Ovine IgA-reactive proteins from Teladorsagia circumcincta infective larvae

Infection of small ruminants with Teladorsagia circumcincta has, until now, been controlled using a combination of pasture management and frequent anthelmintic treatments. Resistance to the commonly used anthelmintics has driven research into the development of a subunit vaccine, encouraged by the demonstration of development of protective immunity in sheep following exposure to this parasite. Local immune effectors in the abomasum, in particular IgA, are thought to play important roles in naturally- and experimentally-acquired immunity. L3s represent the first contact of this pathogen with the host immune system and, herein, the presence of L3 antigen-specific IgA was demonstrated in abomasal mucus from immune sheep. This antibody source was used to immunoaffinity purify and identify IgA-reactive molecules present in L3s. We identified 155 different proteins in this way, including a number of activation-associated secretory proteins, venom allergen-like-type proteins, detoxifying enzymes, galectins and a suite of other potential vaccine candidate molecules. Levels of immunoaffinity-enriched L3 antigen-specific IgA in gastric lymph from previously-infected sheep were statistically significantly higher (P = 0.004) than those measured in helminth-free sheep and a statistically significant negative correlation (P = 0.005, r_s = −0.565) was identified between immunoaffinity-enriched L3 antigen-specific IgA levels in efferent gastric lymph and total T. circumcincta burden measured at necropsy. In addition, a statistically significant positive correlation (P = 0.007, r_s = 0.534) was measured between immunoaffinity-enriched L3 antigen-specific IgA levels in efferent gastric lymph and the percentage of inhibited L4s enumerated at necropsy. These results indicate that the purified antigens contain components that could be strongly considered as vaccine candidates.     

Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta

Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30 h and adult worms for 4-6 h. Ammonia was the main excretory product, with about 20% urea. For the first 4-6 h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30 h. An initial external ammonia concentration of 600 μM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets.     

Benzimidazole resistance allele haplotype diversity in United Kingdom isolates of Teladorsagia circumcincta supports a hypothesis of multiple origins of resistance by recurrent mutation

Polymorphisms in the isotype I β-tubulin gene are important genetic determinants of benzimidazole (BZ) resistance in a number of parasitic nematode species including Teladorsagia circumcincta, a major gastrointestinal nematode of sheep. This study investigates the genetic diversity at this locus in a BZ-resistant isolate of T. circumcincta (MTci5) derived from a sheep farm in the United Kingdom (UK) that was open to animal, and therefore parasite, migration. Pyrosequencing was used to determine the frequency of single nucleotide polymorphisms (SNPs) known to be associated with BZ resistance. This was followed by a combination of single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing to sample allelic diversity in a 276 bp fragment immediately surrounding the isotype I β-tubulin F200Y mutation. The genetic diversity at this locus was extremely high, with seven different haplotypes found to contain the resistant F200Y polymorphism in this single resistant isolate. Genotyping by SSCP interfaced with pyrosequencing demonstrated that the P200^Y mutation is also present on multiple haplotypes in two other BZ-resistant T. circumcincta isolates from the UK. This contrasts with much lower levels of haplotype diversity in BZ-resistant alleles present in T. circumcincta isolates from French goat farms that are closed to parasite migration. Taken together with our knowledge of T. circumcincta population genetic structure, these results are most consistent with multiple independent origins of resistance and mixing of alleles due to the large amount of livestock movement in the UK.     

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。     

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.     

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.     

In silico and in vitro comparative activity of novel experimental derivatives against Leishmania major and Leishmania infantum promastigotes

This in silico and in vitro comparative study was designed to evaluate the effectiveness of some biurets (K1 to K8) and glucantime against Leishmania major and Leishmania infantum promastigotes. Overall, eight experimental ligands and glucantime were docked using AutoDock 4.3 program into the active sites of Leishmania major and Leishmania infantum pteridine reductase 1, which were modeled using homology modeling programs. The colorimetric MTT assay was used to find L. major and L. infantum promastigotes viability at different concentrations of biuret derivatives in a concentration and time-dependent manner and the obtained results were expressed as 50% and 90% of inhibitory concentration (IC₅₀ and IC₉₀). In silico method showed that out of eight experimental ligands, four compounds were more active on pteridine reductase 1. K3 was the most active against L. major promastigotes with an IC₅₀ of 6.8 μM and an IC₉₀ of 40.2 μM, whereas for L. infantum promastigotes was K8 with IC₅₀ of 7.8 μM. The phenylethyl derivative (K7) showed less toxicity (IC₅₀s > 60 μM) in both Leishmania strains. Glucantime displayed less growth inhibition in concentration of about 20 μM. In silico and especially docking results in a recent study were in accordance with the in vitro activity of these compounds in presented study and compound K3, K2 and K8 showed reasonable levels of selectivity for the Leishmania pteridine reductase 1.     

