Combined colour deconvolution and artificial intelligence approach for region-selective immunohistochemical labelling quantification: The example of alpha smooth muscle actin in mouse kidney.

Immunohistochemical (IHC) localisation of protein expression is a widely used tool in pathology. This is semi-quantitative and exhibits substantial intra- and inter-observer variability. Digital approaches based on stain quantification applied to IHC are precise but still operator-dependent and time-consuming when regions of interest (ROIs) must be defined to quantify protein expression in a specific tissue area. This study aimed at developing an IHC quantification workflow that benefits from colour deconvolution for stain quantification and artificial intelligence for automatic ROI definition. The method was tested on 10 whole slide images (WSI) of alpha-smooth muscle actin (aSMA) stained mouse kidney sections. The task was to identify aSMA-positive areas within the glomeruli automatically. Total aSMA detection was performed using two channels (DAB, haematoxylin) colour deconvolution. Glomeruli segmentation within the same IHC WSI was performed by training a convolutional neural network with annotated examples of glomeruli. For both aSMA and glomeruli, binary masks were created. Co-localisation was performed by overlaying the masks and assigning red/green colours, with yellow indicative of a co-localised signal. The workflow described and exemplified using the case of aSMA expression in glomeruli can be applied to quantify the expression of IHC markers within different structures of immunohistochemically stained slides. The technique is objective, has a fully automated threshold approach (colour deconvolution phase) and uses AI to eliminate operator-dependent steps.

Mycoplasma bradburyae sp. nov. isolated from the trachea of sea birds.

In the search for mollicutes in wild birds, six Mycoplasma strains were isolated from tracheal swabs taken from four different species of seabirds. Four strains originated from three Yellow-legged gulls (Larus michahellis) and a Cory's shearwater (Calonectris borealis) from Spain, one from a South African Kelp gull (Larus dominicanus), and one from an Italian Black-headed gull (Chroicocephalus ridibundus). These Mycoplasma strains presented 99 % 16S rRNA gene sequence similarity values with Mycoplasma (M.) gallisepticum. Phylogenetic analyses of marker genes (16S rRNA gene and rpoB) confirmed the close relationship of the strains to M. gallisepticum and M. tullyi. The seabirds' strains grew well in modified Hayflick medium, and colonies showed typical fried egg morphology. They produced acid from glucose and mannose but did not hydrolyze arginine or urea. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas, presenting spherical to flask-shaped cells with an attachment organelle. Gliding motility was also observed. Furthermore, serological tests, MALDI-ToF mass spectrometry and genomic studies demonstrated that the strains were different to any known Mycoplasma species, for which the name Mycoplasma bradburyae sp. nov. is proposed; the type strain is T158T (DSM 110708 = NCTC 14398).

Histological Evaluation of Resected Tissue as a Predictor of Survival in Horses with Strangulating Small Intestinal Disease.

Strangulating small intestinal disease (SSID) in horses carries a poor prognosis for survival, especially following resection of ischaemic tissue. The margins of a resection are principally based on visual appraisal of the intestine during surgery. We hypothesized that histological evaluation of resected tissue may identify occult changes indicative of prognosis. Small intestinal samples from 18 horses undergoing resection for SSID and 9 horses euthanised for reasons unrelated to gastrointestinal pathology were utilised. Histological appearance was used to generate a 'total damage score' (TDS) for the control tissue, grossly normal tissue at oral and aboral extremities (sections OR1 and AB1) of the resected intestine, and oral and aboral extremities of visually abnormal tissue (sections OR2 and AB2) from SSID horses. The relationship between TDS and long-term post-operative survival was investigated. TDS was not different between control tissues and OR1 and AB1 sections. Five surgical cases were alive at follow-up, the longest follow-up time being 2561 days. Based on the median scores for SSID cases versus controls, cut-off values were generated to evaluate post-operative survival versus TDS. Only OR2 TDS was significantly associated with survival, with a higher (worse) score indicating longer survival. More severe tissue insult may expedite rapid progression to surgery, improving post-operative outcomes.

Quantifying acute kidney injury in an Ischaemia-Reperfusion Injury mouse model using deep-learning-based semantic segmentation in histology.

