An in vivo and in vitro assessment of TOR signaling cascade in rainbow trout (Oncorhynchus mykiss)

In mammals, feeding promotes protein accretion in skeletal muscle through a stimulation of the insulin- and amino acid- sensitive mammalian target of rapamycin (mTOR) signaling pathway, leading to the induction of mRNA translation. The purpose of the present study was to characterize both in vivo and in vitro the activation of several major kinases involved in the mTOR pathway in the muscle of the carnivorous rainbow trout. Our results showed that meal feeding enhanced the phosphorylation of the target of rapamycin (TOR), PKB, p70 S6 kinase, and eIF4E-binding protein-1, suggesting that the mechanisms involved in the regulation of mRNA translation are well conserved between lower and higher vertebrates. Our in vitro studies on primary culture of trout muscle cells indicate that insulin and amino acids regulate TOR signaling and thus may be involved in meal feeding effect in this species as in mammals. In conclusion, we report here for the first time in a fish species, the existence and the nutritional regulation of several major kinases involved in the TOR pathway, opening a new area of research on the molecular bases of amino acid utilization in teleosts.     

Antioxidant,anti-inflammatory and healing potential of ethyl acetate fraction of Bauhinia ungulata L. (Fabaceae) on in vitro and in vivo wound model

The present work aimed to investigate the antioxidant, anti-inflammatory and wound healing potential of ethyl acetate fraction from Bauhinia ungulata L. (FABU) on in vitro and in vivo models. Wound healing assay using human lung adenocarcinoma A549 cell line was employed to evaluate the ability of FABU in modulating cell migration. In addition, a surgical wound model in C57BL/6 mice was used to study the healing potential of FABU incorporated into gel carbomer 940 (Carbopol®). Evaluation of lipid peroxidation, inflammatory and anti-inflammatory mediator gene expression, rate of wound closure, and histological analysis were done. FABU significantly reduced the gap area in in vitro wound healing assay, 24 h after treatment. In the animal model, FABU at 0.5% topically applied once-daily for 5 days to the surgical wounds significantly reduced the lesion area. Moreover, it significantly decreased the levels of lipid peroxidation in the lesions and decreased the relative gene expression levels of IL-1β and TNF-α in the injured region. In conclusion, our study suggests that Bauhinia ungulata can effectively promote the wound healing, probably by regulating the inflammatory environment during the early stages of the process.

Graphic abstract

     

Evaluation of an in vitro and in vivo model for experimental infection with Leishmania (Viannia) braziliensis and L. (V.) peruviana

Leishmania (Viannia) braziliensis and L. (V.) peruviana are two parasite species characterized by a very different pathogenicity in humans despite a high genetic similarity. We hypothesized previously that L. (V.) peruviana would descend from L. (V.) braziliensis and would have acquired its 'peruviana' character during the southward colonization and adaptation of the transmission cycle in the Peruvian Andes. In order to have a first appreciation of the differences in virulence between both species, we evaluated an in vitro and in vivo model for experimental infection. A procedure was adapted to enrich culture forms in infective stages and the purified metacyclics were used to infect macrophage cell lines and golden hamsters. The models were tested with 2 representative strains of L. (V.) braziliensis from cutaneous and mucosal origin respectively and 2 representative strains of L. (V.) peruviana from Northern and Southern Peru respectively. Our models were reproducible and sensitive enough to detect phenotypic differences among strains. We showed in vitro as well as in vivo that the L. (V.) braziliensis was more infective than L. (V.) peruviana. Furthermore, we found that in vitro infectivity patterns of the 4 strains analysed, were in agreement with the geographical structuring of parasite populations demonstrated in our previous studies. Further work is needed to confirm our results with more strains of different geographical origin and their specific clinical outcome. However, our data open new perspectives for understanding the process of speciation in Leishmania and its implications in terms of pathogenicity.     

An antibody-based affinity chromatography tool to assess Cu,Zn superoxide dismutase (SOD) G93A structural complexity in vivo

‘Conformational diseases’ are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1^G93A mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue-specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody-based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSOD^G93A rats provided evidence about heterodimers rSOD–hSOD^G93A formation in both extracts. Moreover, when characterized by 2-DE and MALDI-TOF/TOF MS, the extracted hSOD^G93A showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post-translational modifications of hSOD1 produced in various tissues.     

