Flow Cytometric Analysis of Internal Calcium Mobilization via a B2-Bradykinin Receptor on a Subclone of PC-12 Cells

Single cell Ca2+ mobilization was studied by nonparametric, quantitative flow cytometry using a sort-selected subclone of PC-12 cells. The response of the parent PC-12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2-BK receptor agonists Lys-BK and Met-Lys-BK (10 pM-1 microM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1-BK receptor agonist Des-Arg9-BK (1 nM-1 microM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2-receptor antagonists D-Arg0-Hyp3-thienyl5,8-D-Phe7-BK and D-Arg0-Hyp3-D-Phe7 (0.1 nM-10 microM) in a concentration-dependent manner. Des-Arg9-Leu8-BK, a B1-receptor antagonist, failed to block the BK responses at 0.1-10 microM. The agonist/antagonist profile of the BK responses indicated that the B2-BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor-mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor-mediated second messenger generation at the single cell level.     

IPSE/alpha-1, a major secretory glycoprotein antigen from schistosome eggs, expresses the Lewis X motif on core-difucosylated N-glycans

Schistosomes are parasitic flatworms that infect millions of people in (sub)tropical areas around the world. Glycoconjugates of schistosomes play a critical role in the interaction of the different developmental stages of the parasite with the host. In particular, glycosylated components of the eggs produced by the adult worm pairs living in the bloodstream are strongly immunogenic. We have investigated the glycosylation of interleukin-4-inducing factor from schistosome eggs (IPSE/alpha-1), a major secretory egg antigen from Schistosoma mansoni that triggers interleukin-4 production in human basophils, by MS analysis of tryptic glycopeptides. Nanoscale LC-MS(/MS) and MALDI-TOF(/TOF)-MS studies combined with enzymatic degradations showed that monomeric IPSE/alpha-1 contains two N-glycosylation sites, which are each occupied for a large proportion with core-difucosylated diantennary glycans that carry one or more Lewis X motifs. Lewis X has been reported as a major immunogenic glycan element of schistosomes. This is the first report both on the expression of Lewis X on a specific schistosome egg protein and on a protein-specific glycosylation analysis of schistosome eggs.     

A Mathematical Model of the Enhanced Permeability and Retention Effect for Liposome Transport in Solid Tumors

The discovery of the enhanced permeability and retention (EPR) effect has resulted in the development of nanomedicines, including liposome-based formulations of drugs, as cancer therapies. The use of liposomes has resulted in substantial increases in accumulation of drugs in solid tumors; yet, significant improvements in therapeutic efficacy have yet to be achieved. Imaging of the tumor accumulation of liposomes has revealed that this poor or variable performance is in part due to heterogeneous inter-subject and intra-tumoral liposome accumulation, which occurs as a result of an abnormal transport microenvironment. A mathematical model that relates liposome accumulation to the underlying transport properties in solid tumors could provide insight into inter and intra-tumoral variations in the EPR effect. In this paper, we present a theoretical framework to describe liposome transport in solid tumors. The mathematical model is based on biophysical transport equations that describe pressure driven fluid flow across blood vessels and through the tumor interstitium. The model was validated by direct comparison with computed tomography measurements of tumor accumulation of liposomes in three preclinical tumor models. The mathematical model was fit to liposome accumulation curves producing predictions of transport parameters that reflect the tumor microenvironment. Notably, all fits had a high coefficient of determination and predictions of interstitial fluid pressure agreed with previously published independent measurements made in the same tumor type. Furthermore, it was demonstrated that the model attributed inter-subject heterogeneity in liposome accumulation to variations in peak interstitial fluid pressure. These findings highlight the relationship between transvascular and interstitial flow dynamics and variations in the EPR effect. In conclusion, we have presented a theoretical framework that predicts inter-subject and intra-tumoral variations in the EPR effect based on fundamental properties of the tumor microenvironment and forms the basis for transport modeling of liposome drug delivery.     

The link between medical conditions and fatal drownings in Canada: a 10-year cross-sectional analysis