This study focuses on ischaemia-reperfusion injury (IRI) in kidneys, a cause of acute kidney injury (AKI) and end-stage kidney disease (ESKD). Traditional kidney damage assessment methods are semi-quantitative and subjective. This study aims to use a convolutional neural network (CNN) to segment murine kidney structures after IRI, quantify damage via CNN-generated pathological measurements, and compare this to conventional scoring. The CNN was able to accurately segment the different pathological classes, such as Intratubular casts and Tubular necrosis, with an F1 score of over 0.75. Some classes, such as Glomeruli and Proximal tubules, had even higher statistical values with F1 scores over 0.90. The scoring generated based on the segmentation approach statistically correlated with the semiquantitative assessment (Spearman's rank correlation coefficient=0.94). The heatmap approach localised the intratubular necrosis mainly in the outer stripe of the outer medulla, while the tubular casts were also present in more superficial or deeper portions of the cortex and medullary areas. This study presents a CNN model capable of segmenting multiple classes of interest, including acute IRI-specific pathological changes, in a whole mouse kidney section and can provide insights into the distribution of pathological classes within the whole mouse kidney section.

TNF-α induced extracellular release of keratinocyte high-mobility group box 1 in Stevens-Johnson syndrome/toxic epidermal necrolysis: Biomarker and putative mechanism of pathogenesis.

Decreased epidermal high-mobility group box 1 (HMGB1) expression is an early marker of epidermal injury in Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Etanercept, an anti-tumor necrosis factor therapeutic, is effective in the treatment of SJS/TEN. The objective was to characterize antitumor necrosis factor-alpha (TNF-α)-mediated HMGB1 keratinocyte/epidermal release and etanercept modulation. HMGB1 release from TNF-α treated (± etanercept), or doxycycline-inducible RIPK3 or Bak-expressing human keratinocyte cells (HaCaTs) was determined by western blot/ELISA. Healthy skin explants were treated with TNF-α or serum (1:10 dilution) from immune checkpoint inhibitor-tolerant, lichenoid dermatitis or SJS/TEN patients ± etanercept. Histological and immunohistochemical analysis of HMGB1 was undertaken. TNF-α induced HMGB1 release in vitro via both necroptosis and apoptosis. Exposure of skin explants to TNF-α or SJS/TEN serum resulted in significant epidermal toxicity/detachment with substantial HMGB1 release which was attenuated by etanercept. Whole-slide image analysis of biopsies demonstrated significantly lower epidermal HMGB1 in pre-blistered SJS/TEN versus control (P < 0.05). Keratinocyte HMGB1 release, predominantly caused by necroptosis, can be attenuated by etanercept. Although TNF-α is a key mediator of epidermal HMGB1 release, other cytokines/cytotoxic proteins also contribute. Skin explant models represent a potential model of SJS/TEN that could be utilized for further mechanistic studies and targeted therapy screening.

Case report: Severe, refractory hypoglycemia in a 9-year-old Brittany Spaniel with renal nephroblastoma.

A 9-year-old female spayed Brittany Spaniel presented for weakness and stumbling, and was diagnosed with severe hypoglycemia. An insulin to glucose ratio was not consistent with insulinoma as a cause for hypoglycemia. Diagnostic imaging (abdominal ultrasound and computed tomography) revealed a large left renal mass and a possible metastatic lesion in the right kidney. Glucagon therapy was initiated, but hypoglycemia was refractory to therapy. A left nephrectomy was performed and hypoglycemia subsequently resolved. Histopathology of the mass was consistent with nephroblastoma and immunohistochemistry for anti-insulin-like Growth Factor-2 (IGF-2) antibody revealed immunoreactivity in over 50% of the neoplastic cells. Chemotherapeutic treatment was initiated with a combined protocol of vincristine and doxorubicin. To the authors' knowledge, this is the first case report documenting the treatment of severe, refractory non-islet cell tumor-induced hypoglycemia in a dog, suspected to be secondary to an IGF-2 secreting nephroblastoma.

Metabolic changes in glioblastomas in response to choline kinase inhibition: In vivo MRS in rodent models.