Investigation on the mechanisms of leukocyte alteration in patients with intolerance to some drugs (with novocaine as a model)

The mechanisms of novocain damaging action on blood cells in persons with increased sensitivity to this drug were studied in the leukocyte alteration test. The leading role of histamine liberated from basophils was established. The increased sensitivity to novocain was shown not to be passively transferred to the cells of healthy donors with patient serum with intolerance to the drug; moreover, the joint action of both cell-mediated and thermolabile humoral factors was found to be necessary for the realization of leukocyte alteration under the action of novocain. The comparison of the information content of a number of methods--skin testing, dosed provocation, leukocyte alteration test and chemiluminescence--for revealing increased sensitivity to novocain in 30 persons with adverse reactions to this preparation registered in their medical history. The two in vitro tests were shown to be comparable in their diagnostic significance with the method of dosed provocation.     

An in vitro model that recapitulates the epithelial to mesenchymal transition (EMT) in human breast cancer

The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells down-regulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In human breast cancer, invasion into surrounding tissue is the first step in metastatic progression. Here, we devised an in vitro model using selected cell lines, which recapitulates many features of EMT as observed in human breast cancer. By comparing the gene expression profiles of claudin-low breast cancers with the experimental model, we identified a 9-gene signature characteristic of EMT. This signature was found to distinguish a series of breast cancer cell lines that have demonstrable, classical EMT hallmarks, including loss of E-cadherin protein and acquisition of N-cadherin and vimentin expression. We subsequently developed a three-dimensional model to recapitulate the process of EMT with these cell lines. The cells maintain epithelial morphology when encapsulated in a reconstituted basement membrane, but undergo spontaneous EMT and invade into surrounding collagen in the absence of exogenous cues. Collectively, this model of EMT in vitro reveals the behaviour of breast cancer cells beyond the basement membrane breach and recapitulates the in vivo context for further investigation into EMT and drugs that may interfere with it.     

Anti-thyroid cancer properties of a novel isoflavone derivative, 7-(O)-carboxymethyl daidzein conjugated to N-t-Boc-hexylenediamine in vitro and in vivo

The incidence of thyroid cancer is up to 3 folds higher in women than in men, suggesting that estrogenic effects may be involved in the pathogenesis of this malignancy. Here, we explore whether or not human thyroid cancer cell growth can be curbed by a novel isoflavone derivative generated in our laboratory, the N-t-Boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc). With the exception of the follicular cancer cell line WRO, estrogen receptor (ER)α mRNA was only marginally expressed in cell lines derived from papillary (NPA), follicular (MRO), anaplastic thyroid carcinoma (ARO) such that the expression of estrogen receptor (ER) βmRNA was more abundant than that of ERα mRNA in these cell types. Estradiol-17β (E2; 0.03-300nmol/l) per se increased proliferation in all four cell-types. The ERβ-specific agonist DPN increased [(3)H]-thymidine incorporation in all four thyroid cancer cell lines, whereas the ERα-specific agonist PPT increased growth only in NPA and WRO. By contrast, cD-tboc, derived from the weak estrogen daidzein, did not cause cell growth and dose-dependently diminished cell growth in all four cell lines via apoptosis and not necrosis, as detected by the release of histone-DNA fragments. The cytotoxic growth inhibitory effect of cD-tboc in these cells was modulated by E2 and the general caspase inhibitor Z-VAD-FMK, and the magnitude of this salvage was cell type-and dose-dependent. When nude mice carrying ARO thyroid xenografts were treated with cD-tboc, tumor volume decreased significantly, and no apparent toxicity was observed. These results suggest that cD-tboc may be a promising agent for therapy of thyroid carcinoma either alone or in combination with existing cytotoxic drugs.     

Prolonged cultures of unstimulated human neutrophils lead to the apparition and persistence of rest-in-plate structures (RIPs) recognized by professional phagocytes in vitro and in vivo

Polymorphonuclear neutrophil cells (PMNs) are known to spontaneously undergo apoptosis and then eliminated by professional phagocytes to prevent inflammation, a process called efferocytosis. However, when efferocytosis is impaired, PMNs will fall into secondary necrosis. Whether this state can persist for a certain period of time is unclear, since most of the studies investigating secondary necrosis are performed within 24 h following induction by a proapoptotic agent. In this study, freshly isolated human PMNs were incubated without addition of exogenous agents in order to force them to undergo apoptosis and then secondary necrosis, an ideal experimental condition to study the behavior of secondary necrotic PMNs in absence of efferocytosis. By monitoring PMN cell morphology over time, we observed that an increasing proportion of cells harbored a ghost-like phenotype. Because these cellular remnants persist in plates for several days, we introduce here the terminology RIPs for ‘rest-in-plate’ structure. Heating of freshly isolated PMNs for 5 min did not lead to the apparition of RIPs over time. In vivo administration of 7-days old RIPs in the murine air pouch model induced a slight inflammation resorbed within 24 h. PKH26-stained RIPs were found to be ingested by professional phagocytes in vitro and in vivo in the murine air pouch and peritonitis models. Therefore, aged-PMNs have the potential to become RIPs in absence of efficient efferocytosis. Fortunately RIPs are recognized by professional phagocytes and, therefore, the concept of resolution of inflammation based on elimination of apoptotic and secondary necrotic PMNs could also be applied to RIPs.     

Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): A novel model system to study oil reserves in nonseed tissues

? Premise of the study: Storage oil (triacylglycerol) accumulates in tissues such as the embryo and endosperm of seeds and the fruit mesocarp, but seldom in underground organs. As a rare exception, cultivated variants of yellow nutsedge (Cyperus esculentus) contain high amounts of both oil and starch in the mature tubers. ? Methods: Biochemical analyses and light and electron microscopy were used to study the accumulation patterns of storage nutrients in developing nutsedge tubers. ? Key results: During the initial phase of tuber development, the conducting rhizome tissue is transformed into a storage compartment, then massive storage reserves accumulate in the tuber. At the beginning of tuber development, a large sugar load coincided with the onset of starch accumulation. Oil accumulation started later, concomitant with a substantial drop in the sugar content. Initially, oil accumulated at a lower rate compared to starch, but the rate later increased; after 6 wk, oil made up 24% of tuber dry mass, while starch made up 32%. Protein concentration changed only a small amount throughout this development. Oil and starch accumulated in the same cells throughout the tubers in a sequential fashion during tuber development. ? Conclusions: The developmental pattern in the build up of storage nutrients in the tubers highlights nutsedge as a novel model plant, having potential to significantly widen our understanding on how synthesis of storage reserves, and in particular oils, is regulated and directed in nonseed tissues such as tubers and roots.     

In vitro susceptibility of T lymphocytes from chimpanzees (Pan troglodytes) to human herpesvirus 6 (HHV-6): a potential animal model to study the interaction between HHV-6 and human immunodeficiency virus type 1 in vivo.

The in vitro susceptibility of several nonhuman primate species to human herpesvirus 6 (HHV-6) was investigated. Only peripheral blood mononuclear cells from chimpanzees (Pan troglodytes) were found permissive to productive infection by HHV-6, indicating that the host range of HHV-6, albeit limited, may not be restricted to Homo sapiens. However, natural HHV-6 infection in chimpanzees, as well as in the other species tested, could not be documented by serological analysis. As previously observed with human cells, HHV-6 infection of chimpanzee peripheral blood mononuclear cells was highly cytopathic and the infected cells exhibited phenotypic features of activated T lymphocytes. Although in humans the majority of HHV-6-infected lymphocytes displayed the CD4 antigen, in chimpanzees a mixed CD4+ and CD8+ phenotype was observed. HHV-6 was also shown to productively coinfect individual chimpanzee T cells with human immunodeficiency virus type 1, resulting in an accelerated induction of cytopathicity. In light of these findings, we propose the utilization of chimpanzees as a potential animal model system to investigate the in vivo interaction between HHV-6 and human immunodeficiency virus type 1 and its relevance to the development of acquired immune deficiency syndrome.     

Use of monocyte/endothelial cell co-cultures (in vitro) and a subcutaneous implant mouse model (in vivo) to evaluate a degradable polar hydrophobic ionic polyurethane

Potential benefits of co-culturing monocytes (MC) with vascular smooth muscle cells have been reported on for tissue engineering applications with a degradable, polar, hydrophobic, and ionic polyurethane (D-PHI). Since the interaction of MC and endothelial cells (EC) within the blood vessel endothelium is also a process of wound repair it was of interest to investigate their function when cultured on the synthetic D-PHI materials, prior to considering the materials' use in vascular engineering. The co-culture (MC/EC) in vitro studies were carried out on films in 96 well plates and porous scaffold disks were prepared for implant studies in an in vivo subcutaneous mouse model. After 7 days in culture, the MC/EC condition was equal to EC growth but had lower esterase activity (a measure of degradative potential), no pro-inflammatory TNF-α and a relatively high anti-inflammatory IL-10 release while the ECs maintained their functional marker CD31. After explantation of the porous scaffolds, a live/dead stain showed that the cells infiltrating the scaffolds were viable and histological stains (May-Grunwald, Trichrome) demonstrated tissue in growth and extracellular matrix synthesis. Lysates from the implant scaffolds analyzed with a cytokine antibody array showed decreased pro-inflammatory cytokines (IL-6, TNF-α, GM-CSF), increased anti-inflammatory cytokines (IL-10, IL-13, TNF-RI), and increased chemotactic cytokines (MCP-1, MCP-5, RANTES). The low foreign body response elicited by D-PHI when implanted in vivo supported the in vitro studies (EC and MC co-culture), demonstrating that D-PHI promoted EC growth along with an anti-inflammatory MC, further demonstrating its potential as a tissue engineering scaffold for vascular applications.     

