Chemotherapeutic agents attenuate CXCL12-mediated migration of colon cancer cells by selecting for CXCR4-negative cells and increasing peptidase CD26.

BACKGROUND: Recurrence of colorectal cancer (CRC) may arise due to the persistence of drug-resistant and cancer-initiating cells that survive exposure to chemotherapy. Proteins responsible for this recurrence include the chemokine receptor CXCR4, which is known to enable CRC metastasis, as well as the cancer-initiating cell marker and peptidase CD26, which terminates activity of its chemokine CXCL12. METHODS: We evaluated the expression and function of CXCR4 and CD26 in colon cancer cell lines and xenografts following treatment with common chemotherapies using radioligand binding, flow cytometry, immunofluorescence, and enzymatic assays. RESULTS: 5-Fluorouracil, oxaliplatin and SN-38 (the active metabolite of irinotecan), as well as cisplatin, methotrexate and vinblastine, each caused decreases in cell-surface CXCR4 and concomitant increases in CD26 on HT-29, T84, HRT-18, SW480 and SW620 CRC cell lines. Flow cytometry indicated that the decline in CXCR4 was associated with a significant loss of CXCR4+/CD26- cells. Elevations in CD26 were paralleled by increases in both the intrinsic dipeptidyl peptidase activity of CD26 as well as its capacity to bind extracellular adenosine deaminase. Orthotopic HT-29 xenografts treated with standard CRC chemotherapeutics 5-fluorouracil, irinotecan, or oxaliplatin showed dramatic increases in CD26 compared to untreated tumors. Consistent with the loss of CXCR4 and gain in CD26, migratory responses to exogenous CXCL12 were eliminated in cells pretreated with cytotoxic agents, although cells retained basal motility. Analysis of cancer-initiating cell CD44 and CD133 subsets revealed drug-dependent responses of CD26/CD44/CD133 populations, suggesting that the benefits of combining standard chemotherapies 5-fluoruracil and oxaliplatin may be derived from their complementary elimination of cell populations. CONCLUSION: Our results indicate that conventional anticancer agents may act to inhibit chemokine-mediated migration through eradication of CXCR4+ cells and attenuation of chemokine gradients through elevation of CD26 activity.

Student Performance on an Objective Structured Clinical Exam Delivered Both Virtually and In-Person.

OBJECTIVE: Passing a milestone objective structured clinical examination (OSCE) is a graduation requirement for the University of Waterloo Pharmacy students. In January 2021, the milestone OSCE was offered concurrently both virtually and in-person, with students being able to choose their desired format. The purpose of this study was to compare student performance between the 2 formats and to identify factors that may have predicted student choice of format. METHODS: Objective structured clinical examination scores for in-person and virtual exam-takers were compared using 2-tailed independent t tests with Bonferroni correction. Pass rates were compared using χ2 analysis. Prior academic performance variables were analyzed to identify predictors of the chosen exam format. Student and exam personnel surveys were used to capture OSCE feedback. RESULTS: A total of 67 students (56%) participated in the in-person OSCE, and 52 students (44%) participated virtually. There were no significant differences in overall exam averages or pass rates between the 2 groups. However, virtual exam-takers scored lower in 2 of 7 cases. Previous academic performance did not predict the choice of exam format. Feedback surveys indicated that the exam organization was perceived as a strength regardless of format, but in-person students felt more prepared for the exam than virtual exam-takers with technical challenges and difficulty navigating station resources being noted as barriers in the virtual offering. CONCLUSION: Virtual and in-person administration of a milestone OSCE resulted in similar student performance, with slightly lower performance on 2 individual case scores with virtual delivery. These results may inform the future development of virtual OSCEs.

A non-inflammatory form of immune competence prevails in acute pre-pubescent malnutrition: new evidence based on critical mRNA transcripts in the mouse.

