Participation in a Prison-Based Training Programme Is Beneficial for Rescue Dogs.

Dogs are often relinquished because of behavioural issues which may be exacerbated in rehoming centres. Prison-based dog training programmes (DTPs) may enhance outcomes for rescue dogs by providing socialisation and training opportunities to improve behaviour, welfare and likelihood of rehoming. We assessed whether dogs benefitted from participation, 1-3 times per week, in a prison-based DTP in which male young offenders learn how to train and care for dogs waiting to be rehomed. Within DTP sessions, there was significant improvement on a range of training tasks (n = 42 dogs). Analyses of videos (n = 17 dogs) in the kennels and a training barn pre- and post-DTP participation showed improvement in some positive behaviours, but no significant change in other behaviours. Subjective ratings by staff of the dogs' behaviour were made (n = 20 dogs). Desirable behaviours (e.g., playful/friendly) increased, and most undesirable behaviours (e.g., frustrated and noisy) decreased. Participation in the DTP did not mitigate all negative behaviours. However, improvements are consistent with enhanced welfare and likelihood of successful rehoming. Prison-based DTPs can be effective in supporting the work of animal rescue organisations to improve outcomes for dogs, while offering people in custody an opportunity to engage in purposeful activity and provide a community service.

Preparing Interns as Teachers: Teaching Fourth-Year Medical Students the Tenets of the One-Minute Preceptor Model.

Introduction: Often, interns are expected to teach medical students early in their residency, but most are not formally taught how to be effective teachers before residency. Currently, there is emphasis on developing teaching skills of residents rather than students before they become residents. Most published student-as-teacher courses are voluntary and do not assess skill acquisition. Methods: We taught 290 fourth-year medical students across two academic years (2020-2022) the tenets of the One-Minute Preceptor (OMP) using a 2-hour workshop during their transition to residency course. A variety of role-play cases allowed students to practice the different parts of the OMP in isolation and combined. Then, we assessed their teaching skills after the workshop using an objective structured teaching exam (OSTE). Results: Two hundred seventy-eight students (96%) completed the self-assessment of their confidence demonstrating the skills of the OMP before and after the workshop. Their confidence improved in all domains, with ps < .001. Additionally, all students successfully demonstrated competency on the OSTE. Discussion: We used a 2-hour workshop based on the OMP to improve fourth-year medical students' confidence in their teaching skills and allow them to demonstrate competence in those skills before starting their intern year.

Practice Doesn't Make Perfect: Clinical Experience With Procedures Does Not Correlate Well With Competence in Third-Year Medical Students.

OBJECTIVE: There is limited data available about factors which promote competence with procedures in medical students. Specifically, the relationship between procedural clinical experience and performance on an assessment is unclear. We sought to determine whether a correlation exists between the amount and type of clinical experience with a procedure and student performance on a standardized assessment of that procedure. DESIGN: Faculty performed standardized assessments of third-year medical students on ten procedures using simulation. We prospectively surveyed students about 3 types of experience (performed, observed, and simulated) with these procedures during their clerkships. We then analyzed whether a correlation exists between student experience and their competency assessment scores using Pearson's correlation. SETTING/PARTICIPANTS: Third-year medical students at the University of Kentucky College of Medicine. RESULTS: In 2018 to 2019, 131 students were assessed on procedural competency with 10 failures. One hundred and twenty students (91.6%) completed the clinical experience survey. Correlations between types of experience and competency scores were small to moderate, with only 5 of 40 being significant. We found no correlation between experience having performed a procedure and competency score. CONCLUSIONS: Overall, we did not find convincing evidence of a correlation between experience with procedures during clerkships and performance on a competency assessment. This suggests other factors may be contributing to procedural competence, which has implications for how educators should develop procedural competence in students.

Mucosal viral infection induces a regulatory T cell activation phenotype distinct from tissue residency in mouse and human tissues.

