Hox-driven conditional immortalization of myeloid and lymphoid progenitors: Uses, advantages, and future potential.

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage-like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation-a signature cellular process of the macrophage-many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation-competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage-like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow-derived macrophages. To supplement the existing data, mass spectrometry-based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow-derived macrophages and the macrophage-like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.

Extracellular cathepsin Z signals through the α5 integrin and augments NLRP3 inflammasome activation.

Respiratory silicosis is a preventable occupational disease that develops secondary to the aspiration of crystalline silicon dioxide (silica) into the lungs, activation of the NLRP3 inflammasome, and IL-1ß production. Cathepsin Z has been associated with the development of inflammation and IL-1ß production; however, the mechanism of how cathepsin Z leads to IL-1ß production is unknown. Here, the requirement for cathepsin Z in silicosis was determined using WT mice and mice deficient in cathepsin Z. The activation of the NLRP3 inflammasome in macrophages was studied using WT and cathepsin Z-deficient bone marrow-derived murine dendritic cells and the human monocytic cell line THP-1. The cells were activated with silica, and IL-1ß release was determined using enzyme-linked immunosorbent assay or IL-1ß bioassays. The relative contribution of the active domain or integrin-binding domain of cathepsin Z was studied using recombinant cathepsin Z constructs and the α5 integrin neutralizing antibody. We report that the lysosomal cysteine protease cathepsin Z potentiates the development of inflammation associated with respiratory silicosis by augmenting NLRP3 inflammasome-derived IL-1ß expression in response to silica. The secreted cathepsin Z functions nonproteolytically via the internal integrin-binding domain to impact caspase-1 activation and the production of active IL-1ß through integrin α5 without affecting the transcription levels of NLRP3 inflammasome components. This work reveals a regulatory pathway for the NLRP3 inflammasome that occurs in an outside-in fashion and provides a link between extracellular cathepsin Z and inflammation. Furthermore, it reveals a level of NLRP3 inflammasome regulation that has previously only been found downstream of extracellular pathogens.

Pathologists aren't pigeons: exploring the neural basis of visual recognition and perceptual expertise in pathology.

Visual (perceptual) reasoning is a critical skill in many medical specialties, including pathology, diagnostic imaging, and dermatology. However, in an ever-compressed medical curriculum, learning and practicing this skill can be challenging. Previous studies (including work with pigeons) have suggested that using reward-feedback-based activities, novices can gain expert levels of visual diagnostic accuracy in shortened training times. But is this level of diagnostic accuracy a result of image recognition (categorization) or is it the acquisition of diagnostic expertise? To answer this, the authors measured electroencephalographic data (EEG) and two components of the human event-related brain potential (reward positivity and N170) to explore the nature of visual expertise in a novice-expert study in pathology visual diagnosis. It was found that the amplitude of the reward positivity decreased with learning in novices (suggesting a decrease in reliance on feedback, as in other studies). However, this signal remained significantly different from the experts whose reward positivity signal did not change over the course of the experiment. There were no changes in the amplitude of the N170 (a reported neural marker of visual expertise) in novices over time. Novice N170 signals remained statistically and significantly lower in amplitude compared to experts throughout task performance. These data suggest that, while novices gained the ability to recognize (categorize) pathologies through reinforcement learning as quantified by the change in reward positivity, increased accuracy, and decreased time for responses, there was little change in the neural marker associated with visual expertise (N170). This is consistent with the multi-dimensional and complex nature of visual expertise and provides insight into future training programs for novices to bridge the expertise gap.

Comparison of Fine Arts- and Pathology-Based Observational Skills Training for Veterinary Students Learning Cytology.

Keen observational skills are essential for veterinarians; however, the development of these skills is not usually an explicit part of the veterinary curriculum. Fine arts-based (FAB) observation training has been shown to improve medical students' observational skills and might also improve veterinary students' observational skills. We compared FAB and pathology-based (PB) observation training in a veterinary cytology course. Students initially wrote a pre-test in which they described two cytology images and one art image, followed by participation in either FAB or PB observation training. Both groups completed a similar post-test immediately after training and a delayed post-test 4 weeks later following instruction in cytology. Differences between groups were noted only in the immediate post-test cytology descriptions. The PB group used significantly more specific vocabulary terms and significantly more accurate observations than the FAB group, suggesting an immediate benefit to the discipline-specific information gained in the PB observation training. In the delayed post-test, results for both groups were similar. The FAB group significantly increased their use of specific vocabulary terms and maintained but did not increase accurate observations following cytology instruction, while accurate observations decreased significantly for the PB group. The FAB group might have been able to generalize their observation skills to the discipline of cytology and to better retain these skills. Neither type of training resulted in both achievement and maintenance of the highest recorded scores for accurate observations. Both FAB and PB training led to improved observational skills, and explicit observation training may be useful for veterinary students.

