Interaction of peptides, peptidomimetics and other drug candidates with the DI/tripeptide transport system in intestinal epithelial cells, using the in vitro caco-2 cell culture system

An uptake system was developed using Caco-2 cell monolayers and the dipeptide, glycyl-[3H]L-proline, as a probe compound. Glycyl-[3H]L-proline uptake was via the di-/tripeptide transport system (DTS) and, exhibited concentration-, pH- and temperature-dependency. Dipeptides inhibited uptake of the probe, and the design of the system allowed competitors to be ranked against one another with respect to affinity for the transporter. The structural features required to ensure or increase interaction with the DTS were defined by studying the effect of a series of glycyl-L-proline and angiotensin-converting enzyme (ACE)-inhibitor (SQ-29852) analogues on the uptake of the probe. The SQ-29852 structure was divided into six domains (A-F) and competitors were grouped into series depending on structural variations within specific regions. Domain A was found to prefer a hydrophobic function, such as a phenyl group, and was intolerant to positive charges and H+ -acceptors and donors. SQ-29852 analogues were more tolerant of substitutions in the C domain, compared to glycyl-L-proline analogues, suggesting that interactions along the length of the SQ-29852 molecule may override the effects of substitutions in the C domain. SQ-29852 analogues showed a preference for a positive function, such as an amine group in this region, but dipeptide structures favoured an uncharged substitution. Lipophilic substituents in domain D increased affinity of SQ-29852 analogues with the DTS. A similar effect was observed for ACE-NEP inhibitor analogues. Domain E, corresponding to the carboxyl group was found to be tolerant of esterification for SQ-29852 analogues but not for dipeptides. Structural features which may increase interaction for one series of compounds, may not have the same effect for another series, indicating that the presence of multiple recognition sites on a molecule may override the deleterious effect of anyone change. Modifying current, poorly absorbed peptidomimetic structures to fit the proposed hypothetical model may improve oral bioavailability by increasing affinity for the DTS. The stereochemical preference of the transporter was explored using four series of compounds (SQ-29852, lysylproline, alanylproline and alanylalanine enantiomers). The L, L stereochemistry was the preferred conformation for all four series, agreeing with previous studies. However, D, D enantiomers were shown in some cases to be substrates for the DTS, although exhibiting a lower affinity than their L, L counterparts. All the ACE-inhibitors and β-lactam antibiotics investigated, produced a degree of inhibition of the probe, and thus show some affinity for the DTS. This contrasts with previous reports that found several ACE inhibitors to be absorbed via a passive process, thus suggesting that compounds are capable of binding to the transporter site and inhibiting the probe without being translocated into the cell. This was also shown to be the case for oligodeoxynucleotide conjugated to a lipophilic group (vitamin E), and highlights the possibility that other orally administered drug candidates may exert non-specific effects on the DTS and possibly have a nutritional impact. Molecular modelling of selected ACE-NEP inhibitors revealed that the three carbonyl functions can be oriented in a similar direction, and this conformation was found to exist in a local energy-minimised state, indicating that the carbonyls may possibly be involved in hydrogen-bond formation with the binding site of the DTS.

Formulation and characterisation of an effective particulate delivery vehicle for the novel sub-unit vaccine antigen, Ag85B-ESAT-6

This research focused on the formation of particulate delivery systems for the sub-unit fusion protein, Ag85B-ESAT-6, a promising tuberculosis (TB) vaccine candidate. Initial work concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyl dioctadecyl ammonium (DDA). These studies demonstrated that addition of the immunomodulatory trehalose dibehenate (TDB) enhanced the physical stability of the system whilst also adding further adjuvanticity. Indeed, this formulation was effective in stimulating both a cell mediated and humoural immune response. In order to investigate an alternative to the DDA-TDB system, microspheres based on poly(DL-lactide-co-glycolide) (PLGA) incorporating the adjuvants DDA and TDB, either alone or in combination, were first optimised in terms of physico-chemical characteristics, followed by immunological analysis. The formulation incorporating PLGA and DDA emerged as the lead candidate, with promising protection data against TB. Subsequent optimisation of the lead microsphere formulation investigated the effect of several variables involved in the formulation process on physico-chemical and immunological characteristics of the particles produced. Further, freeze-drying studies were carried out with both sugar-based and amino acid-based cryoprotectants, in order to formulate a stable freexe-dried product. Finally, environmental scanning electron microscopy (ESEM) was investigated as a potential alternative to conventional SEM for the morphological investigation of microsphere formulations. Results revealed that the DDA-TDB liposome system proved to be the most immunologically efficient delivery vehicle studied, with high levels of antibody and cytokine production, particularly gamma-interferon (IFN-ϒ), considered the key cytokine marker for anti-mycobacterial immunity. Of the microsphere systems investigated, PLGA in combination with DDA showed the most promise, with an ability to initiate a broad spectrum of cytokine production, as well as antigen specific spleen cell proliferation comparable to that of the DDA-TDB formulation.