Regulatory T cells (Tregs) mediate immune homeostasis, yet also facilitate nuanced immune responses during infection, balancing pathogen control while limiting host inflammation. Recent studies have identified Treg populations in non-lymphoid tissues that are phenotypically distinct from Tregs in lymphoid tissues (LT), including performance of location-dependent roles. Mucosal tissues serve as critical barriers to microbes while performing unique physiologic functions, so we sought to identify distinct phenotypical and functional aspects of mucosal Tregs in the female reproductive tract. In healthy human and mouse vaginal mucosa, we found that Tregs are highly activated compared to blood or LT Tregs. To determine if this phenotype reflects acute activation or a general signature of vaginal tract (VT)-residency, we infected mice with HSV-2 to discover that VT Tregs express granzyme-B (GzmB) and acquire a VT Treg signature distinct from baseline. To determine the mechanisms that drive GzmB expression, we performed ex vivo assays to reveal that a combination of type-I interferons and interleukin-2 is sufficient for GzmB expression. Together, we highlight that VT Tregs are activated at steady state and become further activated in response to infection; thus, they may exert robust control of local immune responses, which could have implications for mucosal vaccine design.

A regulatory T cell signature distinguishes the immune landscape of COVID-19 patients from those with other respiratory infections.

Despite recent studies of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), little is known about how the immune response against SARS-CoV-2 differs from other respiratory infections. We compare the immune signature from hospitalized SARS-CoV-2­infected patients to patients hospitalized prepandemic with influenza or respiratory syncytial virus (RSV). Our in-depth profiling indicates that the immune landscape in SARS-CoV-2 patients is largely similar to flu or RSV patients. Unique to patients infected with SARS-CoV-2 who had the most critical clinical disease were changes in the regulatory T cell (Treg) compartment. A Treg signature including increased frequency, activation status, and migration markers was correlated COVID-19 severity. These findings are relevant as Tregs are considered for therapy to combat the severe inflammation seen in COVID-19 patients. Likewise, having defined the overlapping immune landscapes in SARS-CoV-2, existing knowledge of flu and RSV infections could be leveraged to identify common treatment strategies.

Anti-PD-1 Checkpoint Therapy Can Promote the Function and Survival of Regulatory T Cells.

We have previously shown in a model of claudin-low breast cancer that regulatory T cells (Tregs) are increased in the tumor microenvironment (TME) and express high levels of PD-1. In mouse models and patients with triple-negative breast cancer, it is postulated that one cause for the lack of activity of anti-PD-1 therapy is the activation of PD-1-expressing Tregs in the TME. We hypothesized that the expression of PD-1 on Tregs would lead to enhanced suppressive function of Tregs and worsen antitumor immunity during PD-1 blockade. To evaluate this, we isolated Tregs from claudin-low tumors and functionally evaluated them ex vivo. We compared transcriptional profiles of Tregs isolated from tumor-bearing mice with or without anti-PD-1 therapy using RNA sequencing. We found several genes associated with survival and proliferation pathways; for example, Jun, Fos, and Bcl2 were significantly upregulated in Tregs exposed to anti-PD-1 treatment. Based on these data, we hypothesized that anti-PD-1 treatment on Tregs results in a prosurvival phenotype. Indeed, Tregs exposed to PD-1 blockade had significantly higher levels of Bcl-2 expression, and this led to increased protection from glucocorticoid-induced apoptosis. In addition, we found in vitro and in vivo that Tregs in the presence of anti-PD-1 proliferated more than control Tregs PD-1 blockade significantly increased the suppressive activity of Tregs at biologically relevant Treg/Tnaive cell ratios. Altogether, we show that this immunotherapy blockade increases proliferation, protection from apoptosis, and suppressive capabilities of Tregs, thus leading to enhanced immunosuppression in the TME.

The human memory T cell compartment changes across tissues of the female reproductive tract.

Memory CD4 T cells in tissues fulfill numerous functions that are critical for local immune homeostasis and protection against pathogens. Previous studies have highlighted the phenotypic and functional heterogeneity of circulating and tissue-resident memory CD4 T cells across different human tissues such as skin, lung, liver, and colon. Comparatively little is known in regard to memory CD4 T cells across tissues of the female reproductive tract (FRT). We examined CD4 T cells in donor-matched vaginal, ecto- and endocervical tissues, which differ in mucosal structure and exposure to external environmental stimuli. We hypothesized that this could be reflected by tissue-specific differences in the memory CD4 T cell compartment. We found differences in CD4 subset distribution across these tissues. Specifically, CD69+CD103+ CD4 T cells were significantly more abundant in vaginal than cervical tissues. In contrast, the transcriptional profiles of CD4 subsets were fairly conserved across FRT tissues. CD69+CD103+ CD4 T cells showed a TH17 bias independent of tissue niche. Our data suggest that FRT tissues affect T cell subset distribution but have limited effects on the transcriptome of each subset. We discuss the implications for barrier immunity in the FRT.