Changes in glioblastoma (GBM) metabolism was investigated in response to JAS239, a choline kinase inhibitor, using MRS. In addition to the inhibition of phosphocholine synthesis, we investigated changes in other key metabolic pathways associated with GBM progression and treatment response. Three syngeneic rodent models of GBM were used: F98 (N = 12) and 9L (N = 8) models in rats and GL261 (N = 10) in mice. Rodents were intracranially injected with GBM cells in the right cortex and tumor growth was monitored using T2 -weighted images. Animals were treated once daily with intraperitoneal injections of 4 mg/kg JAS239 (F98 rats, n = 6; 9L rats, n = 6; GL261 mice, n = 5) or saline (control group, F98 rats, n = 6; 9L rats, n = 2; GL261 mice, n = 5) for five consecutive days. Single voxel spectra were acquired on Days 0 (T0, baseline) and 6 (T6, end of treatment) from the tumor as well as the contralateral normal brain using a PRESS sequence. Changes in metabolite ratios (tCho/tCr, tCho/NAA, mI/tCr, Glx/tCr and (Lip + Lac)/Cr) were used to assess metabolic pathway alterations in response to JAS239. Tumor growth arrest was noted in all models in response to JAS239 treatment compared with saline-treated animals, with a significant reduction (p < 0.05) in the F98 model. A reduction in tCho/tCr was observed with JAS239 treatment in all GBM models, indicating reduced phospholipid metabolism, with the highest reduction in 9L followed by GL261 and F98 tumors. A significant reduction (p < 0.05) in the tCho/NAA ratio was observed in the 9L model. A significant reduction in mI/tCr (p < 0.05) was found in JAS239-treated F98 tumors compared with the saline-treated animals. A non-significant trend of reduction in Glx/tCr was observed only in F98 and 9L tumors. JAS239-treated F98 tumors also showed a significant increase in Lip + Lac (p < 0.05), indicating increased cell death. This study demonstrated the utility of MRS in assessing metabolic changes in GBM in response to choline kinase inhibition.

TNF-α‒Mediated Keratinocyte Expression and Release of Matrix Metalloproteinase 9: Putative Mechanism of Pathogenesis in Stevens‒Johnson Syndrome/Toxic Epidermal Necrolysis.

Stevens‒Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are severe cutaneous adverse drug reactions characterized by widespread keratinocyte cell death and epidermal detachment. At present, there is little understanding of how the detachment occurs or how it is abrogated by the TNF-α inhibitor etanercept, an effective SJS/TEN treatment. RNA sequencing was used to identify upregulated transcripts in formalin-fixed paraffin-embedded SJS/TEN skin biopsies. Epidermal matrix metalloproteinase 9 (MMP9) expression was assessed by immunohistochemistry in skin biopsies and cultured human skin explants exposed to serum from patients with cutaneous adverse drug reactions. TNF-α‒induced MMP9 expression and activity and its abrogation by etanercept were determined using the HaCaT immortalized keratinocyte cell line. Epidermal MMP9 expression was significantly higher in SJS/TEN skin (70.6%) than in healthy control skin (0%) (P = 0.0098) and nonbullous skin reactions (10.7%) (P = 0.0002). SJS/TEN serum induced significant MMP9 expression and collagenase activity in healthy skin explants, which was reduced by etanercept. Etanercept was also able to negate the TNF-α‒induced MMP9 expression in the HaCaT cell line. Data suggest that elevated epidermal MMP9 expression and collagenase activity are a putative pathogenic mechanism in SJS/TEN, which is limited by etanercept. Modulation of MMP9 expression and activity represents, to our knowledge, a previously unreported therapeutic target for the treatment of SJS/TEN.

Use of deep learning for the classification of hyperplastic lymph node and common subtypes of canine lymphomas: a preliminary study.

Artificial Intelligence has observed significant growth in its ability to classify different types of tumors in humans due to advancements in digital pathology technology. Among these tumors, lymphomas are quite common in dogs, despite studies on the application of AI in domestic species are scarce. This research aims to employ deep learning (DL) through convolutional neural networks (CNNs) to distinguish between normal lymph nodes and 3 WHO common subtypes of canine lymphomas. To train and validate the CNN, 1,530 high-resolution microscopic images derived from whole slide scans (WSIs) were used, including those of background areas, hyperplastic lymph nodes (n = 4), and three different lymphoma subtypes: diffuse large B cell lymphoma (DLBCL; n = 5), lymphoblastic (LBL; n = 5), and marginal zone lymphoma (MZL; n = 3). The CNN was able to correctly identify 456 images of the possible 457 test sets, achieving a maximum accuracy of 99.34%. The results of this study have demonstrated the feasibility of using deep learning to differentiate between hyperplastic lymph nodes and lymphomas, as well as to classify common WHO subtypes. Further research is required to explore the implications of these findings and validate the ability of the network to classify a broader range of lymphomas.