Smart microsystems for cell manipulations

This thesis documents the design, manufacture and testing of a passive and non-invasive micro-scale planar particle-from-fluid filter for segregating cell types from a homogeneous suspension. The microfluidics system can be used to separate spermatogenic cells from testis biopsy samples, providing a mechanism for filtrate retrieval for assisted reproduction therapy. The system can also be used for point-of-service diagnostics applications for hospitals, lab-on-a-chip pre-processing and field applications such as clinical testing in the third world. Various design concepts are developed and manufactured, and are assessed based on etched structure morphology, robustness to variations in the manufacturing process, and design impacts on fluid flow and particle separation characteristics. Segregation was measured using image processing algorithms that demonstrate efficiency is more than 55% for 1 µl volumes at populations exceeding 1 x 107. the technique supports a significant reduction in time over conventional processing, in the separation and identification of particle groups, offering a potential reduction in the associated cost of the targeted procedure. The thesis has developed a model of quasi-steady wetting flow within the micro channel and identifies the forces across the system during post-wetting equalisation. The model and its underlying assumptions are validated empirically in microfabricated test structures through a novel Micro-Particle Image Velocimetry technique. The prototype devices do not require ancillary equipment nor additional filtration media, and therefore offer fewer opportunities for sample contamination over conventional processing methods. The devices are disposable with minimal reagent volumes and process waste. Optimal processing parameters and production methods are identified with any improvements that could be made to enhance their performance in a number of identified potential applications.

Developing solid particulate vaccine adjuvants - surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

Developing solid particulate vaccine adjuvants:surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

Influence of Egr-1 in cardiac tissue-derived mesenchymal stem cells in response to glucose variations

Mesenchymal stem cells (MSCs) represent a promising cell population for cell therapy and regenerative medicine applications. However, how variations in glucose are perceived by MSC pool is still unclear. Since, glucose metabolism is cell type and tissue dependent, this must be considered when MSCs are derived from alternative sources such as the heart. The zinc finger transcription factor Egr-1 is an important early response gene, likely to play a key role in the glucose-induced response. Our aim was to investigate how short-term changes in in vitro glucose concentrations affect multipotent cardiac tissue-derived MSCs (cMSCs) in a mouse model of Egr-1 KO (Egr-1-/-). Results showed that loss of Egr-1 does not significantly influence cMSC proliferation. In contrast, responses to glucose variations were observed in wt but not in Egr-1 -/- cMSCs by clonogenic assay. Phenotype analysis by RT-PCR showed that cMSCs Egr-1-/- lost the ability to regulate the glucose transporters GLUT-1 and GLUT-4 and, as expected, the Egr-1 target genes VEGF, TGFβ-1, and p300. Acetylated protein levels of H3 histone were impaired in Egr-1-/- compared to wt cMSCs. We propose that Egr-1 acts as immediate glucose biological sensor in cMSCs after a short period of stimuli, likely inducing epigenetic modifications. © 2014 Daniela Bastianelli et al.

Temperature, salinity, methane concentration, oxidation rates and methanotrophic cell number at Svalbard seeps in summer 2011 and 2012 (cruises POS419 and MSM21/4)

Large amounts of the greenhouse gas methane are released from the seabed to the water column where it may be consumed by aerobic methanotrophic bacteria. This microbial filter is consequently the last marine sink for methane before its liberation to the atmosphere. The size and activity of methanotrophic communities, which determine the capacity of the water column methane filter, are thought to be mainly controlled by nutrient and redox dynamics, but little is known about the effects of ocean currents. Here, we report measurements of methanotrophic activity and biomass (CARD-FISH) at methane seeps west of Svalbard, and related them to physical water mass properties (CTD) and modelled current dynamics. We show that cold bottom water containing a large number of aerobic methanotrophs was rapidly displaced by warmer water with a considerably smaller methanotrophic community. This water mass exchange, caused by short-term variations of the West Spitsbergen Current, constitutes a rapid oceanographic switch severely reducing methanotrophic activity in the water column. Strong and fluctuating currents are widespread oceanographic features common at many methane seep systems and are thus likely to globally affect methane oxidation in the ocean water column.