A differential regulatory T cell signature distinguishes the immune landscape of COVID-19 hospitalized patients from those hospitalized with other respiratory viral infections.

SARS-CoV-2 infection has caused a lasting global pandemic costing millions of lives and untold additional costs. Understanding the immune response to SARS-CoV-2 has been one of the main challenges in the past year in order to decipher mechanisms of host responses and interpret disease pathogenesis. Comparatively little is known in regard to how the immune response against SARS-CoV-2 differs from other respiratory infections. In our study, we compare the peripheral blood immune signature from SARS-CoV-2 infected patients to patients hospitalized pre-pandemic with Influenza Virus or Respiratory Syncytial Virus (RSV). Our in-depth profiling indicates that the immune landscape in patients infected by SARS-CoV-2 is largely similar to patients hospitalized with Flu or RSV. Similarly, serum cytokine and chemokine expression patterns were largely overlapping. Unique to patients infected with SARS-CoV-2 who had the most critical clinical disease state were changes in the regulatory T cell (Treg) compartment. A Treg signature including increased frequency, activation status, and migration markers was correlated with the severity of COVID-19 disease. These findings are particularly relevant as Tregs are being discussed as a therapy to combat the severe inflammation seen in COVID-19 patients. Likewise, having defined the overlapping immune landscapes in SARS-CoV-2, existing knowledge of Flu and RSV infections could be leveraged to identify common treatment strategies. HIGHLIGHTS: The immune landscapes of hospitalized pre-pandemic RSV and influenza patients are similar to SARS-CoV-2 patientsSerum cytokine and chemokine expression patterns are largely similar between patients hospitalized with respiratory virus infections, including SARS-CoV-2, versus healthy donorsSARS-CoV-2 patients with the most critical disease displayed unique changes in the Treg compartmentadvances in understanding and treating SARS-CoV-2 could be leveraged for other common respiratory infections.

Maternal exposure to soy diet reduces atheroma in hyperlipidemic F1 offspring mice by promoting macrophage and T cell anti-inflammatory responses.

BACKGROUND AND AIMS: Maternal hypercholesterolemia has been implicated in earlier onset of atherosclerotic lesions in neonatal offspring. In this study, we investigated whether maternal exposure to soy protein isolate (SPI) diet attenuated the progression of atherosclerosis in F1 offspring. METHOD: Pregnant apolipoprotein E knockout (Apoe-/-) female mice were fed SPI diet until postnatal day 21 (PND21) of the offspring (SPI-offspring). SPI-offspring were switched at PND21 to casein (CAS) diet until PND140. Mice fed CAS throughout their lifetime (gestation to adulthood) were used as controls (CAS-offspring). RESULTS: Atherosclerotic lesions in the aortic sinuses were reduced in SPI-offspring compared with CAS-offspring. Total serum cholesterol levels in CAS-offspring or dams were comparable to levels in their SPI-counterparts, suggesting that alternative mechanisms contributed to the athero-protective effect of maternal SPI diet. Aortic VCAM-1, MCP-1, and TNF-α mRNA and protein expression, and expression of macrophage pro-inflammatory cytokines was reduced in SPI-offspring. Interestingly, CD4+ T cells from SPI-offspring showed reduced IFN-γ expression (Th1), while the expression of IL-10 (Th2/Treg), and IL-13 (Th2) was increased. DNA methylation analyses revealed that anti-inflammatory T cell-associated Gata3 and Il13 promoter regions were hypomethylated in SPI-offspring. These findings suggest that anti-inflammatory macrophage and T cell response may have contributed to the athero-protective effect in SPI-offspring. CONCLUSIONS: Our findings demonstrate that gestational and lactational soy diet exposure inhibits susceptibility to atherosclerotic lesion formation by promoting anti-inflammatory responses by macrophages and T cells.

Minimal PD-1 expression in mouse and human NK cells under diverse conditions.

PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.

Effects of Vitamin D Receptor Knockout and Vitamin D Deficiency on Corneal Epithelial Wound Healing and Nerve Density in Diabetic Mice.

