The chemiluminescence response of bivalve haemocytes: utility in screening for immunomodulators and as a biomarker

Resistance to infectious diseases in bivalves depends primarily on the vigour and efficacy of haemocyte-dependent antimicrobial defence mechanisms. Like other phagocytes, haemocytes seem to rely on oxygen-independent (lysosomal hydrolases, lysozyme) and oxygen-dependent (reactive oxygen species) mechanisms to destroy ingested microorganisms. The generation of cytotoxic oxyradicals by haemocytes can be precisely quantified by means of a simple chemiluminescence (CL) assay using luminol or other CL probes. Tributyltin (TBT), and other environmental contaminants, at sublethal levels, will produce dose-dependent suppression of CL activity in haemocytes exposed in short-term, in vitro assays. Presumably, this suppression would find expression as impaired host defence capability. In fact, TBT has been shown to exacerbate progression and lethality of Perkinsus marinus infections in the oyster, Crassostrea virginica. This suggests that CL assays on haemocytes exposed in vitro to single agents or complex mixtures might be useful in screening for aquatic immunomodulators. Statistically significant alterations in CL responses of haemocytes withdrawn from bivalves exposed to xenobiotics in the laboratory or field are more difficult to identify because of high inter-animal variation; however, the use of haemocyte CL as a biomarker of effect merits further investigation.     

Effects of Tolypocladium cylindrosporum and its secondary metabolites, efrapeptins, on the immune system of Galleria mellonella larvae

Interactions between Tolypocladium cylindrosporum (Deuteromycetes), its metabolite, efrapeptins, and the insect immune defense were investigated in vivo and in vitro. In the different phagocytosis studies, Bacillus cereus spores which had been labelled with fluorescein-isothiocyanate (FITC) were used. In vitro studies showed that efrapeptins inhibit phagocytic activity of Galleria mellonella (Lepidoptera: Pyralidae) haemocytes. The response was dose-related. Efrapeptins significantly reduced the number of nodules formed in response to an injection of zymosan supernatant. Phenoloxidase (PO) activation system contained in haemocyte lysate (HLS) was not affected by efrapeptins. In vivo studies when larvae were injected with efrapeptins also revealed that efrapeptins did not affect PO activities and total haemocyte count (THC) after 1 and 6 h post-injection. However, 12 h post-injection there was a significant inhibition of PO activities in HLS. There was also no significant reduction of PO activities and THC when larvae were injected with Tolypocladium cylindrosporum spores until 24 h post-injection. However, PO activities were suppressed and THC reduced 48 h post-treatment of larvae with spores. This study suggests that efrapeptins may interfere with the ligand-receptor interactions that are likely to occur at the plasma membrane of specific haemocytes.     

Phycomyces blakesleeanus car B mutants: Their use in assays of phytoene desaturase

Strains of car B (phytoene-accumulating) mutants of Phycomyces blakesleeanus have been characterized with respect to their carotene contents, in vitro formation of isoprenoids from [2-¹⁴C] mevalonic acid and their ability to produce [¹⁴C]phytoene in situ for use in coupled assays of phytoene desaturase activity. All strains produced predominantly (15-Z)-phytoene both in vivo and in vitro. Other isoprenoids were produced by cell extracts including squalene, sterols, prenyl diphosphates and prenyl alcohols. The addition of 1% Tween 60 to crude cell extracts of the mutants partially restored wild type carotenogenic activity and also altered the proportions of other isoprenoids formed. However, in a cytosolic fraction of the car B mutant, the addition of 1% Tween 60 did not result in the production of any carotenoid from phytoene. This fraction was the most effective source of [¹⁴C] phytoene for use in coupled assays of phytoene desaturase activity.     