Using Fine Arts-Based Training to Develop Observational Skills in Veterinary Students Learning Cytology: A Pilot Study.

Arts-based training has been shown to improve medical students' observational skills. Veterinarians also need keen observational skills. Student veterinarians are expected to develop their observational skills; however, this training is usually not an explicit part of the veterinary curriculum. The impact of arts-based observation training has not been investigated in veterinary students learning cytology. In this pilot study, we compared student descriptions of art and cytology images before and immediately after receiving arts-based observation training. After 10 hours of cytology instruction, we again tested students' observational skills and asked for feedback via a survey. Pre-tests and post-tests were scored following a rubric based on expert descriptions of the images. Scores for art image descriptions were higher for both the immediate and delayed post-tests compared to the pre-test (p < .05). Scores for cytology image descriptions were higher for the immediate post-test than the pre-test, but this difference was not significant. Despite 10 hours of cytology instruction between post-tests, scores for cytology image descriptions were lower for the delayed post-test than the immediate post-test, but again, this difference was not significant. Student feedback on the arts-based observation training was positive. Overall, our results suggest that arts-based training may improve student observational skills, although context could be important, as the improvement in description was only significant for art images. Further investigation with a larger cohort of students and a control group that does not receive arts-based training would be valuable.

Development of and Validity Evidence for a Canine Ocular Model for Training Novice Veterinary Students to Perform a Fundic Examination.

Indirect fundoscopy is challenging for novice learners, as patients are often intolerant of the procedure, impeding development of proficiency. To address this, we developed a canine ocular simulator that we hypothesized would improve student learning compared to live dogs. Six board-certified veterinary ophthalmologists and 19 second-year veterinary students (novices) performed an indirect fundic examination on the model and live dog. Prior to assessment, novices were introduced to the skill with a standardized teaching protocol and practiced (without feedback) with either the model (n = 10) or live dog (n = 9) for 30 minutes. All participants evaluated realism and usefulness of the model using a Likert-type scale. Performance on the live dog and model was evaluated in all participants using time to completion of task, performance of fundic examination using a checklist and global score, identification of objects in the fundus of the model, and evaluation of time spent looking at the fundus of the model using eye tracking. Novices (trained on simulator or live dogs) were compared in fundic examination performance on the live dog and identification of shapes in the model. In general, experts performed the fundic examination faster (p ≤ .0003) and more proficiently than the novices, although there were no differences in eye tracking behavior between groups (p ≥ .06). No differences were detected between training on simulator versus live dog in development of fundoscopy skills in novices (p ≥ .20). These findings suggest that this canine model may be an effective tool to train students to perform fundoscopy.

Elder abuse protocols: identifying key features and establishing evidence for their use and effectiveness.

Written protocols may guide staff responses to elder abuse. Their effectiveness is unclear, as protocols are generally unevaluated. This project aimed to review, evaluate, and update the Alliance for the Prevention of Elder Abuse: Western Australia's 2013 Protocol. A critical literature review identified key features of elder abuse protocols. Three focus groups (N = 19), and an online survey (N = 44) examined the usefulness and effectiveness of the 2013 Protocol and identified improvements. Seventy percent of survey respondents (n = 31) were familiar with the Protocol; all found it useful. Suggested updates included: example policies and resources; screening tools, signs, indicators, and guided risk assessment; further direction and training for front-line workers; and information about issues for diverse and vulnerable groups. An updated Protocol was developed and will be supported by stakeholder education. Ongoing maintenance is required in a changing service context, and future versions could be online and include features to support responses.

The cooling compound icilin attenuates autoimmune neuroinflammation through modulation of the T-cell response.