Hearing the Child's Voice: Their Lived Experience in the Pediatric Intensive Care Unit

Background: More than 200,000 children are admitted annually to Pediatric Intensive Care Units (PICUs) in the US. Research has shown young children can provide insight into their hospitalization experiences; child reports rather than parental reports are critical to understanding the child’s experience. Information relating to children’s perceptions while still in the PICU is scarce. Aims: The purpose of this qualitative study was to investigate school age children’s and adolescents’ perceptions of PICU while in the PICU; changes in perceptions after transfer to the General Care Unit (GCU); differences in perceptions of school age children/adolescents and those with more invasive procedures. Methods: Interviews were conducted in PICU within 24-48 hours of admission and 24-48 hours after transfer to GCU. Data on demographics, clinical care and number/types of procedures were obtained. Results: Participants were 7 school age children, 13 adolescents; 10 Hispanic; 13 males. Five overarching themes: Coping Strategies, Environmental Factors, Stressors, Procedures/Medications, and Information. Children emphasized the importance of peer support and visitation; adolescents relied strongly on social media and texting. Parent visits sometimes were more stressful than peer visits. Video games, TV, visitors, and eating were diversional activities. In the PICU, they wanted windows to see outside and interesting things to see on the ceiling above them. Children expressed anticipatory fear of shots and procedures, frustration with lab work, and overwhelming PICU equipment. Number of child responses was higher in PICU (927) than GCU (593); the largest difference was in Environmental Factors. Variations between school age children and adolescents were primarily in Coping Strategies, especially in social support. Number of GCU procedures were the same (8 children) or greater (2 children) than PICU procedures. Discussion: Admission to PICU is a very stressful event. Perceptions from children while still in PICU found information not previously found in the literature. Longitudinal studies to identify children’s perceptions regarding PICU hospitalization and post-discharge outcomes are needed.

Decadal-to-centennial scale variations during the Holocene reconstructed from sediment core D13902

Sea surface temperature (SST), marine productivity, and fluvial input have been reconstructed for the last 11.5 calendar (cal) ka B.P. using a high-resolution study of C37 alkenones, coccolithophores, iron content, and higher plant n-alkanes and n-alkan-1-ols in sedimentary sequences from the inner shelf off the Tagus River Estuary in the Portuguese Margin. The SST record is marked by a continuous decrease from 19C, at 10.5 and 7 ka, to 15C at present. This trend is interrupted by a fall from 18C during the Roman and Medieval Warm Periods to 16C in the Little Ice Age. River input was very low in the early Holocene but increased in the last 3 cal ka B.P. in association with an intensification of agriculture and deforestation and possibly the onset of the North Atlantic Oscillation/Atlantic Multidecadal Oscillation modes of variability. River influence must have reinforced the marine cooling trend relative to the lower amplitude in similar latitude sites of the eastern Atlantic. The total concentration of alkenones reflects river-induced productivity, being low in the early Holocene but increasing as river input became more important. Rapid cooling, of 1-2C occurring in 250 years, is observed at 11.1, 10.6, 8.2, 6.9, and 5.4 cal ka B.P. The estimated age of these events matches the ages of equivalent episodes common in the NE Atlantic- Mediterranean region. This synchronicity reveals a common widespread climate feature, which considering the twentieth century analog between colder SSTs and negative North Atlantic Oscillation (NAO), is likely to reflect periods of strong negative NAO.

Detecting cell assemblies in large neuronal populations

Recent progress in the technology for single unit recordings has given the neuroscientific community theopportunity to record the spiking activity of large neuronal populations. At the same pace, statistical andmathematical tools were developed to deal with high-dimensional datasets typical of such recordings.A major line of research investigates the functional role of subsets of neurons with significant co-firingbehavior: the Hebbian cell assemblies. Here we review three linear methods for the detection of cellassemblies in large neuronal populations that rely on principal and independent component analysis.Based on their performance in spike train simulations, we propose a modified framework that incorpo-rates multiple features of these previous methods. We apply the new framework to actual single unitrecordings and show the existence of cell assemblies in the rat hippocampus, which typically oscillate attheta frequencies and couple to different phases of the underlying field rhythm

