Leishmania tarentolae: Utility as an in vitro model for screening of antileishmanial agents

Primary screens for antileishmanial compounds use Leishmania species pathogenic to humans that must be handled under biosafety conditions that cannot be adopted or guaranteed everywhere. Leishmania tarentolae, a parasite isolated from the gecko Tarentolae annularis, has not been considered pathogenic to humans. Promastigotes of L. tarentolae have been previously used as a eukaryotic expression system for the production of recombinant proteins and in the amplification of genes involved in resistance to antileishmanial drugs. To validate the use of this Leishmania species in the screening of antileishmanial drugs, the sensitivity of axenic and intracellular amastigotes of L. tarentolae was compared to the sensitivity showed by Leishmania species causative of human leishmaniasis. The ability of L. tarentolae to grow as axenic amastigotes is first described while its ability to infect several mammalian cells has been confirmed. L. tarentolae amastigotes offer a suitable model for the in vitro screening of compounds for antileishmanial activity.     

Neolignan Licarin A presents effect against Leishmania (Leishmania) major associated with immunomodulation in vitro

Leishmaniasis’ treatment is based mostly on pentavalent antimonials or amphotericin B long-term administration, expensive drugs associated with severe side effects. Considering these aforementioned, the search for alternative effective and safe leishmaniasis treatments is a necessity. This work evaluated a neolignan, licarin A anti-leishmanial activity chemically synthesized by our study group. It was observed that licarin A effectively inhibited Leishmania (Leishmania) major promastigotes (IC₅₀ of 9.59 ± 0.94 μg/mL) growth, by inducing in these parasites genomic DNA fragmentation in a typical death pattern by apoptosis. Additionally, the neolignan proved to be even more active against intracellular amastigotes of the parasite (EC₅₀ of 4.71 ± 0.29 μg/mL), and significantly more effective than meglumine antimoniate (EC₅₀ of 216.2 ± 76.7 μg/mL) used as reference drug. The antiamastigote activity is associated with an immunomodulatory activity, since treatment with licarin A of the infected macrophages induced a decrease in the interleukin (IL)-6 and IL-10 production. This study demonstrates for the first time the antileishmanial activity of licarin A and suggests that the compound may be a promising in the development of a new leishmanicidal agent.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

In vitro cultivation of Schistosoma japonicum-parasites and cells

Schistosomiasis is a serious parasitic zoonosis caused by blood-dwelling flukes of the genus Schistosoma. Understanding functions of genes and proteins of this parasite is important for uncovering this pathogen's complex biology, which will provide valuable information to design new strategies for schistosomiasis control. Effective applications of molecular tools reported to investigate schistosome gene function, such as inhibitor studies and transgenesis, rely on the developments of in vitro cultivation system of this parasite and cells. Besides the in vitro culture studies dealing with Schistosoma mansoni, there are also numerous excellent studies about the in vitro cultivation of Schistosoma japonicum, which were performed by Chinese researchers and published in Chinese journals. Nearly every stage of the life-cycle of S. japonicum, including miracidia, mother sporocysts, cercariae, schistosomula, and egg-laying adult worms, was employed for developing in vitro cultivation methods, being accompanied by the introduction of several media and supplements that helped to improve culture conditions. It was not only possible to generate mother sporocysts from miracidia in vitro, but also to obtain adult worms from cercariae through in vitro cultivation. The main obstacles to complete the life cycle of S. japonicum in the lab are the transition from mother sporocysts to cercariae, and the production of fertilized and completely developed eggs by adult worms generated in vitro. With regard to cells from S. japonicum, besides established isolation protocols and morphological observations, media optimizations were conducted by using different chemical reagents, biological supplements and physical treatment. Among these, mutagens like N-methyl-N-nitro-N-nitrosoguanidine and the addition of extracellular matrix were found to be able to induce mitogenic activities. Although enzyme activities or the level of silver-stained nucleolar region associated protein in cultured cells indicated still suboptimal conditions, the achievements made point to the possibility of reaching the aim of establishing cell lines for S. japonicum. Both the improvements of the in vitro culture of larval and adult worms of S. japonicum as well as the access of cells of this parasite provide excellent advances for research on this important parasite in the future.     