The synthetic supercooling drug, icilin, and its primary receptor target, the cation channel transient receptor potential (TRP) melastatin-8 (TRPM8), have been described as potent negative regulators of inflammation in the colon. The aim of this study was to determine whether the anti-inflammatory action of icilin could potentially be used to treat autoimmune neuroinflammatory disorders, such as multiple sclerosis (MS). During experimental autoimmune encephalomyelitis (EAE)-a CD4+ T cell-driven murine model of MS-we found that both wild-type (WT) and TRPM8-deficient EAE mice were protected from disease progression during icilin treatment, as evidenced by delays in clinical onset and reductions in neuroinflammation. In vitro, icilin potently inhibited the proliferation of murine and human CD4+ T cells, with the peripheral expansion of autoantigen-restricted T cells similarly diminished by the administration of icilin in mice. Attenuation of both TRPM8-/- and TRP ankyrin-1-/- T-cell proliferation by icilin was consistent with the WT phenotype, which suggests a mechanism that is independent of these channels. In addition, icilin treatment altered the expressional profile of activated CD4+ T cells to one that was indicative of restricted effector function and limited neuroinflammatory potential. These findings identify a potent anti-inflammatory role for icilin in lymphocyte-mediated neuroinflammation and highlight clear pleiotropic effects of the compound beyond classic TRP channel activation.-Ewanchuk, B. W., Allan, E. R. O., Warren, A. L., Ramachandran, R., Yates, R. M. The cooling compound icilin attenuates autoimmune neuroinflammation through modulation of the T-cell response.

Reviewing the extent of rural and remote considerations in elder abuse policy: A scoping review.

OBJECTIVE: To identify national and international research and literature focussed on policy responses to elder abuse in rural and remote communities to generate recommendations for Australian responses. DESIGN: Scoping literature review. SETTING: Australia (some international policy also included). PARTICIPANTS: National and international elder abuse policy documents relating to rural and remote responses were reviewed. Seventy-two stakeholders were invited to contribute literature that may have been missed in the scoping review. MAIN OUTCOME MEASURE(S): Identification of the extent to which issues for rural and remote communities were discussed in elder abuse policy. RESULT: Of the 13 Australian policy documents reviewed, only four made mention of rural and remote communities, though this was generally only in regard to increase in vulnerability. No mention of these communities were identified in reviewed global policy. One document focussing on abuse in rural and remote Indigenous communities highlighted the importance of developing culturally appropriate responses hinged on community collaboration and consultation. CONCLUSION: Consideration of rural and remote communities in elder abuse policy is generally not meaningful. It is recommended that future policy development includes recognition of the distinct features of elder abuse in rural and remote communities, as well as how these features may impact on prevention and responses in these locations. These features include geographical isolation, lack of access to services and transportation, confidentiality and privacy issues, and the need for culturally sensitive approaches to address elder abuse for Indigenous people which take into account the impact of historical disenfranchisement.

Addressing elder abuse in rural and remote communities: social policy, prevention and responses.

This article reports the findings of an international scoping literature review focussed on social policy, prevention, and service responses to elder abuse in rural and remote communities. Forty-seven articles were included in the review, and fourteen types of prevention or service responses were identified in the literature, including several Indigenous-specific initiatives. The review highlighted the need for meaningful inclusion of rural and remote communities in elder abuse policy, as well as the importance of community consultation and consideration of the local context in developing prevention and response initiatives for these communities.

Changes in Student Perceptions and Study Strategies Over Time in a Veterinary Clinical Pathology Course Using Case-Based Instruction.

Veterinary students are challenged to develop new, nonlinear ways of thinking as they learn diagnostic reasoning skills. To support this process, we use real-life cases in our clinical pathology course. Changes in student perceptions regarding the use of cases and changes in study strategies over time have not been previously investigated or compared to student grades. Students participated in three voluntary online surveys that included 4-point Likert scale questions and open-ended questions on the helpfulness of cases for learning and study strategies used during the course. We used Friedman tests to detect any differences in perceptions over time; McNemar's test and Wilcoxon signed-rank tests were used to detect any differences in study strategies over time. Fisher's exact tests were used to examine the association between the Likert scale responses and grades in quartiles. Before beginning the course, 29% of students responded that cases were very helpful to their learning, with similar responses for helpfulness in applying course material and grasping important concepts. There was a significant trend of increasing positivity over the duration of the course, with 74% responding that cases were very helpful at the end of the course. The most-reported study strategy was working individually on cases before the midterm (74% of students), and the most helpful study strategy was attending class regularly (88% reported it as very helpful). Study strategies did not change significantly over time. Overall, perceptions and study strategies did not vary significantly with grades.

Quantifying Novice and Expert Differences in Visual Diagnostic Reasoning in Veterinary Pathology Using Eye-Tracking Technology.

Visual diagnostic reasoning is the cognitive process by which pathologists reach a diagnosis based on visual stimuli (cytologic, histopathologic, or gross imagery). Currently, there is little to no literature examining visual reasoning in veterinary pathology. The objective of the study was to use eye tracking to establish baseline quantitative and qualitative differences between the visual reasoning processes of novice and expert veterinary pathologists viewing cytology specimens. Novice and expert participants were each shown 10 cytology images and asked to formulate a diagnosis while wearing eye-tracking equipment (10 slides) and while concurrently verbalizing their thought processes using the think-aloud protocol (5 slides). Compared to novices, experts demonstrated significantly higher diagnostic accuracy (p <.017), shorter time to diagnosis (p <.017), and a higher percentage of time spent viewing areas of diagnostic interest (p <.017). Experts elicited more key diagnostic features in the think-aloud protocol and had more efficient patterns of eye movement. These findings suggest that experts' fast time to diagnosis, efficient eye-movement patterns, and preference for viewing areas of interest supports system 1 (pattern-recognition) reasoning and script-inductive knowledge structures with system 2 (analytic) reasoning to verify their diagnosis.