Background:Drowning accounts for hundreds of preventable deaths in Canada every year, but the impact of preexisting medical conditions on the likelihood of death from drowning is not known. We aimed to describe the prevalence of pre-existing medical conditions among people who fatally drowned in Canada and evaluate the risk of fatal drowning among people with common pre-existing medical conditions.Methods:We reviewed all Canadian unintentional fatal drownings (2007–2016) in the Drowning Prevention Research Centre Canada’s database. For each fatal drowning we established whether the person had pre-existing medical conditions and whether those conditions contributed to the drowning. We calculated relative risk (RR) of fatal drowning stratified by age and sex for each pre-existing medical condition using data from the Canadian Chronic Disease Surveillance System.Results:During 2007–2016, 4288 people fatally drowned unintentially in Canada, of whom one-third had a pre-existing medical condition. A pre-existing medical condition contributed to drowning in 43.6% (n = 616) of cases. Fatal drowning occurred more frequently in people with ischemic heart disease (RR 2.7, 95% confidence interval [CI] 2.5–3.0) and seizure disorders (RR 6.3, 95% CI 5.4–7.3) but less frequently in people with respiratory disease (RR 0.12, 95% CI 0.10–0.15). Females aged 20–34 years with a seizure disorder had a 23 times greater risk than their age- and sex-matched cohort (RR 23, 95% CI 14–39). In general, fatal drowning occurred more often while people were bathing (RR 5.9, 95% CI 4.8–7.0) or alone (RR 1.99, 95% CI 1.32–2.97) and less often in males (RR 0.92, 95% CI 0.88–0.95) or in those who had used alcohol (RR 0.72, 95% CI 0.65–0.80), among those with pre-existing medical conditions.Interpretation:The risk of fatal drowning is increased in the presence of some preexisting medical conditions. Tailored interventions aimed at preventing drowning based on pre-existing medical conditions and age are needed. Initial prevention strategies should focus on seizure disorders and bathtub drownings.

Drowning is an important cause of death in Canada and some aspects of its epidemiology have been characterized.^1,2 Identifying and addressing risk factors for fatal drowning can save lives. For example, after research showed a considerable proportion of child drownings occurred in unsecured household pools and the effectiveness of pool fencing at reducing these deaths, legislative changes were introduced in Quebec.^3–5Although studies evaluating risk factors frequently focus on modifiable environmental or behavioural factors (e.g., infant bath seats, supervision status or alcohol and drug use),^6–11 limited research has evaluated the relation between pre-existing medical conditions and the risk of drowning.Studies that have evaluated the association between pre-existing medical conditions and drowning are limited by small sample sizes and narrow focus on certain conditions (e.g., autism spectrum disorder, epilepsy), or are age-specific (e.g., children).^12–17 The association between various pre-existing medical conditions and drowning in different age groups is not well understood. This information could assist with the development of targeted drowning prevention strategies and prioritizing of resources spent on prevention.Forty-four percent of Canadian adults have at least 1 chronic disease, which suggests that millions of Canadians with conditions that potentially impair their heart, lungs or brain participate in water activities.¹⁸ Furthermore, swimming and aquatic fitness is often encouraged for those with chronic illness to promote health.^19,20 However, the public and physicians should be aware of pre-existing medical conditions that might place people at a higher risk of drowning, so that appropriate precautions can be taken to ensure safety while participating in aquatic activities.We sought to describe pre-existing medical conditions by age group among people who fatally drowned in Canada and to evaluate the risk of fatal drowning among people with common conditions to inform future public health interventions.     

Amplified expression of tumor necrosis factor receptor in cells transfected with Epstein-Barr virus shuttle vector cDNA libraries

As an approach to isolate the cell-surface receptor for tumor necrosis factor (TNF), we have developed transfectants of human B-lymphoblastoid cells (UC cells) that overexpress the TNF receptor. These transfectants were isolated from UC cells transfected with cDNA libraries of HeLa or NG108 cells constructed in the mammalian expression vector EBO-pcD. This vector contains the Epstein-Barr virus origin of replication (ori-P) plus the EBNA-1 gene conferring replication function to ori-P and, therefore, the ability to replicate autonomously within the transfected cell (Margolskee, R.F., Kavathas, P., and Berg, P. (1988) Mol. Cell. Biol. 8, 2837-2947). Cells overexpressing the TNF receptor were identified and separated by the binding of fluoresceinated TNF and flow cytometric selection. Scatchard analysis of 125I-TNF binding data revealed a single class of high affinity receptors with a dissociation constant (Kd) of 0.2 to 2 nM and a receptor density of about 150,000 per cell, an increase of approximately 150-fold over UC cells. Cross-linking of receptor-ligand with bis-sulfosuccinimidyl suberate followed by polyacrylamide gel electrophoresis gave estimates of 87 and 104 kDa for the size of the complex. Based on its ability to bind TNF, a 68-kDa receptor protein was identified in cell extracts enriched for the receptor by using immobilized wheat germ agglutinin and TNF affinity chromatography. The difference in the estimated size of the receptor and the receptor-ligand complexes demonstrates that TNF binds to the receptor as a monomer or a dimer. Analysis of cDNA sequences conferring receptor amplification in transfectants revealed that plasmid DNA was present at 30 or more copies per cell, most likely integrated into the genomic DNA or organized into high molecular weight catenanes, and autonomously replicating units could not be recovered. Therefore, while this vector was useful in generating stable receptor-amplified cells, it was not maintained as a recoverable episome.     

Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters

BACKGROUND: Human epidermis is renewed throughout life from stem cells in the basal layer of the epidermis. Signals from the surrounding keratinocytes influence the differentiation of the stem cells, but the nature of the signals is unknown. In many developing tissues, signalling mediated by the transmembrane protein Delta1 and its receptor Notch1 inhibits differentiation. Here, we investigated the role of Delta-Notch signalling in postnatal human epidermis. RESULTS: Notch1 expression was found in all living epidermal layers, but Delta1 expression was confined to the basal layer of the epidermis, with highest expression in those regions where stem cells reside. By overexpressing Delta1 or Delta(T), a truncated form of Delta1, in primary human keratinocytes and reconstituting epidermal sheets containing mixtures of Delta-overexpressing cells and wild-type cells, we found that cells expressing high levels of Delta1 or Delta(T) failed to respond to Delta signals from their neighbours. In contrast, wild-type keratinocytes that were in contact with neighbouring cells expressing Delta1 were stimulated to leave the stem-cell compartment and initiate terminal differentiation after a few rounds of division. Delta1 promoted keratinocyte cohesiveness, whereas Delta(T) did not. CONCLUSIONS: We propose that high Delta1 expression by epidermal stem cells has three effects: a protective effect on stem cells by blocking Notch signalling; enhanced cohesiveness of stem-cell clusters, which may discourage intermingling with neighbouring cells; and signalling to cells at the edges of the clusters to differentiate. Notch signalling in epidermal stem cells thus differs from other progenitor cell populations in promoting, rather than suppressing, differentiation.     

Probiotics: from myth to reality. Demonstration of functionality in animal models of disease and in human clinical trials

The enteric flora comprise approximately 95% of the total number of cells in the human body and are capable of eliciting immune responses while also protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract (GIT) may also be implicated in the pathogenesis of several chronic conditions such as inflammatory bowel disease (IBD). The University College Cork-based Probiotic Research Group has successfully isolated and identified lactic acid bacteria (LAB) which exhibit beneficial probiotic traits. These characteristics include the demonstration of bile tolerance; acid resistance; adherence to host epithelial tissue; and in vitro antagonism of potentially-pathogenic micro-organisms or those which have been implicated in promoting inflammation. The primary objective of this report is to describe the strategy adopted for the selection of potentially effective probiotic bacteria. The study further describes the evaluation of two m embers of the resulting panel of micro-organisms (Lactobacillus salivarius subsp. salivarius UCC118 and Bifidobacterium longum infantis 35624) under in vitro conditions and throughout in vivo murine and human feeding trials. Specifically, an initial feeding study completed in Balb/c mice focused upon (i) effective delivery of the probiotic micro-organisms to the GIT and evaluation of the ability of the introduced strains to survive transit through, and possibly colonise, the murine GIT; (ii) accepting the complexity of the hostile GIT and faecal environments, development of a method of enumerating the introduced bacterial strains using conventional microbiological techniques; and (iii) assessment of the effects of administered bacterial strains on the numbers of specific recoverable indigenous bacteria in the murine GIT and faeces. Additional research, exploiting the availability of murine models of inflammatory bowel disease, demonstrated the beneficial effects of administering probi otic combinations of Lactobacillus salivarius UCC118 and Bifidobacterium longum infantis 35624 in prevention of illness-related weight loss. A further ethically-approved feeding trial, successfully conducted in 80 healthy volunteers, demonstrated that yoghurt can be used as a vehicle for delivery of Lactobacillus salivarius strain UCC118 to the human GIT with considerable efficacy in influencing gut flora and colonisation.     

Energy In-Equivalence in Australian Marsupials: Evidence for Disruption of the Continent’s Mammal Assemblage,or Are Rules Meant to Be Broken?

The energy equivalence rule (EER) is a macroecological hypothesis that posits that total population energy use (PEU) should be independent of species body mass, because population densities and energy metabolisms scale with body mass in a directly inverse manner. However, evidence supporting the EER is equivocal, and the use of basal metabolic rate (BMR) in such studies has been questioned; ecologically-relevant indices like field metabolic rate (FMR) are probably more appropriate. In this regard, Australian marsupials present a novel test for the EER because, unlike eutherians, marsupial BMRs and FMRs scale differently with body mass. Based on either FMR or BMR, Australian marsupial PEU did not obey an EER, and scaled positively with body mass based on ordinary least squares (OLS) regressions. Importantly, the scaling of marsupial population density with body mass had a slope of −0.37, significantly shallower than the expected slope of −0.75, and not directly inverse of body-mass scaling exponents for BMR (0.72) or FMR (0.62). The findings suggest that the EER may not be a causal, universal rule, or that for reasons not yet clear, it is not operating for Australia’s unique native fauna.