In vitro activity of pacifastin-like inhibitors in relation to their structural characteristics

Information on the structural characteristics and inhibitory activity of the pacifastin family is restricted to a handful of locust pacifastin-related inhibitors. In this report the optimization of a bacterial recombinant expression system is described, resulting in the high yield production of pacifastin-like inhibitors of the desert locust. Subsequently, the relative inhibitory activity of these peptides towards mammalian, locust and caterpillar digestive peptidases has been compared. In general, the enzyme specificity of locust pacifastin-like inhibitors towards trypsin- or chymotrypsin-like peptidases corresponds to the nature of the P1-residue at the reactive site. In addition, other structural characteristics, including specific core interactions, have been reported to result in a different affinity of pacifastin members towards digestive trypsin-like enzymes from mammals and arthropods. One remarkable observation in this study is a specifically designed pacifastin-like peptidase inhibitor, which, unlike other inhibitors of the same family, does not display this specificity and selectivity towards digestive enzymes from different animals.     

Amelioration of intracerebroventricular streptozotocin induced cognitive impairment by Evolvulus alsinoides in rats: In vitro and in vivo evidence

Evolvulus alsinoides, also known as Shankpushapi, is a commonly used traditional medicine for enhancing memory. We evaluated the in vitro free radical scavenging and enzymes [acetylcholinesterase, butyrylcholinestrase, glycogen synthase kinase-3-β (GSK-3-β), rho kinase (ROCK II), prolyl endopeptidase (PEP), catechol-O-methyl transferase (COMT) and lipoxygenase (LOX)] inhibitory activities of aqueous and hydro-alcoholic extracts of E. alsinoides. Hydro-alcoholic extract of E. alsinoides demonstrated more free radical scavenging activity as compared to aqueous extract. Hydro-alcoholic extract also showed higher cholinesterase, GSK-3-β, ROCK II, PEP, COMT and LOX enzyme inhibitory activities as compared to aqueous extract. Phytochemical analysis revealed more flavanoids in hydro-alcoholic extract as compared to aqueous extract but no significant difference in phenolic content of the two extracts was observed. Based on in vitro data, hydro-alcoholic extract (100, 300 and 500 mg/kg, p.o.) was selected for in vivo study in intracerebroventricularly injected streptozotocin (STZ) induced cognitive impairment in male Wistar rats. Elevated plus maze, passive avoidance and Morris water maze were used for assessment of cognitive function on 14th, 21st and 28th day after STZ injection. Oxidative stress parameters (malondialdehyde, reduced glutathione, nitric oxide levels and superoxide dismutase activity), cholinergic dysfunction and rho kinase (ROCK II) expression were studied in cerebral cortex and hippocampus of rat brain at the end of the study. Hydro-alcoholic extract of E. alsinoides dose dependently prevented STZ induced cognitive impairment by reducing the oxidative stress, improving cholinergic function and preventing the increase in rho kinase expression. The results suggest an anti-Alzheimer potential of hydro-alcoholic extract of E. alsinoides.     

双色真藓的组织培养和发育研究

对双色真藓(Bryum dichotomum Hedw.)的孢子发育过程及愈伤组织的诱导和培养进行了研究。结果表明,双色真藓孢子萌发和原丝体发育属于典型的真藓型。将双色真藓原丝体接种在含有2.0 mg L-1的硅酸钠和3.0 mg L-1 6-BA的MS固体培养基上,可诱导双色真藓原丝体分化为愈伤组织。愈伤组织在含有2.0 mg L-1的硅酸钠、1.0 mg L-12,4-D和1.0 mg L-1 6-BA的MS固体培养基上可以长期继代培养。而愈伤组织在含有2.0 mg L-1的硅酸钠、1.0 mg L-1 2,4-D和1.0 mg L-1 6-BA的MS液体培养基中可以悬浮培养,且生长迅速,培养28 d达到接种鲜重的9.25倍。     

Leishmania major: In vitro and in vivo anti-leishmanial effect of cantharidin

Cantharidin is a natural poisonous compound secreted by male blister beetles. The effect of different doses of cantharidin on Leishmania major (MRHO/IR/75/ER) were investigated both in vitro (promastigote and amastigote viability) and in experimentally-infected BALB/c mice (skin lesions) using ointment or soluble cantharidin. In this study, cantharidin with concentrations of 0.5, 1, 2, 5, 10, 20 and 50 μg/ml inhibited the growth of L. major promastigotes after 24 h and the resultant inhibition levels were 39.22%, 41.95%, 49.88%, 54.78%, 58.01%, 68.30% and 80.04%, respectively. After 72 h, the mean number of amastigotes per macrophage in the culture using 2 μg/ml of cantharidin, (the 50% inhibitory concentration dose (IC₅₀)), was 1.2 while in the control group it was 2.7. In order to perform the inflammatory blister technique, 500 μg of cantharidin were solved in 25 μl of DMSO to show the formation of the blister which leads to treatment of cutaneous leishmaniasis. Using the blister technique, the small lesions (<5 mm) healed after one session. Two weeks of topical treatment with 0.1% cantharidin ointment was an effective method for treating cutaneous leishmaniasis in infected BALB/c mice.     

Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.     

Axenization and optimization of in vitro growth of clonal cultures of Tetratrichomonas gallinarum and Trichomonas gallinae

A rapid and simple procedure was established to obtain clonal axenic cultures of Tetratrichomonas gallinarum and Trichomonas gallinae and to optimize their in vitro growth conditions. Medium 199 was used for axenization of two genetically different clones of T. gallinarum and T. gallinae. Six different media were used to optimize the growth behaviour of axenically grown parasites: Medium 199, TYM, TYI-S-33, Hollander fluid (HF), Trichomonas vaginalis (TV) and modified TV media. The highest cell yields for both axenic clones of T. gallinarum were obtained in modified TV medium without antibiotics. The maximum numbers of trophozoites of T. gallinae were obtained in an optimized HF medium. This study demonstrated that axenic cultures for T. gallinarum and T. gallinae could be obtained avoiding the migration technique through a V-tube. Following axenization and optimization, both clones of T. gallinarum and T. gallinae could be propagated both aerobically and anaerobically.     

Teladorsagia circumcincta: Survival of adults in vitro is enhanced by the presence of a mammalian cell line

Adult Teladorsagia circumcincta survival and motility in vitro was examined in a range of different cell culture media, supplements and gas mixes. Under optimum conditions, worms survived for 14 days, exhibiting high motility for 9 days and egg production for 72 h. Optimum conditions involved co-culture of worms with a HeLa cell line in a supplemented cell medium (CEM) and an atmosphere containing 10% CO₂, 5% O₂ 85% N₂, 65% humidity at 37 °C. The incubation medium consisted of Minimum Essential Medium with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamax and 1% penicillin-neomycin-streptomycin cocktail mix. Compared with optimum conditions, incubation in CEM alone, cell conditioned CEM, RPMI alone, Medium 199 alone, reduced CO₂ or O₂, or when cells were replaced with Escherichia coli, both survival and motility were reduced. Optimum conditions for adult T. circumcincta maintenance for culture, anthelmintic testing or generation of excretory/secretory products are described.     

In vitro and in vivo digestion of octenyl succinic starch

This study aimed to understand effects of octenyl succinic anhydride (OSA) modification of normal corn (NCS) and high-amylose corn (HA7) starch on their enzymatic hydrolysis rates. After modification with 3% and 10% OSA, resistant starch (RS) contents of the cooked OS-NCS increased from 0.8% of the control starch to 6.8% and 13.2% (Englyst Method), respectively, whereas that of the cooked OS-HA7 decreased from 24.1% to 23.7% and 20.9%, respectively. When the cooked NCS, HA7 and OS (10%)-HA7 were used to prepare diets for rats at 55% (w/w) starch, RS contents of the diets were 1.1%, 13.2% and 14.6%, respectively. After feeding to the rats, 20.2–31.1% of the starch in the OS (10%)-HA7-diet was not utilized in vivo and was found in rat feces, which was substantially larger than that of the HA7-diet (≤4.9%) and NCS-diet (≤0.2%). The body weights of the rats, however, remained similar between different groups.     

Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model

We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca²⁺ levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca²⁺ release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo.     

Leishmania major: Effect of protein kinase A and phosphodiesterase activity on infectivity and proliferation of promastigotes

Effect of modulators on protein kinase A (PKA) activity, promastigote growth and their ability to infect peritoneal macrophages was monitored. PKA inhibitors reduced [Protein Kinase Inhibitor (PKI) - 56%; H89 - 54.5%] kemptide phosphorylation by Leishmania major promastigote lysates, while activators increased phosphorylation (8-CPT-cAMP - 88%; Sp-cAMPS-AM - 152%). Activation was specifically inhibited by PKI. Phosphodiesterase inhibitors also increased kemptide phosphorylation (dipyridamole - 171%; rolipram - 106%; and 3-isobutyl-1-methyl-xanthine - 154%). Parasite proliferation was significantly retarded (200 nM H89; 100 μM myristoylated-PKI) or completely inhibited (500 nM H89) by culturing with PKA inhibitors. Incubation with dipyridamole or Sp-cAMPS-AM also inhibited proliferation. Brief treatment (2 h) with either H89, myristoylated-PKI, dipyridamole or Sp-cAMPS-AM reduced initial macrophage infection at days 1 and 2 (>40%) and on day 3 (>78% only for 100 μM myr-PKI). Characterization of leishmanial cAMP mediated signal transduction pathways will serve as the basis for the new drug design.     