Expression patterns of sirtuin genes in porcine preimplantation embryos and effects of sirtuin inhibitors on in vitro embryonic development after parthenogenetic activation and in vitro fertilization

We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1-3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1-3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1-3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques.     

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.     

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.     

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.     

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.     

Reproducible reference frame for in vitro testing of the human vertebrae

Definition of an anatomical reference frame is necessary for in vitro biomechanical testing. Nevertheless, there is neither a clear recommendation, nor consensus in the literature concerning an anatomical reference frame for in vitro testing of the human vertebrae. The scope of this work is to define a reference frame for the human vertebrae for in vitro applications. The proposed anatomical reference frame relies on alignment of well-defined points on the endplates, and on two landmarks on the posterior wall. The repeatability of the proposed alignment procedure has been tested in vitro by 5 operators, on 7 specimens. Furthermore, the feasibility and repeatability of the proposed procedure was assessed in silico, using CT-scans of the same specimens.     

The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A.Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine.In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.     

Novel in vitro and in vivo models to study central nervous system infections due to Acanthamoeba spp.

Acanthamoeba granulomatous encephalitis is a serious human infection with fatal consequences. The most distressing aspect of Acanthamoeba granulomatous encephalitis is the limited improvement in mortality. The underlying neurobiology is at present not well understood and treatment options are often of limited efficacy. There is therefore a real need to obtain more knowledge regarding the pathogenesis and pathophysiology of Acanthamoeba granulomatous encephalitis and to develop new chemotherapeutic approaches. However, the difficulties in using mammalian models to study this infection have hindered our search for therapeutic interventions. Recent availability of the blood-brain barrier, in vitro and use of locust as an in vivo model will undoubtedly allow us to investigate disease pathogenesis, mechanisms of parasite traversal across the blood-brain barrier and new drug therapies. It is argued that the models described here can offer several advantages in terms of speed, cost, technical convenience, and ethical acceptance. Furthermore, they are extremely valuable tools to discriminate molecules participating from both sides of the host-parasite interaction and will generate potentially useful leads in the identification of new potential drugs, as well as testing drug toxicity.     

Reproducible and controllable light induction of in vitro fruiting of the white-rot basidiomycete Pleurotus ostreatus

Fruiting is a crucial developmental process in basidiomycetes yet the genetic and molecular factors that control it are not yet fully understood. The search for fruiting inducers is of major relevance for both basic research and for their use in industrial applications. In this paper, an efficient and reproducible protocol for controlled fruiting induction of Pleurotus ostreatus growing on synthetic medium is described. The protocol is based on the control of light intensity and photoperiod and permits the life cycle for this fungus to be completed in less than two weeks. The fruiting bodies produced by this method release fertile spores after 4-5 d of culture. Our results indicate that fruiting induction is solely dependent on the illumination regime and that it occurs long before the available nutrients are depleted in the culture. This protocol will greatly facilitate molecular and developmental biology research in this fungus as it avoids the need for complex culture media based on lignocellulosic materials or the use of chemical inducers.     

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.     

The effect of light quality on leaf production and development of in vitro-cultured plants of Alternanthera brasiliana Kuntze

We investigated the influence of light quality on the leaf development of Alternanthera brasiliana Kuntze (Amaranthaceae) grown in vitro. Growth parameters including specific leaf mass, thickness, and leaf density were lowest in plants grown under red light. Blue light induced the largest number of leaves/plant, and the largest thickness and area of the leafblade. Green and red lights induced the smallest leaf areas. The thickness of the abaxial-face epidermis and spongy parenchyma of the plants was significantly reduced in plants grown under red light. The thickness of the palisade parenchyma and upper epidermis were significantly increased in plants grown under blue light, compared to the other fluorescent-light treatments. The specific spectral band also influenced the differentiation of mesophyll cells. In the dark and under red light, the mesophyll was homogenous; and in the dark and under green light, the leaves were more compact. Under blue light, the cells displayed the characteristic palisade morphology. The results showed that the increase of a specific parenchyma type was related to a specific spectral band. All spectral-quality treatments reduced the numbers of stomata and trichomes. The results for green light were in some respects similar to those for red light, and in other respects similar to those for blue light, probably because phytochromes and cryptochromes are green-light receptors. This study indicated that Alternanthera plants have strong morphological plasticity induced by light. The results suggest that high-quality Alternanthera can be achieved by culturing the plants in vitro under a combination of blue and red light.