Glutathione concentrations in the hard clam,Mercenaria mercenaria,following laboratory exposure to lead (a potential model system for evaluating exposure to carcinogens and toxins)

This study determined whether M. mercenaria retain Pb from sea water and whether endogenous GSH acts as an important primary response modulator of heavy metal detoxification. Lead accumulation in M. mercenaria may be related to the rate of endogenous formation of GSH. Glutathione concentrations decrease with increasing early exposure to Pb and increase after continued acute exposure. M. mercenaria do not accumulate Pb but appear to reach an equilibrium with their environment. GSH formation may protect the hard clam from accumulating excess Pb by forming insoluble sulfide adducts with Pb and excreting these complexes.     

Nanoencapsulation (in vitro and in vivo) as an efficient technology to boost the potential of garlic essential oil as alternatives for antibiotics in broiler nutrition

The addition of essential oil (EO) as chitosan encapsulated can increase the efficiency of these oils in broiler feeding. Therefore, the objective of the current research was to explore the antibacterial and antioxidant potential of garlic essential oil (GEO) (free vs. nanoencapsulated) and their effects on performance, gene expression of mucin2, microbial, and morphology of intestine in broilers. A total of 900 1-day-old male broilers (Ross 308) were assigned to six dietary treatments (0, 100, and 200 mg/kg free GEO and 0 (contain of chitosan), 100 and 200 mg/kg nanoencapsulated GEO) with a 2 × 3 factorial arrangement based on completely randomized design. Garlic essential oil encapsulation with chitosan significantly enhanced antibacterial and antioxidant parameters. At 100 mg/kg nanoencapsulated GEO had significant (P < 0.01) advantages in improving BW gain (BWG) (22–42 and 0–42) and feed conversion ratio (FCR) (0–42). Maximum feed intake (FI) was also associated with the control group (P < 0.05). Broilers fed on 100 mg/kg of nanoencapsulated GEO showed higher villi length and width relative to other treatments and villi length to crypt depth ratio as well (P < 0.01). The nanoencapsulation process of GEO (P < 0.01) affected the Lactobacilli population in the digesta of ileo-caecum and mucin2 gene expression. In broiler chickens, the tested EO, especially nanoencapsulated type, enhanced more evaluated parameters. Because of its ideal properties, nanoencasulating with chitosan may also be an effective and inexpensive way to protect bioactive compounds and improve GEO effects in broiler chickens.     

The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O(6)-(4-bromothenyl)guanine

The O(6)-methylguanine-DNA-methyltransferase (MGMT) inactivator O(6)-benzylguanine (O(6)-beG) is currently under clinical investigation as a potential tumour-sensitising agent. In clinical trials its use has been associated with increased myelotoxicity and a reduced maximum tolerated dose (MTD) for BCNU. Thus the concept of myeloprotection by gene therapy with an O(6)-beG-insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O(6)-(4-bromothenyl)guanine, PaTrin-2), which shows potential advantages over O(6)-beG in terms of higher activity against wild-type MGMT and oral formulation. The use of PaTrin-2 has also been associated with increased myelotoxicity in clinical trials and thus PaTrin-2 may also be a candidate for use in conjunction with mutant MGMT gene transfer in genetic chemoprotective strategies. However, its activity against mutant MGMTs has not been reported. We show here that the P(140)K mutant of MGMT is highly resistant to inactivation by PaTrin-2. Furthermore, we show that a human haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMT(P140K) is highly resistant to the cytotoxic effects of PaTrin-2 in combination with the methylating agent temozolomide, and that cells expressing MGMT(P140K) can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMT(P140K) exhibit haemopoietic resistance to PaTrin-2/temozolomide, which results in in vivo selection of gene-modified cells. All of these effects were comparable to those also achieved using O(6)-beG/temozolomide. Thus our data show that MGMT(P140K) is a suitable candidate for chemoprotective gene therapy where PaTrin-2 is being used in conjunction with temozolomide.     