Translating tissue culture results into animal models: the case of Salmonella typhimurium

Investigators use both in vitro and in vivo models to better understand infectious disease processes. Both models are extremely useful in research, but there exists a significant gap in complexity between the highly controlled reductionist in vitro systems and the largely undefined, but relevant variability encompassing in vivo animal models. In an effort to understand how Salmonella initiates disease at the intestinal epithelium, in vitro models have served a useful purpose in allowing investigators to identify molecular mechanisms responsible for Salmonella invasion of host cells and stimulation of host inflammatory responses. Identification of these molecular mechanisms has generated hypotheses that are now being tested using in vivo models. Translating the in vitro findings into the context of an animal model and subsequently to human disease remains a difficult challenge for any disease process.     

Application and prospects of high-throughput screening for in vitro neurogenesis

Over the past few decades, high-throughput screening (HTS) has made great contributions to new drug discovery. HTS technology is equipped with higher throughput, minimized platforms, more automated and computerized operating systems, more efficient and sensitive detection devices, and rapid data processing systems. At the same time, in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes. However, challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional (3D) in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions. Here, we review the applications of HTS technologies on in vitro neurogenesis, especially aiming at identifying the essential genes, chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids. We also discuss the developmental tendency of HTS technology, e.g., so-called next-generation screening, which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

In vitro steroid metabolic studies in human testes II: Metabolism of cholesterol, pregnenolone, progesterone, androstenedione and testosterone by testes of an estrogen-treated man

The effect of long-term in vivo estrogen treatment on in vitro steroidogenesis by the testes of a young man was investigated. In vitro incubation of testicular tissue of this man with ³H-pregnenolone, ³H-progesterone, ³H-androstenedione and ³H-testosterone demonstrated suppression of 17-hydroxylase activity, with little or no effect of the treatment on Δ⁵-3β-hydroxysteroid oxidoreductase, 5a-reductase and aromatase. Increased 20-hydroxysteroid oxidoreductase activity was observed. Determination of intratesticular steroid concentrations led to similar conclusions.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Studies on the chromosomes of human lymphocytes treated with diazepam in vitro

Human lymphocytes were exposed in vitro to various concentrations of the psychotherapeutic agent diazepam, using two different techniques for culturing the lymphocytes, a whole-blood and a macro procedure. There was no evidence for a damaging effect of diazepam on the lymphoblast chromosomes at any concentration or exposure time studied with either technique of culture. The significance of results obtained in vitro on chromosome-breaking effects of chemical agents in commercial use is discussed, and the importance of some technical details in conducting such experiments is pointed out.     

Enhancement of myeloid cell growth by benzene metabolites via the production of active oxygen species

In low concentrations, benzene and its metabolite hydroquinone are known to have diverse biological effects on cells, including the synergistic stimulation with GM-CSF of hematopoietic colony formation in vitro, stimulation of granulocytic differentiation in vitro and in vivo, and general suppression of hematopoiesis in vivo. These chemicals are also known to be active in the induction of active oxygen species. We used several assays to determine the effects of benzene metabolites (hydroquinone, benzenetriol, benzoquinone) and active oxygen species (xanthine/xanthine oxidase) on cell growth and cell cycle kinetics of the human myeloid cell line HL-60. HL-60 cells treated with these chemicals for 2 h in PBS showed increased growth over untreated controls in a subsequent 18h growth period in complete media. Incorporation of ³H-thymidine was also increased proportionately by these treatments. Catalase treatment abrogated the increased cell growth of all chemicals, suggesting an oxidative mechanism for the effect of all treatments alike. Cell cycle kinetics assays showed that the growth increase was caused by an increased recruitment of cells from G₀/G₁ to S-phase for both hydroquinone and active oxygen, rather than a decrease in the length of the cell cycle. Benzene metabolite's enhancement of growth of myeloid cells through an active oxygen mechanism may be involved in a number of aspects of benzene toxicity, including enhanced granulocytic growth and differentiation, stimulation of GM-CSF-induced colony formation, apoptosis inhibition, and stimulation of progenitor cell mitogenesis in the bone marrow. These effects in sum may be involved in the benzene-induced “promotion” of a clonal cell population to the fully leukemic state.     