The declining inflammatory immune competence of acute (i.e. wasting) pre-pubescent protein-energy malnutrition has been regarded as reflecting an unregulated immunological disintegration. Recent evidence, however, suggests that malnutrition stimulates a regulated immunological reconfiguration to achieve a non-inflammatory form of competence, perhaps offering protection against autoimmune reactions - the 'Tolerance Model'. Our objective was to determine the influence of acute pre-pubescent malnutrition on the expression of genes critical to tolerogenic regulation. Male and female C57BL/6J mice, initially 19 d old, consumed a complete purified diet either ad libitum (age-matched controls) or in restricted daily quantities (mimicking marasmus), or consumed an isoenergetic low-protein diet ad libitum (mimicking incipient kwashiorkor) for 14 d (six animals per dietary group). Gene expression in the spleen, typically an inflammatory organ, and in the small intestine, a site designed for non-inflammatory defence, was assessed by real-time quantitative RT-PCR, and normalised to ß-actin. In the spleen of the malnourished groups, both IL-10 and transforming growth factor-ß1 mRNA expression increased compared with controls (P < 0.05), whereas mRNA expression of IL-12p40 decreased (P < 0.05). Conversely, malnutrition exerted no influence on the expression of mRNA for these cytokines in the small intestine (P>0.05). Moreover, forkhead box P3 mRNA expression, indicative of cell-based tolerogenic potential, was sustained in both the spleen and intestine of the malnourished groups (P>0.05). Thus, despite limited supplies of energy and substrates, the spleen shifted towards a non-inflammatory character and the intestine was sustained in this mode in advanced pre-pubescent weight loss. These findings provide the first support for the Tolerance Model at the level of mRNA transcript expression.

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells.

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)2D3 traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)2D3 called 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D3-MARRS expression modulates 1,25(OH)2D3 activity in breast cancer cells. Relative levels of 1,25D3-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D3-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)2D3 in MCF-7 cells, a ribozyme construct designed to knock down 1,25D(3)-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D3-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)2D3 ( IC(50) 56+/-24 nM) compared to controls (319+/-181 nM; P<0.05). Reduction in 1,25D3-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)2D3. Knockdown of 1,25D3-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D3-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)2D3 in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D3-MARRS expression or activity as anticancer agents.

Nuclear translocation of the 1,25D3-MARRS (membrane associated rapid response to steroids) receptor protein and NFkappaB in differentiating NB4 leukemia cells.

1,25 Dihydroxyvitamin D(3) (1,25D(3)) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D(3), which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D(3)-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NFkappaB). In unstimulated cells, 1,25D(3)-MARRS can be co-immunoprecipitated with antibodies directed at NFkappaB, and NFkappaB is co-precipitated when antibodies against 1,25D(3)-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D(3)-MARRS and NFkappaB begin translocating to the nucleus within minutes of co-stimulation with 1,25D(3) and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D(3)-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NFkappaB and other factors with which it may be interacting.

Partners in pharmacy: An intraprofessional educational event with pharmacy and pharmacy technician students.

BACKGROUND AND PURPOSE: Upon graduation and licensing, pharmacists work very closely with pharmacy technicians. Despite this, opportunities for learning together as students are limited. We developed and implemented a pilot intraprofessional event for pharmacy and pharmacy technician students. The purpose of this study was to evaluate the perceived value and learner confidence through analysis of participant feedback. EDUCATION ACTIVITY AND SETTING: Pharmacy students from the University of Waterloo School of Pharmacy and pharmacy technician students from Lambton College participated in an intraprofessional event that included a three-station practice objective structured clinical exam (OSCE) and a case discussion regarding a methadone dispensing error, followed by a facilitated debrief. Upon completion of the event, students were invited to complete an online feedback questionnaire. FINDINGS: Twenty-one pharmacy students and 22 pharmacy technician students participated in the event. Twenty-one students completed the questionnaire, for a response rate of 49%. The majority of respondents agreed or strongly agreed that the event enhanced learning and confidence in working together to provide interprofessional care. Students seemed to find the OSCE to be particularly valuable. Feedback suggestions for improvement indicated a desire for more activities and time allocated to the event. SUMMARY: We designed and implemented a pilot intraprofessional event that was well-received by pharmacy students and pharmacy technician students. This supports the development of future similar events.

The Design and Impact of an Interprofessional Education Event Among Pharmacy and Nursing Students in Palliative Care-RnRx.