Using a 1-D mixing model to assess the potential impact of year-to-year changes in weather on the habitat of vendace (Coregonus albula) in Bassenthwaite Lake, Cumbria

1. Bassenthwaite Lake in Cumbria is one of only two English lakes containing a population of vendace (Coregonus albula). The spatial distribution and survival of this fish is strongly influenced by the temperature and oxygen content of the water. In summer, this fish moves into deeper, colder water but avoids areas where the oxygen content is low. 2. In recent years, there has been a dramatic decline in the number of vendace found in the lake, a trend that may have been exacerbated by a succession of warm summers. Bassenthwaite only becomes stably stratified during calm, warm periods when a significant proportion of the deep water becomes anoxic. 3. Here, a one dimensional (1‐D) process‐based temperature‐oxygen model is used to simulate the year‐to‐year variations in the severity of these ‘extreme events’. The model is validated using field measurements acquired in the 1990s and used to predict the range of depths accessible to the vendace. 4. An empirical, weather‐driven model is then used to ‘hindcast’ the mixing characteristics of the lake in the 1980s and estimate the proportion of the habitat lost during warm, calm summers. These simulations show that periods of stable thermal stratification have become increasingly common in recent years. In the 1980s, only one ‘extreme event’ was identified but four such events were recorded in the 1990s. 5. The results are discussed in relation to the conservation status of the species and the potential effect of climate change on its survival in the English Lake District.     

Osteoconductive and bioresorbable composites based on poly(L,L-lactide) and pseudowollastonite: from synthesis and interfacial compatibilization to in vitro bioactivity and in vivo osseointegration studies

This contribution reports on the elaboration of novel bioresorbable composites consisting of pseudowollastonite (psW) (a silicate-based polycrystalline ceramic (α-CaSiO(3))) and poly(L,L-lactide) as a valuable polymeric candidate in bone-guided regeneration. These composites were prepared by direct melt-blending to avoid the use of organic solvents harmful for biomedical applications. Amphiphilic poly(ethylene oxide-b-L,L-lactide) diblock copolymers synthesized by ring-opening polymerization were added to psW-based composites to modulate the bioactivity of the composites. The bioactivity of the composites was first evaluated by monitoring the release of bioactive Ca(2+) and (SiO(4))(4-) ions as well as the concomitant formation of hydroxyapatite on the material surface after soaking them in physiological fluid. Subsequently, the composites were studied in vitro to evaluate their cytotoxicity in the presence of SaOS-2 osteoblastic cells and in vivo to assess their osteoconductivity in an orthotopic rat tibia model. This study provides a first insight into the use of direct melt-blended psW-poly(L,L-lactide) composites for bone-regeneration applications.     

Evaluating the toxicity of environmental concentrations of waterborne chromium (VI) to a model teleost, Oncorhynchus mykiss: a comparative study of in vivo and in vitro

Toxic effects of environmental concentrations (50, 100, and 200μg/L) of waterborne chromium (VI) were evaluated in rainbow trout by comparison of in vitro and in vivo assays. Multiple biomarkers were measured including oxidative stress indices and antioxidant response parameters in liver and brain, as well as Na(+)-K(+)-ATPase in gill. Superoxide dismutase (SOD) and glutathione reductase (GR) activities were significantly induced (1.54-fold and 1.37-fold, respectively) in fish brain in vivo, but no significant differences were observed in any other biomarker or in vivo test group. Oxidative stress was apparent in vitro as significantly higher levels of oxidative indices, with the highest induction of TBARS and CP found in brain at 200μg/L Cr(VI) (2.41-fold and 1.95-fold, respectively), and SOD and GR activities and reduced glutathione in brain were significantly inhibited (65%, 44%, and 36%, respectively). In vitro Na(+)-K(+)-ATPase activity in gill was also significantly inhibited at concentrations of 100 and 200μg/L (69% and 45%, respectively). Short-term exposure to environmental concentrations of Cr(VI) does not therefore evoke marked effects in fish in vivo. Based on the present results, a set of in vitro tests with tissue homogenate can be evoked more remarkable effects by the lower concentrations of Cr(VI) than in vivo, which could provide some useful information and might be a potential alternative approach for monitoring heavy metal pollution in aquatic environments. However, it needs more detailed studies in other area, such as hormonal response or genotoxicity, before these findings could be applied in the field investigation.