitti in vitro from the cercaria to pairing ovigerous adults, using methods and media previously reported for cultivation of Schistosoma mansoni. Growth of S. douthitti in vitro was much more rapid than that of S. mansoni, but the cultures achieved a similar stage of maximal development. Cultured S. douthitti worms were smaller than those from animals and eggs were inviable. We determined that females of this species, known to produce eggs in unisexual animal infections, will also do so in culture in the absence of males.     

The in vitro culture of the blood stages of Plasmodium berghei

Plasmodium berghei was cultured in medium 199 with 10% serum at various temperatures. At 31° and 37°C the parasite numbers decreased. Experiments at 15°C achieved consistent multiplication rates greater than one, with a maximum of three-fold.     

In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica

Significant protection against infection with 10 or 30 metacercariae of Fasciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components, presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established.     

Peppermint (Mentha piperita) inhibits microbial biofilms in vitro

Microbial biofilms have become increasingly problematic in the food processing and medical industries where they cause food and surface contamination. Biofilms have also been implicated as the cause of serious infections in humans as their occurrence makes it difficult to treat common infections and the likelihood of recurrent infections is high. Due to emerging resistance, conventional control methods are fast becoming ineffective. In this study, the use of a selection of commercial plant extracts is investigated. The inhibitory effects of eight herbal extracts on the development of microbial biofilms was investigated against clinical and reference strains of Pseudomonas aeruginosa and Candida albicans. The antimicrobial activity was investigated on the planktonic forms using the minimum inhibitory concentration assay. The extracts that showed the highest antimicrobial activity against the two test organisms were Echinacea angustifolia (cone flower), Mentha piperita (peppermint) and Rosmarinus officinalis (rosemary) with minimum inhibitory concentration values between 0.38 and 2.5 mg/ml. The crystal violet assay was used to assess the effect of pre-treating a surface with plant extracts on cell attachment and the extent of biofilm development following exposure to extracts (biofilm biomass). Most of the extracts reduced microbial colonization by at least 50%. In contrast, preformed biofilms were less responsive to the majority of extracts, thus growth inhibition was more difficult to achieve. Mentha piperita was the only extract that showed some antibiofilm activity against both pathogens.     

Effects of freezing on biological membranes in vivo and in vitro

Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.     

Systematic analysis of the Diplostomidae and Strigeidae (Trematoda)

A systematic analysis of the genera in the Diplostomidae and Strigeidae was made using the Proterodiplostomidae as the outgroup. The Proterodiplostomidae was the family with the greatest preponderance of primitive characters and its monophyly was supported by the unique paraprostate gland. However, no character state supported the monophyly of the Diplostomidae sensu Dubois, 1970. That paraphyletic taxon was composed of 3 monophyletic groups: the pseudosuckerless Neodiplostomidae n. fam. had the most primitive character states of the 3 and its monophyly was based on characters in the neascus/neodiplostomulum metacercariae; the Bolbophoridae n. fam., with pseudosuckers, had its monophyly supported by characters present in the newly named prodiplostomulum metacercaria; and the emended Diplostomidae, also with pseudosuckers, had the most derived states and its monophyly was supported by characters present in the diplostomulum. The presence of pseudosuckers united the Bolbophoridae n. fam., the emended Diplostomidae, and the Strigeidae as a monophyletic assemblage. The Strigeidae had the most derived characters of these 3 taxa and its monophyly was supported by characters in the tetracotyle and the cup-shaped forebody and bilobed tribocytic organ of the adult. In general, the adult stages of these strigeoid families showed very conserved morphology and it was the metacercariae that possessed the innovations. The conserved adult morphology was typical of what one might expect if the intramolluscan stages were analyzed. Thus, the data were concordant with the view that the mollusc and vertebrate definitive host were the original hosts to the Digenea and that the second intermediate host and metacercaria were more recently intercalated. More specifically, the phylogeny of these groups suggested that ancestral bisegmented strigeoids originally infected reptiles, they subsequently radiated into birds with which they coevolved extensively, and on 5 separate occasions they radiated into mammals. The radiations into mammals were, by all available evidence, preceded by second intermediate host shifts from fish to amphibians.     