A role for cathepsin Z in neuroinflammation provides mechanistic support for an epigenetic risk factor in multiple sclerosis.

BACKGROUND: Hypomethylation of the cathepsin Z locus has been proposed as an epigenetic risk factor for multiple sclerosis (MS). Cathepsin Z is a unique lysosomal cysteine cathepsin expressed primarily by antigen presenting cells. While cathepsin Z expression has been associated with neuroinflammatory disorders, a role for cathepsin Z in mediating neuroinflammation has not been previously established. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in both wildtype mice and mice deficient in cathepsin Z. The effects of cathepsin Z-deficiency on the processing and presentation of the autoantigen myelin oligodendrocyte glycoprotein, and on the production of IL-1ß and IL-18 were determined in vitro from cells derived from wildtype and cathepsin Z-deficient mice. The effects of cathepsin Z-deficiency on CD4+ T cell activation, migration, and infiltration to the CNS were determined in vivo. Statistical analyses of parametric data were performed by one-way ANOVA followed by Tukey post-hoc tests, or by an unpaired Student's t test. EAE clinical scoring was analyzed using the Mann-Whitney U test. RESULTS: We showed that mice deficient in cathepsin Z have reduced neuroinflammation and dramatically lowered circulating levels of IL-1ß during EAE. Deficiency in cathepsin Z did not impact either the processing or the presentation of MOG, or MOG- specific CD4+ T cell activation and trafficking. Consistently, we found that cathepsin Z-deficiency reduced the efficiency of antigen presenting cells to secrete IL-1ß, which in turn reduced the ability of mice to generate Th17 responses-critical steps in the pathogenesis of EAE and MS. CONCLUSION: Together, these data support a novel role for cathepsin Z in the propagation of IL-1ß-driven neuroinflammation.

Development of a novel series of non-natural triaryl agonists and antagonists of the Pseudomonas aeruginosa LasR quorum sensing receptor.

Bacterial chemical communication, through a process called quorum sensing (QS), plays a central role in infection in numerous bacterial pathogens. Quorum sensing in Pseudomonas aeruginosa employs a series of small molecule receptors including the master QS regulator, LasR. In this study we investigate a non-natural triaryl series of LasR ligands using a combination of structure activity relationship studies and computational modeling. These studies have enabled the identification of key structural requirements for ligand binding and have revealed a new strategy for inducing the therapeutically relevant antagonism of LasR.

NADPH oxidase modifies patterns of MHC class II-restricted epitopic repertoires through redox control of antigen processing.

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow-derived macrophages, but not dendritic cells, to process and present the I-A(b)-immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-A(b) binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-A(b) mice that were deficient in the p47(phox) or gp91(phox) subunits of NOX2 were partially protected from MOG-induced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4(+) T cells in the CNS, despite eliciting a normal primary CD4(+) T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4(+) T cell-driven autoimmune disease processes.

Educational interventions to improve cytology visual diagnostic reasoning measured by eye tracking.

The teaching of visual diagnostic reasoning skills, to date, has been conducted in a largely unstructured apprenticeship manner. The purpose of this study was to assess if the introduction of two educational interventions improved the visual diagnostic reasoning skills of novices. These were (1) the active use of key diagnostic features and (2) image repetition. A pre-test and post-test research design was used to compare the two teaching interventions to a traditional teaching group and an expert group using eye tracking as an assessment method. The time to diagnosis and the percentage of time spent viewing an area of diagnostic interest (AOI) were compared using independent t-tests, paired t-tests, and analysis of covariance (ANCOVA). Diagnostic accuracy as a dichotomous variable was compared using Chi-square tables. Students taught in an active-learning manner with image repetition behaved most like experts, with no significant difference from experts for percentage of time spent in the AOIs and a significantly faster time to diagnosis than experts (p<.017). Our results from the educational interventions suggest a greater level of improvement in the eye tracking of students that were taught key diagnostic features in an active-learning forum and were shown multiple case examples.

Illuminating trajectories of adolescent thriving and contribution through the words of youth: qualitative findings from the 4-H Study of Positive Youth Development.