Effects of dietary forage particle size and concentrate level on fermentation profile, in vitro degradation characteristics and concentration of liquid- or solid-associated bacterial mass in the rumen of dairy cows

This study investigated effects of dietary forage particle size (PS) and concentrate level (CL) on fermentation profiles of particle-associated rumen liquid (PARL) and free rumen liquid (FRL), in vitro degradation characteristics and concentration of bacterial mass attached to the solid or fluid rumen digesta phase in dairy cows. The experiment was a 4 × 4 Latin square design with four late-lactation dairy cows in four 23 day periods. Cows were restrictively fed (17 kg dry matter (DM)/d) one of four diets varying in the theoretical PS (6 and 30 mm) of grass hay and in the levels (approximately 200 and 550 g/kg, DM basis) of a cereal-based concentrate. Proportion of large particles (>6 mm) and the content of structural fibre in the diet increased by reducing dietary CL and, particularly, by increasing hay PS. This effect was not reflected by changes in mean total volatile fatty acid concentration or pH in the rumen. However, cows fed high concentrate diets had pH of 5.28 and 5.37 in PARL at 3 h after the last meal, when fine or long chopped hay was offered. The low pH may indicate a depression of the capacity of PARL to degrade fibre in vitro. Gas production in vitro of concentrate increased with the high concentrate diet at 12 h, suggesting that amylolytic capacity was affected only in early phases of fermentation. In addition, elevating dietary CL appeared to shift ruminal fermentation outputs from propionate to butyrate and valerate. Inclusion of coarsely chopped hay to a high concentrate diet does not appear to bring advantages due to increased structure in restrictively fed dairy cows. In addition, results suggest that the response of pH in PARL is more sensitive to dietary changes (i.e., forage PS and CL) than the response in FRL, and so PARL might be better to evaluate the risk of ruminal disfunction in dairy cows.     

A review on disease transmission studies in relationship to production of embryos by in vitro fertilization and to related new reproductive technologies

This paper addresses the circumstances of germplasm contamination and updates on transmission of pathogenic agents by embryos produced in vitro and by associated techniques. It has been shown that some pathogenic agents might have been associated with the follicular oocytes and oviductal cells, collected for in vitro fertilization (IVF), resulting in infected embryos. Experimental introduction of pathogenic agent with oocytes or infected semen into the IVF system allows, in most cases, for the fertilization of eggs and for the production of some transferable quality embryos. Rendering of oocytes and embryos free of infectious pathogens, using the standard sequential washing or enzymatic treatment, is inconsistent and more difficult in the presently used in vitro fertilization system as compared to in vivo produced embryos.     

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 μg/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the ‘sleep peptide.’     

Fungal antagonists of the plant pathogen Rhizoctonia solani: selection, control efficacy and influence on the indigenous microbial community

A broad spectrum of fungal antagonists was evaluated as potential biocontrol agents (BCAs) against the soil-borne pathogen Rhizoctonia solani using a new combination of in vitro and in vivo assays. The in vitro characterisation of diverse parameters including the ability to parasitise mycelium and to inhibit the germination of Rhizoctonia sclerotia at different temperatures resulted in the selection of six potential fungal antagonists. These were genotypically characterised by their BOX-PCR fingerprints, and identified as Trichoderma reesei and T. viride by partial 18S rDNA sequencing. When potato sprouts were treated with Trichoderma, all isolates significantly reduced the incidence of Rhizoctonia symptoms. Evaluated under growth chamber conditions, the selected Trichoderma isolates either partly or completely controlled the dry mass loss of lettuce caused by R. solani. Furthermore, the antagonistic Trichoderma strains were active under field conditions. To analyse the effect of Trichoderma treatment on indigenous root-associated microbial communities, we performed a DNA-dependent SSCP (Single-Strand Conformation Polymorphism) analysis of 16S rDNA/ITS sequences. In this first assessment study for Trichoderma it was shown that the pathogen and the vegetation time had much more influence on the composition of the microbiota than the BCA treatment. After evaluation of all results, three Trichoderma strains originally isolated from Rhizoctonia sclerotia were selected as promising BCAs.     