Diabetic keratopathy occurs in ∼70% of all people with diabetes. This study was designed to examine the effects of vitamin D receptor knockout (VDR-/-) and vitamin D deficiency (VDD) on corneal epithelial wound healing and nerve density in diabetic mice. Diabetes was induced using the low-dose streptozotocin method. Corneal epithelial wounds were created using an Algerbrush, and wound healing was monitored over time. Corneal nerve density was measured in unwounded mice. VDR-/- and VDD diabetic mice (diabetic for 8 and 20 weeks, respectively) had slower healing ratios than wild-type diabetic mice. VDR-/- and VDD diabetic mice also showed significantly decreased nerve density. Reduced wound healing ratios and nerve densities were not fully rescued by a supplemental diet rich in calcium, lactose, and phosphate. We conclude that VDR-/- and VDD significantly reduce both corneal epithelial wound healing and nerve density in diabetic mice. Because the supplemental diet did not rescue wound healing or nerve density, these effects are likely not specifically related to hypocalcemia. This work supports the hypothesis that low vitamin D levels can exacerbate preexisting ophthalmic conditions, such as diabetes.

B Cells and T Follicular Helper Cells Mediate Response to Checkpoint Inhibitors in High Mutation Burden Mouse Models of Breast Cancer.

This study identifies mechanisms mediating responses to immune checkpoint inhibitors using mouse models of triple-negative breast cancer. By creating new mammary tumor models, we find that tumor mutation burden and specific immune cells are associated with response. Further, we developed a rich resource of single-cell RNA-seq and bulk mRNA-seq data of immunotherapy-treated and non-treated tumors from sensitive and resistant murine models. Using this, we uncover that immune checkpoint therapy induces T follicular helper cell activation of B cells to facilitate the anti-tumor response in these models. We also show that B cell activation of T cells and the generation of antibody are key to immunotherapy response and propose a new biomarker for immune checkpoint therapy. In total, this work presents resources of new preclinical models of breast cancer with large mRNA-seq and single-cell RNA-seq datasets annotated for sensitivity to therapy and uncovers new components of response to immune checkpoint inhibitors.

Vitamin D receptor and metabolite effects on corneal epithelial cell gap junction proteins.

Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)2D3 and 24R,25(OH)2D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR). The purpose of this study was to specifically determine the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR-/-) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)2D3 or 24R,25(OH)2D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca++ (Ca++i) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)2D3 and 24R,25(OH)2D3. Cx30 and Cx43 protein levels were also significantly increased in VDR-/- MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)2D3 and 1,25(OH)2D3. Ca++i was not affected by 1,25(OH)2D3 or 24R,25(OH)2D3 in HCEC or MPCEC. We conclude that both 1,25(OH)2D3 and 24R,25(OH)2D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Ca++i.

Identification of a Locus in Mice that Regulates the Collateral Damage and Lethality of Virus Infection.

Arenaviruses can cause severe hemorrhagic disease in humans, which can progress to organ failure and death. The underlying mechanisms causing lethality and person-to-person variation in outcome remain incompletely explained. Herein, we characterize a mouse model that recapitulates many features of pathogenesis observed in humans with arenavirus-induced hemorrhagic disease, including thrombocytopenia, severe vascular leakage, lung edema, and lethality. The susceptibility of congenic B6.PL mice to lymphocytic choriomeningitis virus (LCMV) infection is associated with increased antiviral T cell responses in B6.PL mice compared with C57BL/6 mice and is T cell dependent. Pathogenesis imparted by the causative locus is inherited in a semi-dominant manner in F1 crosses. The locus includes PL-derived sequence variants in both poorly annotated genes and genes known to contribute to immune responses. This model can be used to further interrogate how limited genetic differences in the host can remarkably alter the disease course of viral infection.

Opportunities for refinement in neuroscience: Indicators of wellness and post-operative pain in laboratory macaques.

Being able to assess pain in nonhuman primates undergoing biomedical procedures is important for preventing and alleviating pain, and for developing better guidelines to minimise the impacts of research on welfare in line with the 3Rs principle of Refinement. Nonhuman primates are routinely used biomedical models however it remains challenging to recognise negative states, including pain, in these animals. This study aimed to identify behavioural and facial changes that could be used as pain or general wellness indicators in the rhesus macaque (Macaca mulatta). Thirty-six macaques scheduled for planned neuroscience procedures were opportunistically monitored at four times: Pre-Operative (PreOp), Post-Operative (PostOp) once the effects of anaesthesia had dissipated, Pre-Analgesia (PreAn) on the subsequent morning prior to repeating routine analgesic treatment, and Post-Analgesia (PostAn) following administration of analgesia. Pain states were expected to be absent in PreOp, moderate in PreAn, and mild or absent in PostOp and PostAn when analgesia had been administered. Three potential pain indicators were identified: lip tightening and chewing, which were most likely to occur in PreAn, and running which was least likely in PreAn. Arboreal behaviour indicated general wellness, while half-closed eyes, leaning of the head or body shaking indicated the opposite. Despite considerable individual variation, behaviour and facial expressions could offer important indicators of pain and wellness and should be routinely quantified, and appropriate interventions applied to prevent or alleviate pain, and promote positive welfare.

