Characterization of colon cancer cell culture based screening assay to study effects of phenolic acids

In Canada, colorectal cancer is the second leading cause of death from cancer in men and the third leading cause of death from cancer in women. Several factors contribute to the development of cancer. Genetic predisposition, diet, and lifestyle habits are some of the major factors for colorectal cancer development. In the diet related factors, epidemiological studies suggest that consumption of whole grains rich in dietary fiber are associated with low incidence of human colon cancer. Recent studies have also shown that, in addition to dietary fiber, the type of dietary fiber and other compounds such as phenolic acids present in cereal grain bran may also have a role to play in colon cancer prevention. In a recent study, eleven major phenolic acids which differed in anti-oxidant activity were identified in wheat bran from wheat varieties belonging to six different market classes. The main objective of this study was to develop an in vitro cell culture based assay system to study the effect of phenolic acids on colon cancer development. Another objective was to study the effect of phenolic acids on selected molecular markers associated with cell proliferation, apoptosis and inflammation. Two well established colon carcinoma cell lines HT-29 and HCT 116 were treated with varying concentrations of fourteen phenolic acids to study their effect on cell survival and proliferation. In addition, immunohistochemical assays were performed on treated cells for two cell proliferation markers (Cyclin D1 and Ki67), an apoptosis marker (Bax) and three inflammatory markers (Beta-catenin, COX-2 and iNOS). Treatment of phenolic acids inhibited the growth of both the cell lines, however the effects varied with phenolic acid and cell line used in the assay. As determined by IC50, the growth of HCT 116 cells was inhibited the most by caffeic, ellagic, and gallic acids with IC50 of 0.22 mM, 0.17 mM, and 0.15 mM, respectively. On the other hand, caffeic, chlorogenic, and gallic acids are most effective in preventing the growth of HT-29 cells, with IC50 at 0.06 mM, 0.28 mM, and 0.30 mM, respectively. Immunohistochemical and Western Blotting studies revealed that phenolic acids differentially affected markers for cell proliferation, apoptosis and cell inflammation. In most of the cases, phenolic acid treatments up-regulated the pro-apoptosis marker Bax, while it down-regulated cell proliferation marker Cyclin D1. The results clearly show that a cell culture based assay can be used to study the effect of phenolic acids or other chemical constituents isolated from plants to study their effect on colon cancer cell lines. Statistical analysis revealed that only in very limited cases, results of molecular markers correlated to cell growth and proliferation. Therefore, to draw firm conclusions, more detailed and extensive studied need to be completed using different phenolic acids, the two cell lines and more replications. However, this study has developed the necessary protocols and provided some indicative results such as most of the phenolic acids induced pro-apoptosis pathway in both the colon cancer lines. Future studies with extracted phenolic acids from wheat bran using the cell culture system optimized in this study can be used to define the role of different wheat varieties in colon cancer prevention

Grouping Pig-Specific Responses to Mitogen with Similar Responder Animals may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal Model

Copyright: © 2014 J. Alex Pasternak, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Peer ReviewedPig peripheral blood-derived mononuclear cells (PBMCs) and lamina propria mononuclear cells (LPMCs) stimulated with mitogens ex vivo can show significant animal-to-animal variation lead to difficulty in interpreting responses in an out-bred animal species. Mixed-cell populations were stimulated ex vivo with 2.5 μg/ml Con A or 2.5 ng/ml PMA plus 250 ng/ml ionomycin (PMAi; (LPCMs only)) or media alone for 72 hours. Supernatants were then tested for cytokine production using a Bioplex assay for porcine IFNα, IFNγ, IL-10, and IL-12. Unstimulated PBMCs had significant levels of IL-10 and the median value for this group decreased in the presence of Con A. Con A did, however, induce production of IFNα and IFNγ, but not IL-12 in this cell population. In contrast, unstimulated and Con A-stimulated LPMCs produced negligible IL-10, IFNα, IFNγ, and the majority of animals’ LPMCs showed negligible IL-12 production in response to Con A. In contrast, LPMCs stimulated with PMAi produced IFNγ suggesting cytokine production is mitogen–specific response. When we tracked animal-specific responses, we observed that discrete subsets of animal’s PBMCs responded to Con A with significantly increased or decreased IL-10 production relative to unstimulated cells. Further, in the LPMCs, some cells produced no IL-12 in response to Con A but showed augmented production in response to PMAi, while others showed production of IL-12 in response to Con A but no response to PMAi. Flow cytometric analysis showed that the PBMCs were a mixture of CD3+ T cells>CD21+ B cells>CD172+ myeloid cells whereas the LPMCs consisted of mainly Cytotoxic T cells and Natural Killer cells. The percentage of CD8α+CD4+ antigen-experienced T cells was greater in the LPMCs relative to the PBMCs. As expected in an out-bred species, animal-specific differences in cytokine production in response to stimulants exist and may confound interpretation of results unless tracked individually