Effect of aging on behaviour of mesenchymal stem cells

Organs whose source is the mesoderm lineage contain a subpopulation of stem cells that are able to differentiate among mesodermal derivatives (chondrocytes, osteocytes, adipocytes). This subpopulation of adult stem cells, called “mesenchymal stem cells” or “mesenchymal stromal cells (MSCs)”, contributes directly to the homeostatic maintenance of their organs; hence, their senescence could be very deleterious for human bodily functions. MSCs are easily isolated and amenable their expansion in vitro because of the research demanding to test them in many diverse clinical indications. All of these works are shown by the rapidly expanding literature that includes many in vivo animal models. We do not have an in-depth understanding of mechanisms that induce cellular senescence, and to further clarify the consequences of the senescence process in MSCs, some hints may be derived from the study of cellular behaviour in vivo and in vitro, autophagy, mitochondrial stress and exosomal activity. In this particular work, we decided to review these biological features in the literature on MSC senescence over the last three years.     

Mapping oxidative DNA damage at nucleotide level

DNA damage induced by reactive oxygen species (ROS) is considered an important intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of the Ligation-mediated polymerase chain reaction (LMPCR) technique, we investigated the in vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50 mM H₂O₂, or purified genomic DNA was treated with 5 mM H₂O₂, 100 μM Ascorbate, and 50 μM, 100 μM, or 100 μM of Cu(II), Fe(III), or Cr(VI) respectively. The damage pattern generated in vivo was nearly identical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-random with guanine bases heavily damaged. Cr(VI) generated an in vitro damage pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor of metal ions (or metal-like ligands). These data show that the local probability of H₂O₂-mediated DNA damage is determined by the primary DNA sequence, with chromatin structure having a limited effect. The data suggest a model in which DNA-metal ion binding domains can accommodate different metalions. LMPCR's unique aspect is a blunt-end ligation of an asymmetric double-stranded linker, permitting exponential PCR amplification. An important factor limiting the sensitivity of LMPCR is the representation of target gene DNA relative to non-targeted genes; therefore, we recently developed a method to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CEE), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplification signal. CEE provided a 24-fold increase in the signal strength attributable to strand breaks plus modified bases created by ROS in the human p53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage induced in human breast epithelial cells exposed to non-cytotoxic concentrations of H₂O₂.     

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis. International Journal for Parasitology 3: 863–868. Ecdysterone injections (0·5 mg/ml) induced sexual reproduction in Opalina sudafricana parasitic in male or female Bufo regularis after 12 days. Hypophysectomy or gonadectomy did not prevent the hormone from producing its effect on the parasites. Ecdysterone did not induce sexual reproduction in the parasites in vitro. Urine of toads injected with ecdysterone induced the opalinids to encyst in vitro.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Activity of dehydroabietic acid derivatives against wood contaminant fungi

The antifungal activity of 10 dehydroabietic acid derivatives with different configuration in A and B rings (cis/trans A/B junction) and different substituents and/or functionalities was evaluated in bioassays in vitro and in situ (pine wood blocks).

The test compounds dissolved in acetone were assayed at several concentrations w/w (test compound/culture medium) against the fungi. The Relative Inhibition (RI) was determined by measuring the radial growth of colonies of the fungi treated with the test compounds by comparison with those of control cultures; the results are expressed as EC₅₀.

The results of bioassays in vitro have shown that hydroxyl and aldehyde functions are required for antifungal activity in this group of compounds and deisopropylation can increase the activity. Our assay of antifungal activity in situ (in pine wood blocks) provides a means to investigate the preservative activities of these antifungal compounds under actual conditions of use.

The dehydroabietic acid derivative cis-deisopropyldehydroabietanol (10) inhibited the growth of several of the fungi tested, in vitro and in situ.

The results obtained in situ with the test compound (10) at 6% and 8% were not significantly different from the reference products and a good level of protection of the wood against the organisms tested was achieved.

The results in wood bioassays present new possibilities in the search for natural new compounds in the wood protection, as an alternative to conventional fungicides.