Be the Qualitative Researcher: A Team-Based, Active Learning Strategy Using Popular Music Lyrics.

ABSTRACT: A team-based, active learning activity was created to facilitate undergraduate nursing students' understanding of qualitative research concepts. Working in small groups, students utilized the full-text lyrics of albums by recording artist Taylor Swift to analyze phrasing, create a qualitative coding scheme, code the data, and create a content and thematic analysis. The students then collaboratively created a Google Slide to present their findings for the class. An anonymous postactivity survey indicated greater student satisfaction with this activity than with traditional didactic lectures.

Effect of biotin on currently used Beckman thyroglobulin assay and newly reformulated thyroglobulin assay not affected by biotin.

OBJECTIVES: Biotin causes negative interference with thyroglobulin measurement using the Access thyroglobulin assay. Recently, Beckman reformulated the thyroglobulin assay to overcome biotin interference. We investigated the effect of biotin on both current and newly formulated assays. METHODS: Four serum pools were prepared using specimens containing various amounts of thyroglobulin. Then aliquots of each pool were supplemented with various amounts of biotin, and thyroglobulin concentrations were measured by both the current and the new assays. In addition, 3 volunteers ingested 10 mg biotin, and specimens were drawn before and 2 hours after taking biotin. Thyroglobulin concentrations before and 2 hours after taking biotin were measured by both assays. RESULTS: In the presence of biotin, thyroglobulin concentrations were reduced significantly using the current assay, but no significant change was observed using the newly formulated assay. We observed similar results in vivo. CONCLUSIONS: The newly formulated thyroglobulin assay by Beckman is free from biotin interference.

Technical Note: Approach to Identify and Eliminate Biotin Interference in Thyroid Function Tests Using Beckman DXI 800 Analyzer by Taking Advantage of Assay Harmonization with Alinity i Analyzer.

OBJECTIVE: We reported significant interference of biotin in FT3 and FT4 assays using Beckman DXI 800 analyzer. Recently we acquired Alinity i analyzer where TSH, FT3 and FT4 assays are not biotin based. We hypothesized that if thyroid function tests on DXI 800 and Alinity i are harmonized, then it is possible to eliminate biotin interference. METHODS: We investigated assay harmonization by analyzing 35 specimens for TSH, FT4 and FT3 using both analyzers. We prepared one serum pool using left-over specimens where thyroid tests were ordered. Then aliquots of the pool were supplemented with various amounts of biotin followed by measuring thyroid function tests again. RESULTS: We observed assay harmonization between both analyzers for TSH, FT3 and FT4 Tests. TSH assay was not affected in the presence of biotin, but FT3 and FT4 values were significantly elevated using DXI 800 analyzer. In contrast, TSH, FT3 and FT4 assays were not affected by biotin using Alinity i analyzer. CONCLUSIONS: Elevated FT3 and FT4 using DXI 800 analyzer may be due to biotin interference which can be eliminated by observing normal values using Alinity i analyzer. However, normal or slightly elevated TSH with elevated FT3 and FT4 using both analyzers may indicate rare type of TSH producing tumor of pituitary, not biotin interference.

Significant Interference of Biotin in Thyroid Function Tests Using Beckman Analyzer: How to Identify such Interferences?

OBJECTIVE: Biotin in elevated concentration interferes with biotin-based immunoassays. We studied biotin interferences in TSH, FT4, FT3, total T4, total T3 and thyroglobulin assays both in vitro and in vivo using Beckman DXI800 analyzer. METHODS: Two serum pools were prepared from left-over specimens. Then aliquots of each pool (and serum control) were supplemented with various amounts of biotin followed by measuring thyroid function tests again. Three volunteers each took 10 mg biotin supplement. We compared thyroid function tests before and 2 h after taking biotin. RESULTS: We observed significant biotin interferences in biotin-based assays (positive interference with FT4, FT3, and total T3 assay but negative interference with thyroglobulin) both in vitro and in vivo but non-biotin-based assays (TSH and total T4) were not affected. CONCLUSIONS: Elevated FT3 and FT4 in the presence of normal TSH is inconsistent with hyperthyroidism and should be followed up with total T3 and T4 test. Significant discrepancy between total T3 (falsely elevated value due to biotin) and total T4 (not affected as the assay is not biotin based) maybe an indication of biotin interference.

Abnormal response to stress and impaired NPS-induced hyperlocomotion, anxiolytic effect and corticosterone increase in mice lacking NPSR1.