Detecting cell assemblies in large neuronal populations

Recent progress in the technology for single unit recordings has given the neuroscientific community theopportunity to record the spiking activity of large neuronal populations. At the same pace, statistical andmathematical tools were developed to deal with high-dimensional datasets typical of such recordings.A major line of research investigates the functional role of subsets of neurons with significant co-firingbehavior: the Hebbian cell assemblies. Here we review three linear methods for the detection of cellassemblies in large neuronal populations that rely on principal and independent component analysis.Based on their performance in spike train simulations, we propose a modified framework that incorpo-rates multiple features of these previous methods. We apply the new framework to actual single unitrecordings and show the existence of cell assemblies in the rat hippocampus, which typically oscillate attheta frequencies and couple to different phases of the underlying field rhythm

From genes to radioresistance in head and neck squamous cell carcinoma

Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.

Leccinum vulpinum Watling induces DNA damage, decreases cell proliferation and induces apoptosis on the human MCF-7 breast cancer cell line

The current work aimed to study the antitumour activity of a phenolic extract of the edible mushroom Leccinum vulpinum Watling, rich essentially in hydroxybenzoic acids. In a first approach, the mushroom extract was tested against cancer cell growth by using four human tumour cell lines. Given the positive results obtained in these initial screening experiments and the evidence of some studies for an inverse relationship between mushroom consumption and breast cancer risk, a detailed study of the bioactivity of the extract was carried out on MCF-7 cells. Once the selected cell line to precede the work was the breast adenocarcinoma cell line, the human breast non-malignant cell line MCF-10A was used as control. Overall, the extract decreased cellular proliferation and induced apoptosis. Furthermore, the results also suggest that the extract causes cellular DNA damage. Data obtained highlight the potential of mushrooms as a source of biologically active compounds, particularly with antitumour activity.

Regulatory T and B cells in asthmatic women: variations from pregnancy to postpartum Treg and Breg: pregnancy to postpartum

BACKGROUND: Allergic asthma and rhinitis are common in pregnancy. The immune mechanisms underlying the effects of pregnancy in asthma and vice-versa are not completely understood. OBJECTIVES: This work aimed to study the evolution of regulatory T and B cells in asthmatic pregnant women, from late pregnancy till postpartum. METHODS: Four groups of women were enrolled for this study: third trimester pregnant women, asthmatic (n=24) and healthy (n=43), and non-pregnant women, asthmatic (n=33) and healthy (n=35). Pregnant women were also evaluated postpartum (>6 weeks after delivery). Blood samples were taken from each woman and flow cytometry was used to characterize circulating regulatory T and B cells. Foxp3 expression was assessed within CD4DimCD25Hi regulatory T cells. RESULTS: In asthmatic and healthy pregnant women, regulatory T cells did not oscillate significantly from pregnancy to postpartum, but CD24HiCD38Hi regulatory B cells, decreased in pregnancy, rose significantly postpartum. Foxp3 expression in regulatory T cells was also impaired during pregnancy in asthmatic and healthy pregnant women, recovering postpartum. Nevertheless, asthmatic pregnant women presented higher Foxp3 expression than healthy pregnant women (p=0.007), probably due to the use of control medication. CONCLUSIONS: Women with controlled asthma present variations in regulatory cell subsets during pregnancy and postpartum. The similar pattern observed for Foxp3 expression and CD24HiCD38Hi regulatory B cells during this period corroborates the interaction established between regulatory T and B cells in immune responses. Considering the immunomodulatory potential of these immune mediators, more studies are needed to evaluate their relation with asthma and rhinitis complications in pregnancy.

Red blood cell rheology in sepsis

Changes in red blood cell (RBC) function can contribute to alterations in microcirculatory blood flow and cellular dysoxia in sepsis. Decreases in RBC and neutrophil deformability impair the passage of these cells through the microcirculation. While the role of leukocytes has been the focus of many studies in sepsis, the role of erythrocyte rheological alterations in this syndrome has only recently been investigated. RBC rheology can be influenced by many factors, including alterations in intracellular calcium and adenosine triphosphate (ATP) concentrations, the effects of nitric oxide, a decrease in some RBC membrane components such as sialic acid, and an increase in others such as 2,3 diphosphoglycerate. Other factors include interactions with white blood cells and their products (reactive oxygen species), or the effects of temperature variations. Understanding the mechanisms of altered RBC rheology in sepsis, and the effects on blood flow and oxygen transport, may lead to improved patient management and reductions in morbidity and mortality.