Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), remains an ongoing global issue for human and animal health. The Bacille Calmette Guerin (BCG) vaccine offers immunity against bTB, however, the mechanisms underlying the heterogenous protective response, including variations across species and age groups requires further investigation. In this study, we focused on dendritic cells (DCs), which are crucial for adaptive immune stimulation following BCG vaccination. By capturing afferent lymph DCs (ALDCs) migrating from the skin, we investigated shifts in DC profiles and potential subset-specific functions in response to BCG vaccination. Single-cell RNA sequencing (scRNA-seq) was performed on samples from Bos taurus calves (n=3) before and after BCG vaccination, capturing the transcriptome of 20,761 individual cells expressing on average 3,036 genes, which were clustered into ALDCs, monocytes, T-cells, B-cells and NK cells. The ALDC subsets were further identified as cDC1 and cDC2. In homeostasis, ALDCs expressing potential subset-specific genes for cDC1, including ENSBTAG00000056208, STX4, NEBL, ADAM23, ART3, and cDC2; FN1, PSPH, FGL2, SHOX2, and WWTR1 were identified. Following BCG vaccination, while both DC subsets exhibited gene expression signatures indicative of antigen-presenting function, migration, and DC maturation, cDC1 showed upregulation of genes consistent with metabolic alterations and lymphocyte recruitment, whereas cDC2 upregulated genes consistent with inflammatory responses. Overall, this study comprehensively describes the transcriptomic landscape of bovine ALDC subsets, providing evidence for the importance of subset-specific genes to BCG vaccination responses, while advancing knowledge on how ALDCs contribute to protective immunity against bTB.

Ovine pulmonary adenocarcinoma (OPA), caused by Jaagsiekte sheep retrovirus (JSRV), is a contagious neoplastic pulmonary disease of sheep. Early OPA diagnosis is challenging as the disease can remain subclinical until advanced disease. Lung ultrasound offers the potential of identifying OPA lesions as small as 1-2 cm in diameter involving the visceral pleural surface. Although the procedure lends itself to screening large commercial flocks, further research is needed to investigate whether commonly used ultrasound features can be used to diagnose and stage OPA disease, potentially before gross lung tumours develop. Using our JSRV experimental model we adopted a semiquantitative approach to grade, at monthly intervals up to 9 months post-JSRV infection, the incidence and progression of B lines, white lung, and irregular pleural lines. Results were compared against non-infected control sheep. While the presence of irregular pleural lines was inconsistent following JSRV infection, white lung developed only in the lung instilled with JSRV, whereas B lines developed in both lungs. These features occurred both in sheep which developed gross tumours, and also in those which did not develop gross tumours. Changes in B lines and white lung ultrasound features in lungs that had no evidence of gross lung tumour development may reflect structural and/or functionally dynamic aspects of the underlying pathophysiology. Future assessment of the direct association between histopathological characteristics and ultrasound image features will provide a foundation to investigate the practical utility of this knowledge, particularly in the context of screening flocks as a means to control this disease.

IntroductionDifferent regions of the small ruminant lung exhibit variable susceptibility to specific lung pathologies. Such susceptibility may be reflected in regional lung radiomic features extracted from computed tomography (CT) images. In this study, we investigated whether region-specific variation in radiomic features exists in ovine lungs and whether these features remain stable over time.MethodsThoracic CT image datasets from 30 young adult sheep were subject to an image segmentation protocol directed at partitioning the lung into individual lobar and sub-lobar segments for radiomic feature analysis. After identifying and removing unstable, non-reproducible, and highly correlated features, 22 features remained and were used as input for principal component (PC) analysis.ResultsThe significance of segment-related influence on PC scores was determined and visualised. For six sheep, successive CT images were acquired at monthly intervals for a period of 9 months in order to assess time-dependent variation in radiomic features. The results indicated that there was a significant difference in radiomic features derived from different lung segments. Visualisation of PC scores highlighted differences between caudodorsal and cranioventral lung, between lobar and sub-lobar segments, and suggested a bias towards one lung or the other. Significant changes in PC scores occurred over time. With few exceptions, largely similar changes occurred across all segments in this regard.DiscussionOverall, our results indicate that although sheep lung radiomic features are influenced by the lung segment of origin, their variation over time is largely consistent throughout the lung. Such influence should be borne in mind when interpreting radiomic features and their changes over time.