Combination of Innate Immune Modulators as Vaccine Adjuvants in Mice

The development of new, effective, and safe vaccines necessarily requires the identification of new adjuvant(s) to enhance the potency and longevity of antigen-specific immune responses. In the present study, we compare the antibody-mediated and cell-mediated immune (CMI) responses within groups of mice vaccinated subcutaneously with ovalbumin (OVA; as an experimental antigen) plus polyphosphazene (an innate immune modulator), Polyinosinic:polycytidylic acid (poly-I:C; (an RNA mimetic) and glycopeptide ARC5 (which is a Toll-like receptor (TLR), TLR2 ligand and PAM3CSK4 analogue) formulated together in a soluble vaccine. We also investigated the effect of a polymeric nanoparticle of ARC4 and ARC7 (which are a novel muramyl dipeptide analogue and a monophosophoryl lipid A (MPLA) analogue, respectively) plus OVA +/− ARC5 as a subcutaneous vaccine in mice. OVA+ARC4/ARC7 nanoparticle +/− ARC5 triggered a robust and balanced Th1/Th2-type humoral response with significant anti-OVA IgA in serum, and significant interferon (IFN)-γ and interleukin (IL)-17 production in splenocytes after 35 days relative to the controls. Formulation of OVA with ARC4/ARC7 nanoparticles should be investigated for inducing protective immunity against infectious pathogens in mice and other species

Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester

Abstract Background Neisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5′-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells. Results We observed that 20.3% (+/− 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner. Conclusions Flow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis

Temporal trends in out-of-hospital cardiac arrest with an initial non-shockable rhythm in Singapore

RESUSCITATION PLUS1

Temporal trends in out-of-hospital cardiac arrest with an initial non-shockable rhythm in Singapore

Aim: Out-of-hospital cardiac arrest (OHCA) with an initial non-shockable rhythm is the predominant form of OHCA in adults. We evaluated its 10-year trends in epidemiology and management in Singapore. Methods: Using the national OHCA registry we studied the trends of 20,844 Emergency Medical Services-attended adult OHCA from April 2010 to December 2019. Survival to hospital discharge was the primary outcome. Trends and outcomes were analyzed using linear and logistic regression, respectively. Results: Incidence rates of adult OHCAs increased during the study period, driven by non-shockable OHCA. Compared to shockable OHCA, non-shockable OHCAs were significantly older, had more co-morbidities, unwitnessed and residential arrests, longer no-flow time, and received less bystander cardiopulmonary resuscitation (CPR) and in-hospital interventions (p < 0.001). Amongst non-shockable OHCA, age, co-morbidities, residential arrests, no-flow time, time to patient, bystander CPR and epinephrine administration increased during the study period, while presumed cardiac etiology decreased (p < 0.05). Unlike shockable OHCA, survival for non-shockable OHCA did not improve (p < 0.001 for trend difference). The likelihood of survival for non-shockable OHCA significantly increased with witnessed arrest (adjusted odds ratio (aOR) 2.02) and bystander CPR (aOR 3.25), but decreased with presumed cardiac etiology (aOR 0.65), epinephrine administration (aOR 0.66), time to patient (aOR 0.93) and age (aOR 0.98). Significant two-way interactions were observed for no-flow time and residential arrest with bystander CPR (aOR 0.96 and 0.40 respectively). Conclusion: The incidence of non-shockable OHCA increased between 2010 and 2019. Despite increased interventions, survival did not improve for non-shockable OHCA, in contrast to the improved survival for shockable OHCA

Incidence and outcomes of out-of-hospital cardiac arrest in singapore and Victoria : a collaborative study

Background: Incidence and outcomes of out-of-hospital cardiac arrest (OHCA) vary between communities. We aimed to examine differences in patient characteristics, prehospital care, and outcomes in Singapore and Victoria. Methods and Results: Using the prospective Singapore Pan-Asian Resuscitation Outcomes Study and Victorian Ambulance Cardiac Arrest Registry, we identified 11 061 and 32 003 emergency medical services-attended adult OHCAs between 2011 and 2016 respectively. Incidence and survival rates were directly age adjusted using the World Health Organization population. Survival was analyzed with logistic regression, with model selection via backward elimination. Of the 11 061 and 14 834 emergency medical services-treated OHCAs (overall mean age±SD 65.5±17.2; 67.4% males) in Singapore and Victoria respectively, 11 054 (99.9%) and 5595 (37.7%) were transported, and 440 (4.0%) and 2009 (13.6%) survived. Compared with Victoria, people with OHCA in Singapore were older (66.7±16.5 versus 64.6±17.7), had less shockable rhythms (17.7% versus 30.3%), and received less bystander cardiopulmonary resuscitation (45.7% versus 58.5%) and defibrillation (1.3% versus 2.5%) (all P<0.001). Age-adjusted OHCA incidence and survival rates increased in Singapore between 2011 and 2016 (P<0.01 for trend), but remained stable, though higher, in Victoria. Likelihood of survival increased significantly (P<0.001) with arrest in public locations (adjusted odds ratio [aOR] 1.81), witnessed arrest (aOR 2.14), bystander cardiopulmonary resuscitation (aOR 1.72), initial shockable rhythm (aOR 9.82), and bystander defibrillation (aOR 2.04) but decreased with increasing age (aOR 0.98) and emergency medical services response time (aOR 0.91). Conclusions: Singapore reported increasing OHCA incidence and survival rates between 2011 and 2016, compared with stable, albeit higher, rates in Victoria. Survival differences might be related to different emergency medical services practices including patient selection for resuscitation and transport.Ministry of Health (MOH)National Medical Research Council (NMRC)Published versionPAROS is supported by grants from the National Medical Research Council (Singapore), Ministry of Health, Singapore. VACAR receives funding from Victorian Government Department of Health

Incidence and Outcomes of Out-of-Hospital Cardiac Arrest in Singapore and Victoria: A Collaborative Study

10.1161/JAHA.119.015981JOURNAL OF THE AMERICAN HEART ASSOCIATION92