NPSR1 is a G protein coupled receptor expressed in multiple brain regions involved in modulation of stress. Central administration of NPS, the putative endogenous ligand of NPSR1, can induce hyperlocomotion, anxiolytic effects and activation of the HPA axis. The role of NPSR1 in the brain remains unsettled. Here we used NPSR1 gene-targeted mice to define the functional role of NPSR1 under basal conditions on locomotion, anxiety- and/or depression-like behavior, corticosterone levels, acoustic startle with prepulse inhibition, learning and memory, and under NPS-induced locomotor activation, anxiolysis, and corticosterone release. Male, but not female, NPSR1-deficient mice exhibited enhanced depression-like behavior in a forced swim test, reduced acoustic startle response, and minor changes in the Morris water maze. Neither male nor female NPSR1-deficient mice showed alterations of baseline locomotion, anxiety-like behavior, or corticosterone release after exposure to a forced swim test or methamphetamine challenge in an open-field. After intracerebroventricular (ICV) administration of NPS, NPSR1-deficient mice failed to show normal NPS-induced increases in locomotion, anxiolysis, or corticosterone release compared with WT NPS-treated mice. These findings demonstrate that NPSR1 is essential in mediating NPS effects on behavior.

Targeting IL-4/IL-13 signaling to alleviate oral allergen-induced diarrhea.

BACKGROUND: Intestinal anaphylaxis (manifested by acute diarrhea) is dependent on IgE and mast cells. OBJECTIVE: We aimed to define the respective roles of IL-4 and IL-13 and their receptors in disease pathogenesis. METHODS: Wild-type mice and mice deficient in IL-4, IL-13, and IL-13 receptor (IL-13R) alpha1 (part of the type 2 IL-4 receptor [IL-4R]) were sensitized with ovalbumin (OVA)/aluminum potassium sulfate and subsequently given repeated intragastric OVA exposures. The IL-4R alpha chain was targeted with anti-IL-4R alpha mAb before or after intragastric OVA exposures. RESULTS: IL4(-/-) (and IL4/IL13(-/-)) mice produced almost no IgE and were highly resistant to OVA-induced diarrhea, whereas allergic diarrhea was only partially impaired in IL13(-/-) and IL13Ralpha1(-/-) mice. IL13Ralpha1-deficient mice had decreased IgE levels, despite having normal baseline IL-4 levels. Intestinal mast cell accumulation and activation also depended mainly on IL-4 and, to a lesser extent, on IL-13. Prophylactic anti-IL-4R alpha mAb treatment, which blocks all IL-4 and IL-13 signaling, suppressed development of allergic diarrhea. However, treatment with anti-IL-4R alpha mAb for 7 days only partially suppressed IgE and did not prevent intestinal diarrhea. CONCLUSION: Endogenously produced IL-13 supplements the ability of IL-4 to induce allergic diarrhea by promoting oral allergen sensitization rather than the effector phase of intestinal anaphylaxis.

FIP1L1/PDGFRalpha synergizes with SCF to induce systemic mastocytosis in a murine model of chronic eosinophilic leukemia/hypereosinophilic syndrome.

Expression of the fusion gene FIP1-like 1/platelet-derived growth factor receptor alpha (FIP1L1/PDGFRalpha, F/P) and dysregulated c-kit tyrosine kinase activity are associated with systemic mastocytosis (SM) and chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES). We analyzed SM development and pathogenesis in a murine CEL model induced by F/P in hematopoietic stem cells and progenitors (HSCs/Ps) and T-cell overexpression of IL-5 (F/P-positive CEL mice). These mice had more mast cell (MC) infiltration in the bone marrow (BM), spleen, skin, and small intestine than control mice that received a transplant of IL-5 transgenic HSCs/Ps. Moreover, intestinal MC infiltration induced by F/P expression was severely diminished, but not abolished, in mice injected with neutralizing anti-c-kit antibody, suggesting that endogenous stem cell factor (SCF)/c-kit interaction synergizes with F/P expression to induce SM. F/P-expressing BM HSCs/Ps showed proliferation and MC differentiation in vitro in the absence of cytokines. SCF stimulated greater migration of F/P-expressing MCs than mock vector-transduced MCs. F/P-expressing bone marrow-derived mast cells (BMMCs) survived longer than mock vector control BMMCs in cytokine-deprived conditions. The increased proliferation and survival correlated with increased SCF-induced Akt activation. In summary, F/P synergistically promotes MC development, activation, and survival in vivo and in vitro in response to SCF.

A dual activation and inhibition role for the paired immunoglobulin-like receptor B in eosinophils.