CLN1 disease is a fatal neurodegenerative condition caused by deficiency in palmitoyl-protein thioesterase 1 (PPT1), for which no disease-modifying therapy exists. The disease affects the entire central nervous system (CNS), necessitating widespread delivery of therapeutics to the brain and spinal cord. Adeno-associated virus (AAV)-based PPT1 gene therapy delivered intrathecally has been tested in mouse models but has shown limited efficacy due to inadequate brain bioavailability. Here, to maximize therapeutic benefit, PPT1 was engineered for improved cross-correction capabilities, packaged in Spark100, a neurotropic AAV capsid, and administered through intracerebroventricular route in neonatal Ppt1-/- mice. This achieved sustained expression of PPT1 protein across the CNS, including key disease-relevant structures, for up to 15 months. It resulted in long-term therapeutic benefits, such as extended lifespan, preserved neurobehavioral function, and prevention of neuropathology, making treated Ppt1-/- mice nearly indistinguishable from wild type. A translatability study in healthy adult sheep, assessing biodistribution of therapeutic in a large and fully developed brain, showed widespread CNS transduction and PPT1 expression with no adverse effects. These studies demonstrate the potential of this approach for treating CLN1 disease and suggest that a similar platform, using a secreted therapeutic protein, might apply to other neurological disorders with broad CNS deficits.

Radiomic feature (RF) analysis of computed tomography (CT) images may aid the diagnosis and staging of ovine pulmonary adenocarcinoma (OPA). We assessed the RF characteristics of OPA tumours in JSRV-infected sheep compared to non-tumour lung tissues, examined their stability over time, and analysed RF variations in the nascent tumour field (NTF) and nascent tumour margin field (NTmF). In monthly CT scans, lung tissues were automatically segmented by density, and lung tumours were manually segmented. RFs were calculated for each imaging session, selected according to stability and reproducibility, and adjusted for volume dependence where appropriate. Comparisons between scans within sheep were facilitated through fiducial registration and spatial transformations. Initially, 9/36 RFs differed significantly from non-tumour lung tissue of similar density. Predominant RF changes included ngtdm_Complexity, glrlm_RunLNUnif_VN, and gldm_SmDHGLE. RFs in lung tumour segments showed time-dependent changes, whereas non-tumour lung tissue of similar density remained consistent. OPA lung tumour RF characteristics are distinct from those of other lung tissues of similar density and evolve as the tumour develops. Such characteristics suggest that radiomic analysis offers potential for the early detection and management of JSRV-related lung tumours. This research enhances the understanding of OPA imaging, potentially informing better diagnosis and control measures for naturally occurring infections.

[This corrects the article DOI: 10.3389/fvets.2020.00439.].

While the term biomarker is thought to have first been used in the 1970s, the concept itself is considered to be much older [...].

Ovine pulmonary adenocarcinoma (OPA) is an infectious, neoplastic lung disease of sheep that causes significant animal welfare and economic issues throughout the world. Understanding OPA pathogenesis is key to developing tools to control its impact. Central to this need is the availability of model systems that can monitor and track events after Jaagsiekte sheep retrovirus (JSRV) infection. Here, we report the development of an experimentally induced OPA model intended for this purpose. Using three different viral dose groups (low, intermediate and high), localised OPA tumour development was induced by bronchoscopic JSRV instillation into the segmental bronchus of the right cardiac lung lobe. Pre-clinical OPA diagnosis and tumour progression were monitored by monthly computed tomography (CT) imaging and trans-thoracic ultrasound scanning. Post mortem examination and immunohistochemistry confirmed OPA development in 89% of the JSRV-instilled animals. All three viral doses produced a range of OPA lesion types, including microscopic disease and gross tumours; however, larger lesions were more frequently identified in the low and intermediate viral groups. Overall, 31% of JSRV-infected sheep developed localised advanced lesions. Of the sheep that developed localised advanced lesions, tumour volume doubling times (calculated using thoracic CT 3D reconstructions) were 14.8 ± 2.1 days. The ability of ultrasound to track tumour development was compared against CT; the results indicated a strong significant association between paired CT and ultrasound measurements at each time point (R2 = 0.799, p < 0.0001). We believe that the range of OPA lesion types induced by this model replicates aspects of naturally occurring disease and will improve OPA research by providing novel insights into JSRV infectivity and OPA disease progression.

Bovine natural killer (bNK) cells are heterogeneous cell populations defined by constitutive expression of the natural cytotoxicity receptor, NKp46 (CD335). Two major subsets of bNK cells, classified by differential expression of CD2, display divergent functions in innate immunity, and are hypothesised to contribute to adaptive immunity following vaccination. Here we characterised phenotypic variation of bNK cells within afferent lymph and lymph node (LN) tissues and between CD2+ and CD2- bNK subsets, and report phenotypic changes induced by BCG vaccination. CD2- bNK cells, which dominate in the afferent lymph and LN, displayed lower expression of the activation marker CD25 within the LN, with CD25+ cells being less than half as frequent as in afferent lymph. Furthermore, we found bNK cells had a lower expression of CD45RB, associated in cattle with naïve cell status, within LN compared to afferent lymph. Following BCG vaccination, bNK cells in afferent lymph draining the vaccination site showed increased CD2-CD25+ frequencies and increased expression of CD25 on CD2+ bNK cells, although the frequency of these cells remained unchanged. In summary, we provide an overview of the phenotype of bNK cells within bovine lymphatic tissues, and provide an indication of how subsets may diverge following BCG vaccination.