Optical Tissue Clearing to Study the Intra-Pulmonary Biodistribution of Intravenously Delivered Mesenchymal Stromal Cells and Their Interactions with Host Lung Cells.

Mesenchymal stromal cells (MSCs) injected intravenously are trapped in the capillaries of the lungs and die within the first 24 h. Studying the biodistribution and fate of labelled therapeutic cells in the 3D pulmonary context is important to understand their function in this organ and gain insights into their mechanisms of action. Optical tissue clearing enables volumetric cell tracking at single-cell resolution. Thus, we compared three optical tissue-clearing protocols (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC), modified stabilised 3D imaging of solvent-cleared organs (s-DISCO) and ethyl cinnamate (ECi)) to evaluate their potential to track the biodistribution of human umbilical cord MSCs expressing the tdTomato fluorescence reporter and investigate how they interact with host cells in the mouse lung. The results showed that although CUBIC clearing is the only method that enables direct imaging of fluorescently labelled MSCs, combining s-DISCO or ECi with immunofluorescence or dye labelling allows the interaction of MSCs with endothelial and immune cells to be studied. Overall, this comparative study offers guidance on selecting an optical tissue-clearing method for cell tracking applications.

Quantitative histologic evaluation reveals different degree of liver atrophy in cachectic and starved dogs.

Cases of neglect in dogs are among the forensic cases submitted most commonly for postmortem examination. Starvation is a form of primary protein-energy malnutrition in which the availability of food is severely restricted or absent; cachexia is a form of protein-energy malnutrition secondary to progressive metabolic derangement during chronic diseases. Despite both conditions leading to an emaciated appearance of the cadaver, discrimination between the two is crucial in forensic cases. We hypothesized that among emaciated dogs, the degree of liver atrophy in starved animals is higher than in cachectic ones, and that this can be investigated microscopically, regardless of the degree of cadaver decomposition. We studied 46 animals: 23 starved, 11 cachectic, and 12 control dogs. Portal tracts were identified by the presence of a bile duct and associated vascular structures recognizable by a thin rim of collagen still visible regardless of the degree of cadaver decomposition. The number of portal tracts per lpf (10×) was used as an indirect measure of atrophy. The number of portal tracts in starved dogs was significantly higher (p < 0.01) compared to both cachectic and control dogs, indicating a higher degree of liver atrophy in starvation. Measuring the density of portal tracts offers a reliable additional tool for discrimination between starvation and cachexia.

Expression of cannabinoid receptors CB1 and CB2 in canine cutaneous mast cell tumours.

Cannabinoid receptors (CB1 and CB2) belong to endocannabinoid system (ECS), which is also composed from endocannabinoids and the enzymatic systems involved in their biosynthesis and degradation. The expression of CB1 and CB2 have been previously identified in normal canine mast cell and in atopic dermatitis. Canine cutaneous mast cell tumours (cMCTs) are among the most common cutaneous neoplasms in dogs and have a highly variable clinical behaviour. Expression of CB1-CB2 was assessed by means of immunohistochemistry in thirty-seven dogs (from 2019 to 2021) with proven histological diagnosis of cMCT. Dogs were divided in two groups according to the Kiupel's grading system: high-grade (HG) cMCT and low-grade (LG) cMCT. A semiquantitative (score 0-3) and quantitative assessment of immunoreactivity (IR) was performed for each case. Our results show that there CB1 and CB2 are highly expressed in LG- cMCT, in contrast to HG- cMCT.

Pulmonary bleeding in racehorses: A gross, histologic, and ultrastructural comparison of exercise-induced pulmonary hemorrhage and exercise-associated fatal pulmonary hemorrhage.