Nearly all reports of interprofessional education (IPE) in palliative care have excluded pharmacy students. This article describes an IPE event between pharmacy and nursing students and assesses its impact on IPE competencies. Second-year nursing students and third-year pharmacy students participated in an evening-long event, focused on a married couple who each require palliative care-one for end-of-life planning and one for chronic disease progression. The impact of the event was assessed using the Interprofessional Collaborative Competency Attainment Scale (ICCAS) and qualitative feedback. Two hundred nine (96.7%) completed the ICCAS, and 16 of the 20 statements of the ICCAS showed large positive effect sizes (Cohen d ≥ 0.8), with the remaining 4 showing moderate positive effect sizes (Cohen d ≥ 0.5). The greatest effect sizes were related to improved awareness of complementary skillsets and knowledge between the professions. Addressing team conflict and including the patient/family in decision-making showed the least improvement. While ongoing interactions are ideal for the development of skills related to conflict and team development, this article demonstrates that even a 1-time activity can have an impact on students' interprofessional care competence.

Impact and Attitudes about Peer Review of Teaching in a Canadian Pharmacy School.

Objective. To evaluate attitudes toward peer review of teaching and its impact on teaching practices and perceptions. Methods. The University of Waterloo School of Pharmacy implemented a peer-review process for its teaching program in 2015. Those reviewed were invited to complete an electronic survey that captured their attitudes toward teaching, attitudes toward peer review, and changes in teaching practices, and to participate in semi-structured follow-up interviews for more in-depth discussion of these issues. Results. Twenty-six (76%) instructors completed the survey. Instructors agreed that peer reviews of teaching are a development opportunity (96%), and 73% were comfortable with the idea of peer review. Over half (58%) indicated that the review made them feel more confident that their teaching strategies were effective, and the same percentage indicated that they planned to make changes to their teaching as a result of the feedback received from the peer review. Only a few instructors indicated that peer review changed their attitudes toward teaching (12%) or increased the value they placed on teaching (34%). Eight instructors (23.5%) participated in the semi-structured interviews. Themes that emerged included: attempts to make the reviewee comfortable during the peer review were successful; the feedback provided to instructors regarding their teaching was positive but not critical enough; there was lack of clarity as to the purpose of the feedback; and instructors planned to make only minor changes to their teaching as a result of the review. Conclusion. Peer review of teaching was well received and feedback was confirmatory in nature but had minimal impact on teaching practices as it was not deemed to be critical enough. Changes to the peer review program are needed to increase its impact on teaching practices.

Addressing professionalism in both formative and summative simulated patient care activities - Experiences with a discretionary deduction approach.

BACKGROUND AND PURPOSE: Encouraging and assessing professionalism among pharmacy students can be challenging. While non-punitive approaches are preferred as part of professional socialization, our program found this insufficient to ensure professional behaviour, especially during ungraded simulation lab activities. EDUCATIONAL ACTIVITY AND SETTING: In Winter 2015, we included a discretionary grade deduction within the assessments applied to a professional practice lab course in order to provide both intrinsic and extrinsic motivation to follow lab policies on professionalism. FINDINGS: A professionalism code was developed and discussed with students, and was also used as a template to guide professional behaviour throughout the course. Students not exhibiting these behaviours in lab could be subject to up to a five percent deduction from their final course grade at the instructor's discretion. DISCUSSION: Instructors considering this strategy are encouraged to introduce it in the first year of the program to ensure consistent expectations throughout the duration of students' training, and to not determine the magnitude of deductions applied until the end of the semester to ensure consistency and consideration of ongoing behaviour. Documentation of actions leading to deductions should be kept to support the decision. SUMMARY: A deduction-only approach to address unprofessional behaviours in addition to discussion offers additional motivation to students to exhibit professionalism across both graded and ungraded educational activities with minimal additional workload for instructors. The strategy has since been adopted across all lab courses in our program and has also been recommended in lecture-based courses.

Evaluation of a Unique Interprofessional Education Program Involving Medical and Pharmacy Students.

Objective. To measure changes in interprofessional competencies among pharmacy and medical students following a half-day event focusing on interprofessional learning. Methods. There were 118 pharmacy students and 28 medical students who participated in the Healthcare Interprofessional Education Day (HIPED) which consisted of three stations (communication, patient interviewing, and prescribing) in which pharmacy and medical students had to work collaboratively. The standardized Interprofessional Collaborative Competency Attainment Survey (ICCAS) was used to evaluate the effectiveness of the program. Results. There were 133 surveys completed for a response rate of 91%. All 20 items measured by the ICCAS showed a significant improvement. The strongest effect sizes were in the collaboration, roles & responsibilities, and collaborative practice/family-centered approach categories. The least robust effects were in the conflict management/resolution category. Conclusion. The HIPED activity was an effective IPE experience. The strong and consistent improvement in all ICCAS scores suggest a framework for pharmacy and medical school training to move from siloed educational experiences to synergistic learning opportunities.