Supplementary Figure from Integrated DNA and RNA Sequencing Reveals Drivers of Endocrine Resistance in Estrogen Receptor–Positive Breast Cancer

EDITORIAL article Front. Vet. Sci., 15 November 2022Sec. Comparative and Clinical Medicine https://doi.org/10.3389/fvets.2022.1086912

The development of robust implantable sensors is important in the successful advancement of personalised medicine as they have the potential to provide in situ real-time data regarding the status of health and disease and the effectiveness of treatment. Tissue pH is a key physiological parameter and herein, we report the design, fabrication, functionalisation, encapsulation and protection of a miniaturised, self-contained, electrochemical pH sensor system and characterisation of sensor performance. Notably for the first time in this environment the pH sensor was based on a methylene blue redox reporter which showed remarkable robustness, accuracy and sensitivity. This was achieved by encapsulation of a self-assembled monolayer containing methylene blue entrapped within a Nafion layer. Another powerful feature was the incorporation, within the same implanted device, of a fabricated on-chip Ag/AgCl reference electrode - vital in any electrochemical sensor, but often ignored. When utilised in vivo, the sensor allowed accurate tracking of externally induced pH changes within a naturally occurring ovine lung cancer model, and correlated well with single point laboratory measurements made on extracted arterial blood, whilst enabling in vivo time-dependent measurements. The sensors functioned robustly whilst implanted, and maintained in vitro function once extracted and together, these results demon-strate proof-of-concept of the ability to sense real-time intratumoral tissue pH changes in vivo.

Abstract Purpose: Endocrine therapy resistance (ETR) remains the greatest challenge in treating patients with hormone receptor–positive breast cancer. We set out to identify molecular mechanisms underlying ETR through in-depth genomic analysis of breast tumors. Experimental Design: We collected pre-treatment and sequential on-treatment tumor samples from 35 patients with estrogen receptor–positive breast cancer treated with neoadjuvant then adjuvant endocrine therapy; 3 had intrinsic resistance, 19 acquired resistance, and 13 remained sensitive. Response was determined by changes in tumor volume neoadjuvantly and by monitoring for adjuvant recurrence. Twelve patients received two or more lines of endocrine therapy, with subsequent treatment lines being initiated at the time of development of resistance to the previous endocrine therapy. DNA whole-exome sequencing and RNA sequencing were performed on all samples, totalling 169 unique specimens. DNA mutations, copy-number alterations, and gene expression data were analyzed through unsupervised and supervised analyses to identify molecular features related to ETR. Results: Mutations enriched in ETR included ESR1 and GATA3. The known ESR1 D538G variant conferring ETR was identified, as was a rarer E380Q variant that confers endocrine hypersensitivity. Resistant tumors which acquired resistance had distinct gene expression profiles compared with paired sensitive tumors, showing elevated pathways including ER, HER2, GATA3, AKT, RAS, and p63 signaling. Integrated analysis in individual patients highlighted the diversity of ETR mechanisms. Conclusions: The mechanisms underlying ETR are multiple and characterized by diverse changes in both somatic genetic and transcriptomic profiles; to overcome resistance will require an individualized approach utilizing genomic and genetic biomarkers and drugs tailored to each patient.

CLN1 disease, also called infantile neuronal ceroid lipofuscinosis (NCL) or infantile Batten disease, is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by administering monthly infusions of recombinant human PPT1 (rhPPT1) to PPT1-deficient mice (Cln1–/–) and CLN1R151X sheep to assess how to potentially scale up for translation. In Cln1–/– mice, intracerebrovascular (i.c.v.) rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1-treated mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1R151X sheep, i.c.v. infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural MRI and neuropathology. This study demonstrates the feasibility and therapeutic efficacy of i.c.v. rhPPT1 ERT. These findings represent a key step toward clinical testing of ERT in children with CLN1 disease and highlight the importance of a cross-species approach to developing a successful treatment strategy.