The accumulation of eosinophils in inflammatory foci is a hallmark characteristic of Th2 inflammation. Nevertheless, the expression of inhibitory receptors such as paired immunoglobulin-like receptor B (PIR-B) and their function regulating eosinophil accumulation have received limited attention. We now report that Pirb was up-regulated in an eosinophil-dependent manner in the lungs of allergen-challenged and interleukin (IL)-13-overexpressing mice. Eosinophils expressed high levels of PIR-B, and Pirb(-/-) mice displayed increased gastrointestinal eosinophils. Consistent with these findings, PIR-B negatively regulated eotaxin-dependent eosinophil chemotaxis in vivo and in vitro. Surprisingly, Pirb(-/-) eosinophils and neutrophils had decreased leukotriene B4 (LTB(4))-dependent chemotactic responses in vitro. Furthermore, eosinophil accumulation was decreased in a chitin-induced model, partially dependent on LTB(4). Mechanistic analysis using a miniphosphoproteomic approach revealed that PIR-B recruits activating kinases after LTB(4) but not eotaxin stimulation. Consequently, eotaxin-activated Pirb(-/-) eosinophils displayed markedly increased extracellular signal-related kinase 1 and 2 (ERK1/2) phosphorylation, whereas LTB(4)-activated eosinophils had reduced ERK1/2 phosphorylation. We provide multiple lines of evidence supporting a model in which PIR-B displays opposing but potent regulatory functions in granulocyte activation. These data change the conventional wisdom that inhibitory receptors are restricted to inhibitory signals; we therefore propose that a single receptor can have dual functionality in distinct cell types after unique cellular signals.

A central regulatory role for eosinophils and the eotaxin/CCR3 axis in chronic experimental allergic airway inflammation.

To clarify the role and regulation of eosinophils, we subjected several key eosinophil-related genetically engineered mice to a chronic model of allergic airway inflammation aiming to identify results that were independent of the genetic targeting strategy. In particular, mice with defects in eosinophil development (Deltadbl-GATA) and eosinophil recruitment [mice deficient in CCR3 (CCR3 knockout) and mice deficient in both eotaxin-1 and eotaxin-2 (eotaxin-1/2 double knockout)] were subjected to Aspergillus fumigatus-induced allergic airway inflammation. Allergen-induced eosinophil recruitment into the airway was abolished by 98%, 94%, and 99% in eotaxin-1/2 double knockout, CCR3 knockout, and Deltadbl-GATA mice, respectively. Importantly, allergen-induced type II T helper lymphocyte cytokine production was impaired in the lungs of eosinophil- and CCR3-deficient mice. The absence of eosinophils correlated with reduction in allergen-induced mucus production. Notably, by using global transcript expression profile analysis, a large subset (29%) of allergen-induced genes was eosinophil- and CCR3-dependent; pathways downstream from eosinophils were identified, including in situ activation of coagulation in the lung. In summary, we present multiple lines of independent evidence that eosinophils via CCR3 have a central role in chronic allergic airway disease.

New aspects of vascular remodelling: the involvement of all vascular cell types.

Conventionally, the architecture of arteries is based around the close-packed smooth muscle cells and extracellular matrix. However, the adventitia and endothelium are now viewed as key players in vascular growth and repair. A new dynamic picture has emerged of blood vessels in a constant state of self-maintenance. Recent work raises fundamental questions about the cellular heterogeneity of arteries and the time course and triggering of normal and pathological remodelling. A common denominator emerging in hypertensive remodelling is an early increase in adventitial cell density suggesting that adventitial cells drive remodelling and may initiate subsequent changes such as re-arrangement of smooth muscle cells and extracellular matrix. The organization of vascular smooth muscle cells follows regular arrangements that can be modelled mathematically. In hypertension, new patterns can be quantified in these terms and give insights to how structure affects function. As with smooth muscle, little is known about the organization of the vascular endothelium, or its role in vascular remodelling. Current observations suggest that there may be a close relationship between the helical organization of smooth muscle cells and the underlying pattern of endothelial cells. The function of myoendothelial connections is a topic of great current interest and may relate to the structure of the internal elastic lamina through which the connections must pass. In hypertensive remodelling this must present an organizational challenge. The objective of this paper is to show how the functions of blood vessels depend on their architecture and a continuous interaction of different cell types and extracellular proteins.

Two "knockout" mouse models demonstrate that aortic vasodilatation is mediated via alpha2a-adrenoceptors located on the endothelium.

UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine]-mediated vasodilator responses were studied on wire myograph-mounted mouse aorta to determine the cells involved, mechanisms of action, and subtypes of alpha(2)-adrenoceptors. In the presence of induced tone, UK-14,304 produced concentration-related vasodilatation that was abolished by rauwolscine, N(omega)-nitro-L-arginine methyl ester (L-NAME), or endothelium removal, indicating that endothelial alpha(2)-adrenoceptors can release nitric oxide. In the alpha(2A)-adrenoceptor knockout mouse and the D79N mouse, a functional knockout of the alpha(2A)-adrenoceptor, these relaxant effects of UK-14,304 were lost, indicating the involvement of the alpha(2A)-adrenoceptor. UK-14,304 could also contract aorta: a small contraction occurred at high concentrations, was enhanced by L-NAME, and was absent in the alpha(1D)-adrenoceptor knockout mouse, indicating activation of the alpha(1D)-adrenoceptor. There was no evidence for a contractile alpha(2)-adrenoceptor-mediated response. A fluorescent ligand, quinazoline piperazine bodipy, antagonized the relaxant action of UK-14,304. This compound could be visualized on aortic endothelial cells, and its binding could be prevented by rauwolscine, providing direct evidence for the presence of alpha(2)-adrenoceptors on the endothelium. Norepinephrine reduced tone in the alpha(1D)-adrenoceptor knockout and controls, an effect blocked by rauwolscine and L-NAME but not by prazosin. This suggests that norepinephrine activates endothelial alpha(2)-adrenoceptors. In conclusion, the endothelium of mouse aorta has an alpha(2A)-adrenoceptor that responds to norepinephrine; promotes the release of nitric oxide, causing smooth muscle relaxation; and that can be directly visualized. Knockout or genetic malfunction of this receptor should increase arterial stiffness, exacerbated by raised catecholamines, and contribute to heart failure.

Actions of paracetamol on cyclooxygenases in tissue and cell homogenates of mouse and rabbit.

BACKGROUND: Paracetamol is a potent analgesic and antipyretic drug, but has only weak anti-inflammatory activity. Unlike aspirin-like drugs, paracetamol does not damage the stomach mucosa or inhibit the aggregation of platelets. The analgesic action of paracetamol is on the central nervous system. In vitro, paracetamol inhibits cyclooxygenase (COX)-1 and -2 in high concentrations but stimulates in low doses. This study examines the stimulation and inhibition of COX-1 and COX-2 in homogenates of mouse and rabbit tissues and in J774.2 cultured mouse macrophages. MATERIAL/METHODS: Mouse and rabbit tissues were removed, homogenised and treated with different concentrations of paracetamol. Prostaglandins (PGs) E2 and I2 were measured in the homogenates to assess the activity of COX-1. Ex vivo synthesis of PGE2 was measured in tissues after treating rabbits with 100 mg/kg paracetamol. J774.2 cultured mouse macrophages treated with bacterial lipopolysaccharide (LPS) to induce COX-2, were treated with varying concentrations of paracetamol and the PGs produced were measured. RESULTS: Low doses of paracetamol stimulated PG production in J774.2 macrophages and stomach mucosa homogenates, but reduced PG production at high concentrations of paracetamol. This stimulation did not occur when co-factors were added. The order of potency of paracetamol on COX-1 or COX-2 in tissue homogenates was as follows: lungs>spleen>brain>J774.2 cells>stomach mucosa. Paracetamol, 100 mg/kg, inhibited COX-1 in stomach mucosa ex vivo much less effectively than in other tissues. CONCLUSIONS: These data support the hypothesis that paracetamol selectively inhibits a COX enzyme which is different from COX-1 or COX-2 and may be a variant of COX-1.

A knockout approach indicates a minor vasoconstrictor role for vascular alpha1B-adrenoceptors in mouse.

Pharmacological analysis alone has failed to clarify the role of the three alpha(1)-adrenoceptor subtypes in modulating vascular tone, due to a lack of sufficiently selective antagonists, particularly for the alpha (1B)-adrenoceptor, and the complexity when three receptor subtypes are potentially activated by the same agonist. We adopted a combined genetics/ pharmacology strategy based on the alpha(1B)-adrenoceptor knockout (KO) mouse. The potency of three alpha(1)-adrenoceptor antagonists vs. phenylephrine was tested in aorta, carotid, mesenteric, and caudal isolated arteries from KO and wild-type (WT) mice. In the KO mouse the pharmacology became straightforward, showing alpha(1D) in two major conducting arteries (aorta and carotid) and alpha(1A) in two distributing arteries (mesenteric and caudal). By combining antagonist pharmacology and genetics, we provide a simplified analysis of alpha(1)-mediated vasoconstriction, demonstrating that alpha(1D) and alpha(1A) are the major subtypes involved in vasoconstriction, with a minor but definite contribution from alpha(1B) in every vessel.