Exercise-induced pulmonary hemorrhage (EIPH) is a common condition of Thoroughbred racehorses that is usually responsible for reduced performance, while exercise-associated fatal pulmonary hemorrhage (EAFPH) is characterized by severe pulmonary bleeding of unknown pathogenesis resulting in sudden death during strenuous exercise. The aim of the study was to characterize and compare anamnestic data together with pulmonary gross, histologic, and ultrastructural findings in racehorses with EIPH (n = 10), EAFPH (n = 10), and control horses (n = 5). No differences in anamnesis were identified between the 3 groups. Grossly cranial lobe reddening and edema scores were significantly more prevalent and severe in the EAFPH group compared with the EIPH and control groups. Histologically, hemorrhage scores were higher in the EAFPH group, while hemosiderophages, iron encrustations of collagen and elastin fibers, and vascular remodeling scores were significantly higher in EIPH group compared with the EAFPH and control groups. In all groups, caudal lung locations exhibited a significantly higher score for vascular remodeling, hemosiderophage accumulation, iron encrustation, and type II pneumocyte hyperplasia when compared with cranial, dorsal, and ventral locations. Ultrastructural analysis of perivascular collagen showed fibrils with significantly larger diameters in the EAFPH group compared with the EIPH group but not compared with the control group. This study demonstrates that lungs of horses that experienced EAFPH show significantly less vascular remodeling and other long-term pulmonary abnormalities that characterize horses with EIPH.

Murine models of renal ischemia reperfusion injury: An opportunity for refinement using noninvasive monitoring methods.

BACKGROUND: Renal ischemia reperfusion injury (R-IRI) can cause acute kidney injury (AKI) and chronic kidney disease (CKD), resulting in significant morbidity and mortality. To understand the underlying mechanisms, reproducible small-animal models of AKI and CKD are needed. We describe how innovative technologies for measuring kidney function noninvasively in small rodents allow successful refinement of the R-IRI models, and offer the unique opportunity to monitor longitudinally in individual animals the transition from AKI to CKD. METHODS: Male BALB/c mice underwent bilateral renal pedicle clamping (AKI) or unilateral renal pedicle clamping with delayed contralateral nephrectomy (CKD) under isoflurane anesthetic. Transdermal GFR monitoring and multispectral optoacoustic tomography (MSOT) in combination with statistical analysis were used to identify and standardize variables within these models. RESULTS: Pre-clamping anesthetic time was one of the most important predictors of AKI severity after R-IRI. Standardizing pre-clamping time resulted in a more predictably severe AKI model. In the CKD model, MSOT demonstrated initial improvement in renal function, followed by significant progressive reduction in function between weeks 2 and 4. Performing contralateral nephrectomy on day 14 enabled the development of CKD with minimal mortality. CONCLUSIONS: Noninvasive monitoring of global and individual renal function after R-IRI is feasible and reproducible. These techniques can facilitate refinement of kidney injury models and enable the degree of injury seen in preclinical models to be translated to those seen in the clinical setting. Thus, future therapies can be tested in a clinically relevant, noninvasive manner.

A text-mining based analysis of 100,000 tumours affecting dogs and cats in the United Kingdom.

Cancer is a major reason for veterinary consultation, especially in companion animals. Cancer surveillance plays a key role in prevention but opportunities for such surveillance in companion animals are limited by the lack of suitable veterinary population health infrastructures. In this paper we describe a pathology-based animal tumour registry (PTR) developed within the Small Animal Veterinary Surveillance Network (SAVSNET) built from electronic pathology records (EPR) submitted to this network. From an original collection of 180232 free text (non-structured) EPRs reported between April 2018 and June 2019, we used specific text-mining methodologies to identify 109895 neoplasias. These data were normalized to describe both the tumour (type and location) and the animal (breed, neutering status and veterinary practice postcode). The resulting PTR, the largest of its kind for companion animals to date, is an important research resource being able to facilitate a wide array of research in areas including surveillance, clinical decision making and comparative cancer biology.

Comparison of effusion cell block and biopsy immunohistochemistry in mesothelial hyperplasia, mesothelioma, and carcinoma in dogs.