The bovine afferent lymphatic cannulation model allows collection of large volumes of afferent lymph and provides an opportunity to study lymphatic cells trafficking from the periphery directly ex-vivo. The technique requires surgical intervention, but influence of the procedure or time post-surgery on cells trafficking in the lymph has not been well documented. Here, we measured the volume of lymph and number of cells/mL collected daily over a two week time-course. Animal to animal variability was demonstrated but no consistent changes in lymph volume or cell density were observed in relation to time post-cannulation. Cell populations (dendritic cells, αβ T-cells, γδ T-cells and NK cells) were analysed by flow cytometry at 1, 3 and 10 days post-cannulation (DPC) and a reduced percentage of γδ T-cells in afferent lymph was observed at 1 DPC. In addition, cell surface molecule expression by afferent lymphatic dendritic cells (ALDC) was assessed due to the key role of these cells in initiating an adaptive immune response. Co-stimulatory molecules CD80 and CD86 were upregulated by CD172a+ve ALDC early in the time-course, suggesting that the cannulation procedure and duration of experiment may impact the activation state of DCs in the naïve host. This should be considered when analysing the response of these cells to vaccines or pathogens.

IL6-like cytokines are a family of regulators with a complex, pleiotropic role in both the healthy organism, where they regulate immunity and homeostasis, and in different diseases, including cancer. Here we summarise how these cytokines exert their effect through the shared signal transducer IL6ST (gp130) and we review the extensive evidence on the role that different members of this family play in breast cancer. Additionally, we discuss how the different cytokines, their related receptors and downstream effectors, as well as specific polymorphisms in these molecules, can serve as predictive or prognostic biomarkers with the potential for clinical application in breast cancer. Lastly, we also discuss how our increasing understanding of this complex signalling axis presents promising opportunities for the development or repurposing of therapeutic strategies against cancer and, specifically, breast neoplasms.

The immense costs in both financial terms and preclinical research effort that occur in the development of anticancer drugs are unfortunately not matched by a substantial increase in improved clinical therapies due to the high rate of failure during clinical trials. This may be due to issues with toxicity or lack of clinical effectiveness when the drug is evaluated in patients. Currently, much cancer research is driven by the need to develop therapies that can exploit cancer cell adaptations to conditions in the tumor microenvironment such as acidosis and hypoxia, the requirement for more-specific, targeted treatments, or the exploitation of 'precision medicine' that can target known genomic changes in patient DNA. The high attrition rate for novel anticancer therapies suggests that the preclinical methods used in screening anticancer drugs need improvement. This chapter considers the advantages and disadvantages of 3D organotypic models in both cancer research and cancer drug screening, particularly in the areas of targeted drugs and the exploitation of genomic changes that can be used for therapeutic advantage in precision medicine.

Novel biomarkers are needed to continue to improve breast cancer clinical management and outcome. IL6-like cytokines, whose pleiotropic functions include roles in many hallmarks of malignancy, rely on the signal transducer IL6ST (gp130) for all their signalling. To date, 10 separate independent studies based on the analysis of clinical breast cancer samples have identified IL6ST as a predictor. Consistent findings suggest that IL6ST is a positive prognostic factor and is associated with ER status. Interestingly, these studies include 4 multigene signatures (EndoPredict, EER4, IRSN-23 and 42GC) that incorporate IL6ST to predict risk of recurrence or outcome from endocrine or chemotherapy. Here we review the existing evidence on the promising predictive and prognostic value of IL6ST. We also discuss how this potential could be further translated into clinical practice beyond the EndoPredict tool, which is already available in the clinic. The most promising route to further exploit IL6ST's promising predicting power will likely be through additional hybrid multifactor signatures that allow for more robust stratification of ER+ breast tumours into discrete groups with distinct outcomes, thus enabling greater refinement of the treatment-selection process.

Development of an anastomotic leak (AL) following intestinal surgery for the treatment of colorectal cancers is a life-threatening complication. Failure of the anastomosis to heal correctly can lead to contamination of the abdomen with intestinal contents and the development of peritonitis. The additional care that these patients require is associated with longer hospitalisation stays and increased economic costs. Patients also have higher morbidity and mortality rates and poorer oncological prognosis. Unfortunately, current practices for AL diagnosis are non-specific, which may delay diagnosis and have a negative impact on patient outcome. To overcome these issues, research is continuing to identify AL diagnostic or predictive biomarkers. In this review, we highlight promising candidate biomarkers including ischaemic metabolites, inflammatory markers and bacteria. Although research has focused on the use of blood or peritoneal fluid samples, we describe the use of implantable medical devices that have been designed to measure biomarkers in peri-anastomotic tissue. Biomarkers that can be used in conjunction with clinical status, routine haematological and biochemical analysis and imaging have the potential to help to deliver a precision medicine package that could significantly enhance a patient's post-operative care and improve outcomes. Although no AL biomarker has yet been validated in large-scale clinical trials, there is confidence that personalised medicine, through biomarker analysis, could be realised for colorectal cancer intestinal resection and anastomosis patients in the years to come.