Differential sensitivity to short-chain ceramide analogues of human intestinal carcinoma cells grown in tumor spheroids versus monolayer culture.

The cytotoxic activity of short-chain (C(2)) ceramide was evaluated in human intestinal carcinoma cells grown as multicellular tumor spheroids versus the same cells cultured as monolayers under closely comparable conditions. A decrease in cell number was seen in monolayer cultures of HT-29, Caco-2, and HRT-18 cells, with an EC(50) (concentration for half-maximal toxicity) of between 13 and 23 microM. However, when the same cells were grown in the multicellular spheroid format, C(2) was markedly less potent in reducing cell number, with an EC(50) of between 44 and 63 microM, representing a 1.9- to 4.9-fold decrease in its potency. The chemotherapeutic agents 5-fluorouracil and cisplatin were equally potent against spheroids and monolayer cultures, indicating that although drug access is a problem in conventionally grown tumor spheroids it is not a problem for spheroids grown under the conditions used in this study. Our results suggest that although ceramide is capable of inducing cell death in intestinal carcinoma cells grown in spheroid culture, its cellular toxicity is constrained by influences that are independent of drug access and may be the consequence of the altered cellular relationships. Carcinoma cell populations show an intrinsically decreased responsiveness to the effects of ceramide when they are grown in a three-dimensional culture format.

Adenosine downregulates DPPIV on HT-29 colon cancer cells by stimulating protein tyrosine phosphatase(s) and reducing ERK1/2 activity via a novel pathway.

The multifunctional cell-surface protein dipeptidyl peptidase IV (DPPIV/CD26) is aberrantly expressed in many cancers and plays a key role in tumorigenesis and metastasis. Its diverse cellular roles include modulation of chemokine activity by cleaving dipeptides from the chemokine NH(2)-terminus, perturbation of extracellular nucleoside metabolism by binding the ecto-enzyme adenosine deaminase, and interaction with the extracellular matrix by binding proteins such as collagen and fibronectin. We have recently shown that DPPIV can be downregulated from the cell surface of HT-29 colorectal carcinoma cells by adenosine, which is a metabolite that becomes concentrated in the extracellular fluid of hypoxic solid tumors. Most of the known responses to adenosine are mediated through four different subtypes of G protein-coupled adenosine receptors: A(1), A(2A), A(2B), and A(3). We report here that adenosine downregulation of DPPIV from the surface of HT-29 cells occurs independently of these classic receptor subtypes, and is mediated by a novel cell-surface mechanism that induces an increase in protein tyrosine phosphatase activity. The increase in protein tyrosine phosphatase activity leads to a decrease in the tyrosine phosphorylation of ERK1/2 MAP kinase that in turn links to the decline in DPPIV mRNA and protein. The downregulation of DPPIV occurs independently of changes in the activities of protein kinases A or C, phosphatidylinositol 3-kinase, other serine/threonine phosphatases, or the p38 or JNK MAP kinases. This novel action of adenosine has implications for our ability to manipulate adenosine-dependent events within the solid tumor microenvironment.

Effects of natural health products on blood pressure.

OBJECTIVE: To review the scientific literature to identify reports of the effects of natural health products (NHPs) on blood pressure. DATA SOURCES: Electronic databases (MEDLINE [1965-May 2004] via PubMed, the Cochrane Library [1995-May 2004], International Pharmaceutical Abstracts [1970-May 2004], Iowa Drug Information Services [1965-May 2004]) were searched using the key words medicine, herbal plants, medicinal plant preparations, phytotherapy, angiosperms/therapeutic use, gymnosperms/therapeutic use, ethnopharmacology, pharmacognosy, blood pressure, hypertension, hypotension, and diuretic. Searches were not limited by date, language, or publication type. Review articles and texts, as well as reference lists of relevant articles, were used to identify additional reports. STUDY SELECTION AND DATA EXTRACTION: Articles (English-language after 1980) were assigned to the following categories: human study, case report, animal study, in vitro study, or theoretical prediction based on chemical constituents. Discussions of mechanisms of action were noted. DATA SYNTHESIS: A comprehensive search of the scientific literature identified NHPs capable of affecting blood pressure. Case reports and clearly defined mechanisms of action provided strong evidence for the ability of ephedra and licorice to increase blood pressure. Coenzyme Q(10) was reported to decrease systolic and diastolic blood pressure, although the mechanism is unclear. The clinical significance of the blood pressure effects of other NHPs is unclear due to lack of conclusive in vivo data, as well as substantial variability in the chemical content of preparations of NHPs. CONCLUSIONS: Among published information, there is little definitive evidence with regard to the impact of NHPs on blood pressure. Additionally, effects may vary in a given patient with the formulation and standardization of a particular product. Until research better characterizes the effect of NHPs on blood pressure, patients should be encouraged to talk with their healthcare provider before starting or stopping any herbal product.