Theory and research in adolescent development have emphasized that contributing to self, others, and community is important to the success of society and predictive of positive youth and later adult development. Despite this emphasis, there is a lack of qualitative and youth-centered research exploring whether adolescents themselves value contribution as part of their daily lives or future goals. Understandings of contribution are, thus, limited in their generalizability. To lessen this gap, we implemented qualitative analyses of open-ended responses from youth in the 4-H Study of Positive Youth Development. We addressed questions about what is meaningful to youth and about their future goals through descriptive and thematic analyses of responses from 56 youth (66% female) who participated in the 4-H Study in each of three grades (6, 9, and 12). Findings indicated that most youth in this study valued acts and/or ideologies of contribution at some point in their adolescence, and several were committed to facets of contribution across grades. The analyses also identified other aspects of these youth experiences (e.g., athletics, family relationships, and academic competencies) that were described as meaningful across adolescence. Findings are discussed in relationship to youth programming aimed at encouraging well-being and contribution in adolescence.

Exploring Categories of Family Violence Across the Lifespan: A Scoping Review.

Family violence may be experienced at any stage of the lifespan; however, these experiences are often understood differently based on the age of the victim and who perpetrates the abuse. The significance of age is evident in the three categories of child abuse, domestic and family violence (DFV), and elder abuse. Each of these categories has its own definition which determines who is considered a victim or a perpetrator, and the behaviors counted as violent and abusive. These definitions influence how practitioners view victim-survivors' experiences of violence, and the subsequent available responses. This article reports the findings of a scoping review of international literature published between 2011 and 2021, which explored how family violence is categorized and defined. The review was conducted as part of a larger study exploring how violence against women in intimate and family contexts is conceptualized and experienced, as well as the available responses. Forty-eight articles were included in the final review, and five categories of violence in family and intimate contexts were identified. These were child abuse, DFV, elder abuse, adolescent-to-parent violence, and sibling abuse. Comparison of definitions across categories found similarities in terms of the relationship between victim and perpetrator, behavior, intention, and harm caused to the victim. Review findings suggest that definitions of various forms of family violence do not differ greatly. Further research is needed to determine whether responses to family violence across the lifespan can and should be streamlined.

Evaluation of a psychoeducation group for children presenting with functional tic-like behaviours.

We describe the implementation and evaluation of an online psychoeducation group for young people experiencing functional tic-like behaviours (FTLBs) - a type of functional neurological disorder (FND). Across six groups, 50 participants completed pre- and post-group goal-based outcomes (GBOs) and 36 participants completed service-user feedback, which gathered qualitative and quantitative data about participants experiences of the group. Young people and their parents reported significant improvement in their GBOs following the group and increased knowledge and confidence in managing FTLBs. The findings highlight that virtual psychoeducation group intervention is an acceptable and effective first step in treatment of young people with FTLBs. We discuss implications for future intervention development.

We describe an online psychoeducation group for young people experiencing functional tic-like behaviours (FTLBs) ­ a type of functional neurological disorder (FND). Across six groups, 50 participants completed goal-based outcomes (GBOs) before and after the intervention. A total of 36 participants completed service-user feedback, which gathered data about participants experiences of the group. Young people and their parents reported significant improvement in their GBOs following the group and increased knowledge and confidence in managing FTLBs. The findings highlight that virtual psychoeducation group intervention is an acceptable and effective first step in treatment of young people with FTLBs. We discuss implications for future intervention development.

Invasion, establishment, and range expansion of two parasitic nematodes in the Canadian Arctic.

Climate warming is occurring at an unprecedented rate in the Arctic and is having profound effects on host-parasite interactions, including range expansion. Recently, two species of protostrongylid nematodes have emerged for the first time in muskoxen and caribou on Victoria Island in the western Canadian Arctic Archipelago. Umingmakstrongylus pallikuukensis, the muskox lungworm, was detected for the first time in 2008 in muskoxen at a community hunt on the southwest corner of the island and by 2012, it was found several hundred kilometers east in commercially harvested muskoxen near the town of Ikaluktutiak. In 2010, Varestrongylus sp., a recently discovered lungworm of caribou and muskoxen was found in muskoxen near Ikaluktutiak and has been found annually in this area since then. Whereas invasion of the island by U. pallikuukensis appears to have been mediated by stochastic movement of muskoxen from the mainland to the southwest corner of the island, Varestrongylus has likely been introduced at several times and locations by the seasonal migration of caribou between the island and the mainland. A newly permissive climate, now suitable for completion of the parasite life cycles in a single summer, likely facilitated the initial establishment and now drives range expansion for both parasites.