BACKGROUND: Determining the cause of effusions is challenging and might require a biopsy. Whether cell blocks from effusions are representative of biopsies requires investigation. A previously developed immunohistochemical panel aids in the differentiation of hyperplastic and neoplastic mesothelium in canine biopsies but has not been investigated in effusions. OBJECTIVES: The study aimed to assess cell blocks as an alternative to biopsies and determine whether immunohistochemistry helps distinguish hyperplastic mesothelium, mesothelioma, and carcinoma. METHODS: Effusions and biopsies were collected from five dogs with mesothelial hyperplasia (group MH), six with mesothelioma (group M), and five with carcinoma (group C). Immunohistochemistry (IHC) for cytokeratin, vimentin, Wilm's tumor protein 1 (WT1), desmin, glucose transporter 1 (GLUT1), and insulin-like growth factor II mRNA-binding protein 3 (IMP3) was performed. Sections were scored for staining intensity and the percentage of positively stained cells. RESULTS: In paired cell blocks and biopsies, vimentin and WT1 staining were positively correlated for intensity and the percentage of positive cells, although not all paired results were identical. The intensity of IMP3 staining in cell blocks was higher in group M than in group C (P = 0.012), and WT1 staining was higher in group MH than in group C (P = 0.020). For biopsies, the intensity of WT1 staining was higher in group MH than in group C (P = 0.031). In group C, WT1 was negative in all cell blocks and biopsies, and desmin was negative in four of five cases. CONCLUSIONS: IHC results for the cell blocks and biopsies were comparable for potentially useful markers, such as WT1, which helped discriminate between groups. IHC provided additional information, although results were not always definitive. Further studies on a larger population are required.

T cell mediated hypersensitivity to previously tolerated iodinated contrast media precipitated by introduction of atezolizumab.

Many adverse reactions associated with immune checkpoint inhibitor (ICI) treatments are immunologically driven and may necessitate discontinuation of the ICI. Herein, we present a patient who had been administered the radio contrast media amidotrizoate multiple times without issue but who then developed a Stevens-Johnson syndrome reaction after coadministration of atezolizumab. Causality was confirmed by a positive re-challenge with amidotrizoate and laboratory investigations that implicated T cells. Importantly, the introduction of atezolizumab appears to have altered the immunologic response to amidotrizoate in terms of the tolerance-elicitation continuum. Proof of concept studies demonstrated enhancement of recall responses to a surrogate antigen panel following in-vitro (healthy donors) and in-vivo (ICI patients) administrations of ICIs. Our findings highlight the importance of considering all concomitant medications in patients on ICIs who develop immune-mediated adverse reactions. In the event of some immune-related adverse reactions, it may be critical to identify the culprit antigen-forming entity that the ICIs have altered the perception of rather than simply attribute causality to the ICI itself in order to optimize both patient safety and treatment of malignancies.

Immunohistochemical expression and prognostic significance of MAGE-A in canine oral malignant melanoma.

Canine oral malignant melanoma (COMM) is considered a chemo-resistant cancer with a poor long-term prognosis. The melanoma-associated antigen A (MAGE-A) genes, which belong to the cancer-testis antigen family, are expressed in several different canine cancers but not in normal somatic tissue. This study evaluates the expression of MAGE-A proteins and their prognostic role in COMM. The study was conducted in 2 parts. During the first part, biopsies from oral malignant melanomas from 43 dogs were examined and immunohistochemically assessed for expression of MAGE-A proteins. For the second part, the association between MAGE-A expression and outcome was assessed using follow-up data which was available for 20 dogs whose primary tumour had been controlled with surgery +/- radiation therapy. MAGE-A proteins were expressed in 88.4% (38/43) of oral malignant melanomas and had a predominantly cytoplasmic expression pattern. Immunopositivity was observed in more than 50% of the cells in 21 dogs (48.8%). Immunostaining intensity was classified as weak, moderate and intense in 16 (37%), 16 (37%) and 6 (14%) cases, respectively. No staining for MAGE-A was seen in 5 dogs (11%). Dogs whose COMM had weak MAGE-A staining intensity had a median survival time (MST) of 320 days while this was 129 days for dogs with moderate and intense immunostaining (p = 0.161). Dogs whose COMM had >50% of positive staining neoplastic cells had an MST of 141 days and dogs with a staining <50% had an MST of 320 days (p = 0.164). MAGE-A expression did not influence survival in our cohort.