Influence of Aloe vera on water absorption and enzymatic in vitro degradation of alginate hydrogel films

This study investigates the influence of Aloe vera on water absorption and the in vitro degradation rate of Aloe vera-Ca-alginate hydrogel films, for wound healing and drug delivery applications. The influence of A. vera content (5%, 15% and 25%, v/v) on water absorption was evaluated by the incubation of the films into a 0.1 M HCl solution (pH 1.0), acetate buffer (pH 5.5) and simulated body fluid solution (pH 7.4) during 24 h. Results show that the water absorption is significantly higher for films containing high A. vera contents (15% and 25%), while no significant differences are observed between the alginate neat film and the film with 5% of A. vera. The in vitro enzymatic degradation tests indicate that an increase in the A. vera content significantly enhances the degradation rate of the films. Control films, incubated in a simulated body fluid solution without enzymes, are resistant to the hydrolytic degradation, exhibiting reduced weight loss and maintaining its structural integrity. Results also show that the water absorption and the in vitro degradation rate of the films can be tailored by changing the A. vera content.     

In vitro study of the aortic interleaflet triangle reshaping

Aortic interleaflet triangle reshaping (AITR) is a surgical approach to aortic valve incontinence that involves placing three stitches at half of the interleaflet triangles height. In this work, the relationship between the actual stitch height and valve functioning, and the safety margin that the surgeon can rely on in applying the stitches were systematically investigated in vitro. AITR surgery was applied to six swine aortic roots placing the stitches empirically at 50%, 60% and 75% of the triangle heights. Then the actual stitch heights were measured and the hydrodynamic performances were evaluated with a pulsatile hydrodynamic mock loop. Actual stitch heights were 45±2%, 61±4% and 79±6%. As compared to untreated conditions, the 50% configuration induced a significant variation in the effective orifice area. With stitches placed at 60%, the mean systolic pressure drop increased significantly with respect to the untreated case, but no significant changes were recorded with respect to the 50% configuration. At 75%, all the hydrodynamic parameters of systolic valve functioning worsened significantly. Summarizing, the AITR technique, when performed in a conservative manner did not induce significant alterations in the hydrodynamics of the aortic root in vitro, while more aggressive configurations did. The absence of a statistically significant difference between the 50% and 60% configurations suggests that there is a reasonably limited risk of inducing valve stenosis in the post-op scenario due to stitch misplacement.     

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.     

Transovarial passage of Leishmania infantum kDNA in artificially infected Rhipicephalus sanguineus

Phlebotomine sand flies are the only proven biological vectors of Leishmania parasites. However, Rhipicephalus sanguineus ticks have long been suspected to transmit Leishmania infantum in studies carried out in laboratory and natural conditions. In the present study, 5 μl of L. infantum promastigotes (1 × 10⁶ cells per ml) was injected into the hemocel through the coxa I of four engorged females (F1, F2, F3 and F4). Control ticks (F5 and F6) were injected with sterile phosphate-buffered saline (PBS) using the same procedure. Then, these females, their eggs, and the originated larvae were tested by real time polymerase chain reaction (real-time PCR) for the presence of L. infantum kinetoplast DNA (kDNA). Females and eggs were tested after the end of the oviposition period (about 5 weeks post-inoculation) whereas larvae were tested about 4 months after the inoculation of females. All artificially infected females were positive for L. infantum kDNA. In addition, two pools of eggs (one from F2 and other from F4) and four pools of larvae (one from each F1 and F4 and two from F2) were positive for L. infantum kDNA. These results showed, for the first time, the transovarial passage of L. infantum kDNA in R. sanguineus ticks, thus suggesting that the transovarial transmission of L. infantum protozoa in ticks is worth to be investigated.     

In silico investigation of novel biological pathways: The role of CD200 in regulation of T cell priming in experimental autoimmune encephalomyelitis

The use of simulation to investigate biological domains will inevitably lead to the need to extend existing simulations as new areas of these domains become more fully understood. Such simulation extensions can entail the incorporation of additional cell types, molecules or molecular pathways, all of which can exert a profound influence on the simulation behaviour. Where the biological domain is not well characterised, a structured development methodology must be employed to ensure that the extended simulation is well aligned with its predecessor. We develop and discuss such a methodology, relying on iterative simulation development and sensitivity analysis. The utility of this methodology is demonstrated using a case study simulation of experimental autoimmune encephalomyelitis (EAE), a murine T cell-mediated autoimmune disease model of multiple sclerosis, where it is used to investigate the activity of an additional regulatory pathway. We discuss how application of this methodology guards against creating inappropriate simulation representations of the biology when investigating poorly characterised biological mechanisms.     

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.