Rhodobacter capsulatus nifA1 promoter: high-GC -10 regions in high-GC bacteria and the basis for their transcription.

It was previously shown that the Rhodobacter capsulatus NtrC enhancer-binding protein activates the R. capsulatus housekeeping RNA polymerase but not the Escherichia coli RNA polymerase at the nifA1 promoter. We have tested the hypothesis that this activity is due to the high G+C content of the -10 sequence. A comparative analysis of R. capsulatus and other alpha-proteobacterial promoters with known transcription start sites suggests that the G+C content of the -10 region is higher than that for E. coli. Both in vivo and in vitro results obtained with nifA1 promoters with -10 and/or -35 variations are reported here. A major conclusion of this study is that alpha-proteobacteria have evolved a promiscuous sigma factor and core RNA polymerase that can transcribe promoters with high-GC -10 regions in addition to the classic E. coli Pribnow box. To facilitate studies of R. capsulatus transcription, we cloned and overexpressed all of the RNA polymerase subunits in E. coli, and these were reconstituted in vitro to form an active, recombinant R. capsulatus RNA polymerase with properties mimicking those of the natural polymerase. Thus, no additional factors from R. capsulatus are necessary for the recognition of high-GC promoters or for activation by R. capsulatus NtrC. The addition of R. capsulatus sigma(70) to the E. coli core RNA polymerase or the use of -10 promoter mutants did not facilitate R. capsulatus NtrC activation of the nifA1 promoter by the E. coli RNA polymerase. Thus, an additional barrier to activation by R. capsulatus NtrC exists, probably a lack of the proper R. capsulatus NtrC-E. coli RNA polymerase (protein-protein) interaction(s).

RNA polymerase subunit requirements for activation by the enhancer-binding protein Rhodobacter capsulatus NtrC.

Rhodobacter capsulatus NtrC is an enhancer-binding protein that activates transcription of the R. capsulatus sigma 70 RNA polymerase, but does not activate the Escherichia coli sigma 70-RNA polymerase at the nifA1 promoter. We utilized R. capsulatus:E. coli hybrid RNA polymerases assembled in vitro to investigate the subunits required for protein-protein interaction with RcNtrC at the nifA1mut1 promoter. Assembly of core Rc alpha beta beta' or hybrid RNA polymerases containing the Rc beta beta' subunits absolutely require the inclusion of an omega subunit, with the Ec omega subunit only partially promoting RNA polymerase assembly. The Rc alpha Ec beta beta' RNA polymerase is not activated by RcNtrC. Moreover, a mutant form of the Rc alpha lacking the alpha C-terminal domain, when assembled with the Rc beta beta'omega and sigma 70 subunits, is activated by RcNtrC. These results suggest that the R. capsulatus alpha subunit is not important for RcNtrC interaction. All hybrid RNA polymerases that contained the Rc beta' were activated by RcNtrC, suggesting that the Rc beta' subunit plays an important role. It is proposed that RcNtrC recruits R. capsulatus sigma 70-RNA polymerase to the promoter through interaction with Rc beta'. RcNtrC interacts with RNA polymerase from a unique position, with dimers centered at -118 bp from the start site. Placing the RcNtrC tandem binding sites on the opposite face of the helix (-113 bp) completely abolished transcription activation. Moving the RcNtrC tandem binding sites 20 bp closer to or further from the promoter significantly reduced activation, again suggesting unique spatial constraints on how RcNtrC interacts with the R. capsulatus RNA polymerase.