Synergistic anti-glioma effect of Hydroxygenkwanin and Apigenin in vitro

Apigenin (AP) and Hydroxygenkwanin (HGK) are two natural flavonoid compounds. Previous studies have already demonstrated the anti-tumor capability of AP. However, it is not clear whether HGK has such property. In the current study, the anti-glioma activities of HGK and its synergistic anti-glioma effects with AP on C6 glioma cells were investigated. In addition, the possible mechanisms were also studied.     

CdTe quantum dots (QDs) improve the affinities of baicalein and genistein for human serum albumin in vitro

Baicalein and genistein were studied for the affinities for human serum albumin (HSA) in the presence and absence of three CdTe quantum dots (QDs) with different sizes. Three typical CdTe QDs with maximum emissions of 535 nm (green-emitting, G-QDs), 598 nm (yellow-emitting, Y-QDs), and 654 nm (red-emitting, R-QDs) were tested. The fluorescence intensities of HSA decreased remarkably with increasing concentration of QDs. Baicalein resulted in an obvious blue-shift of the λ_em of HSA from 340 to 334 nm. However, the extents of blue-shifts induced by baicalein and genistein in the presence of QDs were much bigger than that in the absence of QDs. The quenching process of baicalein for HSA was easily affected by the QDs size than that of genistein. QDs increased the quenching constant from 136.97% to 162.24% for baicalein. However, QDs only increased the quenching constants from 20.56% to 32.23% for genistein. G-QDs, Y-QDs, and R-QDs increased the affinities of baicalein for HSA about 3.02%, 6.38% and 9.40%. G-QDs, Y-QDs, and R-QDs increased the affinities of genistein for HSA about 2.56%, 13.46% and 19.44%. The binding affinities of baicalein and genistein for HSA increased with increasing QDs size.     

Ancylostoma caninum: Pharyngeal activity in vitro

One hundred Ancylostoma caninum, in groups of 10 in a special apparatus, were offered dog blood and serum, NaCl, Krebs-Ringer bicarbonate, polyvinylpyrrolidone, intestinal epithelial extracts, heated serum, and dialyzed and nondialyzed fractions of serum. The worms' rate of suction was measured. They sucked actively only in blood, serum, and nondialyzed fraction of serum. These findings suggest that dog serum contains one or more macromolecules which stimulate the worm to suck actively.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Validation of the organic electrochemical transistor for in vitro toxicology

Background

The gastrointestinal epithelium provides a physical and biochemical barrier to the passage of ions and small molecules; however this barrier may be breached by pathogens and toxins. The effect of individual pathogens/toxins on the intestinal epithelium has been well characterized: they disrupt barrier tissue in a variety of ways, such as by targeting tight junction proteins, or other elements of the junctions between adjacent cells. A variety of methods have been used to characterize disruption in barrier tissue, such as immunofluorescence, permeability assays and electrical measurements of epithelia resistance, but these methods remain time consuming, costly and ill-suited to diagnostics or high throughput toxicology.

Methods

The advent of organic electronics has created a unique opportunity to interface the worlds of electronics and biology, using devices such as the organic electrochemical transistor (OECT), whose low cost materials and potential for easy fabrication in high throughput formats represent a novel solution for assessing epithelial tissue integrity.

Results

In this study, OECTs were integrated with gastro-intestinal cell monolayers to study the integrity of the gastrointestinal epithelium, providing a very sensitive way to detect minute changes in ion flow across the cell layer due to inherent amplification by the transistor.

Major conclusions

We validate the OECT against traditional methods by monitoring the effect of toxic compounds on epithelial tissue. We show a systematic characterization of this novel method, alongside existing methods used to assess barrier tissue function.

General significance

The toxic compounds induce a dramatic disruption of barrier tissue, and the OECT measures this disruption with increased temporal resolution and greater or equal sensitivity when compared with existing methods. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.