"You Think You're Helping Them, But They're Helping You Too": Experiences of Scottish Male Young Offenders Participating in a Dog Training Program.

Interaction with animals can be beneficial to humans and animal-assisted interventions (AAIs) are increasingly popular in a range of contexts. Dog training programs (DTPs) are the most popular form of AAI in custodial contexts; prisoners often have multiple needs and DTPs seem to facilitate a diverse range of positive outcomes, including improvements in well-being, behavior, and offending behavior. However, evidence on the efficacy of prison-based DTPs is still limited and these evaluations often lack detail or methodological rigor. We examined the experiences of male young offenders (N = 70) using thematic analysis of semi-structured interviews conducted following completion of a DTP. The themes that emerged indicated a broad range of inter-related experiences and positive outcomes. The most prevalent theme related to their experiences with Dogs (including feelings and attitudes), and there were perceived improvements categorized as: Positive Effects (including mood and well-being), Motivation, Charitable Purpose, Self-Efficacy, Improved Skills, Impulsivity, and Emotional Management. These themes mapped well onto outcomes previously identified in research on DTPs, and to the program's core aims of improving behavior, educational engagement, employability, and well-being. The diversity and nature of these themes indicates that DTPs have considerable potential to engage and benefit those individuals with multiple needs, such as young offenders, and ultimately to achieve positive long-term outcomes with significant social, health, and economic impact.

Treg depletion potentiates checkpoint inhibition in claudin-low breast cancer.

Claudin-low breast cancer is an aggressive subtype that confers poor prognosis and is found largely within the clinical triple-negative group of breast cancer patients. Here, we have shown that intrinsic and immune cell gene signatures distinguish the claudin-low subtype clinically as well as in mouse models of other breast cancer subtypes. Despite adaptive immune cell infiltration in claudin-low tumors, treatment with immune checkpoint inhibitory antibodies against cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death receptor 1 (PD-1) were ineffective in controlling tumor growth. CD4+FoxP3+ Tregs represented a large proportion of the tumor-infiltrating lymphocytes (TILs) in claudin-low tumors, and Tregs isolated from tumor-bearing mice were able to suppress effector T cell responses. Tregs in the tumor microenvironment highly expressed PD-1 and were recruited partly through tumor generation of the chemokine CXCL12. Antitumor efficacy required stringent Treg depletion combined with checkpoint inhibition; delays in tumor growth were not observed using therapies that modestly diminished the number of Tregs in the tumor microenvironment. This study provides evidence that the recruitment of Tregs to the tumor microenvironment inhibits an effective antitumor immune response and highlights early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint blockade antibodies in specific subtypes of cancer that are heavily infiltrated with adaptive immune cells.

Facial expression: An under-utilised tool for the assessment of welfare in mammals.

Animal welfare is a key issue for industries that use or impact upon animals. The accurate identification of welfare states is particularly relevant to the field of bioscience, where the 3Rs framework encourages refinement of experimental procedures involving animal models. The assessment and improvement of welfare states in animals depends on reliable and valid measurement tools. Behavioral measures (activity, attention, posture and vocalization) are frequently used because they are immediate and non-invasive, however no single indicator can yield a complete picture of the internal state of an animal. Facial expressions are extensively studied in humans as a measure of psychological and emotional experiences but are infrequently used in animal studies, with the exception of emerging research on pain behavior. In this review, we discuss current evidence for facial representations of underlying affective states, and how communicative or functional expressions can be useful within welfare assessments. Validated tools for measuring facial movement are outlined, and the potential of expressions as honest signals is discussed, alongside other challenges and limitations to facial expression measurement within the context of animal welfare. We conclude that facial expression determination in animals is a useful but underutilized measure that complements existing tools